Characterization of assembly of recombinant type IV collagen α3, α4, and α5 chains in transfected cell strains

Abstract

Background. Alport syndrome is caused by mutations in type IV collagen α3, α4, and α5 genes. Immunohistochemical analyses of kidney sections from normal individuals and Alport syndrome patients have suggested that the α3(IV), α4(IV), and α5(IV) chains form a heterotrimer in the glomerular basement membrane (GBM) and that a defect in any one of the chains disrupts the assembly of the three chains, resulting in Alport syndrome. Methods. We established stable transformants of HEK293 cells that expressed mouse α3(IV) and/or α4(IV) and/or α5(IV) chains. Using cell extracts and culture media of these cells, experiments were performed to determine whether or not the α3(IV) and α4(IV) chains were coimmunoprecipitated with the α5(IV) chain. Moreover, we examined complex formation of mutant α5(IV) chain containing either a deletion or substitution mutation with the α3(IV) and α4(IV) chains. Results. The established cell strains were named according to their transfected α(IV) chains. The α3(IV) and α4(IV) chains were coimmunoprecipitated with the α5(IV) chain in α345 cells but not in α35 and α45 cells. These chains were not coimmunoprecipitated with the α5(IV) chain, which lacked either a collagenous domain or NC1 domain. The ability of the α5(IV) chain with either a G1182R or C1573R substitution, corresponding to previously reported mutations in Alport syndrome patients, to form a complex with α3(IV) and α4(IV) chains was diminished. Conclusion. The findings indicate that α3(IV), α4(IV), and α5(IV) chains form a complex, which is a heterotrimer, and that a defect in complex formation might be one of the molecular mechanisms underlying the pathogenesis of Alport syndrome.