Functional analyses of mutant recessive GUCY2D allelesidentified in Leber congenital amaurosis patients: protein domaincomparisons and dominant negative effects

[摘要] Purpose: Recessive mutations in GUCY2D, the gene encoding theretinal guanylyl cyclase protein, RetGC-1, have been shown to causeLeber Congenital Amaurosis (LCA), a severe retinal dystrophy. Thepurpose of this study was to determine the functional consequences ofselected mutations in GUCY2Dlinked to LCA. The mutationsinvestigated in this study map to the catalytic domain (P858S, L954P)and the extracellular domain (C105Y, L325P) of RetGC-1.Methods: All four mutations were introduced into the in vitroexpression plasmid, pRC-CMV human RetGC-1, and expressed in HEK-293cells. We assayed the abilities of the mutant cyclases to generate cGMP(basal activity), and to be activated by guanylyl cyclase activatingproteins (GCAP-1 and GCAP-2). Additionally, we co-expressed thecatalytic domain mutations (P858S and L954P) with a wild-type allele totest for dominant negative effects on wild-type RetGC-1.Results: The P858S and L954P mutations, both in highly conservedresidues of the catalytic domain of RetGC-1, severely impair basal,GCAP-1, and GCAP-2 stimulated catalytic activity of the enzyme. Inaddition, when co-expressed with the wild-type allele, both catalyticdomain mutations act as dominant negative proteins and reduce theactivity of wild-type RetGC-1. The basal activities of the C105Y andL325P mutants are unaltered, but GCAP-1 and GCAP-2 stimulated cyclaseactivities are reduced approximately 50%.Conclusions: GUCY2D mutations from LCA patients have distinctfunctional consequences on RetGC-1 catalytic activity in vitro. Ouranalyses showed that the catalytic domain mutations cause a markedreduction in cyclase activity, while the extracellular domain mutationsmoderately reduce activity. The catalytic domain mutant alleles causedominant negative effects, indicating that the functionality of RetGC-1is compromised even in heterozygotes. This is consistent withabnormalities in cone electroretinograms (ERGs) detected in obligateheterozygous GUCY2D parents that carry the L954P mutation.

[学科分类] 生物化学/生物物理