Expression of Wild-Type and Modified Proα Chains of Human Type I Procollagen in Insect Cells Leads to the Formation of Stable [α1(I)]2α2(I) Collagen Heterotrimers and [α1(I)]3 Homotrimers but Not [α2(I)]3 Homotrimers

Abstract Insect cells coinfected with a baculovirus coding for the proα1(I) chain of human type I procollagen and a double promoter virus coding for the α and β subunits of human prolyl 4-hydroxylase produced homotrimeric [proα1(I)]3procollagen molecules. The use of an additional virus coding for the proα2(I) chain led to the formation of a heterotrimeric molecule with the correct 2:1 ratio of proα1 to proα2 chains of type I procollagen (proα1(I) and proα2(I) chains, respectively), unless the proα1(I) chain was expressed in a relatively large excess. Replacement of the sequences coding for the signal peptide and the N propeptide of the proα1(I) chain with those of the proα1(III) chain increased level of expression of the proα1(I) chain, whereas no similar effect was found when the corresponding modification was made to the virus coding for the proα2(I) chain. Molecules containing such modified N propeptides were found to be processed at their N terminus more rapidly than those containing the wild-type propeptides. TheT m of the type I collagen homotrimer was similar to that of the heterotrimer, both values being about 42–43 °C when determined by circular dichroism. The wild-type proα2(I) chain formed no homotrimers. Replacement of the C propeptide of the proα2(I) chain with that of the proα1(I) chain or proα1 chain of type III procollagen (proα1(III) chain) led to the formation of homotrimers, but the α2(I) chains in such molecules were completely digested by pepsin in 1 h at 22 °C. The data thus suggest that, in addition to control at the level of the C propeptide, other restrictions may exist at the level of the collagen domain that prevent the formation of stable homotrimeric [proα2(I)]3 molecules in insect cells.