Evidence that the collagen in the culture medium of Chinese hamster lung cells contains components related at the primary structural level to the alpha1(V) collagen chain

The collagenous protein synthesized by cultured Chinese hamster lung (CHL) cells and present in the culture medium has been isolated after limited pepsin digestion and differential salt precipitation. Molecular size analysis of this material indicates that the CHL cell medium collagen contains chains which exhibit an apparent molecular mass of approximately 85,000 Da. When chromatographed on CM-cellulose under denaturing conditions, the reduced and alkylated CHL cell medium collagen chains elute slightly after the human alpha1(I) chain but well before the pepsin-derived alpha1(V) chain, which is the constituent chain present in the CHL cell cellular matrix collagen. Analysis of the peptides derived by CNBr cleavage of the CHL medium collagen chains by chromatography on CM-cellulose reveals, however, that these chains contain peptides which correspond both in size and in chemical properties to those derived from the alpha1(V) collagen chain, but clearly lack two peptides (alpha1(V)-CB4 and alpha1(V)-CB5) which are normally present in pepsin-derived alpha1(V) chains. Furthermore, analysis of the CHL cell culture medium collagenous material obtained without pepsin digestion indicates the presence of collagenous chains that exhibit after reduction a molecular mass of approximately 160,000 Da, which is smaller than the proposed size of the pro alpha1(V) collagen chain. These results demonstrate that the collagenous protein present in the culture medium of CHL cells is directly related at the primary structural level to the alpha1(V) collagen chain, and it is postulated that this material represents the large fragment derived from a collagenase cleavage of the [pro alpha1(V)]3 molecules present in the cell layer. Furthermore, these results and previous reports indicate that the only identifiable genetic type of procollagen chain synthesized by this cloned cell line in culture corresponds to the pro alpha1(V) chain.