Carbohydrate Sulfates - American Chemical Society

3 Sulfate Ester Groups as Potential Informational Regulators in Glycoproteins P. W. K E N T , C. J. C O L E S , J. R. C O O P E R , and N. R. M I A N

Downloaded by UNIV LAVAL on July 13, 2016 | http://pubs.acs.org Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

Glycoprotein Research Unit, Durham University, Durham D H 1 3 L H , England

The presence of ester sulphates i n carbohydrate macromolecules i s a feature widespread both i n plants and animals. Considerable speculation exists about the biological functions played by the acidic ester groups, whether these groups solely serve to augment the anionic character of the macromolecule concerned or whether they exert more specific and perhaps more subtle biological effects. The purpose of this paper i s to consider these questions, in particular i n terms of sulphated macromolecules present in animal tissues. The p a r a l l e l aspects i n plants are no less important, bearing i n mind the location of sulphated polysaccharidic material i n c e l l wall structures, their potential role i n relation to water-retaining mechanisms and their capacity to interact with other macromolecules. Sulphated Glycosaminoglycans In animals, relevant macromolecules examined i n greatest detail have been the sulphated glycosaminoglycans (mucopolysaccharides) associated with connective tissues eg. chondroitin sulphates, keratosulphate, heparan sulphate, derman sulphate. In p r i n c i p l e , the sulphate ester groups are located i n defined positions on oligosaccharide chains of regular repeating sugar sequences and these i n turn are attached by a l k a l i -label linkages to a protein (Table I ) . Thus i n chondroitin 4-sulphate, sulphate ester groups are envisaged i n the ideal situation as being attached to C-4 of every N-acetylgalactosaminyl residue and to C-6 of the same aminosugars i n chondroitin 6-sulphate. Nevertheless considerable variations i n practice have been noted i n the actual extent of sulphate between chondroitin sulphate types of different biological o r i g i n . Chondroitin sulphate from shark cartilage, for example, exhibits the abnormally high sulphate content of 33.7% while that from bovine c o r t i c a l bone has 10.3% sulphate.

0-8412-0426-8/78/47-077-029$05.00/0 © 1978 American Chemical Society

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.


Composition of glycosaminoglycans

D-glucosamine

D-gluco samine

D-galactose

D-glucuronic a c i d or L-iduronic acid

Keratan sulphate

Heparin

sulphate

D-glucuronic a c i d o r L-iduronic acid

D-galacto samine

L-iduronic acid or D-glucuronic a c i d

Dermatan sulphate

Heparan

D-galacto samine

D-glucuronic a c i d

Chondroitin 6-sulphate

D-gluco samine

D-galacto samine


Chondroitin 4-sulphate

D-gluco samine


Hyaluronic acid

D i s a c c h a r i d e repeating u n i t Hexosamine Hexuronic a c i d

Table I .

O-sulphate and N-sulphate

O-sulphate and N-sulphate

0-sulphate

0-sulphate

0-sulphate

0-sulphate

Sulphate


D-xylose, D-galactose


D-mannose, D-fucose, s i a l i c acid, D-galacto samine




Other sugar r e s i d u e s , i n c l u d i n g those i n the l i n k a g e r e g i o n

H M

>

d

>

κ! ϋ

w ο

> w

η

GO Ο


3.

K E N T ET AL.

Sulfate Ester Groups

31

I n c o n t r o v e r t i b l e such e s t e r sulphate can c o n t r i b u t e to the e l e c t r o p h o r e t i c a n i o n i c c h a r a c t e r , and under experimental c o n d i t i o n s a t l e a s t , endow the macromolecule with dye-binding p r o p e r t i e s . Studies on the induced c o t t o n e f f e c t s o f a n i o n i c dye-mucopolysaccharide complexes i n the a d s o r p t i o n band o f the dye eg. methylene blue (Stone, 1964* 1965) have been i n t e r p r e t e d on the b a s i s o f polymer conformations, i n d i c a t i v e of both random and h e l i c a l forms (Hirano and Onodera, 1 9 6 7 ) · Whereas c h o n d r o i t i n ( s u l p h a t e - f r e e ) e x h i b i t e d random conformation, c h o n d r o i t i n 4-sulphate ( l i k e derman sulphate and hyaluronate) had h i g h degrees o f h e l i c a l s t r u c t u r e s o f the L e f t Screw sense. By c o n t r a s t , c h o n d r o i t i n 6-sulphate, h e p a r i n and h e p a r i t i n sulphate were h e l i c a l conformations o f the R i g h t Screw sense. I n t e r e s t i n g l y , c h o n d r o i t i n polysulphate ( c h o n d r o i t i n 4 * 6 sulphate) had the dye-binding c h a r a c t e r i s t i c s o f c h o n d r o i t i n 4-sulphate. A d d i t i o n a l s u l p h a t i o n o f c h o n d r o i t i n 6-sulphate (to OH-4 o f the aminosugar r e s i d u e s ) on the other hand l e a d s to conformational i n v e r s i o n . I n the h e p a r i n s e r i e s , de Ν-sulphation o f t h a t m a t e r i a l o r o f h e p a r i t i n sulphate d i d not b r i n g about change o f conformation, though complete desulphation caused the h e l i c a l s t r u c t u r e to disappear. The primary s t r u c t u r e p l a y s a d e c i s i v e p a r t i n conformational determination, the l i n k e d p o s i t i o n o f each e s t e r sulphate being important i n r e l a t i o n to hydrogen-bonding c a p a b i l i t y . Elegant X-ray a n a l y t i c a l s t u d i e s by Rees (1969) and by A t k i n s and Laurent (1973) have e s t a b l i s h e d f i n e d e t a i l s o f ordered conformation o f these mucopolysaccharides. Detailed reviews o f these aspects have been presented by Kirkwood (1974) and Rees (1975). The presence o f e s t e r sulphate groups i n these d e f i n e d and r e i t e r a t e d p o s i t i o n s i n sequence nevertheless does not n e c e s s a r i l y impair the a c t i o n o f dégradative enzymes. H y d r o l y t i c endohexosaminidases eg. hyaluronidase a c t r e a d i l y on c h o n d r o i t i n 4 - and 6-sulphates, as on hyaluronate, g i v i n g corresponding d i s a c c h a r i d e and t e t r a s a c c h a r i d e products. In g e n e r a l , i t would be concluded t h a t i n these i n s t a n c e s the i n - b u i l d i n g o f e s t e r sulphates i n a r e g u l a r p e r i o d i c i t y does not seemingly i n f l u e n c e the metabolic s t a b i l i t y nor t h e i r immunological p r o p e r t i e s , but r a t h e r the macromolecular shape and, by i n f e r e n c e , t h e i r i n t e r - m o l e c u l a r a s s o c i a t i o n s . Sulphated G l y c o p r o t e i n s E s t e r sulphates are however widely found i n other s i t u a t i o n s , e s p e c i a l l y i n a wide v a r i e t y o f sulphated g l y c o p r o t e i n s (Table I I ) . These were f i r s t shown to e x i s t i n g a s t r o - i n t e s t i n a l e p i t h e l i a l mucins and c o r n e a l g l y c o p r o t e i n s . The degree o f sulphate here, too, i s v a r i a b l e , though a value



G-i tract

Human G a s t r i c carcinoma

Human G a s t r i c tissue

Human G a s t r i c juice

A.

Source

Sialic acid

5.8 3-2 3.1 3.8

2.5

2.9 1.3 2.3

8.5

Sulphate

2.1 3.8 6.5 7.1

5.1

4-3 4-4 4.9

1.8 7-4

21.1 1.4

28.9 30.2 28.5

13.6 10.2 10.2 32.2

13.6 10.2 10.2

34.1

-

22.6

16.2

17.3

Galactose

26.3 25.4 25.0 22.0

GlcN GalN

2.0 1.9 1.9 2.3

Hexosamine

36.2 35.8 32.8 33.2

18.2 17.3 15.4

Fucose Ref.

Kimoto e t a l (1968 )

) ) M a r t i n e t a l (1968)

M a r t i n e t a l (1967)

) ) Hakkinen e t a l (1965 ) )

(g/lOOg d r y wt)

Table l i a Composition o f E p i t h e l i a l Sulphated G l y c o p r o t e i n s


w

H

>

c!

X

« o

> o


submaxillary gland

References

Pig Colonic muco sa

1.3 3.0 6.5 7.4 7.4 8.8 7.0 5.5

+0 0.06 1.84

3.2 3.8 3.8 3.4

11.8 24-5

Sialic

52.3 51.2 48.1 21.3 22.4 21.4 29.3

5.6 3.2 5.2 11.2 8.8 21.4 5.5

( 2 ) Slomiary and Meyer (1972)

( 4 ) Inoue and Yosizawa (1966)

( 3 ) Coles and Kent (1977)

32.1

18.0 24.5

6.2 5.0 12.5

Hexosamine

Fucose

(1966)

acid

3 2 2 3

0.9 0.97 0.75

-

_

GlcN GalN

21.7 25.6 23.2 20.4

13.9 14.6 13.0

45

9.4 19.6

Galactose

L

)

(2)

(

8.0 ) 8.7 )

8.0 )

)

u

'

) (3)

)

(

Ref

55.4 ) , ) 40.0 )

Peptide

(g/lOOg d r y wt)

of E p i t h e l i a l Sulphated G l y c o p r o t e i n s (Pooled p r e p a r a t i o n s )

3.5

Sulphate

Composition

: (1) Katzman and E y l o r

Pig Duodenal tissue

Pig g a s t r i c mucosa

G-i t r a c t

Pig

Source

Table l i b .


CO 00

-*

o Si

es

8"

C/3

M H W H


34

CARBOHYDRATE SULFATES

of about 2$ i s common i . e . about 20 sulphate residues per 100,000 d a l t o n s . Unlike the mucopolysaccharides, g l y c o p r o t e i n s show l i t t l e evidence of ordered sugar-repeating sequences and the task o f a l l o c a t i n g exact p o s i t i o n s to such sulphate residues i s c o n s i d e r a b l e . I n a study o f g l y c o p r o t e i n s (sulphate content between 2.8 and 5 · 9$) o f p i g g a s t r i c mucosa, Slomiany and Meyer (1972) showed that a t l e a s t one N - a c e t y l glucosamine residue was sulphate, a t p o s i t i o n 6 , and l o c a t e d i n c l o s e proximity to the peptide attachment ( F i g . l a ) . T h i s m a t e r i a l i s f r e e from u r o n i c and s i a l i c a c i d s . A s i m i l a r arrangement has been i n d i c a t e d i n the sheep e p i t h e l i a l g l y c o p r o t e i n i n which s i a l i c a c i d residues are a l s o present (Kent, 1 9 7 0 ) . ( F i g . l b ) . The p i g g a s t r i c g l y c o p r o t e i n e x h i b i t s strong (A 4- H) blood group a c t i v i t y and i t i s apparent that a s i n g l e sulphate e s t e r group remote from the immuno-determinant groups has no demonstrable e f f e c t on the blood group a c t i v i t y . The l a r g e m a j o r i t y o f sulphated g l y c o p r o t e i n s have not y e t been investigated i n s u f f i c i e n t d e t a i l to a s s i g n the s t r u c t u r a l p o s i t i o n of the sulphate r e s i d u e s . I n t h i s r e s p e c t , the chemical study o f 3 5 s - l a b e l l e d g l y c o p r o t e i n s obtained by biochemical i n c o r p o r a t i o n techniques, and w e l l c h a r a c t e r i s e d by customary p h y s i c a l means, o f f e r s s u b s t a n t i a l advantages. I t remains a matter f o r s p e c u l a t i o n whether i n c e r t a i n o f these cases the presence of the small number o f e s t e r sulphate groups nevertheless a c t as ' i n f o r m a t i o n a l b l o c k s ' e i t h e r of immunologically important s i t e s o r o f substrate s p e c i f i c i t y i n enzymic degradation. Semi-synthetic

Studies

Means o f gathering i n f o r m a t i o n about these important p o s s i b i l i t i e s can be envisaged i n other ways. I n p a r t i c u l a r , the e f f e c t s are open to i n v e s t i g a t i o n s o f i n t r o d u c i n g sulphate groups, ^ S - l a b e l l e d otherwise, i n t o a w e l l - c h a r a c t e r i z e d g l y c o p r o t e i n by chemical means. While i t must be i n e v i t a b l y expected t h a t m u l t i p l e s i t e s w i l l be e s t e r i f i e d even with low degrees o f s u l p h a t i o n , nevertheless s u f f i c i e n t l y s e l e c t i v e chemical and enzymic techniques are now a v a i l a b l e t o make e x p l o r a t i o n worthwhile. A p a r t i c u l a r study has been made o f the g l y c o p r o t e i n s obtained from pooled p i g duodenal t i s s u e by papain d i g e s t i o n and f r a c t i o n a t i o n i n i t i a l l y by i o n exchange ( B e r t i l l i n i e t a l . 1 9 7 1 ) · The i n i t i a l mixed glycopeptides had a sulphate content between 1.04$ and 1.11$ f o r a range o f successive p r e p a r a t i o n s , while the s i a l i c a c i d content v a r i e d between 1.9$ and 2 . 6 $ . F r a c t i o n a t i o n by a n i o n i c exchange chromatography (Watman DE52) was e l u t e d with sodium acetate b u f f e r followed by a sodium chloride gradient ( F i g . 2 ) . T h i s enabled s i x components to be o

r



Gal

3 )

GalNAc

I ?

Fuc

Fuc

(4)

?

Gal

(6) .Gal.

(6) 4 Gal.

X-CHAIN

Gal(44)

Fuc (16)

GalNAc

GalNAc

Gal Gal-

I T

GlcNAc 16 Gal Y-CHAIN

-(GlcNAc)

Sulfate (47)

a

sulfated glycoproteins

SER

OR

THREO

PEPTIDE

H)

Gal—i- G l c N A c — ^ G l c N A c — G a l — ^ - G l c N A c

6-SULFATE (3)

Suggested structure of the carbohydrate chains of hog stomach blood group (A +

H

Fuc I 2(

Fuc

(3)

Abbreviations: Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, ^-acetylgalactosamine.

Figure lb. Proposed oligosaccharide structure of desialated colonic goblet cell glycoprotein H (sheep). Numbers in parentheses are number of residues per average molecule. For each polypeptide, 30 main X-chain oligosaccharides are present, each bearing two or three side Y-chains (taken from Kent (1970)).

Figure la.

(6)

G a l N A c — G a l — G l c N A c — Gal [2 ft

(4)



36

CARBOHYDRATE

SULFATES

separated (Table I I I ) . The p r i n c i p a l f r a c t i o n (A) was a nearn e u t r a l PAS-t- m a t e r i a l accounting f o r 45$ of the o r i g i n a l m a t e r i a l , f o l l o w e d by two l e s s e r , but more a c i d i c g l y c o p r o t e i n s (C and D). I n a d d i t i o n , a c h o n d r o i t i n sulphate f r a c t i o n (F) and h y a l u r o n i c a c i d (E) were p r e s e n t . Of the g l y c o p r o t e i n components, a l l three showed some (A and H) blood group a c t i v i t y and the t h i r d component (D) had i n a d d i t i o n an e s t e r sulphate content of 1.84$ (Table I V ) . I n a s t r u c t u r a l examination of the p r i n c i p a l glycopeptide A, a l k a l i n e borohydride cleaved the o l i g o s a c c h a r i d e r e s i d u e s , with concomitant d e s t r u c t i o n of s e r y l and t h r e o n y l residues of the peptide c h a i n and q u a n t i t a t i v e formation of N-acetylgalactosaminoL The o l i g o s a c c h a r i d e side chains were of a t l e a s t three types, t e r m i n a t i n g i n s i a l i c a c i d , fucose and g a l a c t o s e , attached to the peptide c h a i n by O - g l y c o s y l r e s i d u e s . The second g l y c o p e p t i d e (C ) d i f f e r e d from A notably by i t s higher content of s i a l i c a c i d s , w h i l s t glycopeptide (D) had both s i a l i c a c i d and sulphate groups w i t h i n i t . Though the s i t e s of s u l p h a t i o n (227 groups per molecule, m.wt. 3.5 x 10^) have not y e t been i d e n t i f i e d p r e c i s e l y , i t i s known from the work of Smith t h a t the groups are not i n the t e r m i n a l regions of the o l i g o s a c c h a r i d e side branches but i n those regions proximal to the carbohydrate-peptide j u n c t i o n s , as i n the other e p i t h e l i a l g l y c o p r o t e i n s mentioned e a r l i e r . Complete s u l p h a t i o n ( i . e . > 96$) o f mixed duodenal g l y c o p e p t i d e p r e p a r a t i o n s (or of the c o n t r i b u t i n g p u r i f i e d GLP-A o r GLP-C) has been achieved ( P r i n o , L i e t t i and P a g l i a l u n g a , 1971). Though the product i s reported to possess some a n t i coagulant a c t i v i t y i n v i t r o , and i t s b i o l o g i c a l p o t e n t i a l as an a n t i - u l c e r agent i s c o n s i d e r a b l e , the l a t t e r property i s common to a number of c h e m i c a l l y sulphated macromolecules eg. dextran sulphate ( R i c k e t t s 1952, R i c k e t t s and Walton 1 9 5 2 ) , amylopectin sulphate ( f o r example, San and Ryan 1 9 7 0 ) , h y a l u r o n i c a c i d sulphate ( P a n t l i t s c h k o et a l . 1951 )> but with the added advantage t h a t the parent p i g g l y c o p e p t i d e d e r i v e s from an e p i t h e l i a l system r e l a t e d to the ABO blood group system. ~Microtechniques have been devised f o r the S-sulphation of the mixed duodenal g l y c o p e p t i d e s , as w e l l as f o r the component f r a c t i o n s , u s i n g 3 5 SO^Cl i n p y r i d i n e . Near q u a n t i t a t i v e s u l p h a t i o n (> 96$ or a v a i l a b l e h y d r o x y l s i t e s ) produced a h i g h l y a n i o n i c d e r i v a t i v e (GLPS) i n which the sugar composition was i n other r e s p e c t s undisturbed. This material (and a l s o sulphated GLP-A) was a modest i n h i b i t o r of p u r i f i e d human and p i g pepsins i n v i t r o (Table V ) . I t a l s o was a powerful i n h i b i t o r of c e l l growth of Swiss 3T3 f i b r o b l a s t s and of c e l l adhesion. I t i s d o u b t f u l however whether t h i s p r o p e r t y i s s p e c i f i c to the d e r i v a t i v e and other r e l a t e d H



A l c i a n Blue

PAS

identity

neutral glycopeptide

16$

45$

neutral glycopeptide

C

A

sulphated glycopeptide

31$

D

3$

E

l 2

chondroitin sulphate e t c .

f o r about

hyaluronic a c i d (E-^)

E

(Component Β i s of low molecular weight and accounts 1$ of the i n i t i a l m a t e r i a l )

$ of i n i t i a l m a t e r i a l

Component



38

T a b l e IV* C o m p o s i t i o n o f P i g D u o d e n a l

Glycopeptides

Çtimoles/g. d r y Component


NANA NGNA

) )

Fucose Gal GalNAc GluNAc

106

D

C

A ) )

342 772 1260 1140

wt)

230 199 812 1170 1140

) )

263 316 721 1240 926

( X y l , Mann, G l u c u r o n i c a c i d not detected)

Asp Thre Ser Glu Pro Gly Ala Val I-Leu Leu His Lys Arg Tyr Phe

55 567 310 85 369 63 151 93 17 66 64

16 39

ND ND

55 533 304 87 331 66 138 88 14 56 59 15 33

ND ND

49 711 290 66 447 67 72 53 34 41 35 12 17

ND ND

( C y s and M e t n o t Sulphate

l e s s than 0.3

7.4

detected)

227


3.


K E N T ET AL.

39

Ε

S o'

5

50m M Sodium Acetate 1-2M NaCI pH 4 0 —— •

A

CO

\

(/) g

ΟΙΟ-

Q _l < 005Downloaded by UNIV LAVAL on July 13, 2016 | http://pubs.acs.org Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch003

^

lOmM Sodium Acetate pH 4 0

Λ ν/Λ α

o^S^

C

F

D

y^

100

500

400 ELUTION VOLUME (ML)

Figure 2. Fractionation of duodenal glycopeptides by ion exchange. in 5mL starting buffer was applied to a column of Whatman DE 52 (120 X 1.25) equilibrated with lOmM sodium acetate, pH 4.0 (starting ionic gradient terminating with 50mM sodium acetate, 1.2M sodium was applied. Flow rate, 10mL/hr.

Table V.

200 mg of GLP anion exchanger buffer). A linear chloride, pH 4.0

E f f e c t of sulphated duodenal glycopeptides on p u r i f i e d

pepsins

( i n c o l l a b o r a t i o n with Dr. W. H. T a y l o r ) H i g h l y p u r i f i e d p i g o r human pepsins were incubated a t 3 7 ° with glycopeptide

(2.5mg/ml).

30 min, pH 4 · 0 ( a c e t a t e ) ; haemoglobin s u b s t r a t e . Values expressed

at % of glycopeptide-free c o n t r o l .

glycopeptide

human * 118.6

native glycopeptide sulphate ( f u l l ) glycopeptide sulphate (33%)

Pig 112

substrate

91

72

inhibition

-

90

inhibition

(average o f 3 experiments)



40


p o l y s u l p h a t e s eg. dextran sulphate behaved s i m i l a r l y , both q u a l i t a t i v e l y and q u a n t i t a t i v e l y ( F i g . 3 ) towards f i b r o b l a s t s i n culture. M i l d a c i d i c h y d r o l y s i s r e s u l t s i n cleavage o f two s i a l i c a c i d d e r i v a t i v e s , separated by t . l . c . on c e l l u l o s e and i d e n t i f i e d as the tetra-O-sulphates o f N-acetylneuraminic a c i d and as the penta-O-sulphate o f N - g l y c o l l y l n e u r a m i n i c a c i d . Sulphated forms of fucose have not y e t been i d e n t i f i e d though presumably a r e cleaved simultaneously. N e i t h e r o f these products a r e o x i d i s e d by sodium metaperiodate though they may be detected by the Svennerholm procedure. Not s u r p r i s i n g , the f u l l y sulphated glycopeptide (GLPS 9 6 $ ) i s not a substrate f o r cholerae neuraminidase and there i s some evidence t h a t i t may be a noncompetitive i n h i b i t o r o f t h i s enzyme towards other s i a l o p r o t e i n s . The i n h i b i t i o n i s not considered t o be s p e c i f i c but r a t h e r a consequence o f the p o l y a n i o n i c c h a r a c t e r o f the substance. I t i s o f i n t e r e s t t o examine the s i t u a t i o n a r i s i n g from much lower degrees o f s u l p h a t i o n o f the g l y c o p e p t i d e and e m p i r i c a l l y two d e r i v a t i v e s r e p r e s e n t i n g 3 4 $ and 1 1 $ s u l p h a t i o n of a v a i l a b l e hydroxyl s i t e s have been prepared. The low s u l p h a t i o n d e r i v a t i v e (GLP-S 1 1 $ ) i s h y d r o l y s a b l e by d i l u t e s u l p h u r i c a c i d , with r e l e a s e o f s i a l i c a c i d s (NANA and NGNA) i n which predominantly one hydroxyl group i s r e p l a c e d by sulphate. (The d e r i v a t i v e s a r e o x i d i s e d by I 0 7 , suggesting t h a t the sulphate e s t e r i n NANA i s a t Cy o r C ^ j . T h i n - l a y e r chromatography o f the h y d r o l y s a t e showed the presence o f a t l e a s t three sulphates and i t appears l i k e l y t h a t a sulphated fucose i s a l s o r e l e a s e d . I t i s estimated t h a t 16$ o f the t o t a l sulphate content i s present i n s i a l i c a c i d and fucose t e r m i n a l r e s i d u e s . U n l i k e the f u l l y sulphated d e r i v a t i v e (GLP-S 9 6 $ ) , d e r i v a t i v e s with lower s u l p h a t i o n had l i t t l e e f f e c t on f i b r o b l a s t c e l l growth o r on p e p s i n a c t i v i t y . The sulphated d e r i v a t i v e (GLP-S 1 1 $ ) a l s o i s not a s u b s t r a t e f o r V. cholerae neuraminidase and indeed shows some i n h i b i t i o n o f the enzymic h y d r o l y s i s w i t h a known s u s c e p t i b l e substrate (Table V I ) . I t i s b e l i e v e d t h a t t h i s i s a competitive i n h i b i t i o n and a t t r i b u t a b l e t o the presence o f O-sulphate r e s i d u e ( s ) i n t e r m i n a l s i a l i c a c i d s , s i n c e the sulphated g l y c o peptide residue a f t e r m i l d a c i d i c h y d r o l y s i s was not such an inhibitor. I t i s o f i n t e r e s t t o c o n s i d e r whether the i n h i b i t i o n can be r e l i e v e d by metabolic i n t e r v e n t i o n o f a p p r o p r i a t e enzymes, and t h i s would suggest a p a r t i c u l a r r o l e f o r the sulphatases, lysosomal systems known t o be capable o f a c t i o n both on g l y c o p r o t e i n and g l y c o l i p i d carbohydrate sulphate e s t e r s . I n a p p r o p r i a t e c o n d i t i o n t h i s step c o u l d represent a f u r t h e r c o n t r o l mechanism r e g u l a t i n g the metabolic turnover o f important t e r m i n a l sugar r e s i d u e s . A p a r a l l e l s i t u a t i o n e x i s t s with 0 - a c e t y l d e r i v a t i v e s o f NANA and NGNA r e s i d u e s i n bovine, p o r c i n e and equine submaxillary gland g l y c o p r o t e i n s (reviewed


3.


KENT ET AL.


2

-2

-I

Ο

+1

+2

Log (^g/ml) Glycopeptide Concentration

Figure 3.

Table VI

Effect of sulfated glycopeptides on 3T3 fibroblast growth

Sulphated D e r i v a t i v e s of P i g Duodenal Glycopeptide

(sodium s a l t s )

(g/lOOg dry wt)

glycopeptide

%

sulphate

-

degree of sulphation

fully sulphated

49 > 96%

5.0

2.3

Gal

24.8

13.4

Fuc

11.0

5.1

Hexosamines

37.5

20.1

NANA ) NGNA )

partly sulphated SI 14.2

partly sulphated S2 4.7 ca.ll$



42

CARBOHYDRATE

SULFATES

by Schauer, Buscher and C a s a l s - S t e n z e l , 1 9 7 4 ) · In. the n a t i v e m a t e r i a l those t e r m i n a l s i a l i c a c i d s c a r r y i n g a 4-O-acetyl s u b s t i t u e n t a r e reported to be i n s e n s i t i v e to V. cholerae neuraminidase and here a l s o an i n t e r e s t i n g r e g u l a t o r y r o l e may be e x e r c i s e d by d e - a c e t y l a s e s . The i n t a c t nature o f t e r m i n a l s i a l i c a c i d s i s now e s t a b l i s h e d as an important i n f o r m a t i o n a l s i g n a l r e g u l a t i n g c e r t a i n forms o f b i o l o g i c a l r e c o g n i t i o n of g l y c o p r o t e i n s . I n p a r t i c u l a r , t h i s new concept o f the r o l e o f carbohydrates i s i n v o l v e d i n r e g u l a t i n g the serum s u r v i v a l time o f plasma g l y c o p r o t e i n s (reviewed by Ashwell and M o r e l l 1 9 7 4 ) · Hydrolysis of t e r m i n a l s i a l i c a c i d s by the a c t i o n o f neuraminidase, even i n p a r t , exposes subterminal sugar galactose^and the consequent a l t e r a t i o n i n molecular i d e n t i t y leads to the removal of the g l y c o p r o t e i n by s p e c i f i c h e p a t i c b i n d i n g s i t e s (Lunney and Ashwell, 1 9 7 6 ; Kawasaki and Ashwell 1 9 7 6 ) . Thus any f u r t h e r m o d i f i c a t i o n of t e r m i n a l sugars by a d d i t i o n a l subs t i t u t i o n c l e a r l y has important i m p l i c a t i o n s f o r t h i s recogn i t i o n process. I t i s a p p r e c i a t e d t h a t as y e t , amongst the g l y c o p r o t e i n s , a d d i t i o n a l s u b s t i t u t i o n by s u l p h a t i o n r a r e l y occurs on e x t e r i o r sugar s i t e s . Presence of these e s t e r groups i n the i n t e r i o r p a r t s o f the carbohydrate side-branches nevertheless can be envisaged as having i n f o r m a t i o n a l s i g n i f i c a n c e of two k i n d s . The f i r s t aspect of t h i s hypothesis a k i n to the above s i t u a t i o n can be considered as i n f l u e n c i n g the s u s c e p t i b i l i t y of the monosaccharide concerned towards exoglycosidases i n the course of metabolic turnover. The second aspect i n v o l v e s the changed acceptor status o f a sulphated monosaccharide residue towards sugar n u l e o t i d e t r a n s f e r a s e s . The p o s s i b i l i t y e x i s t s t h a t these s p e c i f i c enzymes may w e l l d i s c r i m i n a t e between the non-sulphated and sulphated residues o f a g i v e n sugar w i t h i n a s i n g l e g l y c o p r o t e i n molecule with s i g n i f i c a n t a l t e r a t i o n i n the p a t t e r n s of b i o s y n t h e s i s o r r e s y n t h e s i s of o l i g o s a c c h a r i d e c h a i n s . Accumulated evidence suggests t h a t i n v i v o sugar residues i n the more e x t e r i o r p a r t s o f the g l y c o p r o t e i n s t r u c t u r e are more r e a d i l y exchangeable than the deeper i n t e r n a l regions o f the molecule. I n t h i s , O-sulphation may be one c o n t r i b u t i n g f eature. The hypothesis t h a t O-sulphation may have i n f o r m a t i o n consequences f o r synthesis and b i o s y n t h e s i s of g l y c o p r o t e i n s i s not s o l e l y r e s t r i c t e d to those processes. The extensive change i n a n i o n i c charge and the conformational e f f e c t o f i n t r o d u c i n g a s i n g l e sulphate e s t e r i n t o a carbohydrate residue i n v o l v e d i n an immunological determinant s i t e may be expected a l s o t o l e a d to a l t e r a t i o n o f antigen-antibody i n t e r a c t i o n s . The o v e r a l l evidence suggests f u r t h e r r o l e s f o r the p a r t


3.

K E N T ET AL.


43

played by low l e v e l s of s u l p h a t i o n i n a v a r i e t y o f processes. I t p o i n t s to the importance o f i d e n t i f y i n g p r e c i s e l y the p o s i t i o n s occupied by O-sulphates i n g l y c o p r o t e i n s and o f f i n d i n g new and s p e c i f i c means o f i n t r o d u c i n g such e s t e r s a t d e f i n e d p o s i t i o n s of o l i g o s a c c h a r i d e s . The sulphatases would appear to be enzyme systems of considerable p o t e n t i a l importance i n respect to c o n t r o l o f i n f o r m a t i o n a l content o f g l y c o p r o t e i n s .


References Ashwell, G. and Morell, A.G. (1974) Advanc. Enzymol. 41 99-128 Atkins, E.D.T. and Laurent, T.C. (1973) Biochem. J. 133,605-606 Bertillini, G., Butti, Α., Piantanida, B., Prino, G., Riva, Α., Rossi, A. and Rossi, S. (1971) Drug Res. 21, 244-248 Hirano, S. and Onodera, K. (1967) Life Sciences 6, 2177-2183 Kawasaki, T. and Ashwell, G. (1976) J. biol. Chem. 251, 1296-1302 Kent, P.W. (1970) Exposés Ann. Biochem. Méd. Sér. 30, 98-120 Kent, P.W. (1974) in Topics in Gastroenterology II (ed.) S.C. Truelove and J. Trowell, Blackwell, Oxford, U.K. pp. 77-99 Kirkwood, S., (1974) Annu. Rev. Biochem. 43, 401-417 Lunney, J. and Ashwell, G. (1976) Proc. Nat. Acad. Sci. USA 73, 341-343 Pantlitscko, M., Schmid, J., Seelich, F. and Kaiser, E. (1951) Monatsh. Chem. 82, 380-386 Prino, G., Lietti, A. and Paglialunga, S. (1971) Drug Res., 21, 918-921 Rees, D.A., (1969) Advanc. Carbohydrate Chem. 24, 267 Ricketts, C.R. (1952) Biochem. J. 51, 129-135 Ricketts, C.R., and Walton, K., (1952) Chem. Ind. 869 Schauer, R., Buscher, H-P., and Casals-Stenzel, J. (1974) in Metabolism and Function of Glycoproteins ed. R.M.S. Smellie and J.G. Beeley Biochem. Soc. Symposium No.20 pp. 87-116 Slomiany, B.L. and Meyer, K., (1972) J. biol. Chem. 247, 5062-5070 Stone, A.L., (1964) Biopolymers 2, 315 Stone, A.L., (1965) Biopolymers 3, 617 Sun, D.C.H., and Ryan, M.L. (1970) Gastroeneterology 58, 756-761 Winterbourne, D.J. and Kent, P.W. (1977) Trans. Biochem. Soc. 5, 439-440 Yosizawa, Z. (1972) in Glycoproteins ed. A. Gottschalk pp. 1000-1018 Elsevier, Amsterdam RECEIVED February 6,

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Carbohydrate Sulfates - American Chemical Society

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