The disulphide-bonded nature of procollagen and the role of the extension peptides in the assembly of the molecule

Abstract

1. The molecular weights of chick tendon and cartilage procollagens, and their constituent polypeptides, were determined by gel filtration and gel electrophoresis. The values obtained are in good agreement and indicate that the mol.wts. of the secreted procollagens (types I and II) and their individual pro-alpha-chains are of the order of 405 000-445 000 and 137 000-145 000 respectively.2. Digestion of tendon procollagen with human rheumatoid synovial collagenase gave products consistent with the presence of large non-helical peptide extensions at both N-and C-termini. Electrophoretic analysis gave apparent mol.wts. of 17 500 and 36 000 for the respective N- and C-terminal extensions of pro-alpha1(I)-and pro-alpha2-chains, and inter-chain disulphide bonds were restricted to the C-terminal location. 3. During the biosynthesis of procollagen by tendon and cartilage cells a close correlation was observed between the extent of inter-chain disulphide bonding and the proportion of procollagen polypeptides having a triple-helical conformation. These processes appeared to commence in the rough endoplasmic reticulum and be completed in the smooth endoplasmic reticulum, but the rate at which they occur in cartilage cells is markedly slower than that found in tendon cells. 4. When the intracellular [14C]procollagen polypeptides present in the rough-endoplasmic-reticulum fractions of tendon and cartilage cells were analysed under non-reducing conditions on agarose/polyacrylamide composite gels, no significant pools of dimeric intermediates were detected. 5. In both cell types, inter-chain disulphide-bond formation occurred even when hydroxylation, and hence triple-helix formation, was inhibited. The presence of pro-alpha1- and pro-alpha2-components in a ratio of 2:1 in the disulphide-linked unhydroxylated procollagen isolated from tendon cells demonstrated that correct chain association occurs in the absence of hydroxylation. This observation is consistent with a model for the assembly of pro-gamma112-chains in which the recognition and selection of pro-alpha1-and pro-alpha2-chains in a 2:1 ratio are directed by the non-helical C-terminal extension peptides of tendon procollagen.

Translation of embryonic-chick tendon procollagen messenger ribonucleic acid in two cell-free protein-synthesizing systems

“…This result correlates well with the synthesis of pro-al and pro-a2 chains in a 2: 1 ratio observed in calvaria (Fessler et al, 1975), tendon cells (Harwood et al, 1977) and in studies with collagen-synthesizing polyribosomes (Kerwar et al, 1972;Vuust, 1975). However, in the wheatgerm system the very faint bands corresponding to bands A and B were not in a 2: 1 ratio.…”

“…However, since the translation products do not undergo hydroxylation in the reticulocyte-lysate system (Table 2), the cell-free products might be expected to migrate in positions corresponding to unhydroxylated pro-a1 and unhydroxylated pro-a2 chains, which have been shown to migrate faster than the corresponding hydroxylated polypeptide in composite agarose/polyacrylamide gels (Harwood et al, 1977). This difference in mobility between hydroxylated and unhydroxylated pro-a chains is also demonstrable in SDS/polyacrylamide gels (Fig.…”

“…This 2:1 ratio is also reflected in the proportions of pro-al and pro-ac2 chains synthesized by chick tendon cells (Harwood et al, 1977). To determine whether this ratio is maintained during the cell-free translation of procollagen mRNA, a comparison was made of the relative amounts of the bands corresponding most closely to pro-cl and pro-,a2 chains, i.e.…”

The nature of the microfibrillar glycoproteins of elastic fibres. A biosynthetic study

“…However, the available evidence algo suggested that glycoprotein MFP I was unlike any type of collagen or procollagen hitherto characterized. In particular, the solubility of glycoprotein MFP I in 30%-saturated (NH4)2SO4 distinguishes it from type-I, -II and -III procollagen species, which are insoluble under such conditions (Olsen et al, 1976;Harwood et al, 1977;Scott et al, 1977;Uitto, 1977). This observation suggests that glycoprotein MFP I is much more highly glycosylated than are the interstitial collagen precursors, a possibility that is supported by several other findings, including the ease with which glycoprotein MFP I is labelled with [3Hlfucose, its avidity for the periodic acid-Schiff reagent and its high content of hydroxylysine (Fig.…”

Fell–Muir Lecture: From collagen chemistry towards cell therapy – a personal journey

“…By removing the tendon extracellular matrix by digestion with bacterial collagenase, tendon fibroblasts are released into suspension (approx 3 · 10 6 cells per embryo) and for 4-6 h in culture they will continue to synthesize and secrete the soluble collagen precursor, procollagen (Dehm et al 1972). This molecule was eventually shown to possess N-and C-terminal extensions to the collagen helix, which confer solubility on the procollagen molecule and play a significant role in helix formation and fibre assembly (Harwood et al 1977;Prockop et al 1979). I was fortunate to join the Prockop laboratory in September 1970, on leave from my position in Manchester, and as a joint post-doc with Dr Nicholas Kefalides we resolved to investigate the synthesis of the collagenous proteins associated with the basement membrane structure which forms the lens capsule.…”