Functional characterization of missense mutations at codon 838 in retinal guanylate cyclase correlates with disease severity in patients with autosomal dominant cone-rod dystrophy

Three different mutations in codon 838 of GUCY2D, the gene for retinal guan ylate cyclase 1, have been linked to autosomal dominant cone-rod dystrophy at the CORD6 locus. To examine the relationship between enzyme activity and disease severity, the three disease-causing substitutions (R838C, R838H an d R838S) and four artificial mutations (R838A, R838E, R838L and R838K) were generated. Assay of GCAP1-stimulated cyclase activity in vitro shows that, compared with wild-type, R838E, R838L and R838K possess only low activity, whereas R838A, R838C, R838H and R838S have activity equal or superior to w ild-type at low Ca2+ concentrations. These four latter mutants showed a hig her apparent affinity for GCAP1 than did wild-type. The Ca2+ sensitivity of the GCAP1 activation was also altered with marked residual activity at hig h Ca2+, the effect increasing: wildtype < R838C < R838H << R838A < R838S. W ithin the photoreceptor, this would result in a failure to inactivate cycla se activity at high physiological Ca2+ concentrations. Amongst the three di sease-associated mutations, the effect correlates directly with disease sev erity. The wild-type and R838H mutant displayed a difference in pH sensitiv ity, with the mutant showing a higher specific activity with pH > 6.0. Site 838 is in the dimerization domain that forms a coiled-coil in the active p rotein, A computer-aided structure prediction of this region indicates that R838 in the wild-type breaks the structure at four helical turns, and ther e is an increasing tendency for the structure to continue for further turns in the order R838C < R838H,S,K << R838E < R838A < R838L.