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

Abstract

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...

Location of procollagen in chick corneal and tendon fibroblasts with ferritin-conjugated antibodies.

“…It is now clear that the pro-al chain obtained from acid-extracted cranial bone procoilagen represents a truncated precursor chain (21). Cleavage of disulfide-bonded regions occurs, presumably catalyzed by acid proteases released during homogenization of bone.…”

Purification, composition, and31P NMR spectroscopic properties of a noncollagenous phosphoprotein isolated from chicken bone matrix

“…below). Pepsin, lysozyme, ribonuclease, and chicken osteocalcin were used after reduction and alkylation [16] to determine elution volumes for proteins of known molecular weight. Blue dextran and dinitrophenyI-L-isoleucine were used to measure the void volume, Vo, and the total bed volume, Vt, respectively.…”

The structure and assembly of procollagen – a review

“…More systematic studies of the procollagen secreted by cultured cells indicated that a substantial fraction o f the protein contained interchain disulfide bonds (1 9-21, 25). When embryonic chick cranial bones were extracted either at neutral pH in the presence of a number of enzyme inhibitors (41) or by rapid extraction in acetic acid (42), essentially all the recently synthesized procollagen was shown t o exist as a high molecular weight product linked by interchain disulfide bonds (Fig. 1).…”

“…in such a way as t o preserve, in so far as possible, the native structure of the protein (41). Since the triple helix is resistant t o pepsin, refolding of procollagen was assayed by determination of the fraction of protein that, after limited cleavage with pepsin, migrated in the position of a chains on sodium dodecyl sulfate acrylamide gel electrophoresis.…”

“…Clearcut experimental evidence for the existence of a higher molecular weight biosynthetic precursor of collagen has come from several sources: 1) studies o f the synthesis and structure of the hydroxyproline-containing material secreted into the medlum by cultured fibroblasts (17)(18)(19)(20)(21)(22)(23); 2) studies o f the synthesis and secretion of procollagen by freshly isolated embryonic chick cells obtained from tendon, cartilage, or lens capsule (24-29); 3) an investigation of a heritable disorder of cattle and sheep (dermatosparaxis), in which an enzymatic defect in the conversion of procollagen t o collagen was found (30)(31)(32)(33)(34)(35); an analogous defect has been identified as a form of the Ehlers-Danlos syndrome in man (36); 4) studies of procollagen synthesis and structure by cranial bone explants (13,(37)(38)(39)(40)(41)(42); and 5) isolation of derivatives of procollagen from tissues of normal animals (43)(44)(45)(46).…”