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 posi tions 48-57 of the N-terminal alpha 2(V) sequence recognized the matur e form of the human alpha 2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protea se inhibitors. It also recognized the pro-alpha 2(V) and pN-alpha(V) c ollagen chains secreted in the cell-culture media of the rhabdomyosarc oma A204 cell line. The pN-alpha collagen chain from this cell line mi grated during electrophoresis with the alpha 2(V) chain obtained from tissues. This demonstrates that the alpha 2(V) chain in tissues is inc ompletely processes and is present as the pN-alpha 2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclon al antibody raised against residues at positions 284-299 of the N-term inal 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 colla gen 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 ana lysis, 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)/a lpha 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 populat ions of type-V collagen molecules consisting of an [alpha 1(V)](2) alp ha 2(V) heterotrimer bearing considerable N-terminal non-triple-helica l 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.