Ns. Greenhill et al., The alpha 1(VIII) and alpha 2(VIII) collagen chains form two distinct homotrimeric proteins in vivo, MATRIX BIOL, 19(1), 2000, pp. 19-28
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 homotrimer
s in cells and tissues. High-performance liquid chromatographic separation
of type VIII collagen isolated from Descemet's membrane consistently demons
trated equimolar quantities of the two chains (alpha 1:alpha 2 1.03 +/- 0.0
2 (S.E.M.); n = 41). The availability of highly specific antibodies for the
two polypeptides has assisted the in vivo characterisation of type VIII co
llagen. Immunoprecipitation of trimeric type VIII collagen from Descemet's
membrane with purified anti-alpha 1(VIII) and anti-alpha 2(VIII) yielded fr
actions that contained only the alpha 1(VIII) and alpha 2(VIII) chains, res
pectively. Cultured human mesangial cells synthesised both polypeptides, bu
t the alpha 1(VIII) chain was found exclusively in the cell pellet, while t
he media contained only the alpha 2(VIII) chain. The RNA from human mesangi
al 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-loc
alise. These results indicate the alpha 1(VIII) and alpha 2(VIII) chains pr
eferentially form pepsin-resistant, homotrimeric molecules and so can exist
as two distinct proteins. (C) 2000 Elsevier Science B.V./International Soc
iety of Matrix Biology. All rights reserved.