RECOMBINANT ALPHA-1(VIII) COLLAGEN CHAINS FORM HOMOTRIMERS IN-VITRO

Type Vm. collagen, a member of the short chain collagen family, is exp ressed in large blood vessels and in the subendothelium and mesangium of the glomerulus, While two genetically distinct type VIII collagen c hains, alpha 1 and alpha 2, have been identified, it is not known whet her type VIII collagen exists as homotrimers or heterotrimers. To dete rmine whether alpha 1(VIII) collagen chains can associate to form homo trimers, a full length human alpha 1(VIII) collagen cDNA was generated by overlap extension PCR and used as a substrate for coupled in vitro transcription/translation. A translation product of 80kDa, the predic ted size of alpha 1(VIII) collagen, was identified by autoradiography of SDS-gels containing radiolabelled translation products. Sensitivity of the in vitro translated protein to digestion with bacterial collag enase and trypsin and the size of the proteins generated by these dige stions provide evidence that alpha 1(VIII) collagen can participate in the formation of homotrimers. (C) 1996 Academic Press, Inc.