Formation of Recombinant Triple-Helical [α1(IV)]2α2(IV) Collagen Molecules in CHO Cells

Abstract

Collagen IV molecules represent a major structural component of basement membranes providing a network of support for the supramolecular structure. Like other collagens, collagen IV forms a triple-helical molecule composed of three α chains. Six different α chains exist for collagen IV, although the most common isoform consists of two α1(IV) and one α2(IV) chain. To understand the molecular mechanism of triple-helical formation of collagen IV, we expressed recombinant α1(IV) and α2(IV) mouse collagen chains in Chinese hamster ovary (CHO) cells. An expression vector containing the full length cDNA for the mouse α1(IV) chain was stably transfected into CHO cells and a cell line, A222, which expressed recombinant α1(IV) chains was selected. These A222 cells were then infected with a retroviral expression vector containing the mouse α2(IV) chain and a cell line, A222-A2, stably expressing both recombinant α1(IV) and α2(IV) chains was obtained. Immunoprecipitation of A222 cell lysates revealed a high level of α1(IV) chain monomer, which was unable to form a homotrimer. Analysis of A222-A2 cell lysates revealed the presence of both monomeric α2(IV) and α1(IV) chains as well as a higher molecular weight collagen IV species. Second dimensional SDS-PAGE analysis demonstrated that the high molecular weight species was a heterotrimer consisting of two α1(IV) and one α2(IV) chain. This heterotrimer collagen IV species was pepsin-resistant indicating the formation of a stable triple-helical structure. Pulse-chase experiments showed that the monomer α1(IV) chain was secreted, but at a much slower rate than the heterotrimer. Together these results demonstrate that the α1(IV) chain is not capable of forming homotrimers and suggest that the coexpression with the α2(IV) chain is necessary to form a triple-helical structure.