Diversity in the processing events at the N‐terminus of type‐V collagen

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Abstract
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-α1(V) and pro-α2(V) chains. The anti-peptide polyclonal antibody raised against positions 48–57 of the N-terminal α2(V) sequence recognized the mature from of the human α2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-α2(V) and pN-α2(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-α2(V) collagen chain from this cell line migrated during electrophoresis with the α2(V) chain obtained from tissues. This demonstrates that the α2(V) chain in tissues is incompletely processed and is present as the pN-α2(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 α1(V) human sequence failed to recognize the mature form of the α1(V) chain while it reacted with the pN-α1(V) collagen chain form. These results suggest that the α1(V) chain undergoes a processing event in the N-terminal region that involves the revmoval 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 α1(V) chain gave, as expected, only sequences corresponding to the α1(V) chain. However, the band previously considered to be the intact α2(V) chain also gave sequences for the α1(V) chain in addition to the α2(V) chain. This result indicates the presence in tissue extracts of a further processed form of α1(V) chain which migrates with the intact α2(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 α1(V)/α2(V) chain ratio of 3:1. These results indicate that the α1(V) chain exists in an additional stoichiometry, different from [α1(V)]2α2(V). We suggest the existence of two different populations of type-V collagen molecules consisting of an [α1(V)]2α2(V) heterotrimer bearing considerable N-terminal non-triple-helical extensions of both α1(V) and α2(V) chains and an [α1(V)]3 homotrimer composed of fully processed α1(V) chains.

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