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. (30th August 2018)

• Record Type: Journal Article
• Authors: Marino, Valerio; Dal Cortivo, Giuditta; Oppici, Elisa; Maltese, Paolo Enrico; D'Esposito, Fabiana; Manara, Elena; Ziccardi, Lucia; Falsini, Benedetto; Magli, Adriano; Bertelli, Matteo; Dell'Orco, Daniele
• Is Part Of: Human molecular genetics. Volume 27:Number 24(2018:Dec. 15)
• Page Start: 4204
• Page End: 4217
• Publication Date: 2018-08-30
• Subjects: Human molecular genetics -- Periodicals; Human chromosome abnormalities -- Periodicals; 572.8
• DOI: 10.1093/hmg/ddy311
• Languages: English
• ISSNs: 0964-6906

Abstract

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 Ca 2+ - or Mg 2+ -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 Ca 2+ ion in the high-affinity site (EF-hand 3, EF3), thus significantly decreasing the ability of GCAP1 to sense Ca 2+ (∼80-fold higher Kd app compared to WT). Both WT and E111V GCAP1 form dimers independently on the presence of cations, but the E111V Mg 2+ -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 Ca 2+ -bound form of E111V GCAP1, in line with the decreased affinity for Ca 2+ . In contrast, a more rigid backbone conformation is observed in the Mg 2+ -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 [Ca 2+ ] (IC50 E111V ∼10 μM), thereby leading to the aberrant constitutive synthesis of cGMP under conditions of dark-adapted photoreceptors.