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Characterization of assembly of recombinant type IV collagen alpha3, alpha4, and alpha5 chains in transfected cell strains.

BACKGROUND
Alport syndrome is caused by mutations in type IV collagen alpha3, alpha4, and alpha5 genes. Immunohistochemical analyses of kidney sections from normal individuals and Alport syndrome patients have suggested that the alpha3(IV), alpha4(IV), and alpha5(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 alpha3(IV) and/or alpha4(IV) and/or alpha5(IV) chains. Using cell extracts and culture media of these cells, experiments were performed to determine whether or not the alpha3(IV) and alpha4(IV) chains were coimmunoprecipitated with the alpha5(IV) chain. Moreover, we examined complex formation of mutant alpha5(IV) chain containing either a deletion or substitution mutation with the alpha3(IV) and alpha4(IV) chains.


RESULTS
The established cell strains were named according to their transfected alpha(IV) chains. The alpha3(IV) and alpha4(IV) chains were coimmunoprecipitated with the alpha5(IV) chain in alpha345 cells but not in alpha35 and alpha45 cells. These chains were not coimmunoprecipitated with the alpha5(IV) chain, which lacked either a collagenous domain or NC1 domain. The ability of the alpha5(IV) chain with either a G1182R or C1573R substitution, corresponding to previously reported mutations in Alport syndrome patients, to form a complex with alpha3(IV) and alpha4(IV) chains was diminished.


CONCLUSION
The findings indicate that alpha3(IV), alpha4(IV), and alpha5(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.