Yoshio Hojima

Pleomorphism in type I collagen fibrils produced by persistence of the procollagen N-propeptide

The assembly of type I collagen and type I pN-collagen was studied in vitro using a system for generating these molecules enzymatically from their immediate biosynthetic precursors. Collagen generated by C-proteinase digestion of pC-collagen formed D-periodically banded fibrils that were essentially cylindrical (i.e. circular in cross-section). In contrast, pN-collagen generated by C-proteinase digestion of procollagen formed thin, sheet-like structures that were axially D-periodic in longitudinal section, of varying lateral widths (up to several microns) and uniform in thickness (approximately 8 nm). Mixtures of collagen and pN-collagen assembled to form a variety of pleomorphic fibrils. With increasing pN-collagen content, fibril cross-sections were progressively distorted from circular to lobulated to thin and branched structures. Some of these structures were similar to fibrils observed in certain heritable disorders of connective tissue where N-terminal procollagen processing is defective. The observations are considered in terms of the hypothesis that the N-propeptides are preferentially located on the surface of a growing assembly. The implications for normal diameter control of collagen fibrils in vivo are discussed.

Cadmium ions inhibit proollagen C-proteinase and cupric ions inhibit procollagen N-proteinase

Procollagen C- and N-proteinases specifically cleave the C- and N-terminal extension propeptides of type I, II and III procollagen molecules. The collagen molecules generated by the enzymes self-assemble into collagen fibrils. We previously observed the inhibition of these enzymes purified from chick tendons by several divalent metals. Here the inhibitory effects of CdCl2, CuCl2, ZnCl2, NiCl2, CoCl2 and Hg(C2H3O2)2 have been studied in detail using crude or purified C- and N-proteinases from chick tendons and sterna. CdCl2 was a strong inhibitor of C-proteinases from both sources, and the inhibition was independent of enzyme purity (I50 = 10-16 microM). In contrast, CuCl2 and ZnCl2 were inhibitory only of purified C-proteinase. With the N-proteinase, CuCl2 was a strong inhibitor, and the inhibition was independent of the purity of the enzyme preparation used (I50 = 14-40 microM). On the other hand, CdCl2 was a moderate inhibitor, and ZnCl2 was a strong inhibitor only of the purified N-proteinase (I50 = 8-17 microM). NiCl2 inhibited crude and purified N-proteinase from sternum (I50 = 23-29 microM) but not from tendon. These results suggest, therefore, that the accumulation of some of these metals in the body may cause suppression of collagen fibril formation in tissues.

Recombinant c-proteinase and processes, methods and uses thereof

The present invention is directed to the isolation and identification of the nucleic acid sequence encoding C-proteinase, the recognition of such proteins activity and applications, and tools, processes, and methods of use thereof.

Effects of Mutations that Change Primary Structure of Collagen on the Self-Assembly of the Protein into Fibrils

We have recently observed that a single base mutation in a gene for type I procollagen converts a glycine residue to cysteine and that the substitution for the glycyl residue has a remarkable effect both on the conformation of the molecule and the morphology of the fibrils that are formed as the mutated procollagen molecule is processed to collagen (Vogel et al., 1987; 1988; Kadler et al., 1988b). The observations have largely been made possible through the development of a new system for examining the self-assembly of collagen de novo (Kadler et al., 1987; 1988a).