Naturally Occurring Anti-Band 3 Antibodies Bind to Protein Rath

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THE JOURNALF BIOLCCICALHEMISTRY0 1993 by The American Society for Biochemistry and Molecular Biology,Inc.

Vol. 268, No. 31, Issue of November 5, pp . 23562-23566, 1993Printed in U.S.A.

Naturally Occurring Anti-band3 Antibodies Bind to Protein RatherThan to Carbohydrate on Band "

(Received fo r publication, July 1, 1993)

Hans U. LutzS, Omar Gianora, Mena Nater, Erich Schweizer, and Pia Stammler

From the Laboratory fo r Biochemistry, Swiss Federal Institute of Technology, ETH-Zentrum,CH 8092 Zurich, Switzerland

Naturally occurring anti-band 3 antibodies were affin-ity purified from pooled human IgG (Sandoglobulin@)(Lutz, H. U., Flepp, R., and Stringaro-Wipf,G. (1984)J.Zmmunol. 133,2610-2618). They boundo the major inte-

gral membrane proteinf human red bloodells and its55-kDa NH2-terminal chymotryptic fragment but notothe carbohydrate-rich 38-kDa fragment on blots. Like-wise, neither an endo-P-galactosidase nor a neuramini-

dase treatment of band 3 on intact red cells reducedtheir binding to the blotted antigen. Lactoferrin (10 pgl

ml) had no significant effect on heir binding to band 3and to its 65-kDa chymotryptic fragment. Even in thepresence of 20 pg/ml lactoferrin anti-band 3 antibodiesbound specifically to chymotrypsin-pretreated and xi-datively stressed red cells. Thus, naturally occurringanti-band 3 antibodies bindo protein rather than car-bohydrate within bandrotein,rrespective ofwhether the antibodies were depleted of anti-idiotypicand other I&-reactive antibodies or ot.

Natura lly occurring (aut0)antibodies have recently gained

considerable interest, since they are, where investigated,xact

copies of the germ line Ig gene information (1,2), whereas

induced antibodies to non-self rep resent highly muta ted ex-pressions of the encoded Ig information. Despite the fact that

natural ly occurring antibodies exist in normal serum at low

concentrations and have affinities eing 2-3 orders of magni-

tude lower than those of induced antibodies, they appear to

overcome their weakness and gain functionality y unconven-

tional routes nvolving bispecificity (3, 4)or stimulation of the

alternative complement pathway (5-7). Evidence suggest thatsome of these natu rally occurring antibodies have a tissue-

homeostatic role. Extensively studied is the potential role of

anti-band 3 antibodies n clearance of senescent and oxida-

tively stre ssed human red cells (for reviews see Refs. 7-9).

Selective recognition an d opsonization of senescent andoxida-

tively stressed redcells appears to nvolve increased anti-band

3 binding and C3b-deposition via the alternat ive complementpathway ( 10 , 11). The molecular prerequisiteb) for increased

anti-band 3 binding to red blood cells is, however, not firmly

established. The reasonsor these uncertainties area) he low

number of antibodies required to tag the damaged redells, (b )

~~ ~ ~ ~~ ~

Grant 0-20-023-90,wiss National ScienceoundationGrant* This work was supported by Swiss Federal Institute of Technology

31-32383.91),nd an award from the Central Laboratory of the SwissRed Cross, Berne all o H.U. L.) and was sponsored bythe Union Bankof Switzerland on behalf of a client. The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore e hereby marked "advertisement" in accordancewith 18U.S.C. Section 1734 olely to indicate this fact.

chemistry, ETH-Zentrum, CH 8092Zurich, Switzerland. Tel.: 0041-1-$ To whom correspondence should be addressed: Laboratory forBio-

632-3009;ax: 0041-1-251-4633.

the experimental difficulties in identifying the actual binding

site of th e few hundred antibodies bound per cell, and ( c ) the

different sources of anti-band 3 antibodies that were either

affinity-purified from pooled IgG (12) or eluted from red cells

(13).Some evidence calls for an endogenous proteolytic modi-

fication of band 3 protein and the occurrence of a COOH-ter-

minal60-kDa polypeptide which hen serve s as senescent cell

antigen (14)with binding sites nvolving amino acids539-554

and 812-830 (15).Most support has beengiven (10,16-19) for

a much simpler hypothesis (12, 201,which suggests that th e

binding site pre-exists within band 3 protein as an exposedantigen whose availability is altered by topologic changes

within the membrane of aging and oxidatively st ressed red

cells. The hypothesis predicts that oligomerization of band 3

protein within the planef the membrane enhances anti-band

3 binding by favoring bivalent binding. It is based on the fact

that the binding sites for anti-band 3 antibodies are equally

present on band 3 protein from young an d old red cells (12).

This binding site isocalized within the 55-kDa NHz-terminal

fragment of band 3 protein which can be generated by treating

intact red cells with chymotrypsin (12).Thus, it may include

the domain ormed by amino acids 39-554, but not the OOH-

terminal domain(21,221.Since the 55-kDa chymotryptic frag-

ment of band 3 protein lacks carbohydrate,t was surprising to

see a recent report that suggested anti-band 3 antibodies tobind to sialylatedN-acetyllactosamine residues within the ar-

bohydrate portion of band 3 protein (23). In an attempt to

clarify this discrepancy we studied the binding properties of

anti-band 3 antibodies t o band 3 protein that was modified by

chymotrypsin or glycosidases.

MATERIALS ANDMETHODS

Naturally Occurring Anti-bund 3 Antibodies-Naturally occurringanti-band 3 antibodies (12)were purified from human IgG essentiallyas described with the following modifications. Pooled human IgG San-doglobulin") was a gift from he Central Laboratory f the Blood Trans-fusion Service SRC Berne. IgG(36 ) was dissolved in300ml of bufferL (100mM NaC1,lO mM phosphate,1mM MgCI2,50 pg/ml PMSF,0.08%sodium azide, 0.05% Nonidet P-40,H 7.41, ialyzed against buffer L,and diluted before use to 25 mg/ml IgG. One preparation of I& was

directly passed througha column containing immobilized band 3 notpreabsorbed). Another preparationf IgG was depleted f material thatbound t o IgG, like rheumatoid factorr anti-idiotypic antibodies(241,ypassing througha column containing immobilized eat-aggregated gGat 38 ml/h. The flow-through was pumped at room temperature and22.8mVh through a column containing immobilized pectrindimer andsubsequently through ne containing band3protein. The flow-throughof the band 3 column represented whole IgG depleted of anti-band 3

antibodies (IgG-). The columns were washed overnight with buffer Lcontaining0.01%Nonidet P-40. ound antibodies wereeluted with 1 M

NaC1, 0.1M glycine, 0.01%Nonidet P-40,H 2.7, eutralized immedi-ately, and dialyzed against PBS containing 0.08% sodium azide. Puri-

fied antibodies were concentratedo less than 1 mg/ml in Centricon 30microconcentrators (Amicon, Beverley,MA) , and the IgG concentrationwas determined (25).

Iodination of Antibodies-Antibodies were labeled with [12611iodine.

Twenty to 100pg of protein were iodinated fo r 45 s with 1 mCi using

23562



Ant i -band 3 Antibodies Do

chloramine T as anoxidant (26). The reaction was stopped with meta-

bisulfite and the proteins gel-filtered over SephadexG-75 (Pharmacia ,

Uppsala, Sweden). The columns and theollecting tubes were prerinsed

with PBS containing 1 mM NaI and 1%gelatin. Labeled antibodies

having a specific activity of 20-40 x lo6 cp d p g were frozen in small

aliquots.

Hum an Red Blood Cells an d Membranes-Human blood, type 0 Rh+,

was collected in citrate-phosphate dextrose, and processed within 1 h

aft er collection. Red cells were freed f white cells essentia lly according

to Ref. 27 as modified in Ref. 28. Red cells were obtained y washing thefiltered cells three times with buffer B (140M NaCl, 5 mM KC1,lO m~phosphate, 0.5 m~ EDTA, 0.05% D-glucose, pH 7.4). Membranes were

prepared by hypotonic lysis with 30 volumes of 5 m~ phosphate, 1 mM

EDTA, pH 7.4, and serine proteases inactivated s outlined (29).

Label ing of Red Cell Carbohydrates-Washed red blood cells were

treated at 15% hematocrit with 15 unitdm 1 suspension of galactose

oxidase (no. 1213.784, Boehringer Mannheim), and imines were formed

with the generat ed aldehyd e groups and [l4C1aniline (Radiochemical

Center, Amersham, Great Britain) in the presencef cyanoborohydride

exactly as described elsewhere (30). This labeling procedure replaces

th e one with [3H]borohydride wh ich generates large amountsf volatile

label.

Zkeatment ofRed Cells with Chymotrypsin n d Glycosidases-Band 3

was cleavedon intact red cellsy incubating red cells t a hematocritof

15% for 90 min a t 37 "C with 300 pg/ml a-chymotrypsin (Sigma). The

reaction was stopped with phenylmethylsulfonyl fluoride (31). Threehundre d pl of packed unlabeled or 14C-labeled red cells were incubated

for 1 h at 37 "C with 20 milliunits, if not otherwise ind icated, of each

enzyme from BoehringerMannheim:ndo-P-galactosidase (no.

982.954),euraminidaseno.080.725), endoglycosidase-H (no.886.424) in 20 mM sodium acetate, 130 mM NaC1, 10 m~ D-glucose, 30

pg/ml phenylmethylsulfonyl fluoride, H 5.8, in a totalolume of 700 pl.

The reactions were stopped y diluting the cells with buffer B and two

washes in bufferB. In order to prevent membrane protein aggregation

during incubationa t low pH, the treatment with glycosidases (32 ) was

modified, primarily by including glucose in the incuba tion mixtu re and

by usi ng 20-30 milli units of endo-/%galactosidase insteadof 75.

Anti-band 3 Bind ing to I ntact R ed Blood Cells-Untreated or chy-

motrypsin-treated red cells were resuspended o a hematocrit of 5% in

a buffer containing0m~ Hepes, 140m~ NaCl, 5 mM KC1,2 mM MgCl,,

1 gAiter D-glucose, pH 7.4, and were incubated for 1 h a t 37 "C at the

given concentrationsof diamide which was issolved shortly before use

(Sigma). Diamide-treated red cells were washed three times with 150mM NaC1, 10mM phosphate, 1g/liter D-glucose and 2 m~ MgCl,, pH 7.4,

and resuspended n he same buffer containing 0.1% human serum

albumin and the ell number determined. Binding f labeled ant i-band

3 or IgG depletedof anti-band 3 (IgG-) (33)hich were lZ5I-iodina ted to

the same specific activity (ranging from 20 to 40 x lo6 cp dp g in dif-

ferent experiments) was studied in parallel. Aliquots containing 2lo s

of red cells were mixed with1ml of wash buffer containing0.1% serum

albumin in Eppendorftubes. Cells were pelleted the supernatants care-

fully removed. Eightyp1 of an incubation mixture containing the wash

buffer, 500,000-600,000cpm of either lZ5I-iodinated anti-band 3r IgG-,

50 mg/ml human serum albumin,0mg/ml whole human IgG, the given

concentration of anti-band 3 antibodies, and where indicated, 20g/ml

lactoferrin were gently mixed with the pelleted cells and the suspe n-

sions incubated for 30 min a t 37 "C. Reactions were stopped by adding

120 plof wash buffer supplemented with 0 mM EDTA and transferr ing

150 pl of the suspensions onto00 pl of a mixtu reof phthala te oils (34).Tubes were centrifuged, and the bottom of the tubes, containing the

pelleted red cells, were cutff and counted n a y counter. Backgrounds

were subtracte d and the rea ding or labeled IgG- was subtract ed from

th at obtained with labeled anti-band 3 antibodies. The remainder was

expressed in ng per O' O red blood cells.

SDS-Po lyacrylamide gel electrophoresis-Samples kep t in 1% SDS

and 5 mM N-ethylmaleimide were thawed and mixed with electropho-

resis buffer containing 40 mM dithiothreito l. Reduction and denatur-

ation was carried out for 30 mint 37 "C. All samples were then alkyl-

ated by adding 50 mM N-ethylmaleimide. Samples were electrophoresed

using amodified version of the Neville (35)gel system (36). The running

gel contained 8% acrylam ide and 2.7% bisacrylamide. Gels were either

electroblotted or stained, dried,exposed to X-Omat x-ray films (Kodak,

Lausanne, Switzerland)or to PhosphorImager screens from Molecular

Dynamics (Sunnyvale,CA). The major redcell membrane polypeptides

were marked according o Ref. 37.

Zmmunoblotting-Electrophoretically separatedpolypeptideswereblotted onto nitrocelluloseas outlined (12). Blots were preincubated for

1 h a t 37 "C to block non-specific binding of labeled antibodies. The

Notindoarbohydrate 23563

blocking solution contained buffer R (0.9% NaCl, 20 mM Tris, 0.08%

sodium azide, pH 7.4) th at was supplemented with 2% gelatin. Blots

were either stained withAmido Black or incubated overnight at room

temperature n buffer R supplemented with 0.75 x lo6 cp dm l lZ5I-

iodinated anti-band 3 antibodies as indicated, 1% gelatin,.04% Triton

X-100. Blots were washed four timesor at least 5min with incubation

buffer and buffer R alone. Quantitationf immunoblots was carried out

from a digitized picture obtained by exposing dried blots o a phospho-

rescence screen which was read by a PhosphorImager from Molecular

Dynamics. The figures either show autoradiographs or the digitizedpictures.

RESULTS ANDDISCUSSION

Naturally occurring anti-band 3 antibodies (anti-band 3 an-

tibodies) have a weak affinity for purified band 3 protein (5-7

x lo6 literdmol, Ref. 33) and do not readily bind to intact red

blood cells, unless the redcells have been oxidatively stressed

(10). The same is trueor chymotrypsin-treated redblood cells

(Fig. 1).This figure shows the specific binding of anti-band 3

antibodies, since red cells were either incubated with labeled

anti-band 3 antibodies or with the same amount of equally

labeled whole IgG depleted of anti-band 3 antibodies in the

presence of 10 mg/ml human IgG and 50 m g h l human serum

albumin. Anti-band 3 antibody binding, shown as the ifferencein bound label and expressed in ng/lOlo cells, increased with

increasing concentrations of diamide used t o stress the red

blood cells. Because of their low affinity anti-band 3 antibody

binding t o diamide-treated cells increased and also became

detectable on cells not treated with diamide, when its concen-

tration was raised beyond the physiological level. Similar re-

sult s were obtained with 10 and 20 pg/ml of anti-band 3 anti-

bodies (not shown). These studies clearly demonstrate binding

of anti-band 3 antibodies to exofacial portions of red blood cells.

Since lactoferrin (10 pg/ml) apparently inhibited binding of

anti-band 3 antibodies to red blood cells to over 90%, when

studied by Beppu et al. (231, the effect of lactoferrin was inves-

tigated. An excess of lactoferrin (20 pg/ml) did not abrogate

anti-band 3 binding t o red cells. Instead, it stimulated inding

to oxidatively st ressed and chymotrypsin-pretreated red cells

L

0 100 200 300 400

phl diamide

1 lpg/ml anti-band 3, o addition

+ lpg/mlanti-band 3, + 20 pg/ml lactoferrin

")- 5.lpg/ml anti-band 3, noaddition

"Q- 5.1 pghl anti-band 3, + 20 pglml lactoferrin

FIG. . Specific binding of anti-band 3 antibodies to chymo-trypsin-pretreated and diamide-treated red blood cells. Anti-band 3 binding was measured as outlin ed under "Materials and Meth-ods" at two concentrations of anti-band 3 antibodies and the indicated

additions. Results were averaged from two independent experimentswith readings th at did not deviate more than 1 ng/l0 Io cells from theaverage.



23564 Anti-band 3 Antibodies Do

A Anti-band 3 binding

preabsorbedon

heat-aggregated

IgG and onspectrinimer not preabsorbed

”“-7”-

55 kD a -

a b cb c

a) purified band 3 protein

b) polypeptides of membranes

c) polypeptides of membranes

from chymotrypsin-treated RBC

B stalned 14C-labelled. ‘7

l * ”2-

3-

Ia b a b

C Antl-band 3 binding

band 3 -55 kDa D

5 (actin) -6 -

a b a b

- lactoferrln + lactofenln

Frc.2.Bindingof anti-band3 antibodiestoband3 rotein andita 55-kDa a-chymotryptic fragment. , purified band3 protein (3.3pg) (39) and10 pg of untreated and chymotrypsin-treated membraneswere electrophoresed after reduction, blotted, and incubated with12RI-

Not Bind to Carbohydrate

at 1.1pg/ml of antibody and inhibited t partially a t 5.1 pglml.

Its differential effect was also found in experimen ts with red

cells th at were not pretreated with chymotrypsin (not shown).

Thus, in contrast o the findingsof Beppu et a l. 123), anti-band

3 antibody binding to intact red cells was most likely not via

carbohydrate under conditions mimicking physiology. To fur-

ther nvestigate he possible reasons for this difference, we

studied the binding specificities of two anti-band 3 antibody

preparations which were purified from pooled human I g C withor without preabsorption on heat-aggregated I g C .

Anti-band 3 antibodies bound to purified band 3 protein, i t s

oligomers, and among red ell membrane proteins primarily o

band 3 protein (Fig. 2A ). Their specificity was the same, irre-

spective of whether the antibodies were preabsorbed on heat-

aggregated I g G o deplete of anti-idiotypic and other gG-reac-

tivecomponents or not (Fig. 2 A ) . Anti-band 3 antibodies

prepared withou t preabsorption generated a noticeable back-

ground on blots, and their binding to I g C was considerably

hig her than tha t of preabsorbed antibodies (not shown). De-

spite these differences both antibody prepara tions bound ex-

clusively to the 55-kDa NH2-terminal. chymotryptic fragment

of band 3 protein (Fig. 2 A ) and not to the carbohydrate-rich

38-kDa fragment which also remained membrane-associated(22) and was pres ent among t he lectrophoresed polypeptides

as verified in Fig. 2B. This figure shows that the 55-kDa frag-

ment was freeof labeled carbohydrate, whereas a zone under-

neath actin (band )and exte nding eyond band 6, correspond-

ing to the 38-kDa fragment of band 3 protein, was heavily

labeled in membranes rom chymotrypsin-treated cells, but not

in those from untreated cells.

The lack of reactivity with carbohydrate was further evident

from the inabilityof lactofemn to inhibit anti-band3 antibody

binding to band 3 protein and its 55-kDa fragment on blots

(Fig. 2C). t inhibited anti-band binding to band 3 protein and

it s 55-kDa fragment insignificantly, by 2-596, when determined

with a PhosphorImager (Molecular Dynamics) rom blots (Fig.

2C). On he other hand, his extremely sensitive echniquerevealed a weak binding to actin, band 6, and also o a diffuse

zone reminiscent of the 38-kDa fragment (seeheauy arrowx in

Fig. 2C). Binding to this fragment was observedwith both

types of anti-band 3 antibody preparations and was somewhat

lower, when blots were ncubatedwith 10 pdml actofemn

(Fig. 2C). lthough we cannot exclude the existence f a minute

fraction of antibodies which bound to carbohydrate, the vast

majority of anti-band 3 antibodies did not bind to carbohydrate

bu t to peptide portions of band 3 protein and its 55-kDa chy-

iodinated anti-band 3 antibodies. Anti-band 3 antibodies were either

purified from human I@ with or without a preabnorption on heat-aggregated I g G and spectrin. The figure shows autoradiographs from

immunoblots performed on l anes loaded with: purified band 3 protein( lane a) , red cell membranes ( l a n e h ) , and red cell membranes from

galactose oxidase and generatedldehydes labeled with [“Claniline. Aa-chymotrypsin-treated red cells lanec) . R , red cells were treated with

samp le of labeled red cells was reated with a-chymotrypsin . Mem-

branes were prepared rom both preparations and 5 pg of protein fromeach were electrophoresed. Half the gel was stain ed the ot her tre atedfor autoradiography. The diffusely labeled areabove band 5 epresents

the carbohydrate containing glucose transport er (38). lane a. mem-branes; lane h, memb ranes from chymotrypsin-treated cells. C, 10pg of

untreated and chymotrypsin-treated membranes were electrophoresedafter reduction, blotted. and incubated with ”“I-iodinated anti-hand 3

antibodies in the absence and the presencef 10pdml lactofemn.Blotswere exposed to a PhosphorImager screen and the digitized picture is

shown with a linear gray tone scale y setting the backgroun d w h i t e )to 20 counts and the maximum gray toneM a r k ) o 1000 counts. lane a .

arrows mark th e zone wher e the 38-kDa fragmen t of band 3 protein

membranes; lane h , membranes from chymotrypsin-treated cells. The

migrates. Similar results were obtained inwo other experimentn withanti-band 3 antibodies either absorbed or not absorbed on heat-aggre-

gated I g G .



Anti-band 3 Antibodies Do Not Bindoarbohydrate 23565

motryptic fragment.

Another argument against binding to carbohydrate comes

from studies with glycosidases. Trea tment of red blood cells

with either endo-P-galactosidase, neuraminidase, or endogly-

cosidase-H did not alte r the exte nt of anti-band 3 antibody

binding to band 3 protein on blotted membrane proteins (Fig.

3).The figure shows resultsor anti-band 3 antibodies purified

by preabsorption; the same wasound with the other anti-band

3 preparation. Anti-band3 antibody binding reached, even af-ter a treatmentof intact red cells with 30 milliunits of endo-8-

galactosidase, 97-1028 of control values (n= 3) .These results

were not due to neffective enzymes, s ince the same amountf

endo-P-galactosidase cleaved 52 to 588 (n = 3) f the band 3

carbohydrate from red cells pretreated with galactose oxidase

followed by [’“Clani line and cyanoborohydride (30) Fig. 4).

Endoglycosidase-H was used as a negative control. Neuramin i-

dase had no effect on anti-band 3 binding (Fig.3) nd a minute

one on the remaining label on band 3 protein (95-96%, n = 2).

On the other hand,t removed sia lic acid from glycophorin a nd

thereby altered the electrophoreticmobility of the I4C-labeled

glycophorin band (see heavy arrow in Fig. 4).These results

contradict those of Beppu et al . (23). he binding of their anti-

band 3 antibodies to oxidatively stressed red cells was appar-ently abolished by endo-p-galactosidase or neuraminidase. In

our hands, neitherndo-p-galactosidase nor neuraminidasede-

stroyed the binding site for anti-band 3 antibodies on band 3

protein . Instead , endo-P-galactosidase shortened those copies

of band 3 protein that had a large carbohydrate chain. This

resulted in a sharpen ing f the zone containing band3 protein

on SDS-polyacrylamide gel electrophoresis (see Fig. 4) nd on

immunoblots incubated with anti-band 3 antibodies (Fig. 3).

The difference between he two set sf dat a cannot e explained

by assuming hat he ir anti-band 3 antibodies, which were

purified withou t preabsorption on heat-aggregated I gG , con-

tained anti-idiotypic antibodies with aspecificity for N-acetyl-

lactosaminyl groups , since ant i-band ntibodies purified from

pooled human I g G without preabsorption had the same bindingproperties a s those isolated following absorption of these sub-

stances. It is furthermore unlikely that anti-bandantibodies

bind to N-acetyllactosamine residues, since these carbohydrate

groups were also found associated with red ell membrane pro-

tein band4.5 diffuse zone in between band 4.1and band (Fig.

2B and Fig. 4), epresentin g the lucose transporte r (38)) hich

did not appear positive on blots incubated with anti-band 3

Anti-band 3 bindlng

a b c d e

FIG.. Binding of anti-band 3 antibodies (preabsorbed) toband 3 protein from red cell membranes treated with glycosi-dases. Red cel ls were treated as outlined with: 20 milliunits of endo-p-galactosidase ( lane a), 0milliunits of endo-13-galactosidase ( f a n eh) ,neuraminidase( lane c), endoglycosidase-H f a n ed ) ,or nothing f a n e e ) .Membranes from these red cel ls were p repared,0pg of protein of each

were electrophoresed, blotted. and incubated with labeled anti-hand 3antibodies. The figure shows a digitized picturef the blot obtained by

a PhosphorImagerwith a lineargray onescale anging from 180

counts for whi le to700

counts for the maximum gray one (hlack).Quan tit atio n of antibody binding tohe band3 egion yielded in percent

of control ( f a n e ) for this blot (one of thre e): 103% for lane a, 100% for

lane h . 105% for lane c . and 102% for lane d .

1%-carbohydrate Coomassle blue

Ia b c d e b c d e

that were “C-labeled within carbohydrates and treated withFIG. . Labeling pattern of red cell membranes from red cells

glycosidases. Redcell carhohydratrswere nhelrd ns outlined. he

cells reatedwithglycosidases as given.andmemhranesprepared.

Membrane proteins from each preparation (20 pflane) were electro-phoresed. The stained and driedgel ICoomassie nlurl was expnsed to aPhosphorImager screen from which a di$ized picture was prepared

with the background set to 55 and the maximum gray tone to20 ounts(I ‘Clcarbohydrate). The rat!v arrow points to ~lycophorin. Red crll s

were treated with: 20 milliunits of endo-(~-galactosidase f a n r neur-aminidase ( l a n e h ) . 20 milliunits of endoglycosidase-H f f a n c I , 30 mil-liunits of endoglycosidase-H tlanc d ~ .r nothinK l lanr cl . Quantitation

of label associated with hand 3 protein yieldrd in prrccnt of control 52for lane a and IOO-IO.? for f a n e s c and d . Similar results wrre ound in

two other experiments inwhich endo-13-galactosidasewas applied at 20and 30milliunits. Both concentrations had th r s n m c ere ct nnd lowrrrdthe ext ent of carbohydrate labeling in hand 3 protein to 42‘;.

antibodies (Fig.2).We cannot exclude the possibility that Rep-

pu’s res ult s are asalid as ours, but within a different context,

since these authors id not use human g G pooled from several

thousands of donors for anti-band 3 purification. Instead. they

used I g G from a rat her limited group of donors having exclu-

sively AB blood type and applied purified antibodies to 0 Rh-

positive red cells. Hence, much of the controversy in th is field

may have its origin in the type of I g G used a s a source of

natural ly occurr ing antibodies and in the protocols applied to

purify antibodies. If we intendto better

understand the role of

naturally occurring antibodies,e have to accept theirligo- or

polyclonality which may be of functional relevance, but we have

to do our best o purify them on a particu lar solated antigen in

order to attempt to get monospecific antibodies. Even then,

some, primarily thoseof the IgM class, will turn out o be bi- or

polyspecific (40,41). nti-band 3 & antibodies appear among

naturally occurring antibodiess some of th e most specific ones

and can easilybe afl nit y purified.

The predominant binding of anti-band 3 antibodies to the

55-kDa NH,-terminal chymotryptic fragmentof band 3 protein

and he esultsobtained withglycosidases call for peptide

rather than carbohydrate structures as potential bindingites.

The findings are not only incompatible with reactivity to car-

bohydrates, but also ith the existence f an extra bindingite

in the region of amino acids 812430 (15. 42).t cannot be

excluded, however, that some anti -band antibodies recognize

a second and separate domain, ast might exist on the 38-kDa

fragment (seeFig. 2C). ts visualization mightbe hampered on

blots, since Kay noticed more than an addi tiveffect on binding

of anti-band 3 ntibodies, obtainedby elution of red cells, when

peptides comprising both domains were offered concomitantly

(15.42). n the other hand, anti-bandbinding to amino acids

812-830 s unlikely, because thepublished structures of band 3

protein contains these amino acidsn a cytoplasmic loop (for a

review see Ref. 21).We currently verify whether binding is

exclusively to exoplasmic loops of the chymotryptic55-kDa

fragment.

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