BIOCHIMtE, 1973, 55, i101-1106.

Studies on the induction of heparin degrading enzymes in Flavobacterium heparinum

II. Structural requirements of the inducer.

Maria E. SILVA and Carl P. DIETRICH,

Depar tamento de B ioqu imica e Farmacologia, Escola Paulista de Medicina, C.P. 20.372, $5o Paulo, S.P., Brasil.

(5-12-1972).

Summary. - - The structural requirements for an inducer of the heparin-degrading system in F. heparinum have been studied. It was concluded that the compound must be composed of an uronic acid (with a free carboxyl group), linked to N-acetyl (or N-sulfate) hexosamine through an c~ 1,4 glycosidic linkage. It was also demonstrated that O- and N-sulfates are not essential for the induction process. The finding that some heparitin sulfates are 3 times more effective as inducers than heparin itself is discussed.

INTRODUCTION.

A FIavobacter ium capable of d e g r a d i n g h e p a r i n was i so la ted f rom the soil by Payza and Korn [1]. It was s h o w later that h e p a r i n is d e g r a d e d to its basic cons t i tuents by the c o n c e r t e d act ion of five enzymes p r e sen t in this Flaoobacter ium [2, 3]. At least th ree of these enzymes , name ly a glucosami- n idase , a g lycu ron idase and a su l famidase are in- duced w h e n the bac t e r i um is g r o w n in the pre- sence of h e p a r i n or oligo- and d i s accha r ide s ob- t a ined f rom h e p a r i n [4]. [Kinetic s tudies of the i n d u c t i o n process i n d i c a t e d that the actual indu- cer of the system is a sul fa ted d i s a c c h a r i d e that is f o r m e d f rom h e p a r i n by the ac t ion of const i tu- t ive enzymes p re sen t in very low levels in non- i n d u c e d F. h e p a r i n u m cells [41.

In o r d e r to gain a bet ter u n d e r s t a n d i n g of the na ture of the i n d u c e r and of this process , it was of in te res t to test the act iv i ty of c o m p o u n d s struc- tura l ly re la ted to h e p a r i n as wel l as of chemica l ly m o d i f i e d h e p a r i n s and the i r p roduc t s as i n d u c e r s of the h e p a r i n - d e g r a d i n g sys tem in F. heparinm, n.

MATERIALS ANID METHODS.

Chemicals. --- Hepa r in was ob ta ined f rom Leder le Labora tor ies , Pear l River . N. Y. Chon- d ro i t in sulfate A/,C and c h o n d r o i t i n sulfate B w e r e gifts f rom the late Dr. A. W i n t e r s t e i n (Hoff- m a n n - L a Roche, Basel, Swi tzer land) . The b a r i u m salt of h e p a r i t i n sulfate (batch 6 RTS) was obtain-

(1) Aided by grants from the Fundaq~o de Amparo h Pesquisa do Estado de Silo Paulo (Projeto BIOQ/ FAPESP) and Conselho Nacional de Pesquisas (CNPq), Brasil.

ed f rom the Up John Co., Kalamazoo, Michigan. This c rude h e p a r i t i n sulfate was conve r t ed to its a m m o n i u m salt and f r ac t iona ted into four differ- ent n m c o p o l y s a c c h a r i d e s : hepa r i t i n sulfates A, B, C, and D, as p rev ious ly de sc r i bed [5]. Apple pec t in was p u r c h a s e d f rom the BDH. The disac- cha r ide f rom pec t in was p r e p a r e d t h r o u g h the ac t ion of a pec t inase of fungal or ig in (see enzymes) . Glucosamine N-sulfate, g lucosamine 2-6 disulfate , and sulfa ted d i s accha r ide s w e r e prepa- red f rom h e p a r i n by deg rada t ion w i t h enzymes f rom F. hepar inum, as p rev ious ly desc r ibed [2]. The L- iduron ic a c i d - c o n t a i n i n g d i s a c c h a r i d e pre- pa red by d e a m i n a t i o n of h e p a r i n was a gift f rom Dr. A. Pe r l in (Univers i ty of Montreal , Canada). D i saccha r ides f rom c h o n d r o i t i n sulfates w e r e pre- p a r e d as desc r ibed by Suzuki et al. [6]. The fo l lowing c o m p o u n d s w e r e also p r e p a r e d :

1. N-desul fa ted h e p a r i n - - 100 mg of h e p a r i n were d isso lved in 10 m i of 0.04 N HC1 and hea ted in sealed tubes at 10,0°C for 180 minutes . The mix tu r e was then d ia lyzed against d is t i l led wa te r and p rec ip i t a t ed w i t h 2 vo lumes of e thanol in the p r e sence of 0.1 ml of 2 M NaCI. The p rec ip i t a t e was w a s h e d w i t h 8,0 p. cent e thanol and dr ied .

2. N-desulfa ted N - d i n i t r o p h e n y l a t e d h e p a r i n - - 50 mg of N-desul fa ted h e p a r i n w e r e d isso lved in 5 ml of water . To this m ix tu r e 1 ml of t r ie thyl - amine and 2 ml of a 1 p. cent so lu t ion of 2,4-dini- t ro f luo robenzene (DNFB) in e thanol were added . The resu l t ing suspens ion was hea t ed for 5 hour s at 60°C. After this p e r i o d 2 ml more of 1 p. cent DNFB solut ion nvere a d d e d to the m i x t u r e and the i ncuba t ion was m a i n t a i n e d at 60°C for 5 hours more. The mix tu re was t hen e v a p o r a t e d to dry-

1102 M. E. Silva and C. P. Dietrich.

ness , s u s p e n d e d in 5 m l of w a t e r , d i a l y z e d a g a i n s t w a t e r a n d p r e c i p i t a t e d w i t h 2 v o l u m e s of e t h a n o l . T h e p r e c i p i t a t e f o r m e d w a s c o l l e c t e d b y c e n t r i - f u g a t i o n , w a s h e d w i t h 8,0 p. c e n t e t h a n o l , a n d d r i e d .

4. N - O - d c s u l f a t e d N - a c e t y l a t e d h e p a r i n m e t h y l e s t e r - - T h e p r o c e d u r e w a s v e r y s i m i l a r to t ha t d e s c r i b e d b y W o l f r o m [7~ a n d R o s e m a n [8]. T h e s t a r t i n g m a t e r i a l w a s t he c o m p o u n d d e s c r i b e d i n i t e m 3. Th i s p r o d u c t (300 rag) w a s d i s s o l v e d in

TABLE ][.

Induction of heparin-degrading enzymes by heparitin sul[ate [ractions.

General structure

Compound

Heparin

Hepari t in sulfate A

Hepari t in sulfate B

Hepar i t in sulfate C

Hepar i t in sulfate D

Crude hepari t in

sulfate

Average M . W .

12,000

170,000

2 5 , 0 0 0

6 , 0 0 0

R'

- -SO- a

- -Ace ty l

--SO-:~

- - SO-:~

- -SO- a

0 " qH2OR" )

R"

--SO-:,

H

H SO-~

SO- 3

SO-~ 3,000

- - Mixture of heparit in sulfates A , B , C, and D

R'"

H --SO-:~

H

H SO-:~

Induction index

1.0

1.1

2.3

3.1

2.0

2.5

F. heparinum cells (30 mg 'wet weight) grown in glucose were incubated wi th 100 ,l~g of hepar in Dr other compounds in t rypt icase soy bro. th-casein-yeast extract m e d i u m in a final volume of 1 ml. After 5 h incubat ion the mix tu re were di luted wi th 5 ml of cold 0.09. M phosphate buffer (pH 7.0) and the cells harves ted and washed. After the th i rd 'wash, the cells were dried and resuspen- ded in 100 ~1 of phosphate buffer and 10 .~l of this suspension incubated wi th 100 .~g o,f hepa r in in 1 mM MgCI2-0.02 M 'phosphate buffer (pH 7.0) in a total vo lume of 20 ~1 for 18 h in the presence of 0.4 !~g of ch loramphenicol . After incuba t ion the mix tures were centr i fuged and the supe rna tan t spotted in W h a t m a n n ° 1 paper and chromatographed . The glucosamiine 2,6-disulfate formed was detected by the silver n i t ra te reagent and quant i ta ted by densi to- metry. Controls f rom cells g rown in glucose were subtracted f rom the results . Glucosamine 2,6-disulfate nvas considered to be the end product of act ivi ty of the hepar inases . Induc t ion irLdex : relat ive a m o u n t of glucosam.i~e 2,6-disulfate formed by induced cells ta~ing the value for cells induced by hepa r in as 1.

3. N - O - d e s u l f a t e d h e p a r i n m e t h y l e s t e r - - T h e o v e r a l l p r o c e d u r e w a s s i m i l a r to t h a t d e s c r i b e d b y W o l f r o m et al. [71. A s u s p e n s i o n of s o d i u m h e p a r i n a t e (5,00 my) i n m e t h a n o l i c h y d r o g e n c h l o r i d e (0-.0.6 M, 5 ml) w a s s t i r r e d v i g o r o u s l y fo r 18 h o u r s at r o o m t e m p e r a t u r e . T h e s u p e r n a t a n t w a s d e c a n t e d a n d the p r o c e d u r e r e p e a t e d t h r e e t i m e s w i t h f r e s h p o r t i o n s of t he a c i d i f i e d m e t h a - nol . T h e p r e c i p i t a t e w a s w a s h e d w i t h m e t h a n o l a n d d r i e d ( y i e l d : 350 rag).

BIOCHIMIE, 1973, 55, n ° 9.

2.5 m l of w a t e r to w h i c h 0.5 m l of m e t h a n o l a n d 2.5 m l of D o w e x 1,X-8 r e s i n (--CO~- f o r m ) w e r e a d d e d . Th i s s o l u t i o n w a s c o o l e d to 0-5°C a n d 1 ml a c e t i c a n h y d r i d e w a s a d d e d d r o p w i s e w i t h ag i t a t i on . Af t e r s t i r r i n g fo r 10 h o u r s , t h e r e s i n w a s f i l t e r ed a n d w a s h e d s e v e r a l t i m e s w i t h w a t e r . T h e f i l t ra te w a s d i a l y z e d 2 d a y s a g a i n s t w a t e r . T h e d i a l y z a t e w a s c o n c e n t r a t e d to a s m a l l e r vo lu- me , p r e c i p i t a t e d th r ice w i t h 2 v o l u m e s of e t h a n o l , w a s h e d w i t h e t h e r a n d d r i e d i n v a c u u m o v e r

Induction of heparinases in F. heparinum.

TABLE I[.

Induction of heparin-deyrading enzymes by chemically modi[ied heparins.

Compound

Heparln

N- Desulfated heparin

N-Desulfated D N P-heparin

N-O-Desulfated N-acetylated

heparin-sodium salt

N-O - Desulfated N-aeetylated

h¢.parin methyl estc

N-O-Desulfatcd heparin methyl ester

--S0-:~

H

- -DNP (*)

--Aeetyl

--Aeetyl

General structure

' OR . . . . I~HR' j N

R"

- -S0- 3

--S0-:~

--S0-~

H

H

H

R"'

--S0-:~

- - SO-:~

--S0-a

H

H

R""

H

H

H

Induction index

• 1.0

0.10

0.10

0.70 H

I --CH a 0.10

i

--CH:f i 0.02

(*) Dinitrophenyl. The experiments ~¢ere performed as described in Table I except that different

inducers were used as indicated.

1103

CaC12 a f fo rd ing a w h i t e sol id (y ie ld : 240 my). The m e t h a n o l y s i s step w a s r epea ted and the pro- duct was i so la ted in the same m a n n e r . The la t te r is n e c e s s a r y s ince comple te desu l fa t ion can on ly be ob ta ined by r epea t ing the m e t h a n o l y s i s w i t h the N-ace ty la ted p roduc t .

5. N-O-desulfa ted N-ace ty la ted h e p a r i n sod ium salt - - T h e c o m p o u n d w a s p r e p a r e d f rom the one desc r ibed in i tem 4 by sapon i f i ca t ion . N-O-desul- fa ted N-ace ty la ted h e p a r i n m e t h y l es ter (2,00 rag) was t r ea ted w i t h a p r e v i o u s l y t i t r a t ed aqueous sod ium h y d r o x i d e solu t ion (0.1 M, 10 ml). After 2 days at room t e m p e r a t u r e a back t i t r a t ion w i t h s t a n d a r d HCI s h o w e d the l i be ra t i on of free ac id c o r r e s p o n d i n g to the free c a r b o x y l groups of the u ron i c ac id in h e p a r i n . The m i x t u r e was concen- t ra ted , d ia lyzed agains t wa te r , p r e c i p i t a t e d tw ice w i t h 2 volumes of e thanol , w a s h e d w i t h e ther a n d d r i ed (yie ld : 150 rag).

Enzymes. - - The h e p a r i n a s e s f rom F. hepari- hum cells w e r e p r e p a r e d as p r e v i o u s l y desc r ibed [3, 41. C h o n d r o i t i n a s e s AC and ABC were pur- chased f rom Miles Labora to i r i e s (Elkhar t , In-

d iana) . A pec t inase of fungal or ig in was o b t a i n e d f rom Sigma Chemica l Company .

Induction of enzymes in F. h e p a r i n u m . - - The me thods for the p r e p a r a t i o n of i n d u c e d F. hepa- rinum cells were the ones p r e v i o u s l y desc r ibed N] .

R E S U L T S AND DISCUSSION.

Induction of the heparin-degrading enzymes in F. h e p a r i n u m by heparitin sul[ate tractions. Results in table I show the ef fec t iveness of hepa- r i t in sul fa tes as i n d u c e r s c o m p a r e d to h e p a r i n . Most of the h e p a r i t i n sulfa tes are be t te r i n d u c e r s of th i s e n z y m i c sys tem t h a n h e p a r i n itself. Al l these c o m p o u n d s are ve ry s imi l a r to h e p a r i n . For ins tance , all of t hem con ta in g lucosamine and u r o n i c ac id in t h e i r s t ruc ture , a n d these sugars are a p p a r e n t l y l i n k e d t h r o u g h a 1,4 g lycos id ic l inkages . The m a i n d i f fe rences be tween these c o m p o u n d s are the sulfate content , mo lecu la r we igh t and in the case of h e p a r i t i n sulfate A, the a lmost comple te absence of sul fa te groups in the

BIOCHIMIE, 1973, 55, n ° 9. 72

1104 M. E. Siloa and C. P. Dietrich.

molecule . F r o m th is it can be conc luded tha t the p re sence of sulfa tes is not essent ia l for the induc- t ion of the h e p a r i n - d e g r a d i n g e n z y m e s . An impor - tant f i n d i n g is tha t the N-acetyl g roups (present in

of t ime, thus p r o m o t i n g the i n d u c t i o n of the hepa- t i n - d e g r a d i n g enzymes for a p r o p o r t i o n a t e l y lon- ger per iod . This poss ib i l i t y wi l l even tua l ly be explored .

TABLE III.

Induction of heparin-degrading enzymes by several natural polysaccharides.

Compound

Heparin

Chondroitiu sUlfate A:I~ = H R" -~- SO-:; C:R --- SO-:: R" = H

Chondroitiu sulfate B

Mactin (mollusc heparin

Pectin

Structure

oso3 o ~s~ - - - ~ N

coo" ~ i

NXAC N

f ~o q ~ 7 _ W , , O H /I -'o-~ . /'

See heparin

[ j cooc~ j o~ ?-%,

N

Induction index

1.0

O. I5

0.10

0.55

0.15

The experiments were performed as described in table I except that different inducers were used as indicated.

h e p a r i t i n sulfate A) can subs t i tu te the N-sulfate groups w i t h o u t loss of the i n d u c t i o n effect iveness. The r eason w h y some h e p a r i t i n s are be t ter indu- cers of the h e p a r i n - d e g r a d i n g enzymes than hepa- r in i tself is not a l toge ther clear. All these com- p o u n d s are deg raded by the i n d u c e d enzymes a n d in the cases of the h e p a r i t i n sulfa tes C and D, form the same type of d i s accha r ides . It h a s been observed p r e v i o u s l y [3] tha t the ve loc i ty of de- g rada t ion of h e p a r i n and h e p a r i n p r o d u c t s by the h e p a r i n a s e s depend on the cha in leng th of these compounds . Fo r ins tance , h e p a r i n is deg raded fas ter by the c rude h e p a r i n a s e s t han the hexa- s a c c h a r i d e or t e t r a s a c c h a r i d e (both ob ta ined by par t i a I d e g r a d a t i o n of h e p a r i n ) . It is possible , then , tha t h e p a r i t i n sulfates C and D w h i c h are smal le r t han h e p a r i n are deg raded at l ower velo- ci ty t han h e p a r i n . Consequen t ly the i r d i saccha- r ide p r o d u c t s wou ld be f o r m e r for a longer p e r i o d

BIOCHIMIE, 1973, 55, n ° 9.

Induction by chemically modified heparins. N-desul fa t ion of h e p a r i n removes its i nduc t ive effect iveness. D i n i t r o p h e n y l a t i o n of the free amino group does not res tore this p r o p e r t y (table II). Never theless , ace ty la t ion of the group res tores a lmost comple te ly the act iv i ty . The che- mica l p r o c e d u r e used for N-ace ty la t ion of h e p a r i n involves also the r emova l of most of the O-sulfate g roups of th is compound . These resul t s thus con- f i rm the ones ob t a ined w i t h the h e p a r i t i n suIfates, i.e., N- and O-sulfates are not essent ia l for the in- duc t ion process . However , a free c a r b o x y l group seems to be essent ia l for the i n d u c t i o n s ince me- t h y l a t i o n of this group removes the i n d u c t i o n ac t iv i ty of the N-ace ty la ted h e p a r i n ( table II).

Induction by some natural polgsaccharides. Other na tu ra l p o l y s a c c h a r i d e s re la ted to h e p a r i n were tes ted as poss ib le i n d u c e r s (tabIe III) . Wi th

Induction of heparinases in F. h e p a r i n u m . 1105

the excep t ion of mac t i n ( w h i c h is a h e p a r i n f rom mollusc) none of the p o l y s a c c h a r i d e s are able to i nduce this enzymic system. Pec t in , w h i c h con- ta ins only u ron i c ac id uni ts l i n k e d by a 1,4 glyco- s id ic l inkages, is not an inducer . Also, c h o n d r o i t i n

Induction of the heparin-degrading enzymes by di- and monosaccharides. Table IV shows that lri- and di-sulfa ted d i s a c c h a r i d e s ob ta ined f rom hepa- r in by enzymic deg rada t ion are as good as hepa- r in as i n d u c e r s of the enzyma t i c system, thus con-

TABLE IV.

Induclion of heparin-degrading enzymes by several related disaccharides.

Compound

Trisulfated disaceharide from heparin (bacterial

degradation)

Disulfated disaccharide from hepaIin (bacterial

degradation)

Disutfated disaccbaride from

heparin (deamination product)

4-Sulfated disaccharide from

CHSO~, A and B (bacte- rial degradation)

6-Sulfated disaecharide from

CHSO 4 C (bacterial degradation)

Disaccharide from pectin (fungal

degradation)

Structure

O 5 0 3 N H S O 3

coo" c,.pso~

C/a j__o o i

d~qH / ' "~',OH !

OS%

coo c ~ . ~ -o "o~o

O H N H A c

c°°- o,, ~w~

OH NHA C

COO C O O - t - 0 . "

r.duetion index

1.3

1.1

0.10

O. 20

0 15

0 1 0

The experiments were performed as described in talde I except that different inducers were used as indicated.

sulfates w h i c h con ta in u r o n i c acid, h e x o s a m i n e and sulfates but p r e sen t ~ 1,3 or 6 1,4 l inkages , are not i nduce r s of the system. These results ind i - cate so far that, bes ides the ~ l inkages , the i n d u c e r has to p r e sen t in its s t ruc ture a N-acetyl or N-sul- fate h e x o s a m i n e and an u r o n i c acid.

BIOCHIMIE, 1973, 55, n ° 9.

f i rming p rev ious resul ts o b t a i n e d w i t h these com- p o u n d s [4]. A chemica l ly d e a m i n a t e d d isaccha- r i d e also p r e p a r e d f rom h e p a r i n is not an i n d u c e r of this system. This resul t again ind ica tes that the p r e s e n c e of a h e x o s a m i n e ( p r o p e r l y N-subst i tuted) seems to be essent ia l for the p rocess of i nduc t ion ,

1 1 0 6 M. E. S i lva and C. P. Die t r ich .

TABLE V.

Induct ion of heparin-degrading enzymes by several monosaceharides.

Compound

Glucosamine 2 ,6 disulfate

Glucosamine N - sulfate

Glucuronic acid

Iduronic acid

Structure

NHSO. 3

" C~H

I J~\~HOH

co6

//~O~H OH

Induction index

0.15

0 .10

0.10

0.10

The e x p e r i m e n t s were p e r f o r m e d as descr ibed in tab le I except tha t d i f ferent inducers ~vere used as ind ica ted .

D i s a c c h a r i d e s c o n t a i n i n g ~ l i n k a g e s , as t h e o n e s p r e p a r e d f r o m t h e c h o n d r o i t i n s , a n d an a 1,4 l in - k e d d i s a c c h a r i d e p r e p a r e d f r o m p e c t i n a r e n o t i n d u c e r s . S u l f a t e d h e x o s a m i n e s ( p r e p a r e d f r o m h e p a r i n b y e n z y m i c d e g r a d a t i o n ) a n d u r o n i c a c i d s a r e u n a b l e to i n d u c e t h e h e p a r i n d e g r a d i n g e n z y m e s , s t r e s s i n g a g a i n t h e i m p o r t a n c e o f t h e a l i n k a g e s i n t h e i n d u c t i v e p r o c e s s ( T a b l e V).

CA)NCLUSIONS.

T h e r e s u l t s p r e s e n t e d i n t h i s p a p e r i n d i c a t e so f a r t h a t t h e m i n i m u m s t r u c t u r a l r e q u i r e m e n t s f o r t h e i n d u c e r o f t h e h e p a r i n d e g r a d i n g e n z y m e s in

coo- CN~ON

(so;)

F. heparinum a r e t h e p r e s e n c e of a n u r o n i c a c i d a n d a N - a c e t y l (o r N- su l f a t e ) h e x o s a m i n e l i n k e d b y a n a 1,4 g l y c o s i d i c l i n k a g e , as s h o w n i n

f i g u r e 1. T h e p o s s i b i l i t y t h a t t h e u n s a t u r a t i o n of

t h e u r o n i c a c i d m o i e t y is a l so e s s e n t i a l f o r t h e i n d u c t i v e a c t i v i t y is n o w u n d e r i n v e s t i g a t i o n .

A cknatoledgmen ts.

We wou ld l ike to express our g ra t i tude to Dr. S. M. C. Die t r ich for help in the p r e p a r a t i o n of this m a n u s - cript .

Les condi t ions de s t ruc tu re pou r l ' i nduc t ion du sys t6me qui d6grade l ' hdpar ine chez F. heparinum out ~t~ dtudi6es. On a dtabli que la subs tance doi t compor- ter u n r6s idu d 'acide u r o n i q u e (dont le g r o u p e m e n t ca rboxyle est l ibre), li~ h une N-ac6tyl- (ou N-sulfa te)- h e x o s a m i n e pa r une l ia i son g lycos id ique a 1,4. On a d6rnontr6 auss i que les su l fa tes des pos i t ions -N et -O rm sont pas essent ie l les ~ ce processus d ' i nduc t ion . Lc fair que cer ta ins h6pa r i t i ne su l fa tes son t des induc- t eurs 3 fois p lus effectifs que l 'h6par ine e l le-m~me, est. comment~ .

REFERENCES.

1. Payza, A. N. & Korn, E. D. (1956) Nature, 177, 88-89. 2. Dietr ich, C. P. (1968) Biochem. J., 108, 647-654. 3. Dietr ich, C. P. (1969) Biochemistry, 8, 2089-209,1. 4. Dietr ich, C. P. (1969) Biochemistry, 8, 3342-3347. 5. Dietr ich, C. P., Nader, H. B., Bri t to, L. R. & Silva,

M. E. (1971) Biochim. Biophys. Acta, 237, 430- 441.

6. Suzuki , S., Saito, H., Yamagata , T., Anno, K., Seno, N., Kawai, Y. & F u r u h a s h i , T. (1968) J. Biol. Chem., 243, 1543-1550.

7. W o t f r o m , M. L., Vercel lot t i , J. R. a Thomas , G. H. S. (1964) J. Org. Chem., 29, 536-539.

8. Roseman , S. ~ Ludo'wieg, J. (1954) J. Am. Chem. Soc,, 76, 301-302.

BIOCHIMIE, 1973, 55, n ° 9.