Identification of noncollagenous sites encoding specific interactions and quaternary assembly of α3α4α5(IV) collagen: Implications for Alport gene therapy

Abstract

Defective assembly of α3α4α5(IV) collagen in the glomerular basement membrane causes Alport syndrome, a hereditary glomerulonephritis progressing to end-stage kidney failure. Assembly of collagen IV chains into heterotrimeric molecules and networks is driven by their noncollagenous (NC1) domains, but the sites encoding the specificity of these interactions are not known. To identify the sites directing quaternary assembly of α3α4α5(IV) collagen, correctly folded NC1 chimeras were produced, and their interactions with other NC1 monomers were evaluated. All α1/α5 chimeras containing α5NC1 residues 188-227 replicated the ability of α5NC1 to bind to α3NC1 and co-assemble into NC1 hexamers. Conversely, substitution of α5NC1 residues 188-227 by α1NC1 abolished these quaternary interactions. The amino-terminal 58 residues of α3NC1 encoded binding to α5NC1, but this interaction was not sufficient for hexamer co-assembly. Because α5NC1 residues 188-227 are necessary and sufficient for assembly into α3α4α5NC1 hexamers, whereas the immunodominant alloantigenic sites of α5NC1 do not encode specific quaternary interactions, the findings provide a basis for the rational design of less immunogenic α5(IV) collagen constructs for the gene therapy of X-linked Alport patients. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.