DIVERSITY IN THE PROCESSING EVENTS AT THE N-TERMINUS OF TYPE-V COLLAGEN

The processing of human collagen type-V chains was studied using anti-peptide polyclonal antibodies raised against peptide sequences at the N-terminal non-triple-helical region of pro-alpha 1(V) and pro-alpha 2(V) chains. The anti-peptide polyclonal antibodies raised against positions 48-57 of the N-terminal alpha 2(V) sequence recognized the mature form of the human alpha 2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-alpha 2(V) and pN-alpha(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-alpha collagen chain from this cell line migrated during electrophoresis with the alpha 2(V) chain obtained from tissues. This demonstrates that the alpha 2(V) chain in tissues is incompletely processes and is present as the pN-alpha 2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclonal antibody raised against residues at positions 284-299 of the N-terminal alpha 1(V) chain while it reacted with the pN-alpha 1(V) collagen chain form. These results suggest that the alpha 1(V) chain undergoes a processing event in the N-terminal region that involves the removal of at least the first 284 residues. Amino acid sequence analysis was performed on cyanogen-bromide-generated or trypsin-generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower-migrating band corresponding to the intact alpha 1(V) chain in addition to the alpha 2(V) chain. This result indicates the presence in tissue extracts of a further processed form of alpha 1(V) chain which migrates with the intact alpha 2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1:1 ratio whereas, after the pepsin digestion to remove non-collagenous regions, two bands were observed with an alpha 1(V)/alpha 2(V) chain ratio of 3:1. These results indicate that the alpha 1(V) chain exists in an additional stoichiometry, different from [alpha 1(V)](2) alpha 2(V). We suggest the existence of two different populations of type-V collagen molecules consisting of an [alpha 1(V)](2) alpha 2(V) heterotrimer bearing considerable N-terminal non-triple-helical extensions of both alpha 1(V) and alpha 2(V) chains and an [alpha 1(V)](3) homotrimer composed of fully processed alpha 1(V) chains.