Mechanisms of collagen trimer formation. Construction and expression of a recombinant minigene in HeLa cells reveals a direct effect of prolyl hydroxylation on chain assembly of type XII collagen.

Collagen types IX, XII, and XIV are characterized by the presence of a highly conserved region comprising the most C-terminal triple helical domain (COL1, approximately 100 residues/chain) and 2 cysteines separated by 4 amino acid residues at the junction between this COL1 domain and the C-terminal non-triple helical domain (NC1). In order to better understand the functions of this conserved domain, we have constructed a recombinant minigene, comprising the sequence coding for an unrelated signal peptide and for the COL1 and NC1 domains of type XII collagen. This construct was placed under the control of the cytomegalovirus promoter and transfected into HeLa cells. The cells expressed the transfected minigene and the secreted chain, called alpha 1 (mini XII), could be detected by immunotransfer with an anti-peptide antibody recognizing an epitope found in the NC1 domain. Under conditions preventing the hydroxylation of prolyl residues (absence of ascorbate or presence of alpha alpha'-dipyridyl), interchain disulfide bridges did not form, while in the presence of ascorbate, disulfide-bonded (alpha 1 (mini XII))3 molecules were secreted. The collagenous nature and triple helical conformation of the trimeric molecule were ascertained by the differential resistances of the COL1 and NC1 domains to trypsin and collagenase digestions, respectively. Our data demonstrate that the NC1 and COL1 domains of type XII collagen contain the information necessary for trimer formation and that, contrary to the fibrillar collagen types, posttranslational modification of the triple helical domain is essential for assembly and disulfide bonding of the chains.