Partial purification of a procollagen C-proteinase. Inhibition by synthetic peptides and sequential cleavage of type I procollagen

Abstract

A procollagen C-proteinase which cleaves the C-propeptides from type I procollagen was purified about 125-fold from membranous bones of chick embryos. As estimated by gel filtration, the enzyme was about 80 000 daltons. When a reaction with modified procollagen was carried out, the enzyme preferentially cleaved the C-propeptides from the pro alpha chains in the order pro alpha 1, pro alpha 1, and then pro alpha 2. The enzyme was inhibited by several metal chelators and high concentrations of dithiothreitol. It was also inhibited by 5% fetal calf serum. A series of inhibitors of serine proteinases and sulfhydryl-containing proteinases were not inhibitory. Four oligopeptides were synthesized with amino acid sequences similar to the amino acid sequences around the sites at which the C-propeptides are cleaved during the conversion of procollagen top collagen in vivo. The peptide Tyr-Tyr-Arg-Ala-Asp-Asp-Ala inhibited the enzyme 35--60% in concentrations of 6--12 mM. Shorter peptides containing the Ala-Asp bond cleaved by the enzyme were less effective. The partially purified enzyme was also found to cleave the C-propeptides from type II and type III procollagens.

Cathepsin D-mediated processing of procollagen: lysosomal enzyme involvement in secretory processing of procollagen.

“…We report here that the cleavage of procollagen with purified cathepsin D is specific for the COOH propeptide and that cleavage occurs at or near the authentic cleavage site when digestion is carried out near pH 6.0. We postulate a role for cathepsin D in the in vivo conversion of procollagen to collagen.…”

“…By using a rapid assay (6,27) to measure radioactive COOH propeptide liberated at pH 6.0, cpm measurements were linear with added enzyme (data not shown). The time course of release showed an apparent lag phase.…”

“…The degree of conversion at pH 6.0 was estimated to be on the order of 1-5%, based upon the radioactivity released in a 4-hr rapid assay (6,27) (30). Intracellular compartmentalization and concentration of cathepsin D and procollagen (31,32) could enable rapid processing of procollagen.…”

“…Conceivably, if cathepsin D and other lysosomal enzymes were to fuse with a secretory granule whose pH was too low (<6.0) or which contained procollagen with a defective triple helix (47,49) [3][4][5][6].…”

“…4 appear to be in disagreement with two lines of earlier work. Several workers have proposed that the procollagen COOH-terminal proteinase is a neutral metalloproteinase (6,27,54,58,59). Most of these studies were performed with enzymes isolated from tendon fibroblast culture media.…”