FINE ANTIGENIC SPECIFICITY OF HUMAN ANTI-DNA AUTOANTIBODIES AGAINST

DNA OF VARYING SIZE AND CONFORMATION

« ^ . i ^ ^ ^

ABSTRACT

Thesis Submitted for the Degree of

Bottor of $I)ilojBiopI)? IN

BIDCHEMISTRY

^ ti. MS'^i ^ 6 \

-,ii KHURSHID ALAM \ < ^ j ^ _ _ . , r ^

DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE

JAWAHARLAL NEHRU MEDICAL COLLEGE ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

1 9 9 2

[ i ]

Antibodies reactive with cellular and nuclear components occur spon­

taneously in patients with systemic lupus erythematosus. The basis for

spontaneous production of anti-native DNA antibodies in systemic lupus

erythematosus remains a puzzling question especially in view of the diffi­

culty in their induction on immunization with DNA in B-conformation.

The anti-dsDNA antibodies seem to be closely related to the disease patho­

genesis through the process of immune complex formation, and their levels

are frequently indicative of the intensity of disease activity. For reasons

that are not best understood, anti-DNA antibodies have always exhibited

polyreactivity with DNA molecules of varying source, size and conformation.

Analysis of plasma DNA from SLE patients have revealed that these

molecules are 100-1000 base pair long and rich in G+C sequences. Such

DNA sequences have the unique potential to undergo B- to Z- or Z-like

transitions under constraint conditions.

In the present study purified native calf thymus DNA was digested

with micrococcal nuclease and fractionated on Sepharose 4B. The approxi­

mate fragment size was determined by standard DNA marker. DNA-(poly-

lysyl-Sepharose ^B) affinity matrix was chosen for the isolation of

homogeneous anti-DNA antibodies from SLE serum. The affinity purified

human anti-dsDNA antibody was found to possess polyreactivity with native

and modified DNA antigens.

Native DNA covalently modified with lysine under UV-A light (320-

UQQ nm) was found to contain one lysine residue per helical turn. By

the photoaddition of one lysine molecule per helical turn of DNA, the

native B-conformation was considerably altered which resulted in the

appearance of neoepitopes leaving much less epitope typical of B~

conformation. Affinity purified anti-dsDNA IgG showed maximum affinity

towards DNA-lysine photoconjugate followed closely by native DNA, The

retention of crossreactivity of affinity purified anti-dsDNA IgG with DNA

-lysine photoadduct strengthen the idea of an alternate antigen for the

induction of antibodies crossreactive with native DNA, Lysine rich histone

[ii]

HI in nucl£03cma thus on nnodification by physical, chemical or environ­

mental agents might form DNA-histone adduct making it imm'jnogenic

and thus generating antibodies crossreactive with native B-conformation.

Hydroxy! radical generated by hydrogen peroxide and ferrous ions

caused single strand breaks in native DNA, cecre?>se in melting tempe.-ature

(Tm) by 6.5 °C and modification of purine and pyrimidine bases. Experi­

mentally induced antibodies against ROS modified DNA exhibited poly-

reactivity, a property commonly associated with SLE anti-DNA antibodies.

Native DNA, RNA and synthetic polynucleotides in B-conformati.^n were

found to be effective inhibitor of induced antibody-immunogen interaction.

The SLE antibody binding with ROS-DNA was better than native DNA,

Similar results v/ere observed in competition-inhibition assay, lecause

of the diminished superoxide dismutase activity in SLE patient: the

excessively generated free radicals could intercict with DNA resulting

in its modification. Cnce the native structure of DNA is modified it

may become immunogenic.

The binding specificity of SLE anti-DNA IgG with varying DNA

conformation was analyzed in polyacrylamide or agarose gel. The altered

electrophoretic mobility in gel retardation assay of A- and B-conformation

complexed with anti-DNA IgG reiterates the antibody binding to these

conformers. The above conformers showed high binding with SLE antibodies

in competition-inhibition experiments.

The E- to Z- transition of poly(dG~dC).poi;(dC-dC) in ' .O M NaC!

was evaluated in terms of "absorption ratio", the average of abcorbance

at 29^ and 296 nm divided by absorbance at 260 nm. This ratio v/as

observed to be 0.322. The high salt induced Z-form of poly(dC-dC). poly

(dG-dC) was stablized by bromination which exhibited Z-DNA characteristics

when brought in physiological buffer. The low salt brominated polyldG-dC;.

poly(dG-dC) exhibited the characteristics of B-DNA. Native DNA under

lov/ or high salt concentration showed "absorbance ratio" greater than

0.3. Under identical conditins the absorbance ratio for small DNA

fragments varied between 0.2 to 0.38, The data indicate that small DNA

fragments have different levels of potentiality to undergo B- to Z- or

Z-like conformational transitions as the fragments will have variation

[iii]

in the G+C and A+T contents. Native DNA or small DNA fragements

(30C bp) brominated uPider lov,/ or high soidum ch lc ide concentrationj

indicated regions of interspersed sequences which has the potentiai \o

undergo B- to Z- or Z-iike transitions. The Z- cr Z-like epitopes on

brominated native DNA and smaij DNA fragments was confirmed by bindirg

^nd inhibition data o.f nnonocional and polyclonai anti-Z-DNA antibcdits.

The effect of varying concentrations of D- and L-polylvfine cr DMA

showed that in native calf thyrp'.'.-r DNA there ar° certain DNA sequences

which are sensitive to elevated le/els of poiyannines =',nd car. underg:

B- to Z- or Z-like transitions. Since the eukaryotic DNA i^ heavily asso­

ciated with protein; rich in lysine, such conformationai transitions nrigh'.

serve as molecular signal for genetic events inside the cell. That the

bromination has changed the typical B-characteristics o ' DMA was elso

proved by decreased p*rcentag« inhibition of SLE anti-dsDNA .'gC- cctiv'ty

by the brominated forms of DNA compared to high percent inhibitior

of SLE anti-dsDNA IgG activity v/hen the same polymers v/ere in a typical

B-conformation. This again shc-vs tnat SLE ar.tibodies are pclyspeciiic

with respect to binding towards varying DNA conformation.

In conclusion, native and small DNA fragments have interspersed

base pair sequences which have the potential to undergo B- to Z- or

Z-like transitions. Irrespective ci salt concentrations, covalent mrdificaticn

(e.g, bromination) induces 2- or Z-li'ce helix in native and small DNA

fragments. That th i epitopes were prese.'ting the features of Z-DNA was

confirmed by anti-Z-D;JA antibodies binding data,, A detailed study of

SLE anti-DNA antibody interaction with modified DNA antigens (DNA-

iysine photoconjugate and hydroxy! radical modified r;)NA) have been carried

out to explore the possibility of participation of these neoantigens in

the etiopathogenesis of SLE. It appears that the polyspecificity of anti-

DNA antibodies is mujh greater than what is anticipated.

FINE ANTIGENIC SPECIFICITY OF HUMAN ANTI-DNA AUTOANTIBODIES AGAINST

DNA OF VARYING SIZE AND CONFORMATION

Thesis Submitted for the Degree of

ISottor of ^IiiloiBiopIip IN

BIDCHEMISTRY

BY

KHURSHID ALAM -s,^v•..

Approved

Prof. RASHID &LI (Supervisor)

DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE

JAWAHARLAL NEHRU MEDICAL COLLEGE ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

1 9 9 2

;(") ''•^'^*T'\

T4593

3 - AUG 1994

CHECKED 1996^

CERTIFICATE

I cer t i fy that the work presented in the fol lowing pages has

been carried out by Khurshid Alam and is suitable for the award

of Ph.D. degree in Biochemistry of the Aligarh Muslim University,

Al igarh.

(RASHID ALI') Professor

Department of Biochemistry J.N. Medical College Aligarh Muslim University ALIGARH 202 002 INDIA

ACKNOWLEDGMENTS

Facing my window, across the road, stands an old and elegant

banyan tree. It soaks rain, filters sun, absorbs chill but provide

shelter and comfort to every passerby. Through these five years be

it a moment of crisis or despair I have received same comfort,

guidance and protection from my supervisor Professor Rashid Ali. I

thank him for giving me an opportunity to be benefitted from the

sunshine of his knowledge and academic brilliance.

My sincere thanks are also due to Professor A, Salahuddin,

Chairman of the department for the provision of necessary

facilities.

I owe heart felt thanks to Drs. W.A. Naqvi, M.A. Qasim, A. Ali

and M.U. Siddiqui for their encouragement and suggestions. Dr. Saad

Tayyab of the Biotechnology unit is also thanked for his

constructive criticism.

Words cannot express my deep sense of gratitude to my teachers

and well wishers Professors A.M. Siddiqui, M, Saleemuddin, S.M.

Hadi, A.H. Siddiqui, Dr. Sheela Shahab and Mr. Shamshad Alam for

their patient listening to my genuine demands.

I am privileged to thank my research colleagues Ms. Sadia

Arjumand, Mr. Moinuddin, Ms. Zarina Arif and Ms. Jahan Ara for their

unmatchable cooperation and day to day scientific discussion.

Thanks are also due to my senior colleagues Dr. Rashid Hasan,

Dr. Najmul Islam, Dr. Amita Gupta, Mr. Naushad Ali and Mrs. Vandana

Sehgal.

Very sincerely I owe heartfelt thanks to my friends Abeeb Alam

Khan, Abul Faiz Faizy, Mohd. Aleem, Arshad Rahman, Imranuddin,

Zubair, Dilshad, Fabeba, Tabassum, Tauseef, Gaity and Lubna for

their love, help and encouragement.

I will be failing in my duty if I do not acknowledge Drs, A.5.

Khan and Shahzad F. Hag of the Nephrology division for providing the

sera of 5LE patients.

I am highly thankful to nonteaching staff of the department

for their constant help. Cooperation of Lab. Assistants M/s Sabu

Khan, Ashfag Ali and Rizwan Ahmad is highly appreciated.

It takes many hands to give the final shape to Ph.D. thesis. I

am thankful to Mr. Mazahir Hussain and Mr. Syed Vigar Husain for

typing and Mr. Nadeemuddin for graphic v/ork. I appreciate the help

given by the staff of central photography section in preparing the

gel photographs.

Lastly, the financial assistance provided by the University

Grants Commission, New Delhi is gratefully acknowledged.

( KHURSHID ALAM )

CONTENTS

Page No.

ABSTRACT

ABBREVIATIONS

LIST OF FIGURES

LIST OF TABLES

INTRODUCTION

EXPERIMENTAL

RESULTS

DISCUSSION

REFERENCES

[i]

[iv]

[v]

[ix]

[1]

[30]

[51]

[116]

[126]

[ i ]

Antibodies reactive with cellular and nuclear components occur spon­

taneously in patients with systemic lupus erythematosus. The basis for

spontaneous production of anti-native DNA antibodies in systemic lupus

erythematosus remains a puzzling question especially in view of the diffi­

culty in their induction on immunization with DNA in B-conformation.

The anti-dsDNA antibodies seem to be closely related to the disease patho­

genesis through the process of immune complex formation, and their levels

are frequently indicative of the intensity of disease activity. For reasons

that are not best understood, anti-DNA antibodies have always exhibited

polyreactivity with DNA molecules of varying source, size and conformation.

Analysis of plasma DNA from SLE patients have revealed that these

molecules are 100-1000 base pair long and rich in G+C sequences. Such

DNA sequences have the unique potential to undergo B- to Z- or Z-like

transitions under constraint conditions.

In the present study purified native calf thymus DNA was digested

with micrococcal nuclease and fractionated on Sepharose ^B. The approxi­

mate fragment size was determined by standard DNA marker. DNA-(poly-

lysyl-Sepharose ^B) affinity matrix was chosen for the isolation of

homogeneous anti-DNA antibodies from SLE serum. The affinity purified

human anti-dsDNA antibody was found to possess polyreactivity with native

and modified DNA antigens.

Native DNA covalently modified with lysine under UV-A light (320-

14OO nm) was found to contain one lysine residue per helical turn. By

the photoaddition of one lysine molecule per helical turn of DNA, the

native B-conformation was considerably altered which resulted in the

appearance of neoepitopes leaving much less epitope typical of B-

conformation. Affinity purified anti-dsDNA IgG showed maximum affinity

towards DNA-lysine photoconjugate followed closely by native DNA. The

retention of crossreactivity of affinity purified anti-dsDNA IgG with DNA

-lysine photoadduct strengthen the idea of an alternate antigen for the

induction of antibodies crossreactive with native DNA. Lysine rich histone

[ i i ]

HI in nucleosome thus on modif ication by physical, chemical or environ­

mental agents might form DNA-histone adduct making i t immunogenic

and thus generating antibodies crossreactive wi th native B-conformation.

Hydroxyl radical generated by hydrogen peroxide and ferrous ions

caused single strand breaks in native DNA, decrease in melt ing temperature

(Tm) by 6.5 °C and modif ication of purine and pyrimidine bases. Experi­

mentally induced antibodies against R05 modified DNA exhibited poly-

react iv i ty , a property commonly associated wi th SLE ant i -DNA antibodies.

Native DNA, RNA and synthetic polynucleotides in B-conformation were

found to be ef fect ive inhibitor of induced antibody-immunogen interact ion.

The SLE antibody binding wi th R05-DNA was better than native DNA.

Similar results were observed in competi t ion-inhibi t ion assay. Because

of the diminished superoxide dismutase act iv i ty in SLE patients, the

excessively generated free radicals could interact wi th DNA resulting

in its modif icat ion. Once the native structure of DNA is modified i t

may become immunogenic.

The binding specif ici ty of SLE ant i -DNA IgG wi th varying DNA

conformation was analyzed in poiyacryiamide or agarose gel. The altered

electrophoretic mobil i ty in gel retardation assay of A- and B-conformation

complexed wi th ant i -DNA IgG reiterates the antibody binding to these

conformers. The above conformers showed high binding wi th SLE antibodies

in competi t ion-inhibit ion experiments.

The B- to Z- transit ion of poly(dG-dC).poly(dG-dC) in 4.0 M NaCl

was evaluated in terms of "absorption rat io" , the average of absorbance

at 29 * and 296 nm divided by absorbance at 260 nm. This rat io was

observed to be 0.322. The high salt induced Z-form of poly(dG-dC). poly

(dC-dC) was stablized by bromination which exhibited Z-DNA characteristics

when brought in physiological buffer. The low salt brominated poly(dG-dC).

poly(dG-dC) exhibited the characteristics of B-DNA. Native DNA under

low or high salt concentration showed "absorbance rat io" greater than

0.3. Under identical conditins the absorbance rat io for small DNA

fragments varied between 0.2 to 0.38. The data indicate that small DNA

fragments have di f ferent levels of potent ial i ty to undergo B- to Z- or

Z-like conformational transitions as the fragments wi l l have variation

[ i i i ]

in the G+C and A+T contents. Native DNA or snnail DNA fragements

(300 bp) brominated under Jew or high soidum chloride concentrations

indicated regions of interspersed sequences which has the potential to

undergo B- to Z- or Z-i ike transitions. The Z- or Z-l ike epitopes on

brominated native DNA and small DNA fragments was confirmed by binding

and inhibit ion data of monoclonal and polyclonal ant i -Z-DNA antibodies.

The ef fect of varying concentrations of D- and L-polylysine on DNA

showed that in native calf thymus DNA there are certain DNA sequences

which are sensitive to elevated levels of polyamines and can undergo

B- to Z- or Z-like transitions. Since the eukaryotic DNA is heavily asso­

ciated wi th proteins rich in lysine, such conformational transitions might

serve as molecular signal for genetic events inside the cel l . That the

bromination has changed the typical B-characteristics of DNA was also

proved by decreased percentage inhibit ion of SLE anti-dsDNA IgG act iv i ty

by the brominated forms of DNA compared to high percent inhibit ion

of SLE anti-dsDNA IgG act iv i ty when the same polymers were in a typical

B-conformation. This again shows that SLE antibodies are polyspecific

with respect to binding towards varying DNA conformation.

In conclusion, native and small DNA fragments have interspersed

base pair sequences which have the potential to undergo B- to Z- or

Z-like transitions. Irrespective of salt concentrations, covalent modif icat ion

(e.g. bromination) induces Z- or Z-l ike helix in native and small DNA

fragments. That the epitopes were presenting the features of Z-DNA was

confirmed by ant i -Z-DNA antibodies binding data. A detailed study of

SLE ant i -DNA antibody interaction wi th modified DNA antigens (DNA-

lysine photoconjugate and hydroxyl radical modified DNA) have been carried

out to explore the possibility of part icipation of these neoantigens in

the etiopathogenesis of SLE. It appears that the poJyspecificlty of an t i -

DNA antibodies is much greater than what is anticipated.

[iv]

ABBREVIATIONS

A-,, . = Absorbance at 260 nm

BSA = Bovine serum albumin

dsDNA = Double stranded DNA

DEAE = DiethylamlnoethyJ

EDTA = Ethylenediaminetetra-acetic acid (disodium salt)

High salt = i^ M sodium chloride

IgG = Immunoglobulin G

Low salt = 0.]5 M sodium chloride

nDNA = Native DNA

PUVA = Psoralen plus ultraviolet A light

R05 = Reactive oxygen species

RT = Room temperature

ssDNA = Single stranded DNA

SLE = Systemic lupus erythematosus

TEMED = N,N,N',N'-tetraethylenediamine

Tm = Helix-coil transition temperature

UV = Ultraviolet

ul = Microliter

[v]

LIST OF FIGURES

Figure No. Page No.

1. Isolation of IgG from an SLE serum by

DEAE Sephacel Chromatography ... 36

2. Evaluation of titer and immunocross-

reactivity of antibodies against human IgG ... 52

3. Direct binding ELISA of antibodies against

human IgG ... 53

*. Melting profile of native DNA ... 5^

5. Fractionation of SLE IgG on Sepliadex G 200... 57

6. Elution profile of DNA digested with

micrococcal nuclease on Sepharose i^B

column ... 60

7. Thermal denaturation curves of DNA

fragments ... 61

8. Immunoaffinity purification of anti-dsDNA

IgG on DNA-[polylysyl-Sepharose ^B] column ... 63

9. Binding of human SLE antibodies to native

DNA and DNA-lysine photoconjugate ... 66

10. Assay of anti-DNA antibodies by ELISA ... 69

11. Inhibition of SLE antibody binding by native DNA, heat denatured DNA and RNA ... 70

12. Inhibition of SLE anti-DNA antibody binding

by Br^ DNA, poly(dG-me^dC) and poly(rG).

poly(dC) ... 71

13. Competition ELISA of SLE anti-DNA anti­

bodies. The competitors were poly(dA).poly

(dT), poly(dG).poly(dC), poly(dA-dG).poly

(dC-dT), double stranded: poiy(dG-dC), poly

(dl-dC) and poly(dA-dU) ..• 73

[vi]

1*. BindingofSLE anti-DNA IgG to A- and B-

conformation analyzed by gel retardation

assay 7f

15. Direct binding assay of SLE auto antibodies

The coating antigens were native DNA,

poly(dA-dT)-8-MOP, poly(dG-dC)-psoralen,

DNA-8-MOP and DNA-psoralen ... 76

16. Circular dichroisnn spectra of poly(dG-dC).

poly(dG-dC) in 0.15 M and ^ M sodium

chloride ... 78

17. Ultraviolet spectra of poly(dG-dC).poly

(dG-dC) and DNA fragments (300 bp) in

0.15 M and ^ M sodium chloride ... 80

18. Ultraviolet difference spectra of poly

(dG-dC).poly(dG-dC) and DNA fragments

in 0.15 M and 4 M sodium chloride ... 82

19. Ultraviolet difference spectra of poly

(dG-dC) brominated in 4 M sodium chloride ... 83

20. Ultraviolet absorption and difference spectra

of native and brominated DNA fragments ... 8^

21. Ultraviolet absorption spectra of native

DNA in presence of D- and L-polylysine ... 87

22. Ultraviolet absorption spectra of native

DNA in presence of D- and L-polylysine ... 88

23. Ultraviolet difference spectra of polylysine

DNA complex ... 91

2'f. Ultraviolet difference spectra of polylysine-

DNA complex ... 92

25. Agarose gel electrophoresis of plasmid DNA

in presence of hydrogen peroxide and

ferrous ions ... 95

[vii]

26. Ultraviolet absorption spectra of native

and hydroxyl radical modified DNA ... 96

27. Hydroxyapatite column chromatography of

native and hydroxyl radical modified DNA ... 97

28. Alkaline sucrose density gradient centrifuga-

tion of native and hydroxyl radical modified

DNA ... 98

29. Melting profile of native and ROS-modified

DNA ... 99

30. Binding of anti-ROS-DNA IgG with native

and ROS-modified DNA ... 103

31. Inhibition of immunogen-antibody interact­

ion by R05-DNA. The competitors were

native DNA, poly(dA-dG).poly(dC-dT);, double

stranded : poly(dl-dC) and poly(dA-dU) ... 105

32. Determination of antibody affinity by

Langmuir plot ... 106

S'f. Direct binding and competition ELISA of

human lupus antibodies binding to native

DNA and ROS-DNA ... 107

35. Inhibition ELISA of anti-Z-DNA antibodies

(Z 22) by poly(dG-dC) and poly(dG-dC)

brominated in 0.15 M and ^ M sodium

chloride ... 109

36. Inhibition of Z 22 binding to Z-DNA by

poly(dA-dG).poly(dC-dT) and poly(dA-dG).

poly(dC-dT) brominated in 0.15 M and ^ M

sodium chloride ... n o

[viii]

37. Inhibition of SLE anti-DNA IgG binding

to native DNA. The competitors were poly

(dA-dG).poly(dC-dT), poly(dA-dG).poly(dC-dT)

brominated in 0.15 M and 4 M sodium

chloride ... I l l

38. Inhibition of SLE ant i-DNA IgG binding to

native DNA. The competitors were native

DNA, native DNA brominated in 0.15 M

and ^ M sodium chloride ... 11^

[ix]

LIST OF TABLES

Table No. Page No.

1. Autoantibodies associated with ciinical findings ... 20

2. Direct binding ELISA of SLE anti-DNA antibodies ... 56

3. Ultraviolet characteristics of immunoglobulin G ... 59

i^. Melting profile of DNA fragments ... 62

5. Lupus autoantibody binding with native and

modified DNA ... 65

6. Binding profile of SLE anti-DNA IgG ... 67

7. Binding inhibition of anti-DNA autoantibodies by

polynucleotides ... 72

8. Binding of SLE anti-DNA IgG by DNA-

Furocoumarin photoadducts ... 77

9. Ultraviolet absorbance characteristics of DNA

polymers under low and high salt ... 81

10. Ultraviolet absorbance characteristics of DNA

polymers brominated under low and high salt ... 86

11. Effect of polylysine on ultraviolet characteristics

of native DNA ... 90

12. Binding of SLE anti-DNA IgG by amines ... 93

13. Ultraviolet and thermal denaturation charac­

teristics of native and hydroxyl radical

modified DNA ... 100

I'f. Percent base modification of DNA by hydroxyl

radical ... 101

[x]

15. Inhibition of the binding of Z 22 and G 10 to

Z-DNA and NBr^ ... 112

16. Binding of SLE anti-DNA IgG by small DNA

fragments brominated under Jow and high salt ... 115

The DNA has been the subject of biochemical investigations almost

from the t ime i t was f i rst isolated from pus cell nuclei (Miescher,

1879). The information burled in DNA in the form of four-unit language

gets amplif ied to 20 unit language of protein by simple permutation

and combination of nucleotides. These events of language translation

produce various types of polypeptides whose function is dictated by the

type of cell where the event is taking place. The utmost importance

of DNA lie in its unique property to adopt mult iple conformations

depending on its bioenvironment. The environment could mean ions,

small molecules or even proteins. These alternate conformations are

likely to play crucial role in gene expression by increased or decreased

synthesis of regulatory proteins which are ut i l ized by the cel l for its

betterment.

Structure and Antigenicity of DNA

3ames D. Watson and Francis Crick proposed their double helical

model for DNA in 1953 on the basis of X-ray di f f ract ion studies. Analysis

of d i f f ract ion pattern showed that DNA can exist in two forms: as

right handed B-DNA, which is stable at a relat ive humidity of about

92 percent and as A-DNA which appears when the relat ive humidity

falls to about 75 percent. There are two kinds of double-ring purine

bases, adenine (A) and guanine (G), and two kinds of single-r ing pyrimidine

bases, cytosine (C) and thymine (T). The base pairs are held together

by hydrogen bonds. Adenine normally is paired wi th thymine by two

hydrogen bonds and guanine is paired wi th cytosine by three bonds.

The two points of attachment of a base pair to its sugar ring do not

lie direct ly opposite each other across the pair, and this is important

for the geometry of a DNA double-helix. The edge of the base pairs

along which the angle between attachment is less than 180 degrees

is called the minor-groove edge, and the one forming an angle larger

than 180 degrees is the major-groove edge. B-DNA has an average of

[2]

10/10.5 base pairs per turn of the helix, wi th a spacing of 3. * A along

the helix axis f rom one base pair to the next. The A-hel ix has closer

to 11 base pairs per turn, and because of the way the t i l ted • base pairs o

are stacked, the distance along the axis is only 2.9 A per base pair.

The right handed B-DNA is about 2 mm thick.

Since the discovery of B-DNA reactive antibodies in human SLE

and its murine equivalent (Robbins et a l . , 1957) and its cl in ical significance

in the disease, the biochemical and immunochemical approach of the

investigators working wi th this molecule have always faced problem

whenever this macromolecule was used as immunogen. Exogenous native

B-DNA has not been immunogenic in experimental animal. Even MRL

mice, which are genetically programmed to produce anti - B-DNA antibodies

"spontaneously", do not respond to exogenous DNA and i t did not result

in increased anti-native DNA antibody production above the level that

resulted from injection of adjuvant alone (which does cause some increase)

(Madaio et a l . , 198^^). Later normal animals immunized wi th DNA, including

DNA complexed wi th adjuvant, show only l imi ted antibody production

which is almost exclusively directed to single stranded DNA (ssDNA)

(Plescia et a l . , 196^*). The f i rst experimental ant i -DNA antibodies appeared

only in 1960, in the form of antibodies to denatured (single stranded)

DNA of the T-even bacteriophages (Levine et a l . , 1960). By contrast,

double stranded RNA, RNA-DNA hybrid, t r ip le helical RNA and DNA

analogues, and double helical polydeoxyribonucleotides that di f fer f rom

average B-DNA structure, such as poly (dG). poly (dC) have been found

to be immunogenic (Stollar 1973, 1975, 1986; Anderson et a l . , 1988).

Injection of histone-DNA complexes yielded some anti-histone but not

ant i -DNA antibodies (Stollar and Ward, 1970); nor did immunization

wi th isolated nucleosomes induce ant i -DNA antibodies (Einck et al . ,

1982). A monoclonal antibody from animal immunized wi th a duplex

DNA, poly (dT-dC). poly (dG-dA) gave a weak cross reaction wi th native

DNA (Lee et a l . , 1985). Recently, injection of bacterial native DNA

into normal mice induced antisera that bound native bacterial but not

mammalian DNA (Gilkeson et al . , 1989b). However, the sera reacted

[3]

with denatured DNA from both sources. Injection of BK virus into rabbits

induced antibody to both native DNA and histone (Flaegstad, 1988).

Antibodies induced by 5-methylcytosine also include some populations

that react wi th a native circular XP 12 phage DNA that is rich in

5-methylcytosine content (Adouard et a l . , 1985). The modif ied portion

of this base would be accessible to antibody in the major groove and

there is evidence to show that a portion of antibody's combining site

can enter the major groove and interact wi th accessible surfaces of

bases (StoUar, 1986). Using a highly sensitive avidin-biotin micro ELISA,

the base specific binding of adenosine antibodies to double stranded

DNA has been reported (Vaishnav and Antony, 1990).

To explain the unexpected presence of ant i -DNA antibodies in

the sera of normal subjects, i t has been proposed that antibodies that

are highly specific for bacterial DNA arise as part of the overall host

response to infections by these organisms. In this sett ing, bacterial DNA

would be recognized as foreign because of the presence of sequence

arrays, structural features, or higher order conformations that are not

usually present in human DNA. Further studies have shown that bacterial

DNA differs f rom mammalian DNA in its content of pyrimidine clusters,

patterns of base methylat ion, and undoubtedly, in many of its coding

sequences (Wells, 1988; Szybalski et a l . , 1966; Weils et a l . , 1977; Cheng

et al . , 1985).

Types of DNA within B-Family

The B-DNA structure per _s£ is a poor immunogen. There are

certain right handed double stranded polydeoxyribonucleotides which

dif fer slightly from the average B-DNA helix and are immunogenic beyond

the adjuvant induced autoantibody expansion (Madaio et a l . , 198^*). The

immunogenicity itself is an indication that they di f fer in structure from

the average B-DNA. Fufther, nearly all of the antibodies generated

recognize those differences almost uniquely; they react wi th immunogen,

but very weakly or not at all with DNA. This again indicates the existence

of structural differences in the helices. The B-form of synthetic

polydeoxyribonucleotides have been found to be immunogenic and recognize

M

the difference between them and native B-DNA. These polymers include

poly(dG). poly(dC) (Stollar, 1970); poly (dT-dC). poly (dG-dA) (Lee et

a l . , 1985); poly (dA). poly (dT) (Diekmann and Zarl ing, 1987; Arnot t

et a l . , 1983); poly (dG-dC) (Zarling et a l . , 1987). The other right handed

structure in natural DNAs are A- and C-DNA which are found on changing

the humidity and salt conditions (Arnott and Hukin, 1972; Arnot t and

Seising, 1975). Double stranded RNA and RNA-DNA hybrid take on the

A- or A- l ike form (Spencer et a l . , 1962; Milman et a l . , 1967). The sequence

(dC-dA) .(dG-dT) is the most abundant purine-pyrimidine dinucleotide

repeat in eukaryotic genome (Weber, 1990). Relatively sliort (approxitTiately

50 bp) stretches of this sequence occur, on average, at a frequency

of once 30 to 100 kb in all eukaryotic genomes thus far examined,

from yeast to human (Hamada et a l . , 1982). Studies on the chromatin

structure of the potential Z-forming sequence (dT-dG) .(dC-dA) has

revealed evidence for an "alternating B" conformation on the nucleosomal

surface (Gross et a l . , 1985). It is possible that the al ternat ive helical

conformation of these sequences yields a dist inct ive structure in the

nucleus that is recognized by proteins functioning in recombination and

replication events. Because these sequences exhibit length polymorphism,

they represent a new rich source of informative genetic markers (Weber

and May, 1989). An "alternating B" structure has been proposed for

poly (dA-dT).poly(dA-dT) in solution based on DNase I cleavage pattern,

proton and 31 p nuclear magnetic resonance (NMR) spectroscopy, and

X-ray fiber d i f f ract ion (Klug et al.,1979). The synthetic deoxyoctanucleotide

d(G-G-G-G-T-C-C-C) crystall izes as an A-type DNA double helix containing

two adjacent G-T base pair mismatches (Kneale et a l . , 1985). Also,

the potential ly Z-DNA forming sequence d(GTGTACAC) crystall izes

as A-DNA (3ain et a l . , 1987). Poly(dG).poly(dC) sequences, under torsional

stress, induce an altered DNA conformation upon neighboring DNA sequences

and such altered DNA structures may play an important functional role

in chromatin (Shigematsu and Kohwi, 1985).

Conformation Variant of B-DNA : Z-DNA

There have been many attempts to propose alternat ive models

for DNA other than right-handed double helical model. In fact . Crick

[5]

and Watson (195^) ruled out the possibility of a left handed structure

for DNA because they could not build a left handed double helical

model for DNA. In 1965, Fuller et al., had indeed built a left handed

B-DNA, but could not build a left handed model for A-DNA. The possibility

of a left handed structure for DNA was ruled out since the transition

R ^ '*A could not be possible in fiber if a left handed model for B-

DNA and a right handed model for A-DNA were assumed.

The discovery of left handed Z-conformation of DNA has led

to a renaissance of interest in DNA structure and polymorphism (Pohl

and 3ovin, 1972; Wang et al., 1979). The extensive research efforts

aimed at understanding the structural details and possible biological

significance of the altered conformation has so far resulted in furnishing

only a limited information (3ovin et al., 1983; Rich et al., 1984; Sehgal

and All, 1990; Hasan et al., 1991; Alam and Ali, 1992). The DNA sequences

shown to promote the formation of left handed Z-DNA ^ vivo and

in vitro have been implicated in increased frequencies of genetic

recombination in bacteria (Murphy and Stinger, 1986), yeast (Treco and

Arnheim, 1986) and mammalian systems (Bullock et al., 1986). Segments

of DNA with alternate d(GpC) sequences are most prone to form

Z-DNA, the next most effective sequence being d(GpT) . Other sequences

in which the perfect alternate is not preserved can also adopt the Z-

conformation (Klysik et al., 1981; Nordheim et al., 1982; Haniford and

Pulleyblank, 1983; Nordheim and Rich, 1983). Nevertheless, a strictly

alternating purine-pyrimidine is neither necessary nor sufficient (McLean

et al., 1986; Singleton et al., 1983; Kilpatrick et al., 198^; Ellison et

al., 1985). It has also been shown that stretches of poly(dG-dC) sequences

do not contribute significantly to the presence of Z-DNA in fixed polytene

chromosomes of Drosophila melanogaster (Nordheim et al., 1986). Membrane

hybridization experiments and screening of data banks have shown that

d(GpT)^ long sequences (30 > n > 1) are generally dispersed in many if

not all genomes. Long d(GpC) sequences are seen less frequenly (Hamada

and Kakamaga, 1982; Hamada et al., 1984; Rich et al., 198^^).

[6]

Z-DNA, first described at the atomic level for the sequence

(dC-dG), by Rich and his colleagues (Wang et al., 1979), is a prototype

left handed double helix whose structural properties differ significantly

from those of classic B-form DNA. Z-DNA contrast with B-DNA not

only in its left handed helical sense, but in twist ('—>12 versus 10.5 0 0

base pairs per turn), helix diameter ( |8 A versus 20 A), number of helical

grooves (1 versus 2) and structural repeat unit (dinucleotide versus

mononucleotide) (Wang et al., 1979; Drew et al., 1980). In addition,

the purine residues of Z-DNA are rotated 180° about the glycosidic

bond from the anti- to the syn-conformation. Jji vitro analysis have

demonstrated that linear duplej< (dG-dC) sequences can adopt the left

handed Z-conformation when subjected to elevated ionic strength (Pohl

and 3ovin, 1972; Patel et al., 1979; Klysik et al., 1981), or modification

of either guanine or cytosine residues (Sage and Leng, 1980; Lafer et

al., 1981; Behe and Felsenfeld, 1981; Klysik et al., 1983). Other DNA

sequences with alternating purine-pyrimidine residues, including poly(dA-

S dT) and poly (dT-dG).poly(dC-dA), exhibit fiber structures that are

compatible with left handed Z-form as well (Arnott et al., 1980). In addition, the B- to Z-transition can be induced when either (dG-dC)

or (dT-dG) . (dC-dA) sequences are embedded in a B-DNA background n n ^ °

in recombinant plasmid or eukaryotic genome (Singleton et al., 1982;

Nordheim and Rich, 1983; Haniford and Pulleyblank, 1983; Gross et

al., 1985). The above sequence and certain others that contain alternating

purine-pyrimidine residues have been shown to adopt the left handed

Z-DNA conformation _iri vitro when subjected to negative torsional stress

or elevated ionic strengths (Gross et al., 1985).

In 198^, Mura and Stollar showed the ability of different lysine rich

histones to cause varying conformational changes in the condensation

of chromatin in DNA regions of highly biased base sequence. Recently,

it has been shown that complexation of poly(dA-dC).poly(dG-dT) with

spermidine and spermine could significantly alter the immunogenicity

of this polynucleotide and produce antibodies that reacted with Z-DNA

[7]

form of brominated poJy(dG-dC).poJy(dG-dC) in addition to the immunizing

antigen. That the immunizing antigen has presented the features of

2-DNA was shown by its binding to monoclonal ant i -Z-DNA antibodies

(Z 22) (Gunnia et al . , 1991).

The results so far have failed to detect Z-conformation in calf

thymus DNA (Ct-DNA). But in 1990, Hasan and Al i reported that when

Ct-DNA was brominated under high salt (kh'\ NaCl) conditions i t presented

the features of Z-DNA. The Z-l ike conformation was retained even

after the removal of salt. The Ct-DNA is heterogeneous in sequence

and microheterogeneity exist in B-structure (Hutta c>t a l . , 198^! Saniord

et al . , 1988). The B-form of poly(dG-dC).poly(dG-dC) lacks most of this

heterogeneity (Drew et a l . , 1981). The biochemical properties of Z-

DNA, cruci forms, bent DNA, anisomorphic DNA, oligopurine-oligopyrimidine

structures, etc., have been the subject of numerous _iri_ v i t ro investigations

(Wells and Harvey, 1987; Wells, 1988; Rich et al . , 198^). Z-DNA cannot

be incorporated within core particles, except at their te rmin i , and

that a transit ion from B- to Z-form _m vivo might result in a signif icantly

altered local placement of nucleosomes (Garner and Felsenfeid, 1987).

Immunochemical studies have shown that Z-DNA is a powerful

immunogen el ic i t ing a highly specific antibody response (Lafer et a l . ,

1981). The Z-conformation of DNA was determined in interbands of

polytene chromosomes (Nordheim et a l . , 1981) and then in banded regions

(Arndt - 3ovin, et a l . , 1983), in nuclei of protozoa (Lipps et a l . , 1983),

cells of rat tissue (Morgenegg et al . , 1983) and in metaphase chromosomes

(Viegas-Pequignot et a l . , 1983). Both polyclonal and monoclonal antibodies

specific for Z-DNA have served as sensitive reagents for the detection

of Z-epitopes by relatively simple antigen-antibody binding assay (Lafer

et a l . , 1981; Malfoy et a l . , 1982; Zarling et a l . , 198^ ; Pohl et a l . , 1982;

Hanau et a l . , 198^). Although, the precise amount of Z-DNA present

in l iving cells has not been established, these experiments have demonstrated

the potential for Z-DNA formation in chromatin and have helped xo

identify the conditions under which the transit ion can occur. Alongwith

[8]

studies of isolated DNA, these experiments have also demonstrated the

role of torsional stress of supercoiling in determining the transition

from B- to Z-DNA (DiCapua et al., 1983; Hill at al., 198^; Robert-

Nicoud et al., 198^ ; Peck et al., 1982; Haniford and Pulleyblank, 1983;

Singleton et al., 1982). With monoclonal antibodies that show selectivity

for particular base sequences in Z-DNA form (Zarling et al., 198^; Moller

et al., 1982; Lee et al., 198^), it becomes possible to explore further

what kind of sequences may or may not be responsible for Z-DNA formation

in the chromosomes.

The qualitative impression from the experiments is that

the prokaryotic DNA seems to form Z-DNA more readily than the eukaryotic

DNA. This may be a consequence of the markedly reduced frequency

of C-G sequences in eukaryotic DNA compared to prokaryotic. The

reduction may be related to the methylation of C-G sequences at 5-

position of cytosine found in eukaryot es but not in prokaryotes. The

spontaneous deamination of methylated cytosine residues leads to the

thymine which would cause considerable biological confusion. The reduction

of C-G sequences in eukaryotic DNA down to approximately 10% of

what is observed in prokaryotic DNA may lead to Z-DNA being found

less frequently in eukaryotic DNA. A notable difference between these

two systems is the presence of the middle repetitive DNA sequences

d(CA/GT) n ^ 50, which occur frequently in eukaryotic systems and

are entirely absent in prokaryotes.

Possible Biological Role of Z-DNA

Conformational diversity of the DNA helix appears to be used

quite extensively by living organisms with regard to protein-DNA recognition

and thus function (Traverse, 1989). In several cases DNA recognition

by proteins seems to rely heavily-if not entirely on DNA conformations

and flexibility as opposed to base sequence £££ _se (Otwinowski et al.,

1988; Goodman and Nash, 1989). Transcription by RNA polymerase is

terminated by left handed (dC-dG) tracts (Peck and Wang, 1985).

[9]

Further work shows that transcription is associated with Z-DNA formation

with a substantial increase in the binding of ant i -Z-DNA antibodies in

metaboiically active permeabilized mammalian cell nuclei (Wit t ig et

al . , 1991). The question that naturally arises is what physiological processes

act in vivo to generate the negative supercoiiing (which faci l i tates Z-

DNA formation) that is continuously being removed by the act iv i ty of

topoisomerase I (Zhang et al , , 1988). Wit t ig et al . (1991) have suggested

that transcription is actually associated with a marked increase in the

leveld of ant i -Z-DNA antibody binding. This may be one of the major

physiological processes that generate negative supercoiiing and thus

stabilizes Z-DNA formation in the intact cel l . Liu and Wang (1987)

have suggested that RNA polymerase movement generates negative super­

coiiing upstream from the transcription site.

Reactive Oxygen Species and Genetic Apparatus

Free radical reactions are irreversible (Pryor, 1966; Nohebel and

Walton, \37^) and ubiquitous in l iving organisms (Harman, 1981, 1986).

Enzymatic reactions serving as sources of free radical reactions (Pryor,

1966; Nohebel and Walton, 197'f) include those involved in the respiratory

chain, in phagocytosis, in prostaglandin synthesis, and in cytochrome

P-^50 system (Harman, 1988), Free radicals also arise in the non-enzymatic

reaction of oxygen with organic compounds as well as those in i t iated

by ionizing radiation (Ara et al . , 1992). iViajor sources of endogenous

oxygen radicals are hydrogen peroxide (Chance et al. , 1979) and superoxide

(Fridovich, 1982) generated as side products of metabolism, and the

oxygen radical burst from phagocytosis after viral or bacterial

infection or the inf lammatory reaction (Hal l iwel l , 1982), Observation

of unique DNA-oxygen free radical reaction products formed in vivo (Floyd

et al . , 1986; Frenkel et al . , 1986; Kasai et al . , 1986) appear to be impor­

tant, not only to demonstrate direct proof of their presence in vivo, but of

greater significance, the lesions formed are

important aspect ref lect ing the mechanism of

[101

oxgen free radical mediated DNA damage. Oxidative damage is,common and DNA ,

proteins and lipids are all at risk and therefore oxygen free radicals

are thought to be linked to many diseases (Pryor, 1987). Normal oxidative-

damage to mitochondrial and nuclear DNA has been observed to be

caused by excited oxygen species, which are produced by radiation or

are by-products of aerobic metabolism. Normal cellular metabolism has

been known to generate oxygen species as hydrogen peroxide, hydroxyl

radical, singlet oxygen, and hydroperoxy radical (Cerrut i , 1985). Moreover,

superoxide radical and hydrogen peroxide are produced by act ivated

phagocytes which are essential for the ki l l ing of many bacterial strain^

(Babior, 1988). Exposure of cells to visible light may enhance normal -8

cell concentrations of hydrogen peroxide (I x 10" M), hyperbaric oxygen

and metabolism of certain dietary carcinogens lead to hydrogen peroxide

acceleration.

The monitoring of macromolecular damage in tissues f rom humans

exposed to chemical substances that interfere wi th DNA is being actually

pursued to explain the origin of antibodies in autoimmune dlsoders.

It is most probable that the etiological basis for oxygen free radicals

as contr ibuting factors in the development of several pathological conditions

resides in their action on the genetic apparatus (Schneider et a i . , 1989).

Oxygen free radicals have been known to cause damage to nucleic acids

(Wallace, 1987; Stoewe and Prutz, 1987) and hydroxyl radicals have

been implicated in the etiology of many human diseases (Hal l iwel l and

Gutteridge, 1989). The excessive concentration of the -OH overcome

normal defense mechanisms and leave the DNA at risk from oxidative

modif ication (Cotgreave et al . , 1988) of deoxyribose, pyrimidines and

purines (Von Sonntag, 1987). The oxidized nucleic acid base 8-hydroxyguano-

sine is present at a level of 130,000 bases in nuclear DNA and per

8,000 bases in mitochondrial DNA (Richter et al . , 1988). The unique

thing about 8-hydroxyguanosine is that i t does not stop repl icat ion:

but i t might be relat ively benign, although it does cause misreading

at 8-hydroxy-2'-deoxyguanosine residue itself and at the neighboring

[11]

base (Kuchio et a l . , 1987). Formation of 8-hydroxy-2-deoxyguanosine

either in DNA or f rom the free nucleoside deoxyguanosine (dG) has

been shown to be mediated by hydroxyl free radicals (Kasai and Nishimura,

198^^; Floyd et a l . , 1986, 1988) as well as by certain other reducing

substances including hydrazines (Kasai and Nishimura, 198^*3), polyphenols

(Kasai and Nishimura, 1984b), and intermediates in carcinogen metabolism.

Recent studies have demonstrated the existence of 2-hydroxyadenine

(isoguanine) in isolated human chromatin after its exposure to assay

mixture of Nickle (II) and Cobalt (II) and hydrogen peroxide (Nackerdien

et al . , 1991). 8-hydroxyadenine have been isolated from the DNA in

cancer of the female breast (Malins and Haimanot, 1991). The origin

of the 'OH that potential ly init iates the base modif icat ion is unclear,

although one possibility is that this radical arises from hydrogen peroxide

generated through the cytochrome P-450-mediated oxidation of estrogen

(Deodutta et a l . , 1991). Hydroxyl radicals produced by copper (II) plus

hydrogen peroxide has been shown to cause site specific DNA damage

at polyguanosines resulting in single and double strand breaks and al terat ion

in the structure of guanosines (Sagripanti and Kraemer, 1989). Guanine

has previously been shown to be the target for af latoxin (Muench et

a l . , 1983), nitrogen mustards (Mattes et a l . , 1986), anti tumor agents

(bleomycin, neocarcinostatin, cis-platin) (D'Andres and Haseltine, 1978), and

antibiot ic (tetracycl in) action (Piette et a l . , 1986).

DNA damage involving • OH would be relat ively favored in diseases

where copper concentration is elevated such as Menke's syndrome (Sone

et al . , 1987), Wilson's disease or certain neoplasms (3endryczko et a l . ,

1986). In normal cells Cu (I) would be oxidized mainly by oxygen with

l i t t le -OH formation and DNA damage. In tumorr cells, the oxidation

of Cud) by hydrogen peroxide would be favored resulting in DNA damage

(Sagripanti and Kraemer, 1989). Trace metals such as copper and iron

which are present in biological system may interact wi th act ive oxygen

species to damage DNA (Sagripanti et a l . , 1987; Imlay et a l . , 1988).

In the presence of trace amounts of transit ion metal (M) ions (Fe ,

Cu ) found in biological system can part icipate readily in Fenton-l ike

[12]

reactions resulting in the prodution of 'OH which are short l ived but

extremely reactive with their bioenvironment,

M ' ^ ^ H2O2 > M ^ " " ' ^ " . OH +.OH

Under normal circumstances the concentration of reduced metal ions

in chromatin would be expected to be low. Consequently, the production

of a high local density of lesions in DNA by . OH radicals derived from

the Fenton reaction wi l l depend on a large proportion of the potential ly

reactive metal ions being in the reduced state or capable of being reduced

by an intr insic mechanism or by reductants introduced from outside the

cell (Jonas et a l . , 1989). Studies have shown that bacterial k i l l ing by

hydrogen peroxides is enhanced when bacteria (Staphylococcus aureus)

are grown for several days in an iron-rich environment in order to raise

intrinsic iron concentrations (Repine et al . , 1981). Measurement of

DNA damage in the presence of an excess of DNA and 1,10-phenanthroline

has been employed as a sensitive assay to measure the avai labi l i ty of

copper ions in human body fluids (Gutteridge, 198^ ; Gutteridge et a l . ,

1985; Evans et a l . , 1989; Dizdaroglu et a l , 1990). Overal l , the DNA

damage seems to be produced by a Fenton-type mechanism in which

transit ion metal ions are cycled by f i rst being reduced by superoxide

and then oxidized by hydrogen peroxide. The DNA damage _in_ vi vo may

be due to a site-specific generation of this • OH upon the DNA itself.

This means that either the DNA has transit ion metal ions bound to

i t _m v i t ro, or that the oxidative stress liberates such metal ions that

rapidly bind to the DNA (Dizdaroglu et a l . , 1987). Chromosomal proteins

may act as radical scavengers in reactions induced by ionizing radiation

(Rotl RotI et a l . , 1974). Moreover, the iron binding proteins transferr in

(present in plasma) and lactoferr in (secreted by neutrophils) are far

from saturated with iron \n_ vi vo (except during iron overload), and

can bind metal ions liberated from other proteins, thus helping to diminish

damaging radical reactions (Hall iwell and Gutteridge, 1986). Despite

[13]

all the protect ive measures taken care by biological system, the free

radicals generation seems to continue because in nature every reaction

is liable to be altered by changes in normal physiological processes.

Recently, Jiang &. Chen (1992) have shown decreased act iv i ty of superoxide

dismutase (SOD) in SLE patients which resulted in elevated levels of

free radicals. The excessive level of free radicals in SLE than those

in normal persons, probably contr ibuting to the production of autoantibodies.

These free radicals may play an important role in the pathogenesis

of SLE possibly due to diminished SOD act iv i ty .

Polyamines-DNA Nexus

Polyamines are ubiquitous polycationic metabolites of prokaryotic

and eukaryotic cells. These polycations are known to play c r i t i ca l roles

in the regulation of major functions in cell growth, cel l d i f ferent iat ion

and biosynthetic pathways (Tabor and Tabor, \32,U; Pegg, 1986, 1988;

Schuber, 1989). Polyamines are found to be associated wi th ribosomes

and membranes. The precise function played by these molecules inside

the cell is not ful ly understood yet they exert an antimutagenic ef fect ,

they also prevent dissociation of 70S ribosomes to 30 S and 50S components,

and they increase the resistance of protoplasts to osmotic lysis. Polyamines

stimulate translation ( init iat ion and elongation) of the peptide chain

and are implicated in maintaining translational f idel i ty , ribosomes structure

and stabil i ty (Marton and Morris, 1987) thus markedly influencing protein

synthesis in both prokaryotes and eukaryotes. In various growing tissues

polyamine synthesis accompanies or precedes nucleic acid synthesis (Heby

et a l . , 1976; McCann et a l . , 1972). The increased synthesis of RNA

also depends on the st imulating ef fect of polyamines on RNA polymerase.

The concentrat ion of polyamines is elevated in the biological fluids

and tissues in a number of disease states such as cancer, sickle cell

anemia, cystic fibrosis, psoriasis and systemic lupus erythematosus (Puri

et al . , 1978; Gunnia et a l . , 1991). Similarly, the polyamine levels in

the spleen cells of MRL-lpr/ lpr mice are about three fold higher than

that in spleen cells of normal mice (Bowlin et a l . , 1986; Thomas and

Messner, 1989).

[1* ]

Spermidine and spermine are known to be distributed widely in

nature. Spermine is the most abundant polyamine found in animal cells

(Tabor and Tabor, 198'f). Spermidine or spermine is essential for the

aerobic growth of Saccharomyces cerevisiae (Balasundaram et a l . , 1991).

The amout of spermidine varies inversely with the amount of Mg

in ribosomes, and 30S and 50S ribosomal subunits remain associated

in the absence of Mg if spermine is present.

Because of protonated amino groups (at physiological pH values),

polyamines are known to bind to negatively charged cellular molecules

such as nucleic acids. The interaction of polyamines wi th nucleic acids

may be responsible in part for the biological functions of polyamines

(Basu et a l . , 1989). Thermophilic bacteria growing under the extremes

of heat are known to produce a variety of polyamines to protect their

genetic material from denaturation (Oshima, 1982). The polyamines

composition of the extreme thermophile depends on growth temperature

(Oshima and Baba, 1981). The thermophil ic cells grown at an optimum

temperature (75 °C) contain two novel tetramines, thermine (Oshima,

1975) and thermospermine (Oshima, 1979). The interact ion of polyamines

with DNA produces profound changes in the structural organization and

react iv i ty of DNA. These include the stabil ization of double stranded

DNA, collapse of DNA into toroids and spheroids (Gosule and Schellman,

1978; Thomas and Bloomfield, 1983), and the induction of lef t handed

Z-DNA conformation of alternating purine-pyrimidine sequences and

plasmids containing blocks of these sequences (Behe and Felsenfeld,

1981; Thomas and Messner, 1988; Thomas et al . , 1991). Spermine and sper­

midine promote aggregation of DNA, increases its melt ing temperature (Tm),

and induce B- to Z-transit ion (Feuerstein and Marton, 1987). Spermine induce

DNA conformation that binds actinomycin D less ef f ic ient ly (D'Orazi et a i . ,

1979). On the contrary, actinomycin D is a very powerful antibiot ic that

forms a complex with native poly(dG-dC).poly(dG-dC) sequences of DNA (3ain

and Sobell, 1972; Sobell and 3ain, 1972). Behe and Felsenfeld (1981)

and Thomas et ai . (1985) demonstrated that polyamines and their acetyl

derivatives were capable of provoking B- to Z-transit ion of poly(dG-

[15]

m^dC).poly(dG-medC). Blocks of (dA-dC).(dG-dT) which are known to

occur widely in nature as a part of transcriptional enhancer elements

of nnany genes undergo B- to Z- to Y- conformational transitions in

the presence of poiyamines, thus obviating the extreme conditions (3M

NaCl; 19% ethanol or high concentrations of NiCU) otherwise used to

attain these conformations _ii2 vitro (Thomas and Messner, 1986). Extremely

sensitive enzyme immunoassay technique have demonstrated that in

the absence of poiyamines, a polynucleotide sequence poly (dG-medC).

poly(dG-medC) does not bind to monoclonal anti-Z-DNA antibody (Z

22) but rapid interaction occurs when poiyamines are added to the system

(Thomas and Messner, 1988). These immunochemical studies, alongwith

UV spectral and circular dichroism studies corroborate the above mentioned

evidence on the binding of poiyamines to the polynucleotides.

In 1982, Klevan and Schumaker noticed that addition of poly-

L-arginine but not poly-L-lysine to poly (dG-dC).poly(dG-dC) in high

salt solution allows the Z-conformation to be retained upon dialysis

to lower salt concentration. This observation suggests that the stabilization

of the Z-DNA form is not due to simple electrostatic factors but may

also involve some specificity in the interaction of arginine side chains

with Z-helix. Analysis of interactions of HI and H5 histones with

polynucleotides of B- and Z-conformations revealed two things. Firstly,

at low protein: DNA ratios, both H) and H5 bound more Z-DNA than

B-DNA. Secondly, the binding of Z-DNA was less sensitive to interfere

by an increase in ionic strength (to 600 mM NaCl). Moreover, H5 histone

caused more profound C D . spectral changes than did HI (Mura and

Stollar, 198'^). These findings demonstrate the ability of different iysine-

rich histones to cause varying conformational changes in the condensation

of chromatin in DNA regions of highly biased base sequence. The occurrence

of H.is characteristic of cells that are inactive in both transcription and

replication (Billet and Hindley, 1972; Urban et al., 1980).

Recent studies further reiterates that natural poiyamines are

capable of altering the immunogenicity of polynucleotides by mechanisms

involving the stabilization of Z-DNA conformation (Gunnia et al., 1991)

[16]

The presence of high levels of polyamines and anti-Z-DNA antibodies

in some lupus patients and autoimmune mice suggest the involvement

of poiyamines in altering the topological s tate of DNA (Sehgal and

Mi, 1990; Alam et al., 1992).

Autoimmunity and Causative Factors

The immune system has tremendous discrimination potential of

self and nonself. The process of autoimmunity begins when the immune

response is started against the self antigens. The laboratory data till

date indicate that an age dependent loss of immune regulation might

be a single most critical factor which is involved in the process of

autoimmunity. It has not been found possible so far, to reproduce the

disease by the transfer of antibody from the patient to normal individual.

There are three models for the induction of a productive autoimmune

process. The first is the autoimmune response to a cross reacting antigen,

which is different from, but cross reactive with autologous determinants,

the second is a genetic predisposition for autoimmunity through regulatory

failure, and the third is the triggerring with bacterial mitogen of potentially

autoimmune B cells, normally held in check by suppressor cells.

Polyclonal stimulation might, in fact, favor antibodies against

autologous macromolecules, since these are present in the milieu of

activated cells and might provide the second stimulus, which results

in the productive response by a polyclonally activated or hyperactive

B cell (McHugh and Bonavida, 1978). There appears to be atleast two

genetic factors that are involved in autoimmune disease. A predisposition

may be controlled by one gene (a contoller T cell or a deviant gene

for stem-cell development) and the disease may be precipitated by an

external event and the reaction of this precipitating event may be under

polymorphic genetic control. Since suppressor T cells appear to be important

elements in the regulation of immune response, it is likely that any

failure in the capacity to generate suppressor T cells may contribute

to autoimmune disease. Also, genetic factors appear to affect the

autoimmune disease of NZB mice and some forms of human SLE (Cleland

[17]

et al , , 1978). However, the level at which the gene controls this operation

remains unknown. It is l ikely that the autoimnnune disease of NZB/NZW

F1 mice is determined by two dominant genes and that one gene of

NZB strain is on chromosome 17, but not close to H-2 (Knight ^nd

Adams, 1975). There is also good evidence that systemic lupus erythe­

matosus is under genetic controls; 5-10% of close relatives of patients

with SLE have lupus (Arnett and Shulman, 1976). Furthermore, mult iple

autoimmune diseases, including SLE, have been associated wi th genetically

determined products of the major histocompatibi l i ty complex (MHC)

in humans, i.e. HLA (Keuning et a l . , 1976; McMichael et a l . , 1976;

Klouda et a l . , 1979; Fye et al . , 1978; Terasaki et a l . , 1976). There is

common occurrence of two HLA-DRW types in SLE patients (HLA-

DRw 2 and HLA-DRw 3), as well as high percentage of individuals

with B-cell alloantigen la-715 (Decker et a l . , 1979). Increased frequencies

of HLA-DR 3, with or without increased frequencies of DR2, have been

found in several small groups of patients with SLE (Stastany, 1978;

Scherak et a l . , 1979; Celada et a l . , 1980; Scherak et a l . , 1980). Sontheimer

et al . (1981) have reported an increased frequency of DR3 in patients

with subacute cutaneous lupus erythematosus, a subset of lupus erythe­

matosus. As in the disease of NZB mice, there appears to be a fami l ia l

abnormality of suppressor cell function in systemic lupus erythematosus

(Miller and Schwartz, 1979; Fauci, 1980).

There is some evidence for an association of autoimmune disease

with low macrophage funct ion, as assessed by carbon blockades Pass well 125

et a l . , 197^^), \n_ vivo clearance of 1 labeled polyvinyl pyrrolidone

(Morgan and Soothil l, 1975) and low af f in i ty antibody (Passwell et a l . ,

197^). It also seems possible that the class of antibody provides the

event that results in transit ion from autoimmunity to autoimmune disease.

In New Zealand mice, the abi l i ty to clear particles was found to decline

with increasing age (Morgan and Soothil l, 1975; Morgan and Steward,

1976). In short, defective macrophage function may be a factor in the

glomerulonephritis of NZB/W mice. It is possible that an age-dependent

decline in the phagocytic function of macrophages may be direct ly involved

[18]

in preferential formation of low af f in i ty antibody (Steward and Powis,

1977) but i t is also possible that poor macrophage function accentuates

the persistence of complex between antigen and low af f in i ty antibody.

There are growing concern about theoretical risk of autoimmune

disease in patients undergoing PUVA treatment for psoriasis, v i t i l igo

and mycosis fungoides. Psoralens are used in the treatmeat of human

skin diseases (Fitzpatr ick et al . , 1982; Parrish et a l . , 1982), for probing

nucleic acid structure (Song and TapJey, 1979; Cimino et a]., 1985;

Parsons, 1980; Rinke et a l . , 1985; Calvet et a l . , 1982; Setyono and Pederson,

198^) and nucleic acid-protein interactions (Cech and Pardue, 1977;

Potter et a l . , 1980; Cimino et a l . , 1985; K i t t le r and Lober, 1988).

Alterations in DNA resulting from the photoreaction could lead to develop­

ment of antibodies to DNA or could precipitate SLE (Hasan et a l . ,

1991). There have been reports of lupus-like syndrome (erythema) fol lowing

PUVA therapy (Mill ins et al . , 1978; Eyason et a l . , 1979). In a recent

study, cel l membrane DNA has been looked as a new suitable target

for psoralen photoaddition (Gasparro et a l . , 1990).

Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a multisystem autoimmune

disease characterized by a myriad of cl inical manifestations and mult iple

autoantibodies (Provost, 1979; Hahn, 1980). The origin of autoantibodies

in SLE is an enigma poised on the verge of solution. The riddle has

been a taxing one because lupus serum contains a bewildering variety

of autoantibodies against nucleic acids (notably DNA), nucleoprotejns,

cell surface antigens and phospholipids. Such extensive diversity in the

serological abnormalities of the disease is await ing a def ini te mechanism

of antigen act ivat ion (Harley and Scofield, 1991; Brigido and Stollar,

1991; Fronek et a l , 1990).

While detection of anti-dsDNA antibodies remains the most reliable

parameter to define the disease, antibodies to DNA have also been

found to be associated with the nephritis of SLE and are thought to

play a pathogenic role therein. Association of other autoantibodies in

[19]

many pathological conditions have been presented in Table 1. It is envisaged

that certain subpopulation of DNA antibodies may be more pathogenic

than others. Considerable evidence has accumulated to show that both

quali tat ive and quanti tat ive differences in the antibodies to DNA are

important in the production of nephritis in SLE patients (Valle et a l . ,

1985). Antibodies to dsDNA are relatively restr icted to IgGl and IgG3

subclasses whilst antibodies to ssDNA were equally distributed throughout

the four subclasses i.e. IgGI , IgG2, IgG3 and IgG^ f.

Granholm and Cavallo (1990) have proposed that prolonged circulat ion

of immune complexes (between DNA and ant i -DNA autoantibodies) due

to various altered immune functions contributes to nephritis in MRL/ lpr

mice. In human SLE, the early deposits of immune complexes (IC) are

found in the mesangium and can be detected even before cl in ical or

laboratory manifestations of renal involvement are evident (Cavallo

et a l . , 1977). Theoretically altered blood cell carrier functions could

at t r ibute to nephritis by allowing large amounts of ICs to remain unbound

(free) in the circulat ion. Such ICs are thought to localize more readily

in tissues because they circulate close to the plasma-endothelial face

and can be precipitated or entrapped in the microcirculat ion (Schifferl i

and Taylor, 1989). Delayed removal of ICs from the circulat ion is thought

to favor their deposition in tissues (Schifferl i and Taylor, 1989). The

biologically act ive, (pathogenic) immune complex systems that have been

implicated in nephritis of autoimmune mice, i.e. DNA- ant i -DNA (Dixon,

1987), gp 70-an t i -gp 70 (reviewed by Cavallo et a l . , 1985) and RNA

polymerase I - an t i -RNA polymerase I (Stetler and Cavallo, 1987), could

lend itself to localization in the kidney as preformed ICs, as ICs formed

in situ, or both. Lupus prone mice, and many immunologically normal

mice, have relat ively high concentrations of a serum glycoprotein of

molecular weight 70 kD (gp 70). This glycoprotein is related structural ly

and immunologically to the envelop glycoprotein of murine retroviruses

(Yoshiki et a l . , 197^; Strand and August, 1976; Lerner et a l . , 1976;

Elder et a l . , 1977; Hara et al . , 1981). It is synthesized in liver and

it behaves like an acute phase reactant (Hara et al . , 1982). More

[201

TABLE 1

Autoantibodies Associated with Clinical Findings '

Autoantibody Clinical finding

Anti-dsDNA

Ant i -P

Ant i -Ro

Ant i -La

Antiphospholipid

Anti-Sm

Anti-nRNP

Nephritis

Psychosis

Lymphopenia Thrombocytopenia Interstitial pneumonitis Photosensitive skin rash Congenital heart block Neonatal lupus dermatitis

Absence of nephritis Congenital heart block Neonatal lupus dermatitis

Intravascular thromboemboli Miscarriage Thrombocytopenia

Isolated central nervous system lupus

Myositis Raynauds' phenomenon Interstitial pneumonitis Capillary dropout Sclerodactyly

*Source: Harley, 3.B. and Scofield, R.H. (1991) 3. Clin. Immunol. 11, 297-316

[21]

importantly only lupus prone mice develop autoantibodies to gp 70. Part

of their circulat ing gp 70 is complexed wi th IgG (Izui et a l . , 1979,

1981a, 1982) and the serum concentration of such complexed (heavy)

gp 70 correlates with the progression of nephritis (Izui et a l . , 1981b,

198^a,b). A small nuclear RNA-protein complex known as Ro/SSA binds

autoantibody from many patients wi th SLE and Sjogrens syndrome (Reichlin,

1981; Tan, 1982; Wasicek and Reichlin, 1982; Reichlin and Wasicek,

1983). The frequent coexistence of autoantibodies to a cellular DNA-

binding protein complex (p70/p80) and to DNA has also been reported

(Reeves, 1985).

Recent findings have indicated that development of autoimmune

processes in SLE is accompanied by autoantigen driven production of

autoantibodies but is preceded and predicted by polyclonal B-cell act ivat ion

(Kl inman, 1990). B-lymphocytes with immunoglobulin receptor for dsDNA

are found in normal individuals as well as in SLE patients. The act ivat ion

of these cells in SLE may be a problem of disordered immunologic

regulation. In human SLE both CD5 ' and CD^"*" B-cells are capable of

ant i -DNA production, although the high af f in i ty IgG ant i -DNA antibodies

appear to come from CD5 ' subset of B-cells (Cesali et a l . , 1989). These

findings are consistent with a model in v^hich autoantibody production

in lupus is ini t iated by polyclonal act ivat ion and perpentuated by autoantigen

specific stimulation Klinman et al . , 1988, 1990). Also, the presence

of anti-receptor antibodies in SLE and related disorders (Bennett et

a l . , 1986a,b) suggests that the DNA receptor may be an important antigen

in the repertoire of autoimmune responses. Indeed, two murine monoclonal

antibody have been generated with react iv i ty towards a 30000 molecular

v/eight DNA binding protein found on the cell surface of human leukocytes

(Bennett et a l . , 1988). SLE patients have decreased numbers of complement

receptors on their erythrocytes as compared to normal individuals (Miyakama

et a l . , 1981).

Polyspecific ant i -DNA autoantibodies (so called natural autoantibodies)

derived from normal mice showed binding to a variety of proteins including

act in, myosin and spectrin. Natural antibodies against tubul in, act in,

[22]

myoglobin, thyroglobulin, fetuin, albumin and transferr in are present in

normal human sera (Avrameas et al . , 1981). Ant i -DNA antibodies

have been found to interact with cytoskeletal proteins (Andre-Schwartz

et a l . , 198^); f ibronectin (Lake et a l . , 1985; Gupta et a l . , 1988) and

heparan sulfate (Termaat et a l . , 1990), cell surface proteins (Jacob et

a l . , 198 *) and different cell types ( I ron et a l . , 198^1; Faaber et a l . ,

1985). Binding with hyaluronic acid, chondroitin sulfate and heparan

sulfate which contain repeating negatively charged units (Faaber et

al . , 1986) have also been observed. Anti-dsDNA antibodies have been

selectively removed from SLE serum by immunoadsorption wi th dextran

sulfate (Kinoshita et a l . , 1989). Many dif ferent Fc-binding proteins exist

in the biological fluids or on the membrane of various cells. Circulat ing

proteins, such as rheumatoid factors (RF) (Nemazee and Sato, 1983),

complement components (Clq) (Dorrington and Klein, 1983), or immuno-

globulin-binding factors (Khayat et al . , 198^*; Pure et a l . , 198^) can speci­

f ical ly interact wi th Fc portion. Studies on human SLE hybridoma autoant i­

bodies have been shown to bind major cytoskeletal f i laments that included

intermediate f i laments, microfi laments, microtubules and centrioles

(Senecal and Rauch, 1988). In the same study, the immunoblot results

showed that the antigenic specif icity of intermediate f i lament reactive

SLE hybridoma antibodies was not restr icted to vimentin (a phosphoprotein

under hormonal regulation) (Browning and Sanders, 1981) but included

cytokeratins and desmin.

Monoclonal IgM ant i-DNA autoantibodies, produced by hybridomas

prepared from lymphocytes of SLE patients have been found to bind

to several di f ferent antigens including ssDNA (Shoenfeld et a l . , 1982,

1983), synthetic polynucleotides (Shoenfeld et a l . , 1982), vimentin (Andre-

Schwartz et a l . , 198^^), platelets (Asano et a l . , 1985), acidic glycolipidf

in platelets (Asano et al . , 1986) and sulfated complex carbohydrates

(Asano et a l . , 1986). These reactivi t ies can be most simply interpreted

as the interaction of an ant i-DNA with a conformational determinant

or charge array in space that can be represented not only by DNA,

but by other molecules as wel l . Besides, DNA could also interact in

[23]

unanticipated ways wittn other polycationic structures in the body to

innpair cel l function or homeostasis. As a polyanion, DNA could display

heparin like act iv i ty and disrupt c lot t ing mechanisms.

Ant i -DNA antibodies in normal control groups have been reported

from t ime to t ime. To explain the unexpected presence of ant i -DNA

antibodies in normal subjects, i t has been proposed to arise as part

of the overall host response to infections by pathogenic bacteria. In

this sett ing, bacterial DNA would be recognized as foreign because

of the presence of sequence arrays, structural features, or higher order

conformations that are not usually present in human DNA. Studies have

clearly shown that bacterial DNA differs f rom mammalian DNA in its

content of pyrimidine clusters, patterns of base methylat ion, and undoubtedly

in many of its coding sequences (WelJs, 1988; Szybalski et a l . , 1966;

Wells et al . , 1977; Cheng et a l . , 1985), Together, these features could

create local regions in bacterial DNA that are structural ly dist inct and

rarely present in mammalian DNA. A role of ant i -DNA as protoantibodies

in an in i t ia l defense against foreign antigenic challenge has been reported

(Schwartz and Stollar, 1985; Pisestky et a l . , 1990). Ant i -DNA antibodies

serve an important role in normal immunity not because they bind DNA,

but because they are polyspecific and can bind non-DNA antigens, especially

bacterial products. The broad reactivi t ies represent a useful in i t ia l defense

against mult iple antigens. Furthermore, these antibodies can be mutated

into high af f in i ty antibodies that bind more exclusively to foreign antigenic

determinants (Naprastek et a l . , 1986). It seems clear that many ant i -

DNA are in the repertoire of normal individuals and have characterist ics

of non-autoantibodies. Thus, the binding to a self antigen, especially

under certain assay conditions, may not constitute a suff icient cr i ter ion

to indicate that an antibody has pathogenic potential , or is a product

of aberrant regulation or abnormal idiotype expression.

In a recent study, the effect of polyamines on the immunogenicity

of polynucleotides have been investigated (Gunnia et a l . , 1991). The

el ic i tat ion of ant i -Z-DNA antibodies by polyamine-polynucleotide complex

suggests a possible involvement of polyamines in the production of ant i -

{.2H]

Z-DNA antibodies in hunnan SLE. The antigenic DNA may be genomic

DNA in the serum, which has been shown to have an abnormally high

guanine-cytosine (G-C) content (Sano and Morimoto, 1982) and an abnormally

high occurrence of alternating purine-pyrimidine segments (Van Helden,

1985). This phenomenon may be significant, since i t has been shown

that segments of alternating pyrimidine-purine residues, part icularly

poly(dG-dC) can undergo a transit ion from B- to Z-conformation (Pohl

and 3ovin, 1972; Behe and Felsenfeld, 1981; Zacharias et a l . , 1982;

Moller et a l . , 1984) and since it has also been shown that the Z-form

of DNA is highly immunogenic (Lafer et a l . , 1983). if, therefore, Z-

DNA segments occur naturally in SLE patients we may expect a greater

production of ant i -Z-DNA antibodies than ant i -B-DNA antibodies (Van

Helden, 1985). That such ant i -Z-DNA antibodies do occur in SLE sera

has been shown (Lafer et a l . , 1983; Sibley et a l . , 1984; Gunnia et a l . ,

1991; Sehgal and A l l , 1990). There are several reports that serum DNase

1 levels are decreased in patients with SLE (Chitrabamrung et a l . , 1981;

Frost and Lachman, 1968). At the same t ime i t has been suggested

that most of immunogenic duplex nucleic acids are nuclease resistant

(Braun and Lee, 1988).

Antibodies to dsDNA play a key role in the pathogenesis of SLE

and therefore considerable research ef for t over the past 25 years has

focused on both the quantitat ive determination and the qual i tat ive

characterization of anti-dsDNA antibodies that are found in the circulat ion

of patients wi th SLE (Zouali and Stollar, 1986; Lau et a l . , 1991; Rauch

et a l . , 1985; Cairns et a l . , 1989; Alam and A l i , 1992; Alam et a l . , 1992).

Analysis has revealed that pathogenic ant i-DNA antibodies of both human

and murine SLE are of IgG isotype and cationic in nature (Ebling dc

Hahn,1980;Datta et a i . , 1987). Ant i -DNA antibodies induced by immunization

of experimental animals wi th nucleic acids are highly specific for

the immunogen (Stollar, 1980; Lafer et al . , 1983), whereas the spontaneously

produced ant i -DNA autoantibodies in SLE tend to crossreact wi th a

variety of ligands (Andrzejewski et a l . , 1981; Pisetsky and Caster, 1982;

Koike et ai . , 1982; Shoenfeld et al . , 1983; Pisetsky et a]. , 1990).

[25]

There is increasing evidence for the mieroheterogeneity of DNA

and the existence along the DNA molecule of diverse structures that

differ in dinnensions and topology from B-DNA,, It has been debated,

whether DNA from various sources differ markedly in their immunogenic

potential. It has been shown that in mice certain bacterial single stranded

DNA (ssDNA) and dsDNA can induce significant anti-DNA responses,

including antibodies to dsDNA (Gilkeson et al., 1989 a,b). In fact immuni­

zation with E_. coli DNA has induced antibodies (Gilkeson et al., 1989b

that resemble spontaneous anti-DNA antibodies of lupus mice in terms

of their recognition of defined antigens (Pisetsky et al., 1990). In the

year 1990, Gilkeson et al., showed that mice immunized with calf thymus

DNA (Ct-DNA) failed to generate an antibody response to either Ct

or E_. coli (Ec) DNA. The antibodies to Ec dsDNA did not crossreact

with Ct dsDNA. However, immunization with Ec dsDNA did produce

antibodies that were reactive with both Ec and C t ssDNA, possibly

reflecting the _m vivo breakdown of some of the dsDNA antigen into

ssDNA, and the induction of an antibody response specific for this form

of DNA. In these experiments the crossreactive antibodies to ssDNA

resembled those found in lupus: in contrast, the antibodies to dsDNA

bound only the immunizing antigen and lacked activity to common dsDNA

determinants that are characteristic of lupus autoantibodies. Fournie

(1988) has postulated that not DNA, but DNA bound to nuclear proteins

could also play a role in the pathogenesis of SLE.

Monoclonal autoantibodies have been prepared that di scriminates

nearly completely between synthetic poly(dG-dC) and poly(dA-dT) and

protects certain restriction nuclease cleavage sites preferentially (Stollar

et al., 1986). Arnott et al. (1983) have described variation in helical

geometry as presentation of a "wrinkled" backbone surface, which is

different in poly(dA-dT) and poly(dG-dC), both of which differ in precise

dimensions from average B-DNA. Murine monoclonal autoantibody, H2^1

binds well to native DNA but also displays several crossreactions that

include denatured DNA, and both the B-DNA and Z-DNA forms of

poly(dG-dC) (Stollar et al, 1986). On the other hand, H2^1 showed no

reactivity in either direct binding or competitive binding assay, with

[26]

several single stranded homopolymers, [poly(A), poly(C), or poly(U)]; double

stranded RNA [poly(A).poly(U) or poly(I).poly(C)]; DNA-RNA hybrid [poly(A).

poly{dT)]; or triple stranded helices [poly{A).poly(l),poiy(l) or poly(A).

poly(U).poly(U)]. Monoclonal autoantibody HZ'tl has a striking ability

to recognize helical structural features ot poly(dA-dT) and polyCdG-

dC) that depend on base sequence. In poly(dA-dT) the adjacent ApT and

TpA segments are not identical, as revealed by two distinct peaks in the

P-NMR spectrunn (Patel et al., 1981); the repeating unit in this polymer

IS a dinucleotide rather than a mononucleotide (Klug et al., 1979; Patel

et al., 1981). H2^l also reacts with cardiolipin, which contains phosphate

groups spaced by 3 carbon atoms but does not have purines or pyrimidines.

This crossreaction of the antibody (H2'*l) indicates that some of the

binding site also interacts with the phosphate backbone. To determine

whether both heavy (H) and light (L) chains of different anti-nDNA

autoantibodies are uniformly involved in binding to DNA, monoclonal

mouse IgG autoantibodies, H241 and 2C10 were mixed with radiolabeled

oligonucleotide and UV (25'l nm) irradiated for 10 min which led to

specific covalent crosslinking of oligonucleotide to both the H and

L chains of H2^1 but only to H chain of 2C10 (Jang and StoUar, 1990).

The dependence of oligonucleotide-protein crosslinking on specific immune

complex formation was demonstrated by the complete absence of crosslink­

ing to non-immune IgG even after 2 h of irradiation; by which time the

IgG molecules themselves had become aggregated.

Therap>eutic Approach to SLE Treatment

Therapeutic approach to the treatment of SLE have included the use

of corticosteriods, various cytotoxic agents, and total body irradiation (Elad

et al., 1986). Because of the profound adverse effect of corticosteriods on

normal functioning of the immune system due to the fundamental drawbacks

of not distinguishing between physiological and pathological immune response,

the recent treatment involves use of normal immunoglobulin G (IgG) (intra­

venous immunoglobulins) (Dietrich and Kazatehkine, 1990; Schwartz, 1990) and

plasmapheresis, a new approach to interrupting the pathogenetic events in

systemic lupus erythematosus (Lewis et al., 1992). In missile therapy

[27]

of SLE (Sasaki et a l . , 1985), ant i- idiotypic antibodies suppressed mult ipl ication

of EBV transformal lymphocytes of SLE patient has been t r ied. It may

be reiterated that despite tremendous development in understanding

the possible mechanism involved in precipi tat ing autoimmune diseases,

most of SLE patients are st i l l receiving corticosteroids.

Biological Implications of Anti-Nucleic Acid Antibodies

Anti-nucleic acid antibodies may serve as non-destructive models

and a precise yardstick for examining protein-nucleic acid interactions.

They may be used to follow structural changes in given nucleic acids

or nucleoproteins. Anti-nucleic acid antibodies may also be ut i l ized as

reagents to detect and quantitate DNA in serum and other body fluids

(released during pathological conditions). The ant i -DNA antibody can

be used for sensitive detection of DNA-containing structures such as

chromatin in di f ferent tissues of man, rat , mouse, chicken, amphibia

and insects. Cel l nuclei of various cell lines such as XLK E-A6 (Xenopus),

RVF-SMC (rat), PTK-2 (rat-Kangaroo) and HeLa (human) show positive

reactions. They can distinguish almost quanti tat ively among members

of di f ferent helical famil ies (Lacour et a l . , 1973; Stollar, 1973, 1975,

1989) and qual i tat ively among dif ferent members of helical class (Lacour,

et a l . , 1973; Guignes and Leng, 1976; Johnston and Stollar, 1978; Nahon-

Merlin et a l . , 1980). As their specificit ies are defined these antibodies

become useful analytical probes, serving as sensitive and specific reagents

for detecting particular structure in complex biological mater ial including

mammalian genome. Experimentally induced antibodies to le f t handed

Z-DNA (Nordheim et a l . , 1981; Lemeunier et a l . , 1982; Arndt-3ovin

et al . , 1983; Kmiec and Holloman, 198^^), double stranded RNA (Stollar

and Stollar, 1970; Mil ler et a l . , 1975) or DNA-RNA hybrids (Rudkin

and Stollar, 1977; Busen et al . , 1982; Alcover et a l . , 1982) have been

able to detect corresponding structures without interference from a

large excess of other nucleic acids. Monoclonal autoantibodies have

been shown to protect certain restr ict ion nuclease cleavage sites on

DNA preferential ly (Stollar et a l . , 1986). Also monoclonal anti-nucleic

[28]

acid antibodies are useful immunochemical tracer of a specific DNA

conformation of utmost biological importance (Anderson et a l . , 1988).

Recent look at the risk factors which are involved in the pathogenesis

of SLE. Genetic, environmental, immune, and hormonal factors acting either

directly or indirectly lead to the production of mediator substances

like autoantibodies, immune complexes, and complement act ivat ion products

which cause tissue destruction and disease expression.

Factors

Genetic

V

Hormonal

Immune ^ — >

Mediators

Autoantibodies

Immune complexes

Complement act ivat ion

Tissue destruction

Environmental

Objective of the Present Study

The non-immunogenic nature of native DNA (B-form) and poly-

specjf icity exhibited by SLE anti-DNA antibodies, withd respect to mult iple

antigen binding, s t i l l poses the puzzling questions regarding their possible

origin. Despite this, elevated levels of anti-nDNA antibodies in act ive

SLE continue to be diagnostic marker of the disease. As the horizon

of polyspecif icity of SLE antibody is expanding, the scienti f ic views

on possible origin of these antibodies is also increasing and the results

so far reported have been unequivocally accepted. Hence, the study

of antigen binding characteristics of ant i -DNA autoantibody is relevant

to specify dist inct ly its polyreactivi ty with native and modified DNA

[29]

antigens of varying size and conformation. Well defined and characterized

ant i-DNA antibody could be uti l ized for monitoring changing DNA confor­

mations during genetic act iv i t ies. In this study purif ied calf thymus

DNA and its fragments of varying size were modified by bromination

in low and high salt concentrations and were evaluated as antigen for

binding wi th ant i-DNA antibodies. Effect of varying concentrations

of D- and L-isomer of polylysine on B- conformation was also evaluated.

The change in DNA conformation was monitored by UV spectroscopy

and binding with ant i -Z-DNA antibodies. The DNA was also modified

with lysine, furocoumarin and hydroxyl radical. Each of the modified

antigen was systematically characterized and investigated for binding

to ant i-DNA IgG.

[30]

Chemicals

Anti-goat, anti-human, anti-mouse and anti-rabbit IgG alkaline

phosphatase conjugates, bovine serum albumin, calf thymus DNA (highly

polymerized), Coomassie Bri l l iant Blue G 250 (5c R 250, dialysis tubing

(of varying diameter), ethidium bromide, Mil l ipore f i l ter {0A5 um pore

size), Fruends' adjuvants, Hoechst dye 33258, 8-methoxypsoralen, methylated

bovine serum albumin, nuclease 51, poly-L-glutamic acid, poly-D-lysine,

poly-L-lysine, trinitrobenzene sulfonic acid (TNBS), Tris-(hydroxymethyl)-

aminomethane, xylene cyanole FF were purchased from Sigma Chemical

Company, U.S.A. Dextran Blue 2000, DEAE Sephacel, DEAE Sephadex

A-50, Ficol l 'fOO, micrococcal nuclease, poly(dG-dC).poly(dG-dC), poly(dG-

me^dC).poly(dG-me^dC), poly(dA-dT).poly(dA-dT), poly(dA-dU).poly(dA-

dU), poly(dI-dC).poly(dI-dC), poly(dA-dG).poly(dC-dT), poly(dA).poly(dT), poly

(dG).poly(dC), poly(rG).poly(dC), poly(dA), poly(dG), poly(dC), poly(dT), poly U,

Sephadex G 200, Sepharose '•B, protein A-Sepharose CL-'fB were the product

of Pharmacia Fine Chemicals, Sweden. Acrylamide, ammonium persulfate,

bisacrylamide, hydroxyapatite, N,N,N' ,N'- tetraethyl methylenediamine, Tween-

20 were obtained from Bio-Rad, U.S.A. Acetic acid, acetoni t r i le, EDTA (di-

sodium salt), glycine, hydrogen peroxide, sodium lauryl sulfate, sucrose,

ammonium sulfate, calcium chloride, methanol, sodium carbonate, sodium

chloride from Qualigens, India. Chloroform and liquid bromine were the pre­

paration of B.D.H. India. Isoamyl alcohol, sodium bicarbonate, zinc chloride

were purchased from Merck, India. Ferrous sulfate and potassium chloride

was from Sarabhai, India. Psoralen was a generous gi f t f rom C D . R . I . Luck-

now. P-nitrophenyl phosphate was obtained from C.S.I.R. Centre for Bio-

chemicals, Delhi. Cyanogen bromide was a product of S.D. f ine chemicals,

India. Acetaldehyde was from Fluka, Switzerland, Bromophenol blue and ly­

sine monohydrochloride was purchased from B.D.H. England. DEAE Cellulose

was obtained from Whatman, England. Perchloric acid and sodium azide were

products of Ferak-Berl in, Germany. ELISA plates were from NUNC Denmark

<5c Dynatech, U.S.A. Diphenylamine and ethanol were chemically pure.

[31]

Equipment

Spectronic 20 (Bauch &. Lomb, U.S.A.), UV-Visible 2^0 spectro­

photometer (Shimadzu, Japan), •spectrofJuorometer RF-5W (Shimadzu,

Japan), microplate reader MR-600 (Dynatech, U.S.A.), gel electrophoresis

apparatus GNA-100 (Pharmacia, Sweden), pH meter L1-10T and conductivity

bridge (ELICO, India), UV lamps having maximum emission at 25 ^ and

365 nm (Vilber-Lourmat, France), desk top microfuge RM-12C (REMI,

India) were the major equipments used in the present study.

Sera

The normal human sera was collected from healthy subjects without

evidence of SLE or other autoimmune disorders and it was ensured that

the subjects were not taking any drug known to induce antinuclear anti­

bodies. The SLE sera was obtained from patients who had elevated levels

of anti-native DNA antibodies and met preliminary criteria of the American

Rheumatism Association (ARA) for the diagnosis of disease (Tan et

al., 1982). All serum samples were heated at 56 °C for 30 min to inactivate

complement which inhibits the formation of, and also dissolves, preformed

antigen-antibody complexes (Miller and Nussenzweig, 1975; Schefferli

et al., 1980). After decomplementation the sera was kept at -20 °C

with 0.02 percent sodium azide as preservative.

Determination of Protein Concentration

Protein was estimated by the method of Lowry et al. (1951)

and Bradford (1976).

Protein Estimation by Folins-Phenol Reagent

The protein estimation by this method utilizes alkali (to keep

the pH high), Cu ions (to chelate proteins) and ta r ta ra te (to keep

the Cu ions in solution at high pH).

[32]

(a) Folin-Ciocalteu reagent

The reagent was purchased from C.S.I.R. Centre for Biochemicals,

Delhi. Before use the reagent was diluted 1:^ wi th high purity deionised

water.

(b) Alkaline copper reagent

The constituents of alkaline copper reagent were

(i) Two percent sodium carbonate in O.IM sodium hydroxide,

(ii) 0.5 percent copper sulphate in 1,0 percent sodium potassium tar tarate.

The working reagent was prepared fresh by mixing components

(i) and (ii) in the rat io of 50:1.

(c) Procedure

Freshly prepared 5.0 ml of alkaline copper reagent was mixed

with 1.0 ml of protein sample. The tubes were incubated at room

temperature for 10 min. One ml of diluted Folin-Ciocalteu reagent was

added followed by 30 min incubation at room temperature. The absorbance

was monitored at 660 nm. The protein content of the unknown sample

was determined from standard plot constructed by using bovine serum

albumin.

Protein Estimation by Bradford Method

This assay is based on the color change that occurs when Coomassie

Bri l l iant Blue G 250, in acid solution, binds strongly to protein hydro-

phobically and at positively charged groups. .>. the environment of these

positively charged groups, protonation is suppressed and a blue color

is observed ()^max = 595 nm).

(a) Dye pa-eparation

Hundred mil l igram of Coomassie Bri l l iant Blue G 250 was dissolved

thoroughly in 50 ml of 95% ethanol. To this solution was added 100ml

of 85% (v /v ) orthophosphoric acid. The resulting solution was diluted

to a f inal volume of 1 l i ter. On every use the dye solution was f i l tered.

[33]

(b) Protein assay

Solution containing 10-100 ug protein in a volume of upto 0.1ml

was pipetted into test tubes. The volume was adjusted to 1.0 ml with

appropriate buffer. Five ml of dye solution was added and the contents

were mixed by vortexing. The absorbance was taken at 595 nm after

two min and before one hr against a reagent blank prepared from 0.1

ml of buffer and 5.0 ml of dye solution.

Purification of DNA

Highly polymerized calf thymus DNA was purified free of proteins,

RNA and single stranded regions (Ali et al., 1985). DNA solution (2mg/ml)

in 0.1 X SSC buffer (0.015 M sodium-citrate, pH 7.3 containing 0.15

M sodium chloride) was mixed with equal volume of chloroform-isoamyl

alcohol (2'*:1 ratio) in a stoppered glass cylinder. The cylinder was sealed

and the contents were mixed end-to-end gently for one hour. The DNA

present in acqueous phase was separated from the organic layer by

low speed centrifugation. The extraction process was repeated once

and DNA was precipitated with two volumes of cold 95% ethanol and

collected on a glass rod. It was dried by pressing against the wall of

the container. The DNA thus obtained was dissolved in 0.1 X SSC buffer

and treated with RNase A {50 ug/ml) for 30 min at 37 °C to digest

RNA in case present. The digested DNA was precipitated, dried and

dissolved in 30 mM acetate buffer pH 5.0 containing 30 mM zinc chloride.

The digested sample was incubated with single strand specific enzyme,

nuclease Si (250 units/mg DNA) at 37 °C for 30 min. The reaction

was terminated by adding one-tenth volume of 0.2 M EDTA pH 8.0.

The purified DNA was extracted twice with chioroform-isoamyi alcohol

dnd reprenpitated with cold 95% ethanol. The DNA precipitate was

dried and dissolved in required buffer.

Determination of DNA Concentration

DNA was estimated by diphenylamine reagent and Hoechst dye

33258.

DU]

DNA Estimation by Burton Method

Color imetr ic estimation of DNA was carried out according to

Burton (1956) using diphenylamine,

(a) Diphenylamine reagent

To 750 mg of recrystall ized diphenylamine was added 50 ml of

glacial acetic acid followed by 0.75 ml of concentrated sulphuric acid.

The reagent was prepared fresh before use.

(b) Assay procedure

Varying amount of DNA in 1.0 ml was mixed wi th 1.0 ml ol

IN perchloric acid. The tubes were incubated for 15 min in a water

bath maintained at 70 °C. One hundred ui of 5.^3 mM acetaldehyde

was added followed by 2.0 ml of diphenylamine reagent. The tubes were

mixed thoroughly and allowed to stand at room temperature for 16-

20 hr for color development. Absorbance was read at 600 nm. The DNA

concentration in the unknown sample was determined from the standard

plot of purif ied calf thymus DNA.

Fluorimetric Assay of DNA

Fluorimetr ic estimation of DNA was carried out according to

Lebarca and Paigen (1979) using Hoechst dye. The dye Hoechst 33258

[C2-[2-(^ hydroxyphenol)-6-benzimidazolyl-6( 1 -methyl- ' f-piperazyl) benzi-

midazole] interacts wi th DNA wi th a many fold increase in fluorescence.

(a) Reagents

(i) 0.05 M sodium phosphate (pH 7A) containing 0.002 M EDTA and

2 M sodium chloride,

(ii) 200 ug/ml of Hoechst 33258 in deionized water.

On day of use, the working dye solution is prepared by diluting

(ii) 200-fold in solution (i).

[33]

(b) Method

Solution containing 0.1-1.0 ug DNA in 0.1 ml final volume was

mixed with 2.k ml of working dye solution. The mixture was incubated

for 5 min at room temperature and fluorescence was read at 'fSO nm

(^ex = 355 nm). The fluorescence of a solution containing dye plus buffer,

but no DNA, was taken as control.

Isolation of IgG

The IgG was purified by ion exchange chromatography, gel filtration

and affinity chromatography.

(a) Preparation of crude immunoglobulins

To 6.5 ml of fresh serum was added dropwise saturated ammonium

sulphate solution allowing each drop to disperse before the next was

added. When the concentration of ammonium sulphate reached about

20% of saturation, the serum had begun to turn milky. Most immunoglobulins

got precipitated by 35-37 percent of saturation. The precipitation was

carried out at ^ °C. The suspension was stirred for 30 min and then

centrifuged at 10,000 rpm for 15 min. The pellet thus obtained was

washed 2-3 times in 35 percent saturated ammonium sulphate. Repeated

washing of the pellet considerably reduced contamination of non-immuno-

globulins and did not lower the yield significantly. Finally the precipitate

was dissolved in Q.0]75 M sodium phosphate buffer, pH 6.8. Ammonium

sulphate was removed by overnight dialysis against 50Q-\Q00 volumes

of 0.0175 M sodium phosphate buffer, pH 6.8. The dialysis fluid was

changed several times at intervals of a few hours.

(b) DEAE Sephacel chromatography

Dialyzed crude immunoglobulins in 0.0175 M sodium phosphate,

pH 6.8, buffer were loaded on DEAE Sephacel column (1.5 cm X 20 cm)

previously equilibrated with the same buffer. The column was eiuted

with 0.0175 M Na-phosphate and fractions were collected until the

absorbance at 280 nm reached base line (Fig. I). First peak of the

chromatogram was found to contain IgG. The homogeneity of isolated

[36]

00

U.'

z < CD (X

o (/) CD <

FRACTION NUMBER

Fig.l. Isolation of IgG from an SLE serum by DEAE Sephacel. Thirty five percent saturated ammonium sulphate precipitated serum proteins were loaded onto the column. The peaK fractions v;ere pooled as IgG.

Inset: Polyacrylamide gel electrophoresis of isolated leG on 7.5% gel.

[37]

IgG was confirmed by single band nnovement in SDS-polyacrylamide

gel (Fig. 1 inset).

(c) Purification of IgG by gel filtration

The serum immunoglobulins were fract ionated on Sephadex G

200 column. Immunoglobulins from serum were precipitated wi th 50

percent saturated ammonium sulphate and processed accordingly to method

described earl ier. The immunoglobulins were f inal ly dissolved and dialysed

against copious volume of 0.05 M Tr is -HCl , pH 8.0 containing 0.15 M

sodium chloride and loaded on a gravity packed Sephadex G 200 column

(2 cm X 75 cm) previously equilibrated wi th the same buffer. The column

was operated at a flow rate of 25 ml/hr and fract ionated immunoglobulins

were monitored for protein at 280 nm. The IgG peak was identi f ied

by absorbance measurements at 278 and 251 nm. A part icular property

of mammalian IgG is that their absorbance at 278 nm is about 2.5-

3.0 higher than at 251 nm, in contrast to other serum proteins where

this rat io is about 1-1.5 (in neutral buffers).

(d) Affinity isolation of IgG

Protein A-Sepharose is a popular and simple tool in the puri f icat ion

of IgG and its subclasses. The antigen-binding region (Fab) is preserved

in its native state as the binding occurs through Fc region of the molecule.

Commercial ly obtained Protein A-Sepharose CL-^fB (1.5 gm) was

swelled in 0.01 M phosphate buffered saline (PBS), pH 7 A at room

temperature for ^8-72 hr. The swelled gel (about 5.0 mi) was poured

into a small column (0.9 cm x 15 cm). The column was washed once

with 0.1 M sodium-citrate, pH 3.0, to elute any bound material and

then equil ibrated with f ive volumes of PBS. Serum diluted wi th an equal

volume of PBS is passed through the column at a rate of about 20

ml/hr. The absorbance of the eff luent is monitored unt i l no protein

leaves the column. The bound IgG is eluted with 0.58% acetic acid

in 0.85% sodium chloride (Goding, 1976). To prevent the ef fect of acidic

elution buffer on IgG, the eluate was collected in 1 M Tr is-HCl, pH

[38]

8.5. Fractions containing IgG was read at 280 nm and concentration

was determined considering I'f O.D.^„„ = 10 mg IgG/ml. ° 280nm ° °

Hydroxyapatite Chromatography of Single and Double Stranded DNA

Hydroxyapatite discriminates nucleic acids endowed with different

secondary structures. Rigid, ordered structures having more affinity

for hydroxyapatite than flexible disordered ones.

The commercial preparation of hydroxyapatite was washed with

0,01 M Na-K phosphate, pH 6.8 buffer to remove fine particles therein.

Hydroxyapatite packed in glass column (1.5 cm x 5 cm) was equilibrated

with 0.01 M Na-K phosphate. Native DNA dialyzed against equilibrating

buffer was adsorbed to the column. The column was washed with ten

volumes of equilibrating buffer, at a fiov/ rate of 10 ml/hr, to wash

any unbound DNA. The bound DNA was eluted with increasing phosphate

molarity of 0.125 and 0.25 M strength. Native DNA got eluted at 0.25

M ionic strength and in the large majority of cases a complete recovery

was obtained, as judged from A_^„ measurement.

DNA denatured by heating for 15 min at 100 °C, at a concentration

of 50-100 ug/ml in 0.01 M Na-K phosphate (pH 6.8) buffer, showed

a chromatographic behaviour quite different from that of native DNA.

The stepwise elution results show that bulk of denatured DNA is eluted

at 0.125 M phosphate molarity. The propci , les of the main fraction

of denatured DNA were those of single stranded DNA. A small fraction

of denatured DNA is eluted at the same phosphate molarity as native

DNA.

Micrococcal Nuclease Digestion of Native Calf Thymus DNA

Deproteinized calf thymus DNA (2 mg/ml) in 0.006 M Tris, 0.1

M sodium chloride, 0.002 M calcium chloride, pH S.O was digested with

micrococcal nuclease (^0 units/mg) for 2.5 min at about 37 °C. The

reaction was terminated by adding one-tenth volume of 0.2 M EDTA,

pH S..0. The sample, after precipitation with cold ethanol was subjected

to nuclease SI digestion (150 units/mg DNA) for 30 min at 37 °C (Ali

[39]

et a l . , 1985). The resulting DNA was reprecipitated, dissolved in TBS

(0.01 M Tris, 0.15 M sodium chloride, pH 8.0) and passed through a

Sepharose 4B colunnn (1.5 cm x W cm) previously equil ibrated wi th

the same buffer. The fall through fractions (^.0 ml) were collected and

monitored for DNA content at 260 nm, using 1.0 O.D. at 260 nm =

50 ug dsDNA/ml. Size of the DNA fragments were analyzed on poly-

acrylamide slab using Lambda DNA digest of EcoR I and Hind III as

size marker.

Modification of DNA

Poly(dG-dC).poly(dG-dC), native DNA and small DNA fragments

were treated for 1 hr wi th k.O M sodium chloride (ef fect ive concentration

for B- to Z-DNA transition) respectively in c i t ra te buffer (0.02 M t r i -

sodium c i t ra te , pH 7.2 containing 0.001 M EDTA) at a f inal concentration

of 100 ug/ml in 3.0 ml total volume. The same amount of polymers

in PBS (0.01 M phosphate buffer, pH 7A containing 0.15 M NaCl) served

as low salt control sample. The absorbance values were recorded at

260, 280, 29 * and 296 nm to evaluate the absorbance rat io. The UV-

absorbance and difference spectra of both high and low salt sample

was recorded.

Nucleic acid polymers were brominated under high and low salt

conditions as described by Lafer et a l . (1981). Bromine saturated water

was prepared by mixing 0.55 ml of liquid bromine in t49.k5 ml of high

purity dist i l led water. Bromine water (7 uL /mL DNA sample) was added

to the DNA sample, wi th quick mixing, and kept in dark for 30 min

at room temperature. Af ter the completion of reaction, excess bromine

was removed by exhaustive dialysis against TBS (0.01 M Tris, pH 7.4

containing 0.15 M NaCl). Native DNA and small DNA fragments brominated

in high and low salt solution were designated as Br4 and BrN respectively.

Absorbance value of brominated DNA was recorded at 260, 280, 29 ^

and 296 nm against TBS, pH 7A. Average of absorption at 294 and

296 nm divided by absorbance at 260 nm was referred to as "absorbance

['fO]

rat io". The ratio obtained for brominated and non-brominated poly(dG-

dC).poly(dG-dC) was used as standard to which the absorbance rat io

for other nucleic acids were compared. The absorption spectra of low

and high salt brominated DNAs were recorded against buffer and compared

against the spectra obtained wi th same concentration of respective DNA

solution. The UV difference spectra was recorded by taking equal amount

of corresponding DNA polymer as control .

DNA-Polylysine Interaction

To nDNA samples (50 ug/ml), varying amount of D- and L-polylysine

(0-25 ug/ml) was added in a total assay volume of 3.0 ml containing

PBS, pH 7.4. The average molecular weight of D- and L-isomer was

13,000 and 1,94,000 respectively. Control samples were prepared by

taking equal amount of nDNA and polylysine separately. A l l assay tubes

were vortexed gently and lef t for 30 min at room temperature. While

recording the UV difference spectra of DNA-polylysine complex, nDNA

in PBS (50 ug/ml) was taken as control .

Preparation of DNA-Lysine Photoconjugate

The photoconjugate was prepared by mixing calf thymus DNA

with lysine (1:3 rat io, w/w) in PBS (0.01 M sodium phosphate, pH 7.4

containing 0.15 M sodium chloride), pH 7.4 followed by overnight incubation

at 4 °C. The mixture was irradiated under UV-A l ight (320-400 nm)

for 30 min in nitrogen atmosphere and dialyzed wi th copious volume

of 0.01 M carbonate-bicarbonate buffer, pH 10.7 to remove lysine molecules

bound electrostat ical ly to phosphate of DNA backbone. The photoadduct

was f inal ly dialyzed against PBS and photobound lysine was estimated

by ninhydrin reagent (Moore and Stein, 1954).

Preparation of DNA-Furocoumarin Photoadducts

DNA in TNE buffer (0.01 M Tris, 0.05 M NaCl, O.OOOi^ M EDTA,

pH 7.6) was mixed wi th psoralen and 8-methoxypsoralen in the rat io

of 3.2:1 (vj/vj, DNA:drug). The mixture kept in dark at room temperature

['fl]

for ^ hr was irradiated for 30 min under 365 nm ultraviolet l ight. Tiie

irradiated samples were extensively dialyzed against 0.01 M plnosphate

buffer, pH 6.8. Finally, the photoadduct was characterized by UV absorption

spectroscopy, thermal denaturation and fluorescence measurements.

DNA Modification by Hydroxy! Radical

The metal ions in DNA solution were chelated by pre-treatment

wi th EDTA for 3 hr at room temperature at a f inal concentration of

Im mol dm in phosphate buffer, pH IM (Blakely et a l . , 1990). Assay

tube containing mixtures of DNA (1515 um bp), ferrous ions (525 um)

and hydrogen peroxide (3030 uM) were incubated for 30 min at room

temperature in 0.01 M phosphate buffer, pH 1 A. Deionized DNA wi th

hydrogen peroxide and ferrous ions separately served as controls. A t

the end of incubation, reaction products were dialyzed exhaustively

against PBS and their UV absorption characteristics were recorded on

Shimadzu UV-Z'fO spectrophotometer.

Alkaline Sucrose Density Gradient Centrifugation

The damage to DNA by hydroxyl radical was analyzed by alkaline

sucrose gradient centr i fugat ion. Native and modified DNA samples (0.1

ml each) were layered separately on top of a ^.8 ml linear 5-20 percent

alkaline sucrose gradient containing 0.8 M sodium chloride, 0.2 M NaOH,

0.01 M EDTA and 0.015 M p-aminosalicylate at pH 12.5 (Fujiwara et

al . , 1977). The samples were centrifuged at 30000 rpm for 1 hr at 20

°C in an SW 50.1 rotor of a Beekman ultracentr i fuge. Af ter centr i fugat ion,

the bottom of the tubes were pierced wi th a needle and contents were

fract ionated by drop collection {^.1 ml each). The absorbance of collected

fractions was monitored at 260 nm.

Absorption-Temperature Scan

Native and modified DNA samples were subjected to heat denaturation

by an electronically controlled heating device. The cuvette temperature

was raised from 30 to 95 °C at a rate of 1 °C/min after ten min

['*2]

equilibration at 30 °C, The change in absorbance at 260 nnn was recorded

with increasing temperature. The helix-coil transition temperature (Tm)

was determined from percent denaturation data which was calculated

as follows:

Percent denaturation - x 100 Ap - A30

Where,

Ay is the sample absorbance at various temperatures.

Ar- is the final absorbance at 95 °C. F A,^ is the absorbance prior to heating.

The change in melting temperature ( A T ) was calculated using the

formula:

A T = Tm - T m "> m

w/u' xC and T S are the melting temperatures of native and modified where, I p-i m b r DNA sample respectively.

DEAE Sephadex A-50 Column Chromatography

Native and modified DNA samples were mixed separately with

perchloric acid (70%) in a stoppered tube and boiled for one hr to release

the bases. The sample, after neutralization, was placed on top of a

DEAE Sephadex A-50 column (1.6 cm x "^^ cm) equilibrated with Tris-

HCl buffer, pH 7.2. The elution was carried out with the same buffer

at a flow rate of 'fO ml/hr. Fractions (2.5 ml) were collected and monitored

at 260 nm. Mixtures of equimolar concentration of bases (A, G, C, T)

were also passed to locate the elution pattern of unmodified bases.

Native and modified bases were identified on the basis of their unique

absorption profile (Hasan and Ali, 1990). The extent of base modification

was calculated by measuring the peak area.

Measurement of Antibody Affinity

One hundred microgram of immune IgG was incubated, in Ependorff

tubes, with varying amount of DNA antigen in an assay volume of 0.2

[ ^3]

mj. The controJ experiment was carried out with the same amount of

antigens and preimmune IgG. The contents were vortexed and incubated

for 2 hr at 37 °C and overnight at k °C. The soluble antigen- antibody

complex was precipitated by adding cold saturated ammonium sulphate.

The assay tubes were spun in microfuge at 8,000 rpm for 2.5 min and

supernatant was careful ly removed. The unbound antigen was estimated

by Hoechst dye 33258 and diphenylamine reagent. The binding data were

analyzed and antibody af f in i ty was calculated (Langmuir, 1918).

Immunization Scheme

(a) Antibodies against normal human IgG

Female rabbits (8-12 months old, weighing 1.0 to 1.5 kg) were

immunized intramuscularly, in hind limbs, wi th freshly prepared emulsion

of purif ied IgG (0.5 ml of 200 ug/ml in PBS, pH 7A) in Freunds' complete

adjuvant in a total volume of 1.0 ml. Blood was collected from rabbit

by cardiac puncture prior to immunization. Similar injections, but wi th

incomplete Freunds' adjuvant, were given at weekly intervals for f ive

weeks. Anti-IgG act iv i ty was evaluated in the immune serum collected

after one week of the last injection.

(b) Antibodies against DNA modified by hydroxy] radical

Female rabbits were immunized wi th radical modified DNA as

described earlier (Hasan and A l i , 1990). The immunizing antigen (100

ug) was mixed wi th MBSA (1:1 rat io, w/w) and emulsif ied wi th equal

volume of Freunds' complete adjuvant for f i rst inject ion. Subsequent

injections were given in incomplete adjuvant. A single animal received

a totaJ of 600 ug antigen in the course of six injections. Antibody act iv i ty

was checked in the immune serum. Preimmune and immune sera were

collected from the rabbit by cardiac puncture.

[f'f]

Immunoaffinity Purification of Antibodies

(a) CNBr activation of Sepharose fB

Fifteen ml of supplied Sepharose 4B was suspended in dist i l led

water, f i l tered and washed with 300 nnl of cold disti l led water on a

sintered glass funnel (porosity G-2). Moist gel (about 10 g) in 10 ml

of sodium carbonate was kept in ice-NaCl bath placed on magnetic

st irrer. One gram cyanogen bromide in 0.8 ml acetonitr i le was slowly

added to the gel w i th gentle st i r r ing. A f te r 12 min, the reaction product

was f i l tered on sintered glass funnel washed with 'fOO ml of cold 0.01 M

sodium bicarbonate and resuspended in 10 ml of the same buffer. The

unreacted CNBr (drained eff luent) was reacted wi th ferrous sulphate

to convert it into harmless ferrocyanide. A l l steps were carried out

in a fume-hood chamber (A l l , 198'i).

(b) PoIy-L-lysine coupling to activated Sepharose 'JB

The method described by Wllchek (1973) was fol lowed. Immediately

after CNBr act ivat ion of gel, equal volume of poly-L-lysine (100 mg

in 10 ml of 0.1 M sodium bicarbonate) was added and kept at ^ °C

for 12 hr wi th slow st i rr ing. The buffer was drained out and the gel

was washed successively with 100 ml each of cold (i) disti l led water

(ii) 0.1 N HCl (i i i) 0.1 M sodium bicarbonate and (iv) dist i l led water t i l l

neutral. Finally the gel was suspended in UO ml of 0.15 M acetate buffer,

pH ^.5. The extent of poly-L-lysine depletion due to covalent coupling

with activated Sepharose ^B was quantif ied by the treatment of drained

eff luent wi th TNBS as described by Habeeb (1966).

(c) Immunoaffinity purification of anti-DNA antibodies

DNA -[Polylysyl -Sepharose ^B] a f f in i ty column was prepared

according to method described earlier (Nicotra et a l . , 1982) wi th l i t t l e

modif icat ion. Twenty ml of polylys yl-Sepharose ifB was packed in a

glass minicolumn (1.6 cm X 5 cm) and equilibrated with acetate buffer.

Purif ied DNA (12.5 ml of 100 ug/ml in acetate buffer) was loaded onto

the gel and unbound material was washed with 50 ml of PBS, pH 7A.

1^5]

Heat decomplemented SLE serum previously dialyzed against PBS was

diluted 1:10 and passed through the colun^in. The unbound antibodies

were washed and bound proteins were eluted with linear ionic strength

gradient of 0.15-3.0 M sodium chloride in 0.01 M phosphate buffer, pi I

7A. Fallthrough material was collected and monitored at 251 nm, 26C

nm, 278 nm and 280 nm. The molar concentration of sodium chloride

in fractions was evaluated by conductance measurements.

(d) Regeneration of affinity column

The same column was regenerated several times by washing

succesively with 50 ml each of the foliowings: (i) distilled water (ii)

0.1 N HCl (iii) 0.1 M sodium bicarbonate (iv) distilled water till neutral.

Gel Electrophoresis

(a) Polyacrylamide slab gel electrophoresis for proteins

Polyacrylamide slab gel electrophoresis was performed under denaturing

conditions as described by Laemmli (1970). The following stock solutions

were prepared:

(i) Acrylamide-bisacrylamide (30:0.8)

Thirty gram acrylamide and 0.8 g bis-acrylamide was dissolved

in a total final volume of 100 ml. The solution was mixed thoroughly,

filtered and stored at ^ "C in amber color bottle.

(ii) Resolving buffer : 3 M Tris-HCl, pH 8.8

A stock solution was prepared by dissolving 36.3 g Tris base in

^ 8.0 ml of 1 N HCl. The contents were mixed properly, pH adjusted

to 8.8 and the final volume brought to 100 ml.

(iii) Stacking gel buffer : 0.5 M Tris-HCl, pH 6.8

6.05 g Tris base was dissolved in 40 ml distilled water t i trated

to pH 6.8 with IN HCl and the volume adjusted to 100 ml with distilled

water.

[t6]

(iv) Electrode buffer : 0.025 M Tris,

0.192 M glycine,

0 .1% SDS

pH 8.3

(v) Sample buffer

Six gm Tris was dissolved in 80 ml disti l led water and pH adjusted

to 6.8 wi th O-phosphoric acid. The volume v/as made to 100 mi with

disti l led water. To 12.5 ml of above solution was added 1 mg bromophenol

blue and 12.5 ml glycerol. The contents were mixed thoroughly prior

to use.

(b) PAGE procedure

The glass plates (19 cm x 16 cm) were soaked in chromic acid

and thoroughly washed with tap water followed by one rinse wi th dist i l led

water and ethanoi. Finally the plate was cleaned with tissue paper soaked

m acetone, air dried and sealed with 1% agarose. The glass plates were

separated by 1.5 mm thick spacer. 7.5% separating gel was layered

with 2.5% stacking gel according to recipe given below (all volumes

are in ml).

Solution Resolving gel Stacking gel

Acrylamide-bisacrylamide 7.5 2.5

Resolving buffer 3.75

Stacking gel buffer - 5.0

10% SDS 0.3 0.2

Disti l led water 16.95 11.3

1.5% Ammonium persulphate 0.025 0.05

The reagents were mixed and poured between glass plates. The

gel was allowed to harden at room temperature. Protein samples

(10-20 ug) in 0.01 M phosphate buffer, pH 7A were mixed wi th sample

buffer in the rat io of ^:1 (w/w). SDS was added to a final concentration

of 2% and protein sample was boiled for 5 min prior to loading. The

electronhoresis was carried out for 6-8 hr at room temperature ?t 70

[47]

Volts. The gel was stained overnight in 0.0.5% Coo massie Bri l l iant

Blue (R 250) nnixed wi th 25% iso-propyl alcohol and 10% glacial acetic

acid. Destaining was carried out in a mixture of 10% acetic acid and

10% methanol.

(c) DNA electrophoresis on polyacrylamide gel

DNA electrophoresis was carried out fol lowing the procedure

ot Sealey and Southern (198.5). The fol lowing buffers were prepared

for DNA gel electrophoresis:

(i) U0% acrylamide in disti l led water

(ii) 2% bisacrylamide in disti l led water

(ui) Resolving gel buffer : 0.9 M Tris-borate, pH 8.3 containing 0.025

M EDTA

(iv) Electrode buffer : 0.09 M Tris-borate, pH 8.3 containing 0.0025

M EDTA

Recipe for 7.5% DNA-PAGE (all volumes in ml)

Solution Resolving gel Stacking gel

W% Acryamide

2% bisacrylamide

Gel buffer

Disti l led water

Ammonium persulphate

TEMED

The DNA sample (0.5-1.0 ug) was mixed wi th one-tenth volume

of dye (30% Ficol l , 0.025% xylene cyanole FF in gel buffer) and electro-

phoresed for 6-8 hr at 70 Volts. The gel was stained with ethidium bromide

(1 ug/ml) for 3U-4G min & DNA bands were visualized under UV l ight.

(d) Agarose gel electrophorisis of DNA

Thirty ml of 1% agarose NA dissolved in electrophoresis buffer

(O.O' M Tris-acetate, pH 8.0 containing 0.002 M EDTA) was poured

on horizontal gel tray (sealed from both the sides) and allowed to harden

for 30-60 min at room temperature. The DNA samples, prepared as

7.5

7.5

i^.O

21.0

50 mg

0.025

1.25

1.25

1.0

6.5

15 mg

0.010

['fS]

described earlier, were put into the wells and eiectrophoresed for 2-

^ hr at 30 mA current in tank f i l led wi th the sanne buffer.

(e) Gel retardation assay

The antibody binding to DNA antigens was analyzed by altered

electrophoretic migrat ion. Mixtures of 0.1 ug of B-DNA fragnnents (average

size 200 bp) and varying amounts of IgG (50-250 ug) isolated from SLE

serum were incubated in a total volume of 20-30 ul of 0.01 M sodium

phosphate buffer, pH 7 A containing 0.15 M sodium chloride for 1-

4 hr at 'f °C. In case of DNA-RNA hybrid, 0.5 ug polymer was incubated

with varying amounts of SLE IgG (25-100 ug). A t the end of incubation,

2 ul dye solution was added to each assay tube. The complex was eiectro­

phoresed on polyacrylamide or agarose gel using Tris-acetate EDTA

(pH 8.0) buffer at room temperature. On completion of electrophoresis,

gels were stained and photographed under UV i l luminat ion. Control assays

were carried out in the presence or absence of normal human IgG and

ant i-DNA psoralen crosslink IgG that has been shown to be non-reactive

towards native DNA (Hasan et a l . , 1991).

Immunological Detection of Antibodies

Immunized rabbit sera and SLE sera were tested for antibodies

by immunodiffusion, counterimmunoelectrophoresis, and enzyme-linked

immunosorbent assay,

(a) Immunodiffusion (ID)

ID was carried out by Ouchterlony diffusion system using glass

petri dishes. Six ml of 0.^ percent molten agarose in PBS (pH 7A) containing

0.1 percent sodium azide was poured onto glass petri dishes and allowed

to solidify at room temperature. Wells, each 5 mm in diameter and

separated by 8 mm distance were cut into liardened agarose gel. Appropriate

concentration of antibody and antigen was placed in the wells and petri

dishes were allowed to stand in moist chamber at room temperature

for ^^8-72 hr. The gels were v/ashed with 5% sodium-citrate for two

[ W ]

hr to remove non-specific precipit in lines. The result was analyzed visually

and photographed.

(b) Counterimmunoelectrophoresis (CIE)

CIE was performed according to the procedure described by Kurata

and Tan (1976) with part ial modif icat ion. Molten agarose (0.6%) in l"!)

mM barbital buffer, pH ZM containing 0 .1% sodium azide was poured

onto 2.5 mm thick glass slides (2.5 cm x 7.5 cm) and allowed to harden

at room temperature. The slides were stored at k "C and used within

a week. The wells, 3 mm in diameter, were cut on the slides at a distance

of 5 mm between the two opposite wells. Antigen (nucleic acid or protein

in PBS, pH 1M) was placed in cathodal well and antibodies (immune

sera or IgG) in the anodal wel l . Electrophoresis was performed for 30

min in 50 mM barbital buffer, pH %M with a current of 3-'f mA per

slide. Non-specific precipit in lines were removed by treatment with

5% sodium-citrate solution.

(c) Enzyme-linked immunosorbent assay (ELISA)

(i) Buffers and substrate

Tris buffer saline (TBS) :

Tris buffer saline Tween-20

(TBS-T)

Bicarbonate buffer

0.01 M Tris,

0.15 M NaCl,

pH lA

0.02 M Tris

0.14^ M NaCl ,

0.00268 M KCl ,

pH 7.4 containing

500 ul Tween-20

0.015 M sodium

carbonate, 0.035 M Na-

bicarbonate, pH 9.6

containing 0.002 M

magnesium chloride.

[50]

Substrate 500 ug p-nitrophenyl

phosphate per ml of

bicarbonate buffer

(ii) Assay procedure

Polystyrene wells v/ere precoated with 100 ul of polylysine (50

ug/ml in water) for ^3 min at room temperature. The wells were emptied

and washed thrice with TBS. Hundred ul nucleic acid antigens (2.5 ug/ml

in TBS) were coated and incubated for 2 hr at room temperature followed

by overnight incubation at 'f °C. The protein antigen was coated at

50 ug/ml in 0.05 M carbonate buffer (pH 9.6) without polylysine treatment

of the wells. Next morning antigen coated v/ells were emptied and washed

four times with TBS-T. Hundred ul of poly-L-glutamate were added

to the antigen wells and washed after 2 hr. This was followed by 0.15

ml of BSA (1.5% in TBS) coating for 2-6 hr to saturate the binding

sites. Antibody to be tested, diluted in 1% TBS-BSA was added to antigen

coated and control wells for 2 hr at room temperature and overnight

at k °C. After adding appropriate alkaline phosphatase conjugate, color

was developed with p-nitrophenyl phosphate. Absorbance measurements

(at ^10 nm) were determined directly from microtiter plates with a

Dynatech MR 600 reader.

(iii) Competition ELISA

The antigen binding specificity of antibody was defined by competition

-inhibition experiments. For the assay, varying concentration of soluble

competitors were incubated with antibody (serum or purified IgG). The

mixture was incubated at room temperature for 2 hr and overnight

at ^ °C, Inhibition of antibody binding in presence of various competitors

was determined by measuring their ability to reduce the resulting absorbance

relative to control assays lacking competitor. Each serum dilution or

competitor was tested in duplicate. The rosult of inhibition were expressed

as percent inhibition.

[51]

Induction in Rabbits of Antibodies Againsi Normal Human IgG

Rabbits immunized wi th normal human IgG produced precipi tat ing

antibodies. The antibodies were characterized by counterimmunoeJectro-

phoresis (CIE), iminunodil'Iusion (ID) and solid phase enzyme immunoassay

(SPEIA). The antibody t i ter was found to be 1:16 by Ouchterlony double

diffusion in agarose gel (Fig. 2a). The specif icity of antigen-antibody

reaction was ascertained by immuno-crossreactivity of induced antibodies

with immunogen, preimmune rabbit IgG and serum albumin (Fig. 2b).

The results showed react iv i ty with immunogen but nonreactivity towards

preimmune IgG and serum albumin. ELISA of immune serum on poly­

styrene plate coated wi th normal human IgG showed a t i te r of > 1:6^00.

The binding of preimmune serum was of very low magnitude (Fig. 3).

Double Strandedness of Native DNA

Purif ied calf thymus DNA was characterized for double strandedness

by physico-chemical methods. Agarose gel electrophoresis of DNA (1 u g)

digested wi th nuclease SI (20 l.U.) showed identical mobil i ty and f luore­

scence on staining with ethidium bromide in comparision to native DNA

(data not given). By contrast, heat denatured DNA incubated under identical

conditions was completely digested by the enzyme. The double strandedness

ol puri f ied DNA was further investigated by chromatography on hydroxy-

apatite. The hydroxyapatite bind both single and double stranded DNA

molecules which are eluted at di f ferent ionic strength. The elution of

purif ied DNA as a single peak at 0.25 M Pi is suggestive of its double

strandedness. Purif ied DNA was also characterized by thermal denaturation.

Thermal denaturation of native DNA showed no signif icant change in

absorbance upto 80 °C. A sharp melting prof i le (Tm = 87.5 °C) (Fig. ^)

reiterated the double strandedness of native DNA.

[52]

(a) (b)

Fig.2(a). Evaluation of titer of anti-human IgG antibodies by Ouchterlony immunodiffusion. The central well (C) contained immunogen and peripheral wells \,2,3,k,5 and 6 represents neat, 1:2, 1:^, 1:8, 1:16 and 1:32 dilution of antiserum.

(b). Immunocrossreactivity of rabbit anti-normial human IgG. The central well (C) contained anti-human IgG whereas peripheral wells 1,2 and 3 represents immunogen, preimmune rabbit IgG and bovine serum albumin respectively.

[53]

E c o

LJU

O z < m a: o CD <

0.4

0 . 3 -

0.2

0.1 ov,.

" /—^ 2,3 2.6 29 32 3,5 3.8

LOG SERUM DILUTION

Fig.3. Direct binding ELISA of anti-human IgG. The wells were coated with human IgG. Immunized ( —•— ) and preimmune (—O—) serum.

[5'f3

lOOh

z g I— <

ID

< Z UJ

o

UJ

o cr Ixl Q.

TEMPERATURE (°C)

Fig.'f. Temperature dependent nnelting prof i le of purif ied native DNA.

[55]

Detection of Anti-Nucleic Acid Antibodies in SLE Sera

The direct binding of SLE antibodies to native DNA was determined

by ELISA. Table 2 shows the titer of serum antibodies investigated

during the course of this study. Further studies were carried out on

the sera which had elevated levels of anti-dsDNA antibodies, a marker

antibody for the diagnosis of SLE.

Fractionation of Serum Immunoglobulins

Serum samples (control or SLE) after precipitation with fifty

percent saturated ammonium sulphate were dialyzed extensively against

0.05 M Tris-HCl, pH 8.0 containing 0.15 M sodium chloride and fractionated

on Sephadex G 200 column. The fractions were read at 280 nm. Two

well resolved protein peaks were identified (Fig. 5). Their absorbance

was monitored at 278 and 251 nm in order to identify IgG peak. Fractions

from 35 - ^3 were taken as IgG as their absorbance ratio (278/251)

was 2.5.

Characterization of Normal Human and SLE IgG

Only antibodies of the IgG class were studied since it has been

found that several cross reactions of anti-DNA antibodies are mainly

associated with IgM antibodies of low avidity. Moreover, they are not

the true representatives of the pathogenic anti-DNA antibodies found

in SLE patients (Edberg and Taylor, 1986).

The DEAE Sephacel purified IgG from control and SLE sera were

found to be nearly free from contamination by other classes of immuno­

globulins as judged by their homogeneous movement in SDS-PAGE under

non-reducing conditions. Purity of the isolated IgG was also characterized

by ultraviolet absorption measurement at 251 and 278 nm. The absorbance

ratio (278/251) was found to be constant for one class of immunoglobulins

derived from rabbit, human and goat. It has been reported that mammalian

IgG absorbance at 278 nm is about 2.5 - 3.0 higher than at 251 nm,

in contrast to other serum proteins wheie this ratio is about 1 - 1.5.

[56]

TABLE 2

Direct Binding of Anti-DNA Autoantibodies to Calf Thymus Double Stranded DNA _

SLE Serum Reciprocal of Antibody Titer

1 200 2 800 3 100 ^ 1600 5 ^00 6 3200 7 200 8 6^00 9 6400 10 1600 11 1600 12 102400 13 3200 14 200 \5 1600 16 3200 17 3200 18 12800 19 6400 20 3200 21 3200 22 3200 23 3200 24 6400 25 800 26 1600 27 3200

Name of SLE patients from v/hum the sera were derived have

been changed into numbers.

[57]

E c o CO rsj

UJ

o z < CD

o CD

in

00

< cr Hi

z < GO CC

o m <

00 20 30 ' .O 50

F R A C T I O N N U M B E R

Fig.5. Fractionation of SLE serum immunoglobulins on Sephadex G 200. F i f ty percent saturated ammonium sulphate precipitated serum proteins were loaded onto the column. Fractions were collected and monitored at 280 nm (—0— ) and absorbance ratio (278/251) (—»—) was evaluated. Fractions f rom 35-^3 were taken as IgG.

[58]

Measurements of absorbance at these two wavelengths may therefore

indicate the fraction(s) containing IgG. Table 3 represents the absorbance

values recorded for material isolated from one of the SLE sera. Similar

values v.'ere obtained for normal human IgG. Every fresh batch of IgG

was tested for purity before further investigations.

Isolation of DNA Fragments

Small DNA fragments were prepared by controlled digestion of

native calf thymus DNA with micrococcal nuclease. The digested samples

were passed through a Sepharose kb column. The fallthrough fractions

were collected and monitored for DNA content at 260 nm (Fig. 6).

Sizie of the DNA fragments was analyzed on non-denaturing polyacrylamide

gel using Lambda DNA digest of EcoR I and Hind III. The average size

of the fragments was in the range of ^100 to 600 base pairs (Fig. 6 inset).

The fragments behaved as double stranded molecules on thermal denatura-

tion.

Tm Dependence on Fragment Size

The DNA fragments of varying size were melted by an electronically

controlled heating device. The denaturation data were replotted to deter­

mine the helix-coil transition temperature (Tm) (Fig. 7). The Tm of

the fragments was found to vary with size. The data presented in Table ^

can most simply be interpreted as inter- fragment variation of (G+C)

and (A+T) residue. The fragments rich in G+C content will resist heating

as compared to A+T rich fragments which denature early in the transition.

Human Anti-Native DNA Autoantibody Binding to B-DNA

B-conformation specific antibodies from human SLE serum were

isolated on affinity column of DNA-[polylysyi~Sepharose ^B]. The SLE

serum was diluted 1:10 in PBS and passed through the column. After

application of linear sodium chloride gradient, the bound material eluted

in three peaks (Fig. 8). Colorimetric estimation and ultraviolet measure-

[59]

TABLE 3

13

14

15

16

17

18

19

20

Ultraviolet Absorption Characteristics of SLE IgG

Fraction Absorbance at Absorbance ratio Number 278/251

251 nm 278 nm

The IgG was fractionated on DEAE Sephacel matrix .

0.200

0.193

0.153

0.23^1

0.161

0.078

0.065

0.061

0.428

0.485

0.358

0.523

0.315

0.164

0.128

0.122

2.14

2.51

2.33

2.23

1.95

2.10

1.96

2.00

[60]

4 i ^0 3 8 36 3(f iZ2>o ZB2L2A

O

Z < CD

o CO CD <

00 20 40

FRACTION NUMBER

60

Fig.6. Sepharose ^B gel filtration elution profile of calf thymus DNA digested with micrococcaJ nuclease. Four ml fractions were collected at a flow rate of ^0 ml/hr.

Inset: Alternate fractions from 2'f to ^2 were analyzed on 7.5% polyacryiamide slab gel alongwith EcoR 1 and Hind III digested Lambda DNA as marker.

[61]

lOOr

g

< D

< Z UJ

o V —

2 UJ CJ

a a

o I— < a t— < z UJ Q »— Z uJ 'O

a: a

TEMPERATURE {°( TEMPERATURE (°C)

Fig.7. Thermal denaturation curves of DNA fragments. The temperature was raised at a rate of I "C/min from 30° to 95 °C. (a) 70 bp, (b) 90 bp, (c) 175 bp, and (d) 300 bp.

[62]

TABLE 4

Melting Temperature and Percent Hyperchromicity of DNA Fragments

Native DNA fragments (average size)

300 bp

175 bp

90 bp

70 bp

Percent duplex melted at

80 OC

10

12

39

55

Percent chromic!

at 95 OC

29.8

40.1

35.0

31.0

hyper-ty

Melting tempera­ture (Tm),

86.0

85.0

80.5

79.5

[63]

4

< CD QL O U^ CD <

r. r

fc

CC' " N J

0.2>^J3^ 1_

00 \0 20 30

FRACTION NUMBER

40

o o z

Fig.8. Immunoaffinity purification of anti-nDNA antibodies on DNA-[poiyiysyJ-Sepharose 'iB] column. Anti-DNA positive SLE serum was passed through the column and bound material was eluted with linear gradient ( ) of sodium chloride (0.15 to 3 M) in 0.1 M Na-Pi buffer, pH 7A. Inset shows the results of Ouchter-lony immunodiffusion. The central well (C) contained anti-human IgG whereas peripheral wells 1,2 and 3 represents corresponding peak.

[6'f]

ments revealed proteins in peak 1 and peak 2 and DNA in peak 3. Tine

absorbance ratio of peak I (278/251) was found to be 2.62 typical of

mammalian IgG. Ouchterlony immunodiffusion of peak fractions wi t l i

anti-human IgG showed precipit in line of complete identi ty wi th peak

1 and non-reactivity with materials from peak 2 and peak 3 (Fig. 8

ifisct). This inturn sliows that protein in peak I is a homogeneous population

of IgG recognizing the native B-conformation of DNA.

SLE Anti-DNA Antibodies Recognize DNA-Lysine Photoadduct

Native calf thymus DNA covalently modified wi th lysine under

UV-A light (320-^00 nm) was shown to bind naturally occurring ant i -DNA

antibodies from human SLE sera (Alam et a l . , 1992). By the photoaddition

of one lysine molecule per helical turn of DNA (unpublished data), the

native B-conformation was considerably altered. The altered molecule

dif fer ing from average B-helix geometry has been found to be immunogenic

in contrast to native DNA.

Direct binding of SLE ant i-DNA antibodies to chemically modified

and photomodified DNA antigens has been presented in Table 3. Besides

nDNA, the naturally occurring antibodies indicated appreciable binding

with Z-DNA and DNA-lysine photoadduct. In another set of experiments

it was found that seventy percent of the SLE sera recognized DNA-

lysine photoadduct better than native DNA adsorbed direct ly on the

polystyrene plate (Fig. 9). The binding pattern of SLE autoantibodies

upto 1:3200 dilution with either of the antigens was similar to those

observed at lower dilution (1:100 dilution). In competi t ive binding experi­

ments anti-dsDNA IgG isolated from af f in i ty matr ix of native DNA-

[polylysyl-Sepharose '>B] showed maximum af f in i ty towards DNA-lysine

photoadduct followed closely by nDNA based on the amount of competitor

required for 50 percent inhibit ion (Table 6).

Human Anti-DNA Autoantibody Binding to A-, B-, and Z-DNA

Serum antibodies from SLE exhibited a broad spectrum of antigen

recognition as demonstrated by their abi l i ty to recognize a variety of

nucleic acid antigens presenting altogether divergent conformations (Alam

[65]

TABLE 5

Binding Characteristics of Naturally Occurring Autoantibodies with Native

and Modified Nucleic Acids

Antigen Reciprocal of antibody t i ter

nDNA \02W0

DNA-lysine photoadduct 51200

Z-DNA 12800

ssDNA 3200

DNA complexed with cobalt hexaammine 1600

Poly(dG-dC)-psoralen photoadduct 800

DNA intercalated with ethidium bromide ^00

[66]

6 c

o

< CD

QD <

SLE SERUM SAMPLE

Fig.9 Binding of human SLE antibodies to native DNA ( | ) and DNA-lysine photoconjugate ( D )• The binding of control serum with either of the antigens was negligible.

[67]

TABLE 6

Relative Binding Profile of Anti-DIMA IgG

Comp)etitor

Competitor concentration for 50% in­hibition (ug/ml)

7.5

5.5

38.0

-

Maximum percent inhibition

at 50 ug/ml

97

91

55

39

21^

nDNA

DNA-Iysine photoadduct

ssDNA

nDNA UV-irradiated

F^NA

[68]

and AJi, 1992). Direct binding ELI5A un polystyrene plates coated with

native calf thymus DNA indicated high binding (titer>l:6^00) with different

dilutions of a typical SLE serum (Fig. 10). While nDNA proved to be

an effective inhibitor of DNA-anti-DNA antibody interaction, RNA (buffalo

thymus) and single stranded DNA showed inhibition at higher competitor

concentration (Fig. 11). Competition ELISA with Z-form of poiy(dG-me dC).

poly(dG-me dC) and high salt brominated calf thymus DNA showed appre­

ciable inhibition in antibody activity (Fig. 12). The latter polymer has

been shown to attain Z- or analogous conformation on bromination in

high salt (Hasan and Ali, 1990). However, poly(dG-dC).poly(dG-dC) in

low salt (B-conformation) showed inhibition similar to nDNA. With former

the maximum inhibition attained was 81 percent and 50 percent inhibition

obtained with only 1.1 ug of inhibitor (Table 7). Autoantibody recognition

of poly(rG).poly(dC), a polymer known to be present in A- or analogous

conformation, resulted in 77 percent elimination of antibody activity

with 10 ug of inhibitor (Fig. 12).

Competition Studies by Polynucleotides

Besides native DNA, certain right handed helical double-stranded

polydeoxyribonucleotides which differ slightly from the average B-DNA

helix were also employed as inhibitors of nDNA-autoantibody interaction.

It was found that the epitope recognition of polymers by the antibody

was of varying degree (Fig. 13).

Gel Retardation Assay

Antibody binding to polynucleotide was studied in poiyacrylamide

and agarose gel. When mixtures of SLE anti-DNA IgG and DNA fragments

(average size 200 bp) were subjected to slab gel electrophoresis under

non-denaturing conditions, the antigen-antibody complexes showed different

degree of retarded mobility (Fig. l^a). With increasing concentration

of antibody the amount of immune complexes, as judged by their fluore­

scence intensity, was found increased whereas the amount of unbound

[69]

E c o

us

< OD a: o CD <

- LOG SERUM DILUTION

Fig.lO. Assay of anti-DNA antibodies by ELISA. The polystyrene plate was coated with native DNA. Normal human serum (—O—) and SLE serum (-•—).

[70]

2

Ul o UJ

a.

0.01 0.1 1.0 10

COMPETITOR , ng /ml

Fig. 11. Inhibition ELISA of SLE ant i-DNA antibodies. The competitors were native DNA (—O—), heat denatured DNA ( — • — ) and RNA (-•)(•—).

[71

2

g

UJ

or ui a

0.01 0,1 1.0 10

COMPETITOR , Jug /ml

Fig.l2. Inhibition ELISA of SLE anti-])NA antibodies. The competitors were Br4 DNA ( - ^ - ) , poly(dG-me5dC).poly(dG-me5dC) ( - • - ) and poly (rG). poly(dC)

[72]

TABLE 7

Inhibition of the Binding of Anti-DNA Autoantibodies

by Polynucleotides

Competitor

nDNA

Poly(dA-dU).poly(dA-dU)

Poly(dG-dC).poly(dG-dC)

Poly(dA-dG).po]y(dC-dT)

ssDNA

PoJy(dA).poJy(dT)

Poly(rG).poly(dC)

Poly(dG-me dC).poIy(dG-me dC)

Br-DNA

RNA

Poly(dI-dC).poly(dI-dC)

Poly(dG).poIy(dC)

Poly(dC)

PolyidG)

Poly(U)

Concentra tion for 50% inhibi­tion (ng/ml)

0.38

1.0

1.1

3.6

3.8

5.3

3.2

0.9

9.5

NIL

NIL

NIL

NIL

NIL

NIL

Maximum Percent Inhibition at 10 ug/ml

77

77

81

65

63

68

77

55 •X-

85

^^

39

27

35

NIL

NIL

% Relative Affinity

100

38

35

I I

10

07

12

42

04

-

-

-

-

-

-

Inhibition at 100 ug/ml.

[73]

2 O

-T

2 U

a: a

0,01 0.1 10

COMPETITOR , ug / m l

Fig.l3. Competit ion ELISA with SLE ant i-DNA antibodies The coating antigen was native calf thymus DNA. The competitors were poly(dG).poly(dC) i--^-), poly(dA). poly(dT) (—•(>-—), po]y(dA-dG).poly(dC-dT) (—O—) and doube stranded: poly (dG-dC) ( — • — ), poIy(dl-dC) (-A-) and poly (dA-dU) (-A-).

[7^]

m^u «iiiiii»

(a) (b)

Fig.l^(a). Binding of SLE IgG to B-DNA fragments as analyzed by gel retardation assay. B-DNA fragments (200 bp) (O.I ug) were incubated with buffer (lane I), 100 ug of normal human IgG (lane 2), 100 ug of anti-DNA-psoralen crosslink IgG (lane 3) or various concentrations of SLE antibodies (50 ug, lane 5; 100 ug, lane 6; 130 ug, lane 7; 200 ug, lane 8; 250 ug, lane 9) for I hr at 'i °C and eiectrophoresed on polyacrylamide gel (2.5% stacking on 7.5% separating). Lane k contained 100 ug of SLE IgG.

(b). Binding of SLE IgG to poly (rG).poly(dC). The hybride (0.5 ug) was incubated with buffer (lane 1), 100 ug of normal human IgG (lane 2) or various concentrations of SLE IgG (25 ug, lane 3; 50 ug, lane ^; 75 ug, lane 5; 100 ug, lane 6) for I hr at ^ °C and eiectrophoresed on 1% agarose.

[75]

DNA fragments indicated proportional decrease in their concentration

(Lanes 5 thru 9). Norn-ial human IgG and anti-DNA-psoraien crosslink

IgCi (Hasan et a l . , 1991) showed no immune complex formation (lanes 2

and 3) and were negative controls.

The antibody binding to polY(rG).poly(dC) was also studied by

agarose gel eletrophoresis (Fig. I'+b). In this case too, retardation in

the mobil i ty of hybrid was observed as a consequence of immune comp-

lex(es) formation (lanes 3 to 6). The fluorescence was proportional to

the size of iininune complex. Normal human IgG incubated under identical

conditions did not show immune complex format ion.

Binding of Anti-DNA Antibodies to DNA- Furocoumarin Photoadducts

Psoralens (furocoumarins) are a class of compounds which can

intercalate between base pairs of double stranded nucleic acids. Upon

UV-A irradiat ion, the intercalated psoralens can photoreact prominently

with pyrimidine bases to yield monoadducts as well as interstrand diadduct

(Song and Tapley, 1979; Cimino et al . , 1985; Parsons, 1980).

Furocoumarins modified native DNA preparations (after extensive

dialysis) were characterized by UV, fluorescence and thermal denaturation

studies. Al l these experiments suggested that furocoumarins were cova-

lently bound to pyrimidine bases of DNA forming diadducts (Hasan et al . ,

1991). Direct binding of 5LE autoantibodies to furocoumarin-DNA adducts

has been shown in Fig. 15. The true recognition of PUVA modified

DNA was evaluated by competi t ion-inhibit ion assay. The DNA-8-M0P

photoadducts were ef fect ive inhibitors of nDNA-ant i -DNA IgG interactions

(Table 8). The level of inhibition by DNA-psoralen and poly(dG-dC)-psoralen

was ^3 and 38 percent respectively.

Characteristics of Brominated Nucleic Acid Polymers

The sodium chloride induced B — > Z transition in case of poly

(dG-dC) can be measured by circular dichroism (Fig. 16 taken from Pohl

and 3ovin, 1972) and ultraviolet absorbance spectroscopy (Pohl, 1983).

The C D . spectra of poly(dG-dC) in high salt showed near inversion

[76]

•4 CD a o a <

a,

F, 3.2 3.B

- LOG S E R U M DILUTION

Fig.] 5. Direct binding assay of SLE antibodies. The polystyrene plate was coated with (a) native DNA (~^ — ) , poly(dA-dT)-8-MOP (—0— ) and poly(dG-dC)-psoralen ( — • — ) (b) native DNA (—•X-—), DNA-8-M0P (—0—) and DNA-psoralen

[77]

TABLE 8

Inhibition of the Binding by DNA-Furocoumarin Photoadducts of SLE

Anti-DNA IgG

Comp>etitor

Maximum percent inhibition

nDNA

n \A -8 -MOP

76

7S

DNA-psoralen if9

Poly(dA-dT)-8-MOP 77

Poly(dG-dC)-psoralen 38

at 50 ug/ml

at 30 ug/ml

[78]

240 260 280 300

WAVEL ENGTH , fim

Fig. 16. Circular dichroism spectra of poly(dG-dC).poly(dG-dC) in low ( ) and high salt ( ) at pH 7.2. The low and high salt represent 0.15 M and U.O M sodium chloride in phosphate c i t rate buffer. Taken from Pahl and 3ovin, 1972.

[79]

with a positive band at 26^ nm and a negative band at 29k nm. Fig. 17

siiows the ultraviolet right to left transit ion of poly(dG-dC) wi th apparent

red shift in absorption spectrum. The change in UV absorption spectrum

resulted in a change of absorbance ratio [{29^+296)/260] from 0.119

in low salt to 0.322 in high salt (Table 9). The red shift observed for

poly(dG-dC) in high salt has been explained by Pohl in 1971. He proposed

that binding of cations to two adjacent base pairs can bring about the

shift of absorption spectrum due to an ion-dipole interact ion. The u l t ra­

violet difference spectra of poIy(dG-dC) in if M sodium chloride showed

positive band maxima at 259 and 29^ nm and minima at 2 *0 nm (Fig. 18).

Unlike poly(dCi-dC), DNA fragments in high salt did not show apparent

red shift but hypochromic ef fect was clearly seen (Fig. 17). The UV

difference spectra of DNA fragments showed positive band wi th absorbance

maximum at 257 nm and minimum at 2W nm (Fig. 18). Native DNA

under high salt conditions exhibited the similar UV and difference spectra.

The high salt induced Z-DNA form of poly(dG-dC) and altered

conformations of native DNA and DNA fragments of varying size was

stabilized by bromination (Moller et al . , 198^). The bromine atoms

have been shown to add predominantly to the C8 position of the guanine

imidazole ring and less frequently to the C5 position of cytosine. The

UV difference spectra of brominated 'Hily(dG-dC) form brought under

low salt conditions exhibited a positive peak at 3100 nm. The negative

band was found to be peaking at 238 - 283 nm (Fig.) 9). Fig. 19 inset (taken

from Moller et al . , 198 1) shoves the C.D. spectra of brominated polymers

with part ial ly converted ( 8 = 0.6) and ful ly converted Z-DNA ( 6= I).

Although both polymers exhibit near inversion of C.D. spectra but part ial ly

converted DNA showed bromination of 22% guanine and 8% cytosine

residues and ful ly converted Z-DNA exhibited 38% guanine and 18%

cytosine modif icat ion. The UV absorption spectra of native DNA fragments

after bromination in low and high salt conditions showed loss in absorbance

at 260 nm with a concomitant increase at 295 nm (Fig. 20a). The >smin

was shifted from 230 nm to 2^0 nm (under low salt) and 2^5 nm (under

high salt) respectively. The difference spectta of native DNA fragments

after bromination in either low or high salt conditions (Fig. 20b) showed

[801

u z < QD cr o

<

\ h

1000^ y i

0

.\''

200 0 300.0 400.0

WAVELENGTH,nm

Fig.17. UV absorption spectra of double stranded poly (dG-dC) in 0.15 M ( ) and if.O M NaCl ( ) and DNA fragments (300 bp) in 0.15 M ( ) and k.O M NaCl ( •) respectively. The spectra were recorded using buffer as control

[81]

TABLE 9

The UV Absorbance Ratio of DNA Polymers Under Low and High Salt Conditions

Polymers

Sodium Chloride concen­tration

Absorbance ratio

260/280 (29^+296)/260

Poly(dG-dC).poly(dG-dC)

Poly(dA-dG).poly(dC-dT)

Native DNA

Native DNA fragments:

70 bp

90 bp

120 bp

175 bp

300 bp

Poly(dG-dC).poly(dG-dC)

Poly(dA-dG).po!y(dC-dT)

Native DNA

Native DNA fragments:

70 bp

90 bp

120 bp

175 bp

300 bp

0.15 M

0.1.; M

0.15 M

0.15 M

k M

i4 M

k M

•^ M

2.0^3

1.865

1.874

1.381

\Ak7

1.502

1.101

1. 57

1.520

1.800

1.836

1.352

l.'i32

1.502

1.378

1.411

0.1 19

0.116

0.113

0.180

0.151

0.166

0.166

0.153

0.322

0.134

0.114

0.222

0.180

0.183

0.195

0.187

[82]

o z < CD cr c I/) CD <

200.0 300.0 400.0

WAVELENGTH , n m

Fig.18. UV difference spectra of poly(dG-dC) ( ) and DNA fragments in ^.0 M NaCI ( ). Respective polymer in 0.15 M NaCI served as corresponding control.

[83]

1 f

O z < QD cr o J) CD <

V \

240 260 2803U0 320

/ "~ \ WAVELENGTH, nm

200.0 300.0 400.0

WAVELENGTH,nm

FigJ9. UV difference spectra of poly(dG-dC) brominated in l^.O M sodium cinloride. Unbrominated poly

(dG-dC) was taken as control . The brominated polymer was brought in low salt buffer before it was subjected to UV measurements.

Inset: Circular dichroism spectra of double stranded poly(dG-dC).poly(dG-dC) in B-form ( ) and brominated polymers with part ial ly converted Z-DNA, 6 =0.6 ( ) and fully converted Z-DNA, 6=1.0 ( ) in 0.01 M sodium-phosphate (pH 7.0) and 0.001 1984.

containine M EDTA.

0.15 M Taken fr om

urn chloride oiler ef al . ,

[m

z < a a. o CD

<

0 ^ 200.0 300.0

WAVEieN'GTH , nm

iOO.O 200.0 300.0

WAVELENGTH, nm

400.0

Fig.20(a), Ultraviolet absorption spectra of native and brominated DNA fragments. DNA fragments in 0.15 M sodium chloride ( ), fragments brominated in 0.15 M ( ) and 4.0 M sodium chloride

(b). UV difference spectra of brominated polymer in low ( ) and high salt ( ) concentrations. Unbrominated DNA fragments served as control.

[85]

partial resemblance with difference spectra of high salt brominated

poly(dG-dC). The positive band was found peaking around 306 nm and

the negative band was found in between 238-288 nm (low salt brominated

polymer) and 238-286 nm (high salt bromin.. icd polymer). Table 9 and

10 summarizes the absorbance ratios for unbrominated and brominated

polymers under low and high salt conditions including native DNA fragments

of varying size.

Effect of Polylysine on DNA Conformation

The effect of polylysine addition on the native B-conformation

was monitored by UV spectroscopy. After the addition of varying amounts

of polylysine to DNA, the complex was mixed thoroughly and left at

room temperature for I hr to attain equilibrium. The UV absorbance

and difference spectra of polylysine-DNA complex was recorded by taking

DNA as control. Effect of both D- and L-polylysine was evaluated to

see if they can cause different levels of effect on native B-conformation

under identical conditions. At low concentrations of poly-L-lysine there

was little increase in absorbance at 260 nm while at around 300 nm

absorbance was significantly higher (Fig, 21a). Also, the )>s min was shifted

by 2 nm from 230 to 232 nm. When the concentration was raised to

10 ug/ml the absorption spectra showed hyperchromicity at 260 nm and

a further increase in absorbance at 300 nm. The ^v min was shifted

towards higher wavelength by li nm. In UV absorption spectra, at 260 nm,

the effect of poly-D-lysine on native DNA was similar to poly-L-lysine

but the absorbance around 300 nm was higher for poly-D-lysine (Fig. 21b).

At 10 ug/ml, the absorbance at 300 nm was 0.1' O and 0.190 for poly-

L-lysine and poly-D-lysine respectively. When polylysine concentration

was further raised to 12.5 ug/ml the absorption spectra of D- and L-

forin were totally different (Fig. 22). At 12.5 ug, poly-L-lysine showed

a decrease in absorbance at 260 nm and increased absorbance at 300

nm. By contrast, poly-D-lysine at the same concentration caused hyper­

chromicity at 260 nm. The high absorbance at ^00 nm could arise as

a result of turbidity in sample at high ligund concentration.

To monitor any significant topological change in native conformation,

[S5]

TABLII 10

The UV Absorbance Ratios of DNA Polymers Brominated Under Low and High Salt Conditions

Polymers

Sodii Chlo cone

j m ride ent-

ration

0.15

0.13

0.]'^

0.15

it M

ti M

k M

li M

M

M

M

M

Absor

260/280

1.790

1.9 9

1.532

2.382

2.189

2.337

1.879

2.111

1.160

1.529

\A2\

1.650

2.216

1.901

1.901

1.6 *8

bance rat io

(29'f+296)/260

0.212

0.203

0.^36

0.221

0.217

0.198

0.311

0.2i4it

0.29^

O.'f'^l

0.321

0.380

0.211

0.295

0.290

0.379

Poly(dG-dC).poly(dG-dC)

Poly(dA-dG).poiy(dC-dT)

Native DNA

Native DNA fragments:

70 bp

90 bp

120 bp

175 bp

300 bp

PoJy(dG-dC).poly(dG-dC)

Poly(dA-dG).poiy(dC-dT)

Native DNA

Native DNA fragments:

70 bp

90 bp

120 bp

175 bp

300 bp

[87]

(b)

3000

WAVtLENGTH,nm

•iOOO 200.0 300.0

WAVELENGTH,nm

400.0

Fig.21. IJV absorption spectra of native DNA in presence of (a) 2.5 ( ), 5.0 (-•-•) and 10 ug/mi ( ) of poly-L-lysine and (b) 2.5 (- ), 5.0 ( ^ and 10 ug/ml ( ) oi poly-D-lysine.( ) represents the UV spectra of nacive DNA.

[88]

2000 300.0

WAVELENGTH ,nm

400.0 200.0 300.0

WAVELENGTH,nm

iiOO.O

Fig.22. UV absorption spectra of native DNA in presence of (a) 12.5 ug/ml ( ) poly-L-lysine and (b) 12.5 ug/ml of poly-D-lysine ( -). ( ) represents the UV spectra of native DNA.

[89]

the absorbance ratios of 260/280 and average of (29^+296)/260 is a

significant parameter. Table 1 ) summarizes the effect of polylysine

concentrations on absorbance ratio. The effect of poiy-D-iysine seem

to be more pronounced. Data analysis by absorbance ratios can give

further insight into the changing DNA conformation as this parameter

was independent of DNA concentrations.

The UV difference spectra of polylysine-DNA complex was recorded

to monitor change in secondary structure. Equal amount of DNA without

polylysine served as control. At low concentrations of poly-L-lysine

the positive peak at 280 nm was very shallow but as the concentration

was raised the sharpness of the peak became more distinct (Fig. 23a),

thereby indicating that native B-conformation is being perturbed by the

added Ijgand and the molecule is heading towards a neoconformation.

On the other hand poly-D-lysine showed a positive peak which showed

maximum at 295 nm and a more steep negative band at 255 nm (Fig, 23b).

Further increase in polylysine concentration gave birth to a new type

of spectra which cannot be solely due to aggregation, if it is there

at all (Fig. 2k). At 25 ug/ml of poly-D-lysine, the difference spectra

exhibited a shallow positive peak at 300 nm and a large negative band

at 248-280 nm. The UV difference of high salt brominated poly(dG-

dC) showed a positive peak at 300 nm and a negative band peaking

between 238-283 nm. Although the high concentration of poly-D-lysine

caused some turbidity (may be due to aggregation) in the solution but

turbidity alone cannot bring about a gross structural change. The binding of

SLE anti-dsDNA IgG to some of the simple amines have been shown

in Table 12.

Characterization of DNA Modified by Hydroxy] Radical

It in an established fact that Fenton's reagent (hydrogen peroxide +

ferrous ions) results in the production of hydroxyl radical (Floyd, 1981).

The radical formation was evident by its ability of causing single strand

breaks in supercoiled DNA. As a result, nicked circular form showed

slow electrophoretic migration compared to fast migrating supercoiled

[90]

TABLE 11

Effect of PolylysJne Addition on the UV Absorbance Characteristics of Native DNA

L-isomer

D-isomer

Microgram polylysine

Nil

2.5

5.0

10.0

12.5

25.0

Nil

2.5

5.0

10.0

12.5

25.0

260/280

1.883

1.805

1.7^6

1.635

1.739

I.^^O

1.896

1.83^

1.717

1.557

1.357

1.089

Absorbance ratio

(29*+296)/260

0.10^

0.12^

0.1if9

0.209

0.164

0.320

0.095

0.119

0.168

0.259

0.388

0.737

[91]

(J z < CD cr o (.1 03

<

200.0 300.0

WAVELENGTH,nm

(00.0 200,0 300.0

WAVEL.ENGTH,nm

;oo.o

Fig.23. Ultraviolet difference spectra of polylysine-DN A complex, (a) at 2.5 ( ), 5.0 ( ^ and 10 ug/ml ( •) of L-polylysine and (b) at 2.5 ( ), 5.0 ( ) and 10 ug/ml ( ) of D-polylysme. Native DNA without polylysine was taKen as control.

[92]

<

a o if) CD <

/•• \

/' \ ^ \

\ \ / / ^\Jf '

-1 200,0 300,0 AOO.O

WAVELENGTH,nm

Fig.2'^. Ultraviolet difference spectra of poJylysine-DNA complex at 12.5 ( ) and 25 ug/ml ( ) of L-polylysine and 12.5 ( ^ and 25 ug/ml ( •) of D-polylysine.

[93]

TABLE 12

Inhibition of Binding by Amines of SLE Anti-DNA IgG to Native DNA

Inhibitors Percent inhibition at 50 ug/ml

Spermine 53

Ethionine UU

Agmatin Sulfate 32

Tryptamine 26

Histamine 24

Tyramine 18

Lysine NI

Poly-D-Jysine Nl

Poly-L-lysine NI

Histone H^A NI

Arginine NI

Putrescine NI

NI - No iniiibition

m]

form in agarose gel eJectrophoresis (Fig. 25).

The ultraviolet absorption scan of modified DNA showed hypo-

chromici ty at 260 nm compared to native DNA under identical conditions

(Fig. 26). That the DNA modified by hydroxyl radical did not loss its

secondary structure is revealed by its desorption from hydroxyapatite

in the molarity region of double stranded DNA (Fig. 27). Native DNA

sedimentation in alkaline sucrose gradient showed a single sharp boundary

of high molecular weight species. Treatment of hydroxyl radical modified

DNA under identical conditions Vv'as found to loose the sharp boundary

pattern and instead i t revealed a sedimentation prof i le quite dist inct

from the one observed for nDNA (Fig. 28). The altered sedimentation

profi le of modified DNA indicates that the single strand breaks are

randomly situated which appeared as DNA fragments of varying size

that sediment more slowly than the intact single strands of alkali denatured

native DNA. Temperature controlled denaturation of native and modified

DNA showed a significant decrease in melting temperature (Fig. 29).

A 6.5 °C decrease of melt ing temperature could be at t r ibuted to the

local helix distort ion caused by base modif ication and part ial unstacking.

The physico-chemical characteristics of ative and modified duplex has

been presented in Table 13.

Native and modified DNA were analyzed on DEAE Sephadex A-50

matrix after acid hydrolysis to separate and quantitate the modified

bases. Column chromatography of modified DNA hydrolysate indicated

RO> induced base modification (Table l'^). The relat ive susceptibil ity

of DNA to modif ication by hydroxyl radical was as follows:

Thymine >• Cytosine y Adenine = Guanine

The immunogenicity of modified DNA v/as investigated in rabbits

immunized wi th modified DNA complexed wi th Freunds' complete/incomplete

adjuvant. Unlike native DNA, hydroxyl radical modified calf thymus

DNA induced high t i ter antibodies. Preimmune serum showed negligible

binding wi th immunogen. DEAE Sephacel purif ied immune IgG showed

[95]

Fig.25. Gel electrophoresis of supercoiled DNA in presence of Fentons' reagent. Lane 1, DNA; Lane 2, DNA + hydi i en peroxide; Lane 3, DNA + hydrogen peroxide + Fe^''"ions.

[96]

in o z < CE cr o CD <

1.000

200.0 300.0 400.0

WAVELENGTH,nm

Fig.26. Ultraviolet absorption spectra of native ( ) and hydroxyl radical modified DNA ( ).

[97]

t c o

LU O Z < CD

d O

QD <

0-8

0.6|-

0.4

0.2

0.125 M

0.8

0 G

0.4

^ n n p ^ ^ ^ ^ " " 9

0.2 -

0,25 M

jnmnQnnQOQQQG

0.125 M -^H*- •0.25M-

0.0 , . ^ 15

• • f f f f f t 10 15 2^

FRACTION NUMBER

35 45

Fig.27. Hydroxyapatite column chromatography of native (—0— ) and modified DNA (—%—). The elution was carried out stepwise using Na-Pi buffer (pH 6.8) of increasing ionic strength.

[98]

t C O

CD il. CJ

'J' CL <

FRACTION NUMBER

Fig.28. Profile showing sedimentation of native ( — 0 — ) and hydroxy! radical modified DNA (-•—) through alkaline sucrose density gradient. The arrow on top shov/s the direction of sedimentation.

[99]

Z

o I—

Q:

Q

o

in a.

TOO

8 0 -

60

40

2 0

00

o 82.5°C • 89,0°C

60 70 80 90

TEMPERATURE (°C)

100

Fig.29. Effect of temperature on the melt ing of native ( -—#— ) and ROS-modified DNA ( — 0 — ). The Tm values are shown on the left corner.

[100]

TABLE 13

Ultraviolet and Thermal Denaturation Characteristics of Native and Modified DNA Under Identical Experimental Conditions

Parameter Native DNA Modified DNA

Absorbance ratio (260/280)

Percent hyperchromicity at 95 °C

Melting temperature (Tm), °C

Onset of duplex melt ing, °C

2.20

36.03

89.0

80.0

1.95

25.10

82.5

714.O

[ l o t ]

TABLE I'f

Modification of Bases in DNA Exposed to Hydroxyl Radicals

Base Modification (%)

Adenine 30.05

Guanine 29.20

Cytosine 38.80

Thymine ^9.25

[102]

high degree of binding with native DNA besides innmunogen (Fig, 30).

Preimmune IgG as control showed negligible binding to these antigens.

To ascertain the antibody specif ici ty, competit ion ELISA with immunogen

and other double stranded nucleic acids were carried out. In compet i t ion-

inhibit ion assay, the induced antibody binding was f i f t y percent inhibited

by the immunogen at a concentration of 0.32 u g/ml (Fig. 31a). The

c ross- reac t iv i t y of induced antibodies wit l i B-c:onforiiiations like native

DNA, poly(dA-dG).poly(dC-dT), poly(dA-dl )).poly(dA-dU) and poly(dl-dC).

poly(dl-dC) was of varying strength (Fig. 31b). The immunogen-antibody

interaction was also analyzed by precipitat ing the complex with saturated

ammonium sulphate. The unbound antigen was estimated in the supernatant

by diphenylamine reagent. Fig. 32 represents that at 50 U g of antigen

concentration all the antibody binding sites were saturated and further

increase in antigen concentration does not improve the binding. Almost

f i f t y percent of the added antigen was bound by the anti-ROS-DNA

antibodies. The induced antibody af f in i ty was evaluated from Langmuir -8

plot (Fig. 33). The antibody af f in i ty was computed to be 2.7x10 M.

To probe the possible role of hydroxyl radical (or ROS in general)

in the pathogenesis of SLE, four sera having di f ferent levels of ant i -

DNA autoantibodies were tested. Binding of these antibodies (at 1:100

di lut ion of serum) wi th native and modified DMA has been depicted

in Fig. 3'fa. The binding pattern of some of these autoantibodies upto

1:6^00 dilution of serum with either of the antigens was similar to

those observed at lower di lut ion. It was found that SLE autoantibodies

recognit ion of modified DNA was better than native DNA under identical

conditions. However, one serum sample (No. 1) showed high binding

with native DNA as compared to modified DNA preparation. Compet i t ion-

inhibit ion assay of SLE autoantibodies with native and modified DNA

as inhibitors have shown that ROS modi.icd DNA is a better inhibitor

of autoantibody-nDNA interact ion. The amount of competitor required

for 50 percent inhibit ion of autoantibody binding was found to be

0.03 ug /ml and 0.13 ug/ml in case of modified DNA and nDNA respectively

[103 ]

£ c o

<

Ui

z <

a.

03

MICROGRAM IgG

Fig.30. Binding of anti-ROS-DNA IgG with native ( Q ) and ROS-DNA ( | ). The bars having absorbance less than 0.2 represent preimmune IgG binding wi th native DNA and imnnunogen.

[10^]

2 O

cr

a

2

o t—

OQ

X

z

z

cr LU a

MICROGRAM INHIBITOR

Fig.3l(a).Inhibition of immunogen-antibody interaction by ROS-DNA. (b) Inhibition of imnnunogen-antibody interaction by native DNA (—-K —) , poly(dA-dG).poly(dC-dT) (—0—), double stranded: poly(dl-dC) ( — • — ) and poly (dA-dU) i-^-).

[105]

z o

z o I— z <

<

o o cr

10 20 30 40 50

MICROGRAM ANTIGEN ADDED

Fig.32. Determination of antigen bound by immune ("" • ) and preimmune IgG (—O—). The assay was performed by quanti tat ive precipit in t i t ra t ion .

[106]

Fig.33- Determination of antibody af f in i ty by Langmuir plot.

[ 1 0 7 ]

o

QD

I 2

Z

,_)

a:

( 1

£ c o

LU o z < CD cr o CD <

1 2 3 4

S L E SERUM SAMPLE

MICROGRAM INHIBITOR

Fig.3'f(a). Binding of human lupus antibodies to ROS-DNA ( | and native DNA ( D ). (b) Inhibition of autoantibody-nDNA interaction by native DNA (—O— ) and ROS-DNA

[108]

(Fig. 34b). Similar results were obtained with otiner SLE serum samples.

Antibody Detection of Z- or Analogous Conformations

Chemical modification with different reagents has been extensively

used as a tool to study the structure and function of nucleic acids in

the free state or in nucleoprotein complexes. Antibodies with predefined

specificity can be used as a yardstick to locate a particular conformation

in a complex biological system such as mammalian genome.

The poly(dG-dC) brominated under high salt conditions was found

to attain Z-conformation. The monoclonal anti-Z-DNA antibody (Z 22)

binding was nearly 80 percent inhibited at a Z-DNA concentration ot

10 Ug/ml (Fig. 35). The fifty percent inhibition was seen at k.25 u g/ml

of Z-DNA. This demonstrates that Z 22 is a highly specific probe for

Z-DNA conformation. Z 22 recognition of native poly(dG-dC) or low

salt brominated poly(dG-dC) was negligible. These antibodies were also

used as tracer of Z- or Z-like epitopes appeared on poly(dA-dG).poly(dC-dT)

brominated under 4 M and 0.15 M sodium chloride. The Z 22 binding

to Z-DNA was inhibited to the extent of \k and 23 percent by high

and low salt brominated poly(dA-dG).poly(dC.-dT) (Fig. 36). These type

oi sequences are widely distributed in mammalian genome and have

potentiality to undergo conformational transition besides formation oi

multistranded structure when the pH of thi,- medium is lowered.

The anti-dsDNA IgG derived from human lupus were also tested

for their binding to poly(dA-dG).poly(dC-dT). it was found that with

alteration in poly(dA-dG).poly(dC-dT) native conformation (as a result

of bromination), the autoantibody binding changes accordingly (Fig. 37).

The anti-DNA antibody binding dropped to 51 percent in low salt and

34 percent in high salt against 80 percent when the polymer was in

its native conformation. Native DNA brominated under low and high

sodium chloride showed that polymers have undergone a conformational

transition that deviates from native B-conformation. The Z- or Z-like

epitopes on brominated polymer was detected by Z-DNA specific monoclonal

(Z 22) and polyclonal (G 10) antibodies (Tal ' 15). The SLE anti-dsDNA

i

20

'.O

or bO

80

[109]

k: - • - »•-

0 01 0.1 1.0

COMPETITOR , Jig /ml

10 20

Fig.35. Inhibition of ant i -Z-DNA antibody (Z 22) binding to Z-DNA by poiy(dG-dC) (—-X-—), poly (dG-dC) bron-iinated in 0.15 M (—O—) and 4 M sodium chloride (—•—).

[110]

z o

2 2

20 r

z

cc UJ

a

0 01 10

COMPETITOR , i ig /ml

10 20

Fig.36. ' " ' I 'b i t .on oi z 22 antibody binding to Z-HNA by poiy(dA-dG) poly(dC-dT) ( - ^ ), poly(dA-dG).poly(dC-dT) brom.nat id in 0 i M ( - ^ - ) and k M sodium chloride ( - 0 - ) .

[ i n ]

2 O

2

U

0 0

20

40

6 0!-

B O r

0.0^ ... J...

0.1 .0 10 30

COMPETITOR , J-ig / m l

Fig.37. Inhibition of SLE anti-DNA IgG binding to native DNA by poly(dA-dG).poly(dC-dT) ( " • " ) , poly(dA-dG).poly(dC-dT) brominated in 0.15 M (—)(•—) and ^ M sodium chloride

[1121

TABLE 15

Inhibition of the Binding of Z22* and GIO* to Z-DNA and NBr^

Antigen on plate Inhibitor

Maximum percent inhibition with

Z22 GIO

Z-DNA

N &r'^•

NBr^f

N BrO.

N Br'f

NBrO.

15

15

62

12

27

22

NT

* Monoclonal mouse anti-Z-DNA antibody

** Polyclonal goat anti-Z-DNA antibody

NT = Not tested

NBr^ & NBrO, 15 :Native DNA brominated under ^ M and 0.15 M sodium chloride respectively.

[113]

IgG showed dif ferent levels of binding with native DNA and its brominated

analogues (Fig. 38). The binding of autoantibodies to bronninated DNA

can be explained in two ways: First ly, the SLE antibodies are recognizing

the remaining B-epitopes on brominated polymers (as not all the sequences

in native DNA are capable of undergoing B — > Z transition) and secondly,

they are exhibit ing polyspecif icity (a feature common to DNA autoanti­

bodies) and hence recognizing two di f ferent conformations. The SLE

anti-dsDNA IgG binding to brominated DNA fragments have been presented

in Table 16. Some of the brominated DNA fragments did not bind ant i -

DNA IgG at a l l . A possible explanation for such non-reactivi ty can be

given if i t is assumed that such DNA fragments have undergone a radical

conformational transit ion, which is possible in small DNA fragments.

The DNA fragments rich in G + C content arc expected to undergo B — > Z

transition more readily against DNA fragment of same length rich in

A + T. Besides, the DNA molecule is a dynamic structure i t would be

expected that this would alternate between these two states (namely

B and Z) passing through many intermediate stages.

[II*]

z o CD

r 2

a

0.01 0.1 1.0 10 5(

COMPETITOR , i i g / m l

Fig.38. Inhibition of SLE anti-DNA IgG binding to native DNA by native DNA (• native DNA brominated in 0.15 M (-^-) and ' M sodium chJoride (-0—).

) and

[115]

TABLE 16

Inhibition of the Binding of SLE Anti-DNA IgG to DNA Fragments of Varying Size Brominated Under Low and High Salt Conditions

Maximum percent inhibition

Inhibitor in low salt brominated in low salt high salt

Native DNA fragments: (average size)

300 bp

175 bp

120 bp

90 bp

70 bp

70

56

51

Ik

66

'l

26

20

NI

NI

Nl

NI

12

36

NI

NI = No inhibit ion

[116]

Autoimmune diseases are a diverse group of conditions characterized

by antibody recognition of normal components of the host. In many

of these diseases, antibodies with self specificity or autoantibodies are

important effectors. These antibodies mediate disease by a variety of

distinct pathological mechanisms. Although anti-DNA antibodies were

discovered more than twenty five years ago and despite the fact that

numerous studies have suggested various aspects of autoimmune phenomena,

spontaneous antibodies to nucleic acids remain puzzling in many aspects.

Questions about the mechanisms leading to their formation, their specifi­

city, their relationship with antibodies directed towards other self consti-

tuentsis still unsolved (Harley and Scofield, 1991; Jacob et al., 1989;

Cairns et al., 1989; Pisetsky et al., 1990; Brendel et al., 1991).

Investigations designed to assess antibody specificity are important

for many reasons. First they permit diagnostic tests to be developed

to detect and monitor disease. activity (Hashizume e t al., 1987). Second,

specificity studies will provide insight as to the nature of autoantigens

that conceivably are required for eliciting spontaneously occurring anti-

DNA antibodies (Brigido and StoUar, 1991), Double stranded DNA (B-

conformation) is unlikely to be involved in SLE pathogenesis in view

of its poor immunogenicity (Madaio et al., 198^; Pisetsky et al., 1990).

Although commonly referred to as a discrete material, DNA displays

a host of structural forms and sequence patterns that can serve as

antigenic determinants (Stollar, 1975; Wells et al., 1980). The structural

diversity and flexibility of DNA is inherent in its role in gene expression

and providing recognition sites for regulatory molecules. A number of

different conformations have been characterized by physiochemical and

immunochemical methods (Lafer et al., 1986; Sanford et al., 1988),

and structurally distinct regions probably exist along such DNA molecule.

This microheterogeneity of structure provides for a multideterminant

antigen.

Antibodies reactive with cellular and nuclear components occur

[117]

spontaneously in SLE. The complex serology of SLE seems to indicate

response to a large number of dif ferent autoantigens (Zouali et al . ,

1988). The binding diversity of lupus autoantibodies seems even greater

when di f ferent nucleic acids and their modified preparations are used

as test antigens (Ali et a l . , 1991; Alam and A l l , 1992; Aiam et d l . ,

1992; Ara et a l . , 1992; Ara and A l i , 1992). Because a mixed picture,

with aspects of wide cross react iv i ty and aspects of select iv i ty, were

observed in the preliminary studies, a more detailed analysis was performed

to probe the basis for ant i -DNA antibody binding to DNA of varying

size and conformation.

The absorption rat io measurement (278/251) was found to be a

quick assessment of IgG purify. IgG fractions having an absorbance ratio

of less than 2.25 were further purif ied by exclusion chromatography

on Sephadex G 200. Removal of proteins other than IgG was thought

necessary in order to define the true binding specif icit ies of ant i -DNA

IgG.

Native DNA and DNA fragments of varying size prepared by

controlled digestion wi th micrococcal nuclease were indeed double stranded

as revealed by sharp melting prof i le. The T i dependence on fragment

size was quite obvious. Moreover the samples were also digested v/ith

single strand specific nuclease S 1 to get rid of single stranded regions

in various DNA preparations.

The immunological recognition of various DNA conformations

by SLE ant i -DNA antibodies is of immense importance in relation to

their polyspecif ici ty. Double stranded DNA has repetit ious antigenic

determinant on the deoxyribose-phosphate backbone whereas most protein

antigens have mult iple but di f ferent antigenic determinants. The binding

specif ici ty of ant i-DNA antibodies with varying DNA conformations

was analyzed by inhibition ELISA in which the abil i ty of a competitor

to block the DNA-autoantibody interaction is assessed. The binding of

SLE antibodies to native B-conformation, polymers di f fer ing from average

B-helix geometry, DNA-RNA hybrid, Z-form of poly(dG-me dC) clearly

demonstrated that native DNA is not always the preferred antigen for

[118]

antibodies in SLE and that both A- and Z-DNA shares immunogenic

epitopes with native DNA. The aitered electrophoretic mobi l i ty in gel

retardation assay of A- and B-conformation complexed wi th ant i-DNA

antibodies reiterates the antibody binding to these conformers. The gel

retardation assay (also known as band-shift assay) is a sensitive technique

to detect DNA-protein interactions. Both A- and Z-DNA have been

reported to be highly immunogenic in experimental animals (Nakazato,

1980; Lafer et a l . , 1981) and the part icipation of these non-B conformations

in the etiology of SLE cannot be ruled out. Double stranded regions

of RNA (as in cruciform) and RNA-DNA hybrids adopt a right handed

double helical form very similar to that of A-DNA and the occurrence

of hybrid is closely linked with transcriptional act iv i ty of the cell (Busen

et al . , 1982). Moreover, Z-conformation is a reversible structural form

of DNA and its role in genetic act ivi t ies like transcription is well docu­

mented (Kmeic et al . , 1983; Peck and Wang, 1985). The existence of

segments of lef t handed helices in native DNA has also been documented

(Ueda et a l . , 1990; Wit t ig et a l . , 1991; 3imenez-Ruiz et a l . , 1991).

Polyamines are known to bind negatively charged macromolecules

such as nucleic acids. The binding of SLE ant i-DNA antibodies to DNA-

lysine photoadduct has further expanded the horizon of antibody poly-

specif ic i ty. DNA molecules di f fer ing from average B-helix geometry

has been found to be immunogenic (Stellar, 1989) and DNA-lysine photo­

adduct has induced high t i ter antibodies (Islam, 1991). The ant i-DNA

IgG purif ied through DNA-(polylysyl-Sepharose ifB) a f f in i ty column was

homogeneous and found to be recognized equally well by native DNA

and DNA-lysine photoconjugate. Moreover, the relat ive binding af f in i ty

of ant i -DNA antibodies to DNA-lysine photoconjugate was appreciabl>

higher than that with native DNA. By the photoaddition of one lysine

molecule per helical turn of DNA, the native B-conformation was consider­

ably altered which has resulted in the generation of high a f f in i ty neo-

epitopes leaving much less epitope typical of B-conformation. The results

conclusively suggest that a f f in i ty purif ied ant i -DNA IgG is indeed recogniz­

ing the altered conformation of DNA-lysine photoconjugate. The retention

[119]

of cross reactivity of affinity purified anti-DNA IgG with DNA-lysine

photoadduct further strengthen the idea of an alternate antigen for the

induction of antibodies cross reactive with native DNA. The DNA-lysine

photoconjugate could be placed on the existing list of al ternate antigens

for the induction of autoantibodies in SLE. Lysine rich histone H1 in

nucleosomes thus on modification by physical, chemical or environmental

agents might form DNA-histone adduct making it immunogenic and thus

resulting in the production of autoantibodies cross reactive with nDNA.

The findings of Mura and Stollar (198'f) have suggested the ability of

different lysine-rich histones to cause varying conformational changes

in native DNA molecules of interspersed repeatitive sequences.

There has been considerable interest on the damaging effect ol

reactive oxygen species (R05), like superoxide radical (O^) hydrogen

peroxide and hydroxyl radical (Fridovich, 1986; Halliwell, 1987) on nucleic

acids, proteins and lipids. These radicals modify DNA at various sites

that includes base damage and cross linking between cytosine and tyrosine

in nucleohistone (Dizdaroglu et al., 1991; Gajewski and Dizdaroglu, 1990).

It has been shown that DNA after exposure to reactive oxygen species

presents a more discriminating antigen than native DNA for the binding

of SLE autoantibodies (Blount et al., 1989). Oxidative DNA damage

resulting from active oxygen species has been hypothesized to play a

critical role in several biological processes including aging, cancer and

autoimmune diseases (Ames, 1983, 1989; Cerruti, 1985; Von Sonntag,

1987). In our experimental conditions, the yield of free radical induced

base modification predominantes over that of single strand breaks in

DNA as revealed by decrease in UV absorbance at 260 nm. The decrease

in Tm value by 6.5 °C with simultaneous decrease in total percent hyper-

chromicity of modified DNA is suggestive of the fact that DNA has

been modified without undergoing major alterations in its secondary structure.

In case of gross changes in the secondary structure of modified DNA

the decrease in Tm value would have been much more than what has

been observed. The alteration in the topological state of native DNA

[120]

has rendered it immunogenic as shown by experimentally induced high

titer antibodies. The polyspecif icity exhibited by immune IgG was evident

from inhibit ion ELISA results. Native DNA, RNA and synthetic poly­

nucleotides in B-conformations besides immunizing antigen were ef fect ive

inhibitors. In an earlier study, native HNA on ul traviolet irradiation

in presence of hydrogen peroxide induced antibodies which showed recogni­

tion for B-conformation (Ara et al . , 1992). The polyspecif icity exhibited

by immune IgG was comparable to well established heterogeneity of

murine and human SLE ant i -DNA antibodies. Naturally occurring ant i -

DNA antibodies of SLE patients showed enhanced binding wi th modified

DNA and binding of ant i -DNA antibodies to native DNA was inhibited

by modified DNA antigen. Since l i t t le is known about the stimulus that

causes the production of ant i-DNA antibodies in SLE, the potential

reactive oxygen mediated DNA damage may provide a new concept

for the production of anti-nucleic acid antibodies in autoimmune disorders.

The spontaneous production of ant i-DNA antibodies in SLE might arise

as a consequence, of antigenic changes in UNA due to damage caused

by hydroxyl radicals. The augmented levels of ROS in tissues is well

related to many pathological conditions (Willson, 1979). Hydrogen peroxide

on diffusion to a vulnerable part of the cell might generate hydroxyl

radicals on reaction wi th ferrous ion, often bound to phosphates of DNA

backbone and to various protein including those associated wi th chromatin

which inturn could modify DNA (Floyd 1981, 1983). The iron promoters

of hydroxyl radical formation exist _m vivo based on the observation

that iron salts never exist " f ree" in biological system; they wi l l always

be found to be associated with proteins, membranes, nucleic acids or

low molecular weight chelating agents.

The conformational and dynamical properties of DNA are strongly

dependent upon both its sequence and microenvironment. It has been

suggested that when a B-DNA segment is flanked by any di f ferent struc­

tural mot i fs (e.g. A, Z or B), there is an increase in overall f lex ib i l i ty

provided by bending and enhanced f luctuations at the conformational

interfaces (Reich et a l . , 1991). In 1972, when Pohl and 3ovin reported

[121]

right handed to left handed conformational transition for poIy(dG-dC)

in high salt, it was presumed that DNA polymers rich in alternating

punne-pyrimidine bases wi l l readily undergo B — > Z transit ion. Later,

it was discovered that a str ict ly alternating purine-pyrimidine is neither

necessary nor suff icient cr i ter ia for Z-DNA formation (McLean et al . ,

1986; Singleton et a l . , 1983; Ki lpatr ick et a l . , 198*^; Ellison et a l . , 1985).

Nordheim et a l . , (1986) showed that stretches of poly(dG-dC) sequences

do not contribute signif icantly to the presence of Z-DNA in f ixed polytene

chromosomes of Drosophila melanogaster. Further studies showed that

the potentially Z-DNA forming sequence d(GTGTACAC) adopts a right

handed double helical conformation belonging to the A-DNA family (3ain

et al . , 1987). In C.D. spectra the Z-form of poly(dG-dC) shows near

inversion with a positive band at 26^ nm and a negative band at 29^

nm. Comparision of C.D. and UV result suggests that red shift in UV

absorption is solely due to B — > Z transit ion. In 0.15 M and U M sodium

chloride concentration the average of absorbance at 294 and 296 nm

divided by absorbance at 260 nm, termed as absorbance rat io, was evaluated

to monitor conformational transit ion in synthetic and native DNA polymers.

For poly(dG-dC) in Z-conformation this value vas computed to be 0.322.

This reference value for prototype Z-DNA was used to monitor conforma­

tional transitions in native DNA, DNA fragments of varying size and

poly(dA-dG).poly(dC-dT). Neither polymers resulted in absorbance ratio

of 0.322 but data in Table 9 clearly indicates that in high salt this

rat io IS changing which could occur as a result of conformational transit ion.

The low and high salt conformations were stabilized by bromination

(Moller et al . , 1984). The bromination had profound ef fect on the absor­

bance ratio of poly(dA-dG).poly(dC-dT), native DNA and DNA fragments.

Native DNA brominated under low or high salt concentration, showed

absorbance ratio greater than 0.3. Under identical conditions the absorbance

ratio for DNA fragments of varying size varied between 0.2 to 0.38.

This inturn shows that DNA fragments of varying size have dif ferent

levels of potential i ty to undergo B- to Z- or Z-l ike conformations as

the fragments wi l l have variation in the G+C and A+T content. Also

native DNA or its fragments might undergo Z- or Z-l ike conformational

[122]

changes on bromination under low or high salt concentrations. The UV

difference spectra of Z-form of brominated poly(dG-dC) had resennblance

with native DNA or DNA fragments brominated under low or high sodium

chloride concentrations. This further reiterates that both in native DNA

and DNA fragments there are regions of interspersed sequences which

has the potential to undergo B-to Z- or Z-l ike transitions upon bromination

in high or low salt conditions.

A variety of cations such as simple amines or polyamines that

ef fect B- to Z-transit ion in poly(dG-me dC) are known to induce the

collapse of native DNA into compact structural forms. These forms

have been visualized by electron microscope as toroids, folded fiber,

rods and spheroids (Gosule and Schellman, 1976, 1978; Chattoraj et al . ,

1978; Widom and Baldwin, 1980; Allison et a l . , 1981). The polyamines

have been shown to stabilize DNA against thermial denaturation, alkaline

denaturation, enzymatic degradation, shear induced strain and induce

in vi t ro condensation of DNA (Vertino et a l . , 1987). The condensation

of naked DNA takes place in a narrow range of polyamine concentration

whereas the condensation of chromatin can occur in the presence of

a wider range of polyamine concentration. The UV absorption spectra

of D- and L-isomers of polylysine indicated their interact ion with DNA

on charge basis. The effect of D-isomer was appreciably higher than

L-isomer. At low concentrations of these isomers the absorbance at

260 nm showed l i t t l e change but the ^ m i n was found to be red shifted

to the extent of 2-k nm. The shift could be at t r ibuted to the presence

of a ligand in DNA solution. The in( i-ase of absorbance around 300

nm IS considered to be an indication of conformational transit ion in

native B-conformation. The UV difference spectra clearly shows that

at low concentrations of either D- or L-isomer, the primary interacting

sites are phosphate groups on DNA backbone but once the charge neutral iza­

tion has been completed the B-conformation is on the verge of changing

conformation wi th further increase in polylysine concentration. The most

significant change in UV difference spectra was seen when poly-D-lysine

concentration was raised to 25 ug/ml from 12.5 ug/ml . On comparision

[123]

the spectra showed the characterist ic of high salt brominated poly(dG-dC)

which assumes 100 percent lef t handed Z-conformation. The data analysis

of polylysine-DNA interaction suggests that in calf thymus DNA there

are certain base pair sequences which are sensitive to changing polylysine

level and can assume Z- or Z-l ike conformations. Since the eukaryotic

DNA is heavily associated with proteins suff ic ient ly rich in lysine, such

conformational transitions might serve as molecular signal for genetic

events inside the cel l . Recently, Gunnia et a l . (1991) have shown that

rabbits immunized wi th poly(dA-dC).poly(dG-dT) complexed wi th spermidine

or spermine produced antibodies reacting wi th Z-DNA in addition to

those binding toward poly(dA-dC).poly(dG-dT) and ssDNA. Antibodies

el ici ted by polynucleotide - polyamine complexes had no react iv i ty tov/ards

polyamines. This finding suggests that polyamines are capable of altering

the immunogenicity of polynucleotides by mechanism involving the stabil iza­

tion of Z-DNA conformations.

The conformational transit ion in native calf thymus DNA, poly(dA-dG).

poly(dC-dT) and DNA fragments of varying size was evaluated by mono­

clonal ant i -Z-DNA antibody (Z22) and polyclonal goat antibody (GIO).

Both Z22 and GIO are immunological probes for the detection of Z-

or analogous conformation. The binding of Z22 was nearly 80 percent

inhibited at a Z-DNA concentration of 10 ug /m l . This demonstrate the

high specif ici ty of Z22 for Z-conformation as poly(dG-dC) (B-form) or

brominated under low salt showed no binding with Z22. Competit ion

experiments with Z22 of poly(dA-dG).poly(dC-dT) brominated in 0.15 M

or ^ M sodium chloride showed that potential i ty of this polymer to undergo

Z-transit ion was l imi ted. Furthermore, competit ion ELISA with SLE ant i -

dsDNA IgG showed that poly(dA-dG).poly(dC-dT) has undergone conforma­

tional transit ion because binding of anti-dsDNA IgG to brominated polymers

has declined signif icantly as compared to binding with native poly(dA-dG).

poly(dC-dT). The Z- or Z-l ike epitopes on native DNA brominated under

low and high sodium chloride concentrations was probed by both monoclonal

(Z22) and polyclonal (GIO) ant i -Z-DNA antibodies. The binding of Z22

and GIO was inhibited by both low and high salt brominated polymers.

[12^]

This finding again reiterates that dispersed sequences of calf thymus

DNA has potential to adopt Z- or Z-like confornriation. This means calf

thymus DNA exhibits microheterogeneity whose conformation is dictated

by its microenvironment. The binding of SLE ant i -DNA IgG to brominated

native DNAs could be explained in two ways:

I. In native DNA not all the epitopes typical of B-conformation

has undergone conformational transit, MI and hence naturally occurring

anti-dsDNA IgG is recognizing the remaining B-epitopes.

I I . A population of SLE anti-dsDNA IgG is exhibit ing polyreact ivi ty

wi th respect to varying DNA conformation and hence Z-l ike epitope

could also interact with antibodies.

To determine, whether reactive structure could be maintained

in smaller fragments of native DNA the fragments of varying size after

bromination in low and high salt were used as competitors of nDNA-

anti-DNA IgG interact ion. Unbrominated native DNA fragments were

used as true competitors of SLE IgG to which inhibit ion values of low

and high salt were compared. The data indicates that binding of ant i -

DNA IgG was signif icantly reduced when the polymer has undergone

conformational transit ion. The level of transit ion seem to be affected

by two factors: namely length of the DNA fragment and percentage

of G-tC and A+T content.

If ant i -DNA antibodies from human and murine SLE are capable

of exhibiting polyspecif icity, the DNA molecule is a step ahead by its

potential i ty to adopt mult iple conformations. The conformational f lex ib i l i ty

of DNA depends on its microenvironment and any unusual structure

which is stable at physiological conditions, suggest that such a structure

might be formed ^H _vi_vo. The polyspecif icity phenomenon of naturally

occurring ant i -DNA antibodies is most likely due to epitope that recur

in di f ferent molecules, an economy of nature whose meaning awaits

vigorous elucidation.

Based on the results of present study, the fol lowing points of

conclusion have been drawn:

1. Native DNA and DNA fragments of varying size, brominated

[125]

under low and high sodium chloride concentrations is both structural ly

and immunologically distinct from B-form DNA. That the bromination

has induced neoepitopes capable of presenting Z- or Z-l ike features

was detected by monoclonal (Z22) and polyclonal (GIO) ant i -Z-

DNA antibodies.

2. In native calf thymus DNA there are dist inctive distribution of

DNA sequence which has the potential to undergo B- to Z- or

Z-l ike transitions upon binding with nucleoproteins rich in basic

residues.

3. The modified nucleic acid antigens may part icipate as antigenic

stimulus for the production of a population of DNA cross reactive

antibodies in SLE patients. The dietary, physical, chemical, or

environmental agents might act, direct ly or indirect ly, as modif ier.

^. The hydroxyl radical modified DNA may play an important role

in the pathogenesis of SLE because of markedly decreased level

of superoxide dismutase in active SLE patients; the excessively

generated free radicals may exert its modifying ef fect on DNA

and thus rendering i t immunogenic.

3. The binding of A- or A- l ike conformation by SLE ant i -DNA antibodies

suggest that such conformations may also act as potential auto-

immunogen for the induction of antibodies cross reactive wi th

native DNA.

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