A novel p.(Glu111Val) missense mutation in GUCA1A associated with cone-rod dystrophy leads to impaired calcium sensing and perturbed second messenger homeostasis in photoreceptors

Abstract

Guanylate Cyclase-Activating Protein 1 (GCAP1) regulates the enzymatic activity of the photoreceptor guanylate cyclases (GC), leading to inhibition or activation of the cyclic guanosine monophosphate (cGMP) synthesis depending on its Ca2+- or Mg2+-loaded state. By genetically screening a family of patients diagnosed with cone-rod dystrophy, we identified a novel missense mutation with autosomal dominant inheritance pattern (c.332A>T; p.(Glu111Val); E111V from now on) in the GUCA1A gene coding for GCAP1. We performed a thorough biochemical and biophysical investigation of wild type (WT) and E111V human GCAP1 by heterologous expression and purification of the recombinant proteins. The E111V substitution disrupts the coordination of the Ca2+ ion in the high-affinity site (EF-hand 3, EF3), thus significantly decreasing the ability of GCAP1 to sense Ca2+ (∼80-fold higher Kdapp compared to WT). Both WT and E111V GCAP1 form dimers independently on the presence of cations, but the E111V Mg2+-bound form is prone to severe aggregation over time. Molecular dynamics simulations suggest a significantly increased flexibility of both the EF3 and EF4 cation binding loops for the Ca2+-bound form of E111V GCAP1, in line with the decreased affinity for Ca2+. In contrast, a more rigid backbone conformation is observed in the Mg2+-bound state compared to the WT, which results in higher thermal stability. Functional assays confirm that E111V GCAP1 interacts with the target GC with a similar apparent affinity (EC50); however, the mutant shifts the GC inhibition out of the physiological [Ca2+] (IC50E111V ∼10 μM), thereby leading to the aberrant constitutive synthesis of cGMP under conditions of dark-adapted photoreceptors.

Constitutive Activation of Guanylate Cyclase by the G86R GCAP1 Variant Is Due to “Locking” Cation-π Interactions that Impair the Activator-to-Inhibitor Structural Transition

“…GCAP1, as well as other NCS operating in phototransduction, shows a typical mobility shift when the apo-form is compared with the Ca 2+ or Mg 2+ -loaded forms [24,35], therefore, we monitored the mobility of all GCAP1 variants characterized here in different Ca 2+ /Mg 2+ loading conditions. In line with previous findings [20,24], all variants displayed lower apparent molecular mass on sodium dodecyl-sulfate polyacrylamide electrophoresis (SDS-PAGE) upon Ca 2+ -binding ( Figure S1), independent on the presence of Mg 2+ . The lower apparent molecular mass of the four GCAP1 variants in the presence of Ca 2+ suggested a similar Ca 2+ -affinity, since the decrease in Ca 2+ -affinity was previously correlated with a less pronounced electrophoretic shift [20].…”

“…In line with previous findings [20,24], all variants displayed lower apparent molecular mass on sodium dodecyl-sulfate polyacrylamide electrophoresis (SDS-PAGE) upon Ca 2+ -binding ( Figure S1), independent on the presence of Mg 2+ . The lower apparent molecular mass of the four GCAP1 variants in the presence of Ca 2+ suggested a similar Ca 2+ -affinity, since the decrease in Ca 2+ -affinity was previously correlated with a less pronounced electrophoretic shift [20].…”

“…Detailed biochemical and biophysical studies reported a similar common molecular mechanism of dysregulation among the majority of GCAP1 variants [20,[27][28][29][30][31][32], namely, a reduced Ca 2+ -affinity due to mutations in EF3 and EF4. On the other hand, some disease-associated mutations resulted in an unaltered Ca 2+ -sensitivity [24,33], suggesting that GC dysregulation may arise from higher affinity for Mg 2+ or from alterations in the long-range allosteric communication between the EF-hands and the target interface [10].…”

“…Mutations in GUCA1A, encoding for GCAP1 result in aberrant GC regulation and in retinal degeneration, a set of progressive diseases involving cones, the macula, and in some cases rods, ultimately leading to blindness [11][12][13]. To date, 20 GCAP1 variants have been associated with retinal dystrophy [14][15][16][17][18][19][20][21][22][23][24][25], with one of the most recently discovered being the G86R mutation, located in the previously mentioned hinge region [26]. Biochemical experiments performed with the GC-GCAP1 reconstituted system in the presence of the G86R mutation showed that the complex forms with higher affinity than the WT and it is unable to decelerate the GC at high Ca 2+ -levels corresponding to the dark-adapted state [26].…”

GCAP neuronal calcium sensor proteins mediate photoreceptor cell death in the rd3 mouse model of LCA12 congenital blindness by involving endoplasmic reticulum stress

“…Synthesis of cyclic GMP (cGMP) is an essential process in photoreceptor cells of the retina, as cGMP is the signaltransducing molecule in the light response 1,2 . Mutations in a number of genes that impair or alter cGMP synthesis in rods and cones have been associated to different forms of blindness [3][4][5][6][7][8][9][10][11][12] . Loss-of-function mutations in the RD3 gene (name from the natural strain of "retinal degeneration 3" mice, rd3 locus mutated) cause Leber's congenital amaurosis 12 (LCA12) 13,14 .…”