Carbohydrate Moieties of the Collagens and Collagen-Like Proteins in

12 Carbohydrate Moieties of the Collagens and Collagen-Like Proteins in Health and Disease

Downloaded by UNIV LAVAL on July 12, 2016 | http://pubs.acs.org Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0080.ch012

WILLIAM T. BUTLER Institute of Dental Research, University of Alabama in Birmingham, Birmingham, AL 35294 The fact that interstital collagens and other collagen-like proteins contain covalently bound carbohydrate has been known for many years. In 1935 Grassmann and Schleich (1) found that hide collagen had equal quantities of firmly bound glucose and galactose, and later studies from this group (2) suggested that the hexoses were bound through O-glycosidic linkages. The stability of the binding of hexoses to collagen was demonstrated by Kühn, et al (3) who found that repeated reprecipitation of citrate-soluble collagen removed all the hexosamine, but only half the hexose. After three reprecipitations, the hexose level was 0.48%, and it remained constant throughout seven additional reprecipitations. This study revealed that hexose, but not hexosamine was an integral part of i n t e r s t i t i a l collagen. Gross, et al (4) examined the amino acid and sugar contents of collagens from a variety of tissues and species. They found that for vertebrate collagens, purification or gelatinization drastically reduced the hexosamine content, but was less effective i n removing hexose. The carbohydrate was identified as glucose and galactose with traces of other hexoses, pentoses and amino sugars. The hexose content was usually less than 1 % for vetebrate and from 3 to 11% i n invertebrate collagens. Similarly Blumenfeld, et al (5) found that ichthyocol (carp swim bladder collagen) contained glucose and galactose but no hexosamine or other sugars. In contrast to the low content of sugars generally found in i n t e r s t i t i a l collagens of vertebrates, the collagen-like proteins of basement membranes contain much higher levels of carbohydrate (6-8). As i n collagen, one type of carbohydrate consists of glucose and galactose units, but heteropolysaccarides with hexoses and hexosamines are also present.

0-8412-0452-7/78/47-080-213$05.00/0 © 1978 A m e r i c a n C h e m i c a l Society

Walborg; Glycoproteins and Glycolipids in Disease Processes ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

214

GLYCOPROTEINS

AND GLYCOLIPIDS IN DISEASE PROCESSES


THE NATURE OF THE LINKAGE P r i o r to 1965 s e v e r a l s t u d i e s on the nature o f the carbohydrate of c o l l a g e n were r e p o r t e d , but the r e s u l t s l e f t many u n c e r t a i n t i e s . The s t u d i e s of B u t l e r and Cunningham (9,10) c l a r i f i e d the nature o f the hexose attached to c o l l a g e n . S t a r t ing w i t h s o l u b l e guinea p i g s k i n c o l l a g e n and d i g e s t i n g to s h o r t e r u n i t s w i t h collagenase and t r y p s i n , glycopeptides were i s o l a t e d . The compositions of the peptides suggested that glucose and galactose might be attached to the 6-hydroxyl group of h y d r o x y l y s i n e . The r e s i s t a n c e of the h y d r o x y l y s i n e to p e r i o d a t e o x i d a t i o n supported t h i s c o n c l u s i o n (9). The nature of the l i n k a g e was c o n c l u s i v e l y shown by the i s o l a t i o n of a glycopeptide w i t h s t o i c h i o m e t r i c amounts of h y d r o x y l y s i n e , glucose, and g a l a c t o s e recovered a f t e r cleavage of the peptide bonds of the c o l l a g e n g l y c o p e p t i d e w i t h 2N NaOH a t 90° f o r 10 hr. The r e s i s t a n c e t o a l k a l i showed that the attachment was 0g l y c o s i d i c ; t h i s c o n c l u s i o n was a l s o supported by the observat i o n t h a t m i l d a c i d h y d r o l y s i s (2N HC1, 110°, 30 min.) r e l e a s e d 2 mol of reducing sugar. A l i n k a g e to the e-amino group of h y d r o x y l y s i n e was r u l e d out by the r e a c t i v i t y of t h i s group to f l u o r o d i n i t r o b e n z e n e and by the e l e c t r o p h o r e t i c m o b i l i t y of the glycopeptide. These experiments thus showed that c o l l a g e n cont a i n e d a d i s a c c h a r i d e c o n s i s t i n g of glucose and g a l a c t o s e attached O - g l y c o s i d i c a l l y to h y d r o x y l y s i n e ( G l c - G a l - H y l ) . STRUCTURE OF THE CARBOHYDRATE MOIETY The s t r u c t u r e of the carbohydrate was e l u c i d a t e d by S p i r o (11) u s i n g collagenous components from glomerular basement membranes. A f t e r i s o l a t i o n of a mixture of glycopeptides c o n t a i n ing h y d r o x y l y s i n e - l i n k e d glucose and g a l a c t o s e , he showed t h a t glucose, but not g a l a c t o s e , was r e a d i l y l i b e r a t e d w i t h 0.1 N ^SO^ a t 100° f o r 2-20 h r . Thus glucose was i n an e x t e r n a l p o s i t i o n , w i t h galactose l i n k e d to h y d r o x y l y s i n e . Next a glucose and g a l a c t o s e - c o n t a i n i n g d i s a c c h a r i d e was obtained a f t e r N - a c e t y l a t i o n and m i l d a c i d h y d r o l y s i s of the glycopept i d e s . Glucose was shown to be attached to C-2 of galactose by s t u d i e s employing p e r i o d a t e and g a l a c t o s e oxidase. This l i n k a g e was cleaved by a-glucosidase but not 3-glticosidase. Next G l c Gal-Hyl was i s o l a t e d a f t e r a l k a l i n e h y d r o l y s i s of g l y c o p e p t i d e s ; the anomeric c o n f i g u r a t i o n of the g a l a c t o s e l i n k a g e to hydroxyl y s i n e was shown to be 3 by i t s s u s c e p t i b i l i t y to 3 g a l a c t o s i d a s e . These experiments thus i n d i c a t e d that G l c - G a l - H y l had the s t r u c ture 2-0-a-D-glucopyranosy1-0-3-D-galac t o p y r a n o s y l h y d r o x y l y s i n e (Figure 1). A s i m i l a r s t r u c t u r e was proposed by K e f a l i d e s (12). The s t r u c t u r e of G l c - G a l - H y l i n c o l l a g e n s from v e r t e b r a t e and i n v e r t e b r a t e sources has been shown to be the same as that found i n basement membranes. Thus Cunningham and Ford (13) observed that glucose was s p l i t from the d i s a c c h a r i d e of guinea -



12.

BUTLER

Carbohydrate

Moieties

of the

Collagens

215

p i g s k i n c o l l a g e n by m i l d a c i d h y d r o l y s i s , w h i l e galactose r e mained attached. They a l s o reported f i n d i n g g a l a c t o s y l h y d r o x y l y s i n e (Gal-Hyl) a f t e r a l k a l i n e h y d r o l y s i s of s k i n c o l l a g e n . Using the amino a c i d a n a l y z e r , S p i r o (14) introduced a separat i o n technique f o r q u a n t i t a t i o n of G l c - G a l - H y l and Gal-Hyl. Chromatography o f a l k a l i n e h y d r o l y s a t e s of s e v e r a l c o l l a g e n samples showed that they contained the same mono- and d i s a c c h a r i d e u n i t s l i n k e d to h y d r o x y l y s i n e as basement membranes. Katzman, jet a l (15) u t i l i z e d G l c - G a l - H y l i s o l a t e d from the sponge and showed that the s t r u c t u r e was that proposed f o r basement membranes by S p i r o (11). The h y d r o x y l y s i n e - l i n k e d d i s a c c h a r i d e i s o l a t e d from sea anenome, sea cucumber, and bovine cornea was shown to have an i d e n t i c a l s t r u c t u r e by a v a r i e t y of techniques (15). Since these e a r l y s t u d i e s , G l c - G a l H y l and Gal-Hyl have been shown to occur i n a v a r i e t y of c o l l a gen and c o l l a g e n - l i k e p r o t e i n s . OCCURRENCE IN THE DIFFERENT COLLAGEN TYPES The d i s c o v e r y that c a r t i l a g e contained a type o f c o l l a g e n d i s t i n c t from t h a t o f s k i n and bone (16) ushered i n a new e r a i n c o l l a g e n biochemistry. We now understand that a t l e a s t three c o l l a g e n s w i t h unique p r o p e r t i e s e x i s t i n the e x t r a c e l l u l a r m a t r i x of connective t i s s u e s (Table I ) . For more d e t a i l s conc e r n i n g these c o l l a g e n s , the reader i s r e f e r r e d t o the review by M i l l e r (17). S c i e n t i s t s have long recognized t h a t the basement membranes c o n t a i n collagenous components (6,18,19). A f i n a l r e s o l u t i o n of the s t r u c t u r e s o f the molecular species present i n basement membranes has not been accomplished. A t l e a s t one o f these appears to have a t r i p l e - h e l i c a l conformation r e f e r r e d t o as [ a l ( I V ) ] 3 (20). However i t i s apparent that s e v e r a l other s t r u c t u r e s are present (21-23). A l l three o f the i n t e r s t i t i a l c o l l a g e n s c o n t a i n hexose attached to h y d r o x y l y s i n e , but type I I c o l l a g e n has a s i g n i f i c a n t l y greater q u a n t i t y of h y d r o x y l y s i n e and o f h y d r o x y l y s i n e l i n k e d carbohydrate (Table I ) . I n general the collagenous p o r t i o n of basement membranes c o n t a i n much higher l e v e l s o f h y d r o x y l y s i n e and of the a s s o c i a t e d glucose and g a l a c t o s e moieties. I t should be noted that other h y d r o x y p r o l i n e - c o n t a i n i n g p r o t e i n s w i t h c o l l a g e n - l i k e ( i . e . Gly-X-Y repeating) sequences e x i s t . For example, the complement p r o t e i n C l q contains s e v e r a l g l y c o s y l a t e d hydroxylysines i n a c o l l a g e n - l i k e sequence (24). The exact l o c a t i o n s o f s e v e r a l o f the carbohydrate m o i e t i e s of the collagens has been documented during the s t u d i e s on the primary s t r u c t u r e s o f the v a r i o u s a chains. One s i t e of g l y c o s y l a t i o n common to the f o u r chains of the three i n t e r s t i t i a l c o l l a g e n s i s Hyl-87 (Figure 2). The amino a c i d sequences around t h i s s i t e are s i m i l a r i n these chains (25-29). The occurrence of the d i s a c c h a r i d e a t t h i s s i t e suggests that i t performs some



2

[al(II)]

[al(III)]

Several

II

III

IV Basement Membranes (Glomerulus, e t c . )

Ubiquitous ( E s p e c i a l l y Prominent i n F e t a l S k i n , Arteries, Cirrhotic Liver)

Cartilage, Intervetebral D i s c , Notocord

Ubiquitous ( S k i n , Bone, Tendon Dentin, etc.)

Tissue D i s t r i b u t i o n

40-50

5-8

20-25

5-15

b

55

2

b

15-30

2.5

a

Hexose Content (Residues/1000)

Data f o r the c o l l a g e n - l i k e p o r t i o n of glomerular basement membranes. Taken from r e f e r e n c e 19.

Glucose and galactose

3

[al(I)] o2

I

3

Molecular Composition

Type

Hydroxylysine Content (Residues/1000)

TABLE I : S t r u c t u r e s and C h a r a c t e r i s t i c s of I n t e r s t i t a l Collagens and C o l l a g e n - L i k e P r o t e i n s of Basement Membranes


BUTLER

Carbohydrate

Moieties of the

Collagens

CH OH 2

HC* V^~° OH

"^v

6H -NH © 2

3

®H N-CH-C-O© 3

CH OH

^

2

HO/KJ VOH

" /

CH OH 2

»yfr—°\H/°

g


HO

old)

° \ Hy H

2

Q

CH2-NH3®

Figure I. The structures of the glucose- and galactose-containing units isolated from the collagens d collagen-like proteins. (A) / ? - D galactopyranosylhydroxylsine (Gal an

hyl),

H/^ OH

(B)

2-O-a-D-glucopyranosyl

O - /? - D- galactopyranosylhydroxyly sine (Glc-Gal-Hyl),

87 - G L Y - L E U - HYP - 6LY - MET - HYL - GLY - H I S - ARG Glc - Gal

a2

- GLY - L E U - HYP - GLY - P H E - HYL - GLY - I L E - ARG 1

Glc - Gal

a I (II)

- GLY - L E U - HYP - GLY - V A L - HYL - GLY - H I S - ARG -

Glc - Gal

a l(III)

- GLY - PHE - HYP - GLY - MET - H Y L - G L Y - H I S - ARG 1

Glc - Gal

Figure 2. Amino acid sequences around the disaccharide-bearing hydroxylysines common to types J, II, and III collagens

old)

174 - G L Y - A L A . A L A - G L Y - A L A - L Y S - GLY - G L U - A L A -

al(III)

- GLY - SER - HYP - GLY - A L A - L Y S - GLY - GLU - V A L -

ol(II)

- G L Y - A L A - HYP - G L Y - A L A - H Y L - G L Y - GLU - A L A •

Gal

o2

- G L Y - A L A - HYP - GLY - PRO - HYL - G L Y - GLU - L E U 1

Gal

Figure 3.

The amino acid sequences of a chains around the monosaccharide-bearing hydroxylysine of a2



218

GLYCOPROTEINS AND

GLYCOLIPIDS IN

DISEASE PROCESSES

f u n c t i o n a l r o l e v i t a l to the three collagens (e.g. i n c r o s s l i n k i n g ) . The a2 chain of type I c o l l a g e n has a Gal-Hyl at p o s i t i o n 174 (26,27) not found i n a l ( I ) or a l ( I I I ) but present i n a l ( I I ) (Figure 3). Although these appear to be the major s i t e s of carbohydrate i n a l ( I ) , a2 and a l ( I I I ) , other p o s i t i o n s may be p a r t i a l l y g l y c o s y l a t e d . For i n s t a n c e s u b i n t e g r a l l e v e l s of Gal-Hyl are found near the COOH-terminus of a l ( I ) i n c a l f s k i n c o l l a g e n (26). As s t a t e d b e f o r e , the a l ( I I ) chain of c a r t i l a g e c o l l a g e n has many more s i t e s of carbohydrate attachment than the above. Studies on the covalent s t r u c t u r e of bovine and c h i c k a l ( I I ) chains (28,30,31) from t h i s l a b o r a t o r y have a l r e a d y l o c a t e d 13 g l y c o s y l a t e d hydroxylysines (Figure 4 ) , although only about 60% of the sequence i s known. These h y d r o x y l y s i n e s are not confined to any one area but occur throughout the chain. Comparison of the p o s i t i o n s of these g l y c o s y l a t e d h y d r o x y l y s i n e s w i t h homologous sequences i n a l ( I ) and a2 shows that they are f r e q u e n t l y occupied by l y s i n e i n the l a t t e r two chains. Thus the three chains have s i m i l a r amino a c i d sequences w i t h p o t e n t i a l s i t e s f o r carbohydrate attachment, but only f o r a l ( I I ) are the postt r a n s l a t i o n a l m o d i f i c a t i o n s ( i . e . h y d r o x y l a t i o n and g l y c o s y l a t i o n ) made. I w i l l r e t u r n to p o s s i b l e explanations f o r the h i g h l e v e l of Glc-Gal-Hyl and Gal-Hyl i n a l ( I I ) i n a l a t e r s e c t i o n . BIOSYNTHETIC ATTACHMENT OF HEXOSE TO PROCOLLAGEN a CHAINS Collagen i s synthesized w i t h i n connective t i s s u e c e l l s by the u s u a l p r o t e i n s y n t h e t i c mechanism 032) but has s e v e r a l f e a t u r e s which are unique (Figure 5). The i n i t i a l form of the three a chains are elongated, compared to that found i n f i b r i l l a r c o l l a g e n (33-36). These " e x t e n s i o n s " which occur on the NH2~and COOH-terminal ends of the c h a i n s , are cleaved from the molecules during or a f t e r e x i t from the c e l l . During and a f t e r the t r a n s l a t i o n of these p r o c o l l a g e n chains on ribosomal complexes, seve r a l p o s t - t r a n s l a t i o n m o d i f i c a t i o n s take p l a c e . L y s y l and p r o l y l residues are hydroxylated to form h y d r o x y l y s y l and h y d r o x y p r o l y l r e s i d u e s , and galactose and glucose m o i e t i e s are attached to c e r t a i n of the h y d r o x y l y s i n e s . Next the three chains of a p r o c o l l a g e n u n i t a s s o c i a t e , i n t e r c h a i n d i s u l f i d e bonds i n the COOH-terminal extensions are formed, and f o l d i n g i n t o a t r i p l e - c h a i n c o l l a g e n h e l i x takes p l a c e . I t i s obvious that the attachment of galactose and glucose to c o l l a g e n must be preceded by h y d r o x y l a t i o n of l y s y l r e s i d u e s . This r e a c t i o n i s c a t a l y z e d by l y s y l hydroxylase (32,29,40), an enxyme which i s s i m i l a r i n many respects to p r o l y l hydroxylase. Each of these mixed f u n c t i o n oxidases r e q u i r e s molecular oxygen, a - k e t o g l u t a r a t e , ascorbate and f e r r o u s i r o n (Figure 6) and each c a t a l y z e s h y d r o x y l a t i o n of r e s i d u e s i n the Y p o s i t i o n of the r e p e a t i n g Gly-X-Y c o l l a g e n sequence (37), though l y s y l hydroxyl a s e w i l l apparently a l s o a c t upon l y s i n e s i n the X p o s i t i o n (40).



12.

BUTLER

Carbohydrate

Moieties

of the

219

Collagens

al(l) Bovine al(ll) Bovine a 2 Bovine

87 GLY - LEU - HYP. - GLY - MET - HYL - GLY - HIS - ARG - GLY - PHE - SER GLY - LEU - HYP • GLY - VAL - HYL - GLY - HIS - ARG - GLY - THR - HYP GLY - LEU - HYP - GLY - PHE - HYL - GLY - ILE - ARG - GLY - HIS - ASN


99 GLY - LEU - ASP - GLY - ALA - LYS - GLY - ASP - ALA - GLY - PRO - A L A GLY - LEU - ASP - GLY - ALA - HYL - GLY - GLU - ALA - GLY - ALA - HYP GLY - LEU - ASP - GLY - LEU - THR - GLY - GLN - HYP - GLY - ALA - HYP


108 GLY - PRO - ALA - GLY - PRO - LYS - GLY - GLU - HYP - GLY - SER - HYP GLY - ALA - HYP - GLY - VAL - HYL - GLY - GLU - SER - GLY - SER - HYP GLY - ALA - HYP - GLY - VAL - HYL - GLY - GLU - HYP - GLY - ALA - HYP

al(l) Rat a 1(11) Bovine a 2 Bovine

174 GLY - ALA - ALA - GLY - ALA - LYS - GLY - GLU '- ALA - GLY - PRO - GLN GLY - ALA - HYP - GLY - ALA - HYL - GLY - GLU - ALA - GLY - PRO GLY - ALA - HYP - GLY - PRO - HYL - GLY - GLU - LEU - GLY - PRO - VAL

al(l) Rot a 1(11) Bovine a 2 Bovine

219 GLY - GLN - HYP - GLY - ALA - LYS - GLY GLY - ILE - HYP - GLY - ALA - HYL - GLY GLY - LEU - HYP - GLY - ALA - HYL - GLY

al(l)Rat al(l 1) Bovine a 2 Bovine

GLY - ALA GLY - PRO GLY - ALA

al(l) Bovine a 1(11) Bovine a 2 Chick

GLY - PHE - HYP - GLY GLY - PHE - HYP - GLY GLY - PHE - HYP - GLY

a 1(1) Bovine al(ll)Bovine a 2 Chick

GLY - LYS GLY - LYS GLY - LYS

al(l) Bovine alODBovine a 2 Chick

531 GLY - ASN • ASP - GLY - ALA - LYS - GLY GLY - THR •• ASP - GLY - PRO - HYL - GLY GLY - PRO - ASP - GLY - ASN - LYS - GLY

al(l) Bovine a 1(11) Bovine a 2 Chick

564 GLY - LEU - HYP •• GLY - PRO - LYS - GLY - ASP - ARG - GLY - ASP - ALA GLY - ILE - ALA - GLY - PRO - HYL - GLY •• ASP - ARG - GLY - ASP - VAL GLY " VAL - HYP - GLY • GLY • LYS - GLY - GLU - LYS - GLU - A L A - HYP

a 1(1) Bovine alODBovine a 2 Chick

573 GLY - ASP - ALA •• GLY - PRO •• LYS •• GLY - A L A - ASP - GLY " A L A - PRO GLY - ASP - VAL •• GLY - GLU • LYS •• GLY - PRO - GLU - GLY - A L A - PRO GLY - ALA - HYP - GLY - LEU ••ARG - GLY - ASP - THR - GLY - ALA - THR

a 1(1) Bovine alODBovine a 2 Chick

603 GLY - ALA - HYP - GLY - ASP - LYS - GLY - GLU -• ALA - GLY - PRO - SER GLY - ASP • VAL - GLY - GLU - HYL - GLY • GLU - VAL - GLY - PRO - HYP GLY - GLY •• ALA - GLY - ASP - ARG - GLY - GLU -• GLY - GLY - PRO - ALA


648 GLY •• GLN - HYP - GLY - ALA - LYS - GLY GLY - GLN - PRO - GLY - ALA - HYL - GLY GLY -• GLU • HYP - GLY - ALA - LYS - GLY


657 GLY - ASP - ALA - GLY - ALA - LYS - GLY - ASP - A L A - GLY - PRO " GLY - GLU - A L A - GLY - GLN - HYL - GLY - ASP - ALA - GLY " ALA GLY - PRO - LYS - GLY - PRO - LYS - GLY - GLU - THR - GLY - PRO -

Figure 4.

-

- HYP - GLY - LEU - GLY - THR - GLY

- ALA - ASN - GLY - A L A - HYP - SER - ALA - GLY - ALA - HYP - ALA - A L A - GLY - LEU - HYP

252 - PRO - LYS - GLY - ASN • SER - PRO - HYL - GLY - ALA - ARG - GLY - LEU - VAL

408 - PRO - LYS - PRO - HYL - PRO - LYS

- GLY - GLY - GLY

• GLY • GLY

- ALA - ALA - GLY - GLU - HYP - ALA - ASN - GLY - GLU - HYP - PRO - THR - GLY - GLU - HYP

420 - ALA - GLY - GLU - ARG - GLY - VAL - HYP - GLY - PRO - HYP - ALA - GLY - GLU - HYL •- GLY - LEU - HYP " GLY - ALA - HYP - HYP •• GLY - GLU " LYS • GLY - ASN • VAL " GLY - L E U - ALA

- ASP - ALA - GLY - ALA - HYP - ALA - ALA - GLY - PRO - ALA • GLU - HYP - GLY - ASN - VAL

- GLU - HYP •• GLY - ASP - ALA - GLU - GLN •• GLY - GLU - A L A •• GLU - ARG •• GLY - PRO - LYS

The sequences around several hydroxy lysines and lysines of al(I), al(II), and a2 chains (taken from Ref. 31)


220

GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES

LOCATION

BIOSYNTHETIC EVENTS

Polysomes Translation of Procollagen Chains


Hydroxylation of Prolyl and Lysyl Residues Glycosylation

Cysternae of Endoplasmic Reticulum Chain Alignment

Helix Formation Disulfide Bond Formation

Figure 5. Intracellular steps in the biosynthesis of procollagen. Individual procollagen a chains are made by the usual protein biosynthetic mechanisms. The chains are then subjected to several post-translational modifications. After release from ribosomal complexes, three chains align, and triple-helices and interchain disulfide bonds at the COOH-terminal extremities are formed. Post-translational modifications cease upon formation of the triple-helix.


12.

BUTLER

Carbohydrate

o -NH-CH-CCH CH CH CH - N H ©

Moieties

of the Collagens

LYSYL HYDROXYLASE

2

2


0 -NH-CH-C CH CH HO-CH CH-I -NH © 2

2

2

2

221

V

2

3

0

0 -NH-CH-CCH CH HO-CH CH -NH * 2

B.

-NH-CH-CGALACTOSYL TRANSFERASE

CH CH, i * O-CH

CH OH

2

2

2

2

3

3

.©

UDP-Gol MN"

H

OH

CH -NH *.© 2

3

Figure 6. Three enzymatic steps in the biosynthetic attachment of hexose to collagen. (A) Formation of hydroxylysyl residues from lysines on the nascent procollagen a chains; (B) attachment of galactose to certain hydroxylysyl residues; and (C) attachment of glucose to certain Gal-Hyl moieties.



222

GLYCOPROTEINS AND

GLYCOLIPIDS IN

DISEASE PROCESSES

R e s u l t s of experiments i n v i t r o (37,38) and u s i n g c e l l s i n c u l ture (41), i n d i c a t e that the h y d r o x y l a t i o n of l y s y l r e s i d u e s does not occur a f t e r t r i p l e - h e l i c a l conformation of the proc o l l a g e n molecules i s formed ( F i g u r e 5). A f t e r h y d r o x y l a t i o n , g a l a c t o s y l t r a n s f e r a s e c a t a l y z e s the t r a n s f e r of g a l a c t o s e from UDP-Gal to c e r t a i n h y d r o x y l y s i n e s (Figure 6 ) ; then the t r a n s f e r of glucose from UDP-Glc to the C-2 of the g a l a c t o s e r e s i d u e s i s c a t a l y z e d by g l u c o s y l t r a n s f e r a s e . Both enzymes r e q u i r e d i v a l e n t c a t i o n s , p e r f e r a b l y manganese and are probably l o c a t e d i n the c i s t e r n a e of the endoplasmic r e t i culum. G l u c o s y l t r a n s f e r a s e has been p u r i f i e d to homogeneity (42) and g a l a c t o s y l t r a n s f e r a s e 1000-fold (43). S i m i l a r to the h y d r o x y l a t i o n r e a c t i o n s , the attachment of carbohydrate to p r o c o l l a g e n chains ceases when they f o l d i n t o a n a t i v e , t r i p l e - h e l i c a l s t r u c t u r e (44). Using c h i c k embryo tendon and c a r t i l a g e c e l l s i n c u l t u r e , O i k a r i n e n , et. a l (45) have shown t h a t the g l y c o s y l a t i o n of h y d r o x y l y s i n e s was d r a m a t i c a l l y a f f e c t e d by the r a t e of t r i p l e - h e l i x formation of the p r o c o l l a g e n chains. For example, when the r a t e of h e l i x formation was i n h i b i t e d by i n c u b a t i o n i n the presence of 0.6 mM d i t h i o t h r e i t o l , the g l y c o s y l a t i o n was more than doubled. Conversely when c a r t i lage c e l l s r e c o v e r i n g from anoxia were used (a c o n d i t i o n known to a c c e l e r a t e t r i p l e - h e l i x f o r m a t i o n ) , there was a marked decrease i n the extent of h y d r o x y l y s i n e formation and an even greater decrease i n g l y c o s y l a t i o n of h y d r o x y l y s i n e r e s i d u e s . FACTORS CONTROLLING HYDROXYLYSINE FORMATION AND GLYCOSYLATION

SUBSEQUENT

The f o r e g o i n g d i s c u s s i o n may help e x p l a i n some observations on the v a r i a t i o n s i n the content and occurrence of h y d r o x y l y s i n e s and g l y c o s y l a t e d h y d r o x y l y s i n e s . The h y d r o x y l y s i n e content of a l ( I ) chains d i f f e r s c o n s i d e r a b l y i n the type I c o l l a g e n s of d i f f e r e n t t i s s u e s . For example a l ( I ) of r a t d e n t i n c o l l a g e n contains about three times as much h y d r o x y l y s i n e as does s k i n a l ( I ) , though these chains have the same primary s t r u c t u r e (4648). This r e l a t i v e i n c r e a s e could be due to a higher a c t i v i t y of l y s y l hydroxylase i n odontoblasts; a l t e r n a t i v e l y a slower r a t e of f o l d i n g of d e n t i n p r o c o l l a g e n a chains i n t o the t r i p l e - h e l i c a l s t r u c t u r e could occur, a l l o w i n g a more complete h y d r o x y l a t i o n of l y s y l r e s i d u e s . The problem w i t h the l a t t e r hypothesis i s that one might expect an increased g l y c o s y l a t i o n of the h y d r o x y l y s i n e s over t h a t i n s k i n a l ( I ) c h a i n s , i f the r a t e of h e l i x formation i s the major f a c t o r c o n t r o l l i n g h y d r o x y l a t i o n and g l y c o s y l a t i o n of nascent p r o c o l l a g e n a chains. However the a d d i t i o n a l hydroxylys i n e s do not appear to have increased l e v e l s of hexose (48). K i v i r i k k o and R i s t e l l i (32) speculate that the d i f f e r e n c e s i n h y d r o x y l y s i n e and g l y c o s y l a t e d h y d r o x y l y s i n e contents of c o l l a g e n s



12.

BUTLER

Carbohydrate

Moieties

of the

Collagens

223

i n d i f f e r e n t t i s s u e s could be due i n p a r t to d i f f e r e n c e s i n the r a t e s of t r i p l e - h e l i x formation during s y n t h e s i s i n the v a r i o u s connective t i s s u e c e l l s . The extensive comparison of the amino a c i d sequences near g l y c o s y l a t e d hydroxylysines i n the a l ( I I ) chain w i t h r e l a t e d sequences i n a l ( I ) and a2 (Figure 4 ) , suggests that the r e l a t i v e l y h i g h l e v e l of carbohydrate i n type I I c o l l a g e n i s not due to d i f f e r e n c e s i n amino a c i d sequences which favor the enzymatic r e a c t i o n s ( i . e . h y d r o x y l a t i o n and g l y c o s y l a t i o n ) . I t appears that almost every l y s i n e i n p r o c o l l a g e n a l ( I I ) chains which i s i n the Y p o s i t i o n of the Gly-X-Y r e p e a t i n g sequences, i s hydroxylated and g l y c o s y l a t e d . On the. other hand, most of these l y s i n e s i n a l ( I ) are e i t h e r hydroxylated to a minimal extent or not at a l l and, are thus not g l y c o s y l a t e d . I t i s c u r i o u s that the extent of h y d r o x y l a t i o n of a2 chains i s greater than that of a l ( I ) , but f o r the most p a r t , t h i s phenomenon i s confined to the N H - t e r m i n a l t h i r d o f the chain. The more extensive h y d r o x y l a t i o n of l y s i n e s and g l y c o s y l a t i o n of the r e s u l t a n t hydroxylysines i n type I I p r o c o l l a g e n could be due to a slower r a t e o f t r i p l e - h e l i x formation. I n s t u d i e s on the b i o s y n t h e s i s o f type I I p r o c o l l a g e n by c h i c k embryo c a r t i l a g e c e l l s , U i t t o and Prockop (49) found that c h a i n a s s o c i a t i o n and t r i p l e - h e l i x formation of p r o c o l l a g e n a l ( I I ) chains r e q u i r e d almost twice as long as that f o r type I c o l l a g e n synthesized by tendon c e l l s . These data t h e r e f o r e support the above hypothesis. A l t e r n a t i v e l y c a r t i l a g e c e l l s may simply have a higher a c t i v i t y f o r l y s y l hydroxylase and the g l y c o s y l t r a n s f e r a s e s . 2

DISEASES AFFECTING THE LEVELS OF COLLAGEN-ASSOCIATED CARBOHYDRATE One o f the symptoms of diabetes i s a t h i c k e n i n g of basement membranes. Beisswenger and S p i r o (50) have reported that d i a b e t i c glomerular basement membranes c o n t a i n increased l e v e l s of h y d r o x y l y s i n e and of h y d r o x y l y s i n e - l i n k e d d i s a c c h a r i d e u n i t s , compared to c o n t r o l specimens. S p i r o (51) a l s o found an i n creased a c t i v i t y of c o l l a g e n g l u c o s y l t r a n s f e r a s e a c t i v i t y i n a l l o x a n - d i a b e t i c r a t kidneys, which was p a r t i a l l y reversed by i n s u l i n a d m i n i s t r a t i o n . Cohen and K h a l i f a (52) s t u d i e d the e f f e c t of diabetes on p r o l y l and l y s y l hydroxylase a c t i v i t i e s i n the r a t glomerulus. Data from r a t s made d i a b e t i c by s t r e p t o z o t o c i n i n j e c t i o n s was compared t o that from age-matched c o n t r o l s and from i n s u l i n - t r e a t e d , s t r e p t o x o t o c i n - d i a b e t i c r a t s . They found that l y s y l hydroxylase a c t i v i t y was s i g n i f i c a n t l y higher i n d i a b e t i c r a t kidneys than i n c o n t r o l animals, but that p r o l y l hydroxylase was unaffected. A d m i n i s t r a t i o n of i n s u l i n reduced the l y s y l hydroxylase a c t i v i t y to normal. These observations suggest that a l t e r a t i o n s o f h y d r o x y l a t i o n and g l y c o s y l a t i o n of l y s i n e s i n basement membrane c o l l a g e n s might r e l a t e t o the nephropathology o f diabetes.



224

GLYCOPROTEINS A N D GLYCOLIPIDS IN DISEASE PROCESSES

I t should be pointed out that the d i f f e r e n c e s i n hydroxylys i n e and g l y c o s y l a t e d h y d r o x y l y s i n e i n d i a b e t i c kidneys seen by Beisswenger and S p i r o (50) have not been noted by other i n v e s t i g a t o r s (53,54). This p o i n t i s t h e r e f o r e one of controversy. Osteogenesis imperfecta congenita (01) i s a genetic disease r e s u l t i n g i n bones of severe f r a g i l i t y . Since c o l l a g e n i s the m a t r i x o r framework onto which a p a t i t e c r y s t a l s a r e l a i d during bone formation, i t has long been assumed that the b a s i c d e f e c t i n 01 i s some d e f e c t i n c o l l a g e n s t r u c t u r e (55). D i r e c t chemical a n a l y s i s of t i s s u e s from 01 p a t i e n t s have shown that bone and d e n t i n c o l l a g e n s have i n c r e a s e d l e v e l s of h y d r o x y l y s i n e (56). Recently T r e l s t a d , ^ t ail (57) reported a more complete biochemi c a l a n a l y s i s of c o l l a g e n from s e v e r a l t i s s u e s of an 01 i n f a n t , and compared the r e s u l t s to these of t i s s u e s from age-matched c o n t r o l s . Hydroxylysine was doubled i n 01 bone c o l l a g e n and increased by 55% i n c a r t i l a g e . I n a d d i t i o n the galactose and glucose l e v e l s of 01 c o l l a g e n i n both t i s s u e s were s h a r p l y i n creased. The data suggest t h a t a t l e a s t one form of 01 i s a s s o c i a t e d w i t h i n c r e a s e s i n g l y c o s y l a t e d h y d r o x y l y s i n e s i n type I c o l l a g e n of bone and type I I c o l l a g e n of c a r t i l a g e . The a c t u a l e f f e c t which increased l e v e l s of h y d r o x y l y s i n e and the a s s o c i a t e d g l y c o s i d e s might have on the s t r u c t u r e and f u n c t i o n of c o l l a g e n s i s unknown a t t h i s time. But i t seems apparent from the foregoing d i s s c u s s i o n that the c o n t r o l of postt r a n s l a t i o n a l m o d i f i c a t i o n s i s c r i t i c a l i n order to i n s u r e that the e x t r a c e l l u l a r products ( i . e . c o l l a g e n monomers, f i b r i l s , and f i b e r s ) a r e able to perform the intended f u n c t i o n s . ACKNOWLEDGEMENT The research from t h i s l a b o r a t o r y r e f e r r e d to i n t h i s p u b l i c a t i o n was supported by United States P u b l i c Health S e r v i c e Grant DE-02670.

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8. 9. 10. 11. 12. 13.


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Carbohydrate

Moieties

of the

Collagens

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Carbohydrate Moieties of the Collagens and Collagen-Like Proteins in

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