INVOLVEMENT OF PROLYL 4-HYDROXYLASE IN THE ASSEMBLY OF TRIMERIC MINICOLLAGEN-XII - STUDY IN A BACULOVIRUS EXPRESSION SYSTEM

We have shown previously that hydroxylation played a critical role in the trimer assembly and disulfide bonding of the three constituent alp ha chains of a minicollagen composed of the extreme C-terminal collage nous (COL1) and noncollagenous (NC1) domains of type XII collagen in H eLa cells (Mazzorana, M., Gruffat, H., Sergeant, A., and van der Rest, M. (1993) J. Biol. Chem. 268, 3029-3032). We have further characteriz ed the involvement of prolyl 4-hydroxylase in the assembly of the thre e alpha chains to form trimeric disulfide-bonded type XII minicollagen in an insect cell expression system. For this purpose, type XII minic ollagen was produced in insect cells from baculovirus vectors, alone o r together with wild-type human prolyl 4-hydroxylase or with the human enzyme mutated in the catalytic site of its alpha or beta subunits or with the individual alpha or beta subunits. When type XII minicollage n was produced alone, negligible amounts of disulfide-bonded trimers w ere found to be produced by the cells. However, coproduction of the co llagen with the two subunits of the wild-type human enzyme dramaticall y increased the amount of disulfide-bonded trimeric type XII minicolla gen molecules. In contrast, coproduction of the collagen with alpha su bunits that had a mutation completely inactivating the human enzyme fa iled to enhance the trimer assembly. These results directly show that an active prolyl 4-hydroxylase is required for the assembly of disulfi de-bonded trimers of type XII minicollagen.