Identification of Noncollagenous Sites Encoding Specific Interactions and Quaternary Assembly of alpha 3 alpha 4 alpha 5(IV) Collagen IMPLICATIONS FOR ALPORT GENE THERAPY

Defective assembly of alpha 3 alpha 4 alpha 5(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 alpha 3 alpha 4 alpha 5(IV) collagen, correctly folded NC1 chimeras were produced, and their interactions with other NC1 monomers were evaluated. All alpha 1/alpha 5 chimeras containing alpha 5NC1 residues 188-227 replicated the ability of alpha 5NC1 to bind to alpha 3NC1 and co-assemble into NC1 hexamers. Conversely, substitution of alpha 5NC1 residues 188-227 by alpha 1NC1 abolished these quaternary interactions. The amino-terminal 58 residues of alpha 3NC1 encoded binding to alpha 5NC1, but this interaction was not sufficient for hexamer co-assembly. Because alpha 5NC1 residues 188-227 are necessary and sufficient for assembly into alpha 3 alpha 4 alpha 5NC1 hexamers, whereas the immunodominant alloantigenic sites of alpha 5NC1 do not encode specific quaternary interactions, the findings provide a basis for the rational design of less immunogenic alpha 5(IV) collagen constructs for the gene therapy of X-linked Alport patients.