B IOSYNTKES E AND STRUCTlTRE OF COLLAGEN*

THAYNE R . DUTSON Texas A&M University

Introduc t ion

Unlike mny other b io logica l macromolecules which have spec i f i c genetic or metabolic a c t i v i t i e s , collagen i s a protein which t o date has been primarily shown t o be of a s t r u c t u r a l nature. Collagen is ubiquitous in t h e animal kingdom and contributes 20 t o 3 6 of t he t o t a l body proteins i n ver tebra tes . Collagen is present throughout the body and is found i n such diverse t i s s u e s a s skin, tendon, bone, cornea, basement membrane and muscle. Its prime importance l i e s i n i t s mechanical propert ies ( t e n s i l e s t r eng th ) and i t s a b i l i t y t o support and innerconnect body t i s sues , making possible organization of t he whole body or organism. Collagen has a l s o been shown t o be important i n connective t i s s u e diseases (Miller and Matukas, 1974; Bailey, Robbins and Bal ian , 1974), nauscle s t rength (Ham, 1969; Cassens, 1971) and i n meat tenderness (Go11 e t al . , 1963; 1964; Herring e t a l . , 1965). i n order t o understand more completely the mechanisms by which t h i s protein is synthesized and organized in to the f i b e r s that a r e s o important i n the s t ruc tu re and function of connective t i s sues considerable emphasis has been placed on collagen research in recent years. In most t i s sues , these collagen f i b e r s occur i n a va r i e ty of forms such a s f i n e fi laments, t h i ck rope-like f ibers or i n laminated sheets of f i b e r s , as depicted i n f igure 1.

Thus,

Collagen Structure

A considerable amount of information has been known about the basic s t ruc tu re of collagen f o r many years (Traub and Piez, 1971; Gallop e t al., 1972) but new information is being continually added (Bailey and Robins, 1976). collagen, is a t r i p l e stranded, coiled c o i l rod-like s t ruc tu re of about 300 a in diameter. The collagen molecule (tropocollagen) contains three polypeptide chains known as CX chains which a r e coiled together i n a superhelix t o form the collagen molecule (Fig. 2 ) . Each of the a chains themselves have a unique amino acid sequence in that every t h i r d residue is glycine and they have the repeating t r ipep t ide gly-x-y, with the x and y frequently being prol ine and hydroxyproline, respect ively (Traub and Piez, 1971). been reported f o r the a1 ( I ) chain with half being from rat skin and half from ca l f skin collagen (Fietzek et e., 1972; Bal ian e t a l . , 1972; Hulmgs e t a l . , 1973; Chien, 1975). t he alpha chains a r e continuous polypeptide chains i n which glycine does

Tropocollagen, the basic molecular un i t of

The complete a m i n o acid sequence of 1052 residues has

It is now c l ea r from t h i s data that

* Presented a t the 29th Annual Reciprocal Meat Conference of the American Meat Science Association, 1976.

337

PRIMARY SEQUENCE

GLY-PRO-Y-GLY-X- Y- GLY-X-HYP-GLY-

TRIPLE HELIX

TWO DIMENSIONAL QUARTER STAGGER

I-3000 %-I

OVERLAP ZONE

OVERLAP ZONE

COLLAGEN

1-3000 %- I

THREE DIMENSIONAL PENTAFIBRIL QUARTER STAGGER

339

indeed occur a t every t h i r d residue in t h e sequence gly-x-y. sequences gly-pro-y and gly-x-hyp occur in almost equal proportions except for t h e short regions a t the N and C terminal ends of t h e a chain, with hydroxyproline being almost exclusively confined t o the y pos it ion (Bailey and Rob ins , 1976 ) .

The

The high content of glycine in the a chains allows r e l a t i v e l y unres t r ic ted ro ta t ion which i s balanced by the sterochemical properties of t he pyrrolidine rings of prol ine and hydroxyproline t h a t d i r e c t t he chains in to a poly-proline I1 he l ix . Another unique property of glycine i s that it has no s ide chain and t h e associat ion of th ree Q chains i n the collagen superhelix places t h e cr carbon atom of every t h i r d residue i n t h e i n t e r i o r of t h e rod-like molecule, allowing the th ree polyproline I1 hel ixes t o f i t nicely in to a t r iple-s t randed superhelix arrangement (Traub and Piez, 1971). The glycine residues on the inside of t he inside of t h e molecules a l s o form hydrogen bonds t o the amino group of t h e peptide bond on adjacent chains. The s ide chains of other residues being on the outside of t h e tropocollagen molecule allowing them t o be involved i n molecular interact ions control l ing the organization of tropocollagen in to the f i b e r s (Bailey and Robins, 1976).

Water is a l s o an important component of nat ive collagen with about 0.5 g of water needed per g of tropocollagen t o maintain i t s nat ive conformation (Chien, 1975; Dehl, 1970). Water is implicated i n i n t e r - chain hydrogen bonds ( Ramanchandran and Chandrasekharan, 1968 ) of collagen which becomes insoluble upon complete dehydration (Yannas and Tobolsky, 1967). We have a l s o noted the decreased s o l u b i l i t y of freeze-dried intramuscular collagen i n our laboratory, with decreases in s o l u b i l i t y being greater i f freeze-dried collagen is stored a t room temperature.

Each tropocollagen molecules a re formed in to f i b r i l s which probably consis t of f i v e tropocollagens arranged in a quarter stagger a r r a ry known as pen ta f ib r i l s (Smith, 1968) which are shown in Figure 2. The hole and overlap regions caused by the quarter stagger ar ray of t h e tropocollagen molecules account f o r the 650 2 per iodic i ty of collagen f i b r i l s found with t h e electron microscope (Figure 2) . This spec i f i c organization of collagen molecules r e s u l t s i n f ibe r s of very l i t t l e s t rength which a r e l a t e r s t ah i l i zed by the formation of i n t e r - and intra-molecular cross links r e su l t i ng i n a continuous s t ruc ture with very high dimensional s t a b i l i t y . The top ic of cross-l inking w i l l be covered i n the following paper by Dr. Mckin.

Collagen Type S

A t the present time, four d i s t i n c t types of collagen have been ident i f ied i n various t i s sues , I, me 11, Ty-pe I11 and Type IV as presented in Table 1 (Miller and Matukas, 1974; Bailey and Robins, 1976; k r t i n L- e t a l . , 1975). A l l four collagen types a r e composed of three a chains which vary only s l i g h t l y in amino acid composition (Miller and Natukas, 1974). Type I, I1 and I11 collagens a r e aggregated

340

POLYSOME

PRO-a CHAINS

0 Y \%

' r) CIlscIf )-0-Gal-Glc

Assembly

COOH

1 OH

0-Gal-GI- ---.. OH 0-Gal

COOH

COOH COOH

b ' Secrht ion Cell Membrane

>. J.

. t

. COOH 0-Gal NH2 a$'&$

N H 2 a

N H 2 L , . COOH

9 Fiber Formation and crosslinking

F;y 3

Table 1. Types of Collagen

T i s sue Type loca t ion

Molecular Dist inct ive compos it ion features

I

I1

I11

Iv

Skin, tendon, bone, muscle

Cart i lage, In te rver tebra l d i sc s

F e t a l skin, card iwascular system, Synovial membrane, cardiac and s k e l e t a l muscle

Basement membrane

[a1('P)]202 ~2 chains, 6 8 Hyl/chain

[ a I ( I I ) ] 3 a1 chains only, 20-25

low carbohydrate

Hyl/chain, lC$ carbo- hydrate

a1 chains only, 6-8 Hyl/ chain, high Hyp and Gly, low carbohydrate,

[CZI(III)]~

CYS

[a1( m)]3 a1 chains only, 60-70 Hyl/chain, high 3-Hyp, low A l a , 15% carbohy- d ra t e

i n f ibrous form while Type IV msintains an amorphous s t ruc ture i n the basement membrane and is apparently unable t o form the f ibrous s t ruc ture associated with other collagen types (Bailey and Robins, 1976). These four unique collagens a r e composed of f i v e d i f f e ren t a chains a1 ( I ) , a1 (II), a1 (111), a1 (IV), and 02, which have been determined t o be d i f f e ren t gene products, however, they probably undergo the same post- t r a n s l a t i o n a l modifications ( h r t i n e t al. , 1975).

Type I collagen is the most completely characterized collagen with t h e complete amino acid sequence being known (Chien, 1975). i s found i n skin, bone, tendon, and muscle. Type I collagen contains two iden t i ca l a1 chains and a d i f f e ren t t h i r d chain, 2 (Piez, 1967) making the molecular configuration [a1 (I)]* 02. The a1 ( I ) chains a r e d i s t i n c t from a1 chains of other collagen types i n amino acid composi- t i o n and sequence (Linsenmayer, 1973a, 1973b; Miller and Matukas, 1969; McClain, 1974; Bailey and Sims, 1976) and Type I collagen is the only collagen known t o contain Q2 chains (Martin e t a1 ., 1975). collagen contains about 6-8 Hydroxylysine residues and has a low carbo- hydrate content, l e s s than 20% of the Hydroxylysine is glycosylated (Spiro, 1969; Bailey and Robins, 1976).

It

Type I

Type I1

Miller and Matukas (1969) were the f i r s t t o ident i fy the presence of a d i f f e ren t collagen type in ca r t i l age which w a s found t o contain th ree iden t i ca l CI chains and have a molecular conf i w a t ion of [a1 ( 11) 13. The three CI chains of Type I1 collagen d i f f e r from a1 ( I ) and 02 chains with respect t o amino acid composition and sequence (Miller and Matukas, 1969, 1974). carti lagenous s t ruc tures of the chick (Miller, 197la, 1971b, 1972, 1973; Toole e t al., 1972), the bovine (Miller and Lunde, 1973; Strawich and Nimni, 1971), and the h m n (Miller 1973), with Type I1 collagen having 20-25 Hydroxylysine residues per 1000 residues and approximately 5 q 0 of the Hydroxylysine being glyco- sylated. Although Ty-pe I1 collagen was f i r s t shown t o be present i n car t i lage , it has recent ly been implicated i n other t i s sues such as in te rver tebra l d i sc s (Bailey and Robins, 1976).

Type I1 collagen has been ident i f ied i n a var ie ty of

e., 1971; Miller and Lunde,

Type I11

Type I11 collagen, and similar collagens, have been ident i f ied from aor ta , muscle t i s sue , synovial f luid, and granulation t i s s u e (Wiedemann e t a l , , 1975; Chung and Miller, 1974; Epstein, 1974; Chung e t - a1 ,*) 1 9 7 6 McClain, 1974; Bailey e t a l . , 1975). These s tudies have indicated t h a t Type I11 collagen is composed of three iden t i ca l 0 chains which d i f f e r i n amino acid composition from a1 (I) , 02, and CUI (11) chains and has the molecular configuration [ a 1 (11)]3. Type I11 collagen contains 6-8 Hydroxylysine residues per 1000 residues and has approximately 15-2& of the Hydroxylysine glycosylated. Type I11 collagen contains higher amounts of Hydroxyproline and contains cystine, which is apparently involved i n d isu l f ide bonds a t t h e C terminal end and contributes t o t h e low s o l u b i l i t y of t h i s collagen.

Type IV collagen, which has not been completely characterized (Bailey and Robins, 1976), w a s ident i f ied by Kefalides (1971, 1973). Type IV collagen is a l s o mde up of three iden t i ca l cr. chains with t h e m o l e c a r configuration [ a I (IV)] 3' collagens (Daniels and Chu, 1975; Kefalides, 1973) indicate t h a t the a1 (IV) chain has a high content of Hydroxylysine (60-70 residues per 1000 residues ), higher amounts of both 3 and 4 Hydroxyproline and some cyst ine. A high amount of carbohydrate ( 8 6 of the Hydroxylysine is glycosylated) is a l s o associated with 'Type IV collagen (Bailey and Robhs , 1976) .

h i n o acid camposition of me IV

Collagen Biosynthes is

The sequence of events i n collagen biosynthesis includes the production of the nacent polypeptide chains (Transcription and t rans - l a t i on ) , a l t e r a t ions on these chains during and a f t e r t rans la t ion , such

343

as glycosylation and hydroxylation, formation of t he chains in a precursor molecule, secret ion of the precursor and i t s conversion t o collagen, and the aggregation of the collagen molecules in to f ibe r s ( f igure 3 )

Although none of the messenger RNA's (m-RNA) t h a t a r e spec i f ic f o r an individual collagen type have been isolated and purif ied, t h e determination that d i f f e ren t collagen types have d i f f e ren t amino acid contents and sequences i n the chains indicate t h a t there are separate m-RNA's f o r each of t he f i v e a chains, a1 (I), 02, a1 (11), CxI (111) and a1 ( N ) (Miller - e t a1 ,*, 1976; Epstein, 1974; Chien, 1975; Martin e t al., 1975; Kefalides, 1973). The m-RNA's f o r synthesis of these chains a r e probably monolystronic based on s i z e of t h e polysomes ( h z a r i d e s and Lukens, 1971; Kerwar e t al., 1972; Diegelman e t a l . , Benveniste, 1973). Data presented b Vuust and Piez (1970) have indicated that the m-RNA's f o r a1 ( I Y and CQ a r e transmitted simul- taneously and that the chains a re assembled by addi t ion of individual amino acids and not as shorter polypeptide chains. t r ans l a t ion of a1 ( I ) and a2 chains has been determined t o be about 200 residues per minute (Vuust and Piez, 1972) and t h e synthesis of nacent collagen chains has been shown t o occur on membrane bound r i b isomes (Goldberg and Green, 1967; Diegelmtm e t al., 1973 ) .

The r a t e of

Hydroxylation and Glycosylat ion

An unusual series of reactions take place i n collagen biosynthesis which alters the s t ruc tu re of two of the amino ac ids a f t e r t h e i r incor- poration i n t o the nacent chain. Lysine and prol ine are hydroxylated by t h e enzymes prol ine hydroxylase and lys ine hydroxylase, respectively, and hydroxylation has been shown t o take place on the nacent chains p r io r t o t h e i r release from the membrane bound ribisomes (Miller and Udenfriend, 1970; Lazarides -- e t al., 1971). require molecular oxygen, ferous iron, a-ketoglutarate, and ascorbic acid (Grant and Prockop, 1972; Miller, 1971; Miller and lvkLtukas, 1974). Molecular oxygen has been shown t o be a source of oxygen f o r the hydroxyl group (Prockop e t al., 1963). undergoes decarboxykt ion upon hydroxylation of the rX chains (Rhoads and Underfriend, 1968). Hydroxylase a c t i v i t y is act ivated by ferous i ron which may serve as a binding s i t e f o r molecular oxygen (Miller and Matukas, 1974). Ascorbic acid probably a c t s as a reducing agent f o r t he hydroxylation react ion and may a l s o be involved i n converting an inactive form of t h e enzyme t o an ac t ive s t a t e (Hutton e t al . , 1967; Stassen e t a l . , 1973).

These two enzymes both

Alpha-ketoglutarate is required and

Hydroxyproline has been shown t o add s tab i l i ty t o the collagen he l ix and that a t physiological temperatures, he l ix formation of t h e procollagen molecule is dependent upon hydroxylation of proline (Sakakibara e t al . , 1973; Murphy and Rosenbloom, 1973) xylated procollagen has been shown t o accumulate in the c e l l suggesting that hydroxylation or that the complete h e l i c a l procollagen is necessary f o r secret ion (Miller and Matukas, 1974; Mwtin e t al. , 1975).

Under hydro-

344

The carbohydrate groups of collagen (glucosylgalactose and galactose ) a r e covalently bonded by 0-glucosidic linkages t o t h e carbon of hydroxy- lysine (Spiro, 1969). Galactose is apparently bound t o hydroxylysine by a galactosyl t ransferase (Spiro and Spiro, 197la) and glucose i s then bound t o the galactose by a glucosyl t ransferase (Spiro and Spiro, 1971b).

Helix Formation and Secret ion

Although t h e -- i n v i t r o aggregation of t h e polyproline he l ix of individual chains i s slow, the synthesis of t h e t r i p l e h e l i c a l pro- collagen molecule is very rapid (Beier and Engel, 1968; Vuust and Piez, 1972). Assembly during synthesis is probably achieved by a reg is te r ing of t h e ends of t he nonpoly-proline h e l i c a l region of the Q! chains, thus locat ing the cor rec t allignment f o r the formation of the t r ip l ehe l ix (Bailey and Robins, 1976). The extension peptides a t the amino terminus (molecular w e i g h t 15000 Daltons) and a t the carboxy terminus (molecular weight 35000 Daltons ) both contain cystine residues, but d i su l f ide bonds between a chains a r e found only a t t h e carboxy terminal ends (Fessler e t al . , 1975; Bailey and Robins, 1976) linkages may be involved i n a rapid aggregation of the C terminal extension peptides on completion of synthesis . Upon completion of he l ix formation, t h e procollagen molecule passes through the golg i t o the c e l l membrane by being packaged i n ves ic les f o r t ransport through the c e l l s . (Diegelmann and Peterkovsky, 1972; Warlich and Bornstein, 1972; Bailey and Robins, 1976).

This indicates t h a t d i su l f ide

This t ranspor t probably involves t h e microtubular system

Due t o t h e f a c t that procollagen with its in t ac t extension peptides w i l l not aggregate t o form collagen fibers ( m r t i n e t a l . , 1975; Bailey and Robins, 1976) these extension peptides must be removed before f i b e r formation can take place outside t h e c e l l . A number of proteases have the a b i l i t y t o remove t h e non h e l i c a l peptides (Martin e t a l . , 1-97?), however, part of these proteases a r e not present or do n o t function under physiological conditions. An enzyme which is capable of removing extension peptides has been isolated from ca l f tendon and other t i s sues (Lapear &., 1971; Bornstein e t al . , 1972). This enzyme, ca l led procollagen peptidase, has been shown t o be dependent upon calcium f o r a c t i v i t y and t o remove extension peptides indicat ing that t h i s enzyme is an endopeptidase (Kohn e t al., 1974). A f t e r cleavage of extension peptides, t h e tropocollagen molecules a l ign themselves i n t o f ibe r s , according t o t h e scheme described previously under s t ruc ture .

Collagen Turnover

Although the r a t e of synthesis of any protein has been shown t o be affected by various fac tors , t he net accumulation of any protein is dependent upon both t h e rate of synthesis and the rate of degradation (Dayton e t al . , 1975; Bergen, 1975). collagen synthesis should include a discussion of collagen turnover.

Therefore, a discussion of

345

Early experiments on collagen turnover have indicated tha t collagen turnover is very slow (Gerber e t al. , 1960; Popenoe and Van Slyke, 1962). Although some studies have indicated high collagen turnover r a t e s in some t i s s u e s and ce r t a in pathological conditions, compared t o other proteins , collagen is s t i l l considered metabolically i n e r t (Bailey and Robins, 1976). does ac tua l ly turnover, an experiment has been conducted i n our laboratory which involved t h e feeding of f3-amboproprionitrile ( E W N ) t o mature ewes. lysyloxidase and thus prevent the crosslinking of newly synthesized collagen, should be ab le t o increase the s o l u b i l i t y of collagen f rom mature animals if collagen is being turned over, due t o t h e f a c t that the newly synthesized collagen w i l l not be crosslinked and thus w i l l have a higher s o l u b i l i t y . Some preliminary r e s u l t s of t h i s exper-nt a r e presented i n t ab le 2. The percent soluble collagen w a s increased over t h e controls w i t h the percent soluble collagen being greater w i t h greater amounts of W N i n the d i e t and longer time on the BAPN treatment., This data indicates t h a t intramuscular collagen is being turned over even in mature animals and that the s t a t e of t h e collagen can be a l t e r ed i n mature animls.

In order t o determine if collagen i n muscles of mature animals

BAPN, a compound which has been shown t o inhibi t t he enzyme

Table 2. Collagen content and s o l u b i l i t y of the L.D. from mature ewes fed BAPN

D a y s on BAPN 20 20 40 40 60 60 Control

BAPN dosage (g/day 1 2 4 2 4 2 4 0

Soluble collagen ($1 4 -3 5 04 5 -8 7 02 5 -6 7.7 4 .1

An enzyme, which has been shown t o a t t ack nat ive collagen under physiological conditions, was o r ig ina l ly isolated from tadpole t i s s u e (Gross a d lap iere , 1962) and has recent ly been found in numerous other t i s sues (Harris and Krane, 1974; Woolley e t a l e , 1975; McCroskery et G., 1975; Wooley e t al., 1976). around p H 7.5 t o 8.5, is act ivated by calcium ions, cleaves native collagen peptides a t one s i t e and cleaves [a1 (1)]2, 02 [a1 (III)]3 collagens f i v e times more rap id ly than [a1 (II)]3. Although the physiological r o l e of t h i s enzyme has not been determined, it could be a fac tor i n the na tu ra l turnover of collagen i n various t i s sues .

This enzyme has an optimal a c t i v i t y

346

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Dennis Campion: Thank you, D r . Dutson. Consistent with the way the session is outlined i n our program, I think w e ' l l r e f r a i n from any questions a t t h i s point and hold them u n t i l the end of the program.

Dr. Phi l McClain, our next speaker, obtained h i s Bachelor's Degree from the University of Missouri, went t o Louisiana S ta t e University f o r h i s Master's Degree, and then completed a Doctorate of Philosophy Degree a t Michigan S t a t e University. D r . & C h i n is current ly a research chemist a t the Protein Nutr i t ion Laboratory, Nutr i t ion I n s t i t u t e , Agriculture Research Service, U .S . Department of Agriculture. A t t h i s time, I ' d l i k e t o present D r . McClaFn who w i l l be speaking t o us on t h e top ic of chemistry of collagen crossl inking .