title: Functional characterization of missense mutations at codon 838 in retinal guanylate cyclase correlates with disease severity in patients with autosomal dominant cone-rod dystrophy.
abstract: Three different mutations in codon 838 of GUCY2D, the gene for retinal guanylate 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 and 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 wild-type at low Ca(2+) concentrations. These four latter mutants showed a higher apparent affinity for GCAP1 than did wild-type. The Ca(2+) sensitivity of the GCAP1 activation was also altered with marked residual activity at high Ca(2+), the effect increasing: wild-type < R838C < R838H << R838A < R838S. Within the photoreceptor, this would result in a failure to inactivate cyclase activity at high physiological Ca(2+ )concentrations. Amongst the three disease-associated mutations, the effect correlates directly with disease severity. The wild-type and R838H mutant displayed a difference in pH sensitivity, 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 protein. A computer-aided structure prediction of this region indicates that R838 in the wild-type breaks the structure at four helical turns, and there is an increasing tendency for the structure to continue for further turns in the order R838C < R838H,S,K << R838E < R838A < R838L.
authors: Department of Molecular Genetics, Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, UK.
authors: S E
authors: SE
authors: Wilkie
authors: R J
authors: RJ
authors: Newbold
authors: E
authors: E
authors: Deery
authors: C E
authors: CE
authors: Walker
authors: I
authors: I
authors: Stinton
authors: V
authors: V
authors: Ramamurthy
authors: J B
authors: JB
authors: Hurley
authors: S S
authors: SS
authors: Bhattacharya
authors: M J
authors: MJ
authors: Warren
authors: D M
authors: DM
authors: Hunt
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002478:Cells, Cultured; D003062:Codon; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051604:Guanylate Cyclase-Activating Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012160:Retina / Q000201:enzymology; D012174:Retinitis Pigmentosa / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D012720:Severity of Illness Index
chemical_list: D000596:Amino Acids; D002135:Calcium-Binding Proteins; D003062:Codon; C496736:GUCA1A protein, human; D051604:Guanylate Cyclase-Activating Proteins; D011994:Recombinant Proteins; D006162:Guanylate Cyclase; D002118:Calcium