Proposed Mechanism for the Biological Assembly of Collagen Triple Helix

Chick cranial-bone procollagen, extracted at neutral pH in the presence of inhibitors of proteolytic enzymes, exists as a triple-stranded protein with disulfide bonds linking all three chains. The biosynthetic precursor function of this procollagen was demonstrated by pulsechase experiments. The ratio of radioactive hydroxyproline to proline in proa chains obtained by reduction and alkylation of the disulfide-bonded precursor was similar to the value determined for proal from acid-extracted procollagen. However, the molecular weight of these chains was higher than that previously determined for proal and proa2, suggesting that extraction of tissue at low pH results in a selective loss of disulfide-bonded regions from procollagen. These findings reconcile several apparently conflicting reports on the nature of procollagen identified in bone and in the medium of cultured fibroblasts and indicate that in both systems procollagen is synthesized as a disulfidelinked protein.There is ample evidence for the existence of a higher molecular weight precursor of the functional collagen monomer (1-5). The term procollagen was applied to the form identified in extracts of embryonic cranial bone to indicate an antecedent role for the molecule in the biosynthesis of collagen (1). Molecular weights of 115,000-120,000 were determined for the constituent proal and proa2 chains of cranial-bone procollagen, in contrast to the molecular weight of 95,000 established for vertebrate collagen-a chains (1, 6, 7). Possible functions for the additional sequences in procollagen include initiation of triple-helix formation, prevention of intracellular fibrogenesis, and intracellular translocation and secretion of the protein (1, 2, 8). The amino-acid composition of the additional sequence in the proal chain of chick-bone procollagen is markedly different from that of fibrous collagen, in keeping with the possibility of a distinct structure and function for this molecular domain (7,9).In contrast to the discrete proa chains obtained by heat denaturation of acid-extracted bone procollagen, collagen precursors identified in the medium of cultured cells contain interchain disulfide bonds. Thus, reduction of culture medium with 2-mercaptoethanol was required to demonstrate the presence of proa chains in the material synthesized by embryonic chick fibroblasts (3, 10). In the absence of reduction, collagen fractions with molecular weights expected of dimers and trimers of proa chains were identified (3,4,(10)(11)(12).The ability to demonstrate a form of procollagen in aceticacid extracts of cranial bone resulted largely from inactivation Abbreviations: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; EDTA, ethylenediamine tetraacetic acid; CMcellulose, carboxymethyl cellulose. * To whom requests for reprints may be addressed. 3521at low pH of the neutral proteolytic activity, procollagen peptidase (1, 13-15). It seemed possible, however, that homogenization and extraction of bone in acetic acid might promote limited proteolysis o...

mentioning

confidence: 88%

Identification of a Disulfide-Linked Procollagen as the Biosynthetic Precursor of Chick-Bone Collagen

Monson

Börnstein

1973

“…Since the reconstitution in vitro of native collagen molecules from unfolded chains does not proceed readily under physiological conditions (22,23), chain association in vivo may require coordinated biosynthesis of complementary chains on adjacent polysomes or the existence of some form of intracellular template. Another possibility, for which precedence exists in the case of pancreatic proteolytic enzymes (24) and insulin (25), is the existence of a zymogen or secretory form of the protein, characterized by the presence of additional peptide sequences (26,27 (16)(17)(18), the additional peptide se- t Determined by amino acid analysis (12). quences in procollagen presumably exist in some conformation other than the collagen helix.…”

Section: Discussionmentioning

confidence: 99%

Evidence for Procollagen, a Biosynthetic Precursor of Collagen

Bellamy

Börnstein

1971

186

Incubation of rat calvaria for short times in the presence of a labeled amino acid revealed the existence of a collagen fraction (procollagen) that functions as a biosynthetic precursor of collagen. Procollagen contains an al-like chain (pre-al) that elutes earlier from CM-cellulose than does rat-bone al and has a molecular weight, estimated by acrylamide gel electrophoresis, of 120,000. A time-dependent conversion of pre-al to al was demonstrated by incubation of calvaria for periods varying from 9 to 60 min and by a pulse-chase experiment. Limited cleavage of procollagen with pepsin resulted in a molecule with a chain resembling al in chromatographic properties, molecular weight, and relative hydroxyproline and proline contents. Thus, conversion of procollagen to collagen is likely to occur in vivo by a proteolytic mechanism. The additional peptide sequences in procollagen may serve to initiate chain association in triple-helix formation, to facilitate molecular transport, and to inhibit intracellular fibrogenesis.The biosynthesis of a functional collagen molecule and its subsequent utilization are critically dependent on a number of processes that follow the polyribosomal assembly of its polypeptide chains. These include the hydroxylation of prolyl and lysyl residues (1, 2), O-glycosylation of certain hydroxylysyl residues (3, 4), association of the three polypeptide chains to form the native triple-helical monomer(5), transport of the collagen molecule to the extracellular space (6), and the specific aggregation (7) and cross-linking (8) of collagen to form the connective tissue fibril.The hydroxylation, glycosylation, and cross-linking of collagen have been the subject of considerable investigation, but less is known of the mechanisms by which chain association leads to the formation of a triple-helical molecule and its secretion from the cell. If we a-e to understand these processes, it is essential to know whether a chains obtained from extracellular collagen by heat dissociation and chromatography (9) are structurally identical with intracellular collagen polypeptides. One possibility, among others, is that additional peptide sequences facilitate triple-helix formation and transport of the collagen molecule, while at the same time inhibiting intracellular fibrogenesis. Such peptide moieties could be removed by an extracellular proteolytic mechanism, which would thereby initiate the transition of the protein from a soluble to a fibrillar form.During studies designed to investigate the kinetics of collagen chain synthesis in calvarial tissue cultures, we observed that recently synthesized, chromatographically isolated, al chains from rat-bone collagen differed from the bulk of extracellular al chains in their position of elution from CMcellulose. Several lines of evidence subsequently demonstrated that such chains are part of a precursor collagen molecule (procollagen) t and that conversion of procollagen to collagen can be simulated in vitro by limited proteolysis with pepsin. METHODSIn vitro synt...

“…In vitro, correct reassembly of the molecule from separated a chains proceeds slowly and inefficiently, and assembled helical molecules tend to come out of solution to form typical collagen fibers. For these reasons it has been proposed that rapid in vivo assembly of collagen molecules and secretion in a soluble form are determined by extra terminal peptides that are later excised (1,2). Supporting this view are recent reports that biosynthetic precursors of both al and a2 chains ("procollagens": "pro al", and "pro a2") may be extracted from embryonic rat (3) and chick (4) calvaria, and may be found in the medium of cultured fibroblasts (5) and cultured embryonic-chick tendon cells (6,7).…”

mentioning

confidence: 99%

Precursors of Collagen Secreted by Cultured Human Fibroblasts

Goldberg

Epstein

Sherr

1972

Soluble precursors of collagen ("procollagens") in the culture medium of human diploid fibroblasts were characterized by molecular sieve chromatography on 6% agarose and by sodium dodecyl sulfateacrylamide gel electrophoresis. Under the denaturing conditions for gels, the following molecular species were identified: a major species of three procollagen chains, a procollagen dimer, and free procollagen chains. Reduction with 2-mercaptoethanol completely dissociated the trimer and dimer, releasing procollagen al and procollagen a2 chains with molecular weights of about 120,000. Both pro al and pro a2 chains could be labeled with [n5Slcystine.In addition, free procollagen chains with molecular weights of less than 120,000, but heavier than native a chains, were identified in gels. Radioactivity in all of the above molecules could be chased into native a chains extracted from extracellular fibers of the cell layers. The data indicate that: (i) collagen is secreted as a disulfidestabilized procollagen trimer with the composition, (pro al)2 pro a2; (ii) noncovalent interactions maintain the three-chain assembly during stepwise enzymatic excisions of N-terminal, cysteine-containing procollagen peptides; and (iii) after such excisions, native helical molecules, (al)2 a2, precipitate as fibers in the cell layers.Collagen is secreted from fibroblasts in a soluble form and precipitates as characteristic fibers in the extracellular space.Native tropocollagen molecules isolated from such extracellular fibers generally consist of two identical al chains and one a2 chain in triple helical assembly. In vitro, correct reassembly of the molecule from separated a chains proceeds slowly and inefficiently, and assembled helical molecules tend to come out of solution to form typical collagen fibers. For these reasons it has been proposed that rapid in vivo assembly of collagen molecules and secretion in a soluble form are determined by extra terminal peptides that are later excised (1, 2). Supporting this view are recent reports that biosynthetic precursors of both al and a2 chains ("procollagens": "pro al", and "pro a2") may be extracted from embryonic rat (3) and chick (4) calvaria, and may be found in the medium of cultured fibroblasts (5) and cultured embryonic-chick tendon cells (6, 7). The procollagens have been characterized as native al and a2 chains with additional terminal peptide sequences of molecular weight about 15,000-20,000.Noncovalently assembled chains might be stabilized by formation of disulfide bonds between cysteine residues in these extra peptide sequences. In bone culture preparations pro al contains 5 or 6 half-cystine residues per chain, but little or no cysteine was detected in pro a2, and no evidence for disulfideAbbreviations: SDS, sodium dodecyl sulfate; CMC, carboxymethyl cellulose. * Address reprint requests to Dept. of Pathology.bond formation between procollagen chains was obtained (2). In contrast, in preparations of cultured fibroblasts (5, 8) and tendon cells (7), data from molecular sieve...