Collagenous sequence governs the trimeric assembly of collagen XII

A minicollagen containing the COLT and NCI domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triple-helical domain in which the cysteines are not involved in inter- bu t in intrachain bonds. In reducing conditions, providing that the triple-he lix is maintained, disulfide exchange between intra- and interchain bonding is observed, suggesting that the triple-helix forms first and that in favo rable redox conditions interchain bonding occurs to stabilize the molecule. This hypothesis is verified by in vitro reassociation studies performed in the presence of reducing agents, demonstrating that the formation of inter chain disulfide bonds is not a prerequisite to the trimeric association and triple-helical folding of the collagen XII molecule. Shortening the COLI d omain of minicollagen XII to its five C-terminal GXY triplets results in an absence of trimers. This can be explained by the presence of a collagenous domain that is too short to form a stable triple-helix. In contrast, the p resence of five additional C-terminal triplets in COLT allows the formation of triple-helical disulfide-bonded trimers, suggesting that the presence o f a triple-helix is essential for the assembly of collagen XII.