The α1(VIII) and α2(VIII) collagen chains form two distinct homotrimeric proteins in vivo

The short chain collagen variant, type VIII, is considered to be comprised of two distinct gene products, the alpha 1 and alpha 2 polypeptide chains. However, recent in vitro translation studies suggest that these chains can form homotrimers. We report here data from biochemical, immunohistochemical and molecular biological experiments, which together provide evidence that alpha 1 and alpha 2 polypeptides of type VIII collagen exist as homotrimers in cells and tissues. High-performance liquid chromatographic separation of type VIII collagen isolated from Descemet's membrane consistently demonstrated equimolar quantities of the two chains (alpha 1:alpha 2 1.03 +/- 0.02 (S.E.M.); n = 41). The availability of highly specific antibodies for the two polypeptides has assisted the in vivo characterisation of type VIII collagen. Immunoprecipitation of trimeric type VIII collagen from Descemet's membrane with purified anti-alpha 1(VIII) and anti-alpha 2(VIII) yielded fractions that contained only the alpha 1(VIII) and alpha 2(VIII) chains, respectively. Cultured human mesangial cells synthesised both polypeptides, but the alpha 1(VIII) chain was found exclusively in the cell pellet, while the media contained only the alpha 2(VIII) chain. The RNA from human mesangial cells and cornea showed message for both chains. However, in peritoneal fibroblast and mesothelial cell RNA, only alpha 1(VIII) mRNA was detectable, demonstrating that the transcription of these two genes was not always co-ordinated. Immunohistochemistry showed that both polypeptides were present in cornea, optic nerve, aorta and umbilical cord but did not always co-localise. These results indicate the alpha 1(VIII) and alpha 2(VIII) chains preferentially form pepsin-resistant, homotrimeric molecules and so can exist as two distinct proteins. (C) 2000 Elsevier Science B.V./International Society of Matrix Biology. All rights reserved.