title: The alpha1(VIII) and alpha2(VIII) collagen chains form two distinct homotrimeric proteins in vivo.
abstract: The short chain collagen variant, type VIII, is considered to be comprised of two distinct gene products, the alpha1 and alpha2 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 alpha1 and alpha2 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 (alpha1:alpha2 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-alpha1(VIII) and anti-alpha2(VIII) yielded fractions that contained only the alpha1(VIII) and alpha2(VIII) chains, respectively. Cultured human mesangial cells synthesised both polypeptides, but the alpha1(VIII) chain was found exclusively in the cell pellet, while the media contained only the alpha2(VIII) chain. The RNA from human mesangial cells and cornea showed message for both chains. However, in peritoneal fibroblast and mesothelial cell RNA, only alpha1(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 alpha1(VIII) and alpha2(VIII) chains preferentially form pepsin-resistant, homotrimeric molecules and so can exist as two distinct proteins.
authors.affiliation: Renal Research Unit, Department of Medicine, Wellington School of Medicine, P.O. Box 7343, Wellington South, New Zealand.
authors.forename: N S
authors.initials: NS
authors.lastname: Greenhill
authors.forename: B M
authors.initials: BM
authors.lastname: Rüger
authors.forename: Q
authors.initials: Q
authors.lastname: Hasan
authors.forename: P F
authors.initials: PF
authors.lastname: Davis
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D003315:Cornea / Q000378:metabolism; D003886:Descemet Membrane / Q000737:chemistry; D006801:Humans; D011233:Precipitin Tests; D011817:Rabbits
chemical_list: D003094:Collagen