Previous studies have shown that a type I procollagen-derived peptide, called pNal(I)-Col 1, selectively inhibits collagen synthesis by fibroblasts in culture and the translation of procollagen mRNA in a rabbit reticulocyte lysate system. We prepared the 10,700-dalton peptide from dermatosparactic calf skin, which contained high levels of incompletely processed type I procollagen, by collagenase digestion. Time-course and dose-response studies showed that the peptide specifically inhibited the translation of procollagen mRNA in preparations of human fibroblast RNA while not affecting the translation of globin mRNA or of other messenger RNAs in fibroblast RNA. After reduction and alkylation, the peptide lost its specificity but became a nonspecific inhibitor of translation. Enzymatic cleavage enabled us to localize the nonspecific activity to a short sequence -Pro-Thr-Asp-Glu, an assignment confirmed by peptide synthesis. Using pactamycin, a specific inhibitor of translational initiation, we showed that the intact peptide acts on procollagen mRNA translation by inhibition of polypeptide chain elongation or termination, or both, whereas the nonspecific inhibitory activity of the unfolded peptide and its derivatives can be attributed to an inhibition of chain initiation. Although the native peptide may function in feedback regulation ofcollagen synthesis, the physiological role ofthe lower molecular weight fragments, if any, is uncertain.Studies of the regulation of collagen synthesis have usually focused on steps in the biosynthetic pathway-e.g., peptidyl hydroxylation by proline and lysine hydroxylases-that are relatively specific for the protein (for a review see ref. 1). However, in circumstances such as viral transformation by Rous sarcoma virus, clear evidence for a reduction in procollagen mRNA levels (2, 3) and in the rate of transcription of procollagen genes (4) has been obtained. In 1979 Wiestner et aL (5) reported that the non-triple-helical aminoterminal extension of the proal chain of type I collagen [termed PNal(I)-Col 1; in this paper designated "extension"] obtained by bacterial collagenase digestion of dermatosparactic bovine pN collagen, inhibited collagen synthesis when added to bovine fibroblasts in culture. The inhibition was partial and affected synthesis of al and a2 chains equally. Noncollagenous protein synthesis was not affected and changes in degree of peptidyl hydroxylation or intracellular degradation were not noted. These studies were pursued by Paglia et aL (6) who demonstrated that the extension retained the capacity to inhibit, relatively specifically, the translation of procollagen mRNA in a cell-free translation system derived from rabbit reticulocytes.These findings were of considerable interest because they implied both specific recognition by fibroblasts ofaprocollagenderived fragment and a means by which the extension, or a derivative thereof, could be translocated into the cytosol of the cell. Indeed, we have documented the binding and uptake of the extension by fib...
Fibroblasts derived from a skin biopsy of a patient with scleroderma in the sclerotic stage were shown to have a higher rate of DNA synthesis, and to synthesize more collagen than fibroblasts from a healthy control. The addition of procollagen peptides to the culture medium of scleroderma fibroblasts almost normalized the collagen synthesis. This observation indicates that the mechanism for the regulation of collagen synthesis by feed back inhibition of prollagen peptides is functioning in this disease. It is suggested that the level of biologically active procollagen peptides is lowered.
The N-terminal extension peptide of type III procollagen, isolated from foetal-calf skin, contains 130 amino acid residues. To determine its amino acid sequence, the peptide was reduced and carboxymethylated or aminoethylated and fragmented with trypsin, Staphylococcus aureus V8 proteinase and bacterial collagenase. Pyroglutamate aminopeptidase was used to deblock the N-terminal collagenase fragment to enable amino acid sequencing. The type III collagen extension peptide is homologous to that of the alpha 1 chain of type I procollagen with respect to a three-domain structure. The N-terminal 79 amino acids, which contain ten of the 12 cysteine residues, form a compact globular domain. The next 39 amino acids are in a collagenase triplet sequence (Gly- Xaa - Yaa )n with a high hydroxyproline content. Finally, another short non-collagenous domain of 12 amino acids ends at the cleavage site for procollagen aminopeptidase, which cleaves a proline-glutamine bond. In contrast with type I procollagen, the type III procollagen extension peptides contain interchain disulphide bridges located at the C-terminus of the triple-helical domain.
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