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

Valerio Marino, Giuditta Dal Cortivo, Elisa Oppici, Paolo Enrico Maltese, Fabiana D'Esposito, Elena Manara, Lucia Ziccardi, Benedetto Falsini, Adriano Magli, Matteo Bertelli, Daniele Dell'Orco

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)4204-4217
Number of pages14
JournalHuman Molecular Genetics
Volume27
Issue number24
DOIs
Publication statusPublished - Dec 15 2018

Fingerprint

Guanylate Cyclase-Activating Proteins
Second Messenger Systems
Missense Mutation
Homeostasis
Calcium
Guanylate Cyclase
EF Hand Motifs
Cyclic GMP
Cations
Inheritance Patterns
Aptitude
Molecular Dynamics Simulation
Cone-Rod Dystrophies
Recombinant Proteins
Hot Temperature
Ions

Cite this

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. / 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.

In: Human Molecular Genetics, Vol. 27, No. 24, 15.12.2018, p. 4204-4217.

Research output: Contribution to journalArticle

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. / 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. In: Human Molecular Genetics. 2018 ; Vol. 27, No. 24. pp. 4204-4217.
@article{3891d36776db4921aea8927075b729fb,
title = "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.",
author = "Valerio Marino and {Dal Cortivo}, Giuditta and Elisa Oppici and Maltese, {Paolo Enrico} and Fabiana D'Esposito and Elena Manara and Lucia Ziccardi and Benedetto Falsini and Adriano Magli and Matteo Bertelli and Daniele Dell'Orco",
year = "2018",
month = "12",
day = "15",
doi = "10.1093/hmg/ddy311",
language = "English",
volume = "27",
pages = "4204--4217",
journal = "Human Molecular Genetics",
issn = "0964-6906",
publisher = "Oxford University Press",
number = "24",

}

TY - JOUR

T1 - 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

AU - Marino, Valerio

AU - Dal Cortivo, Giuditta

AU - Oppici, Elisa

AU - Maltese, Paolo Enrico

AU - D'Esposito, Fabiana

AU - Manara, Elena

AU - Ziccardi, Lucia

AU - Falsini, Benedetto

AU - Magli, Adriano

AU - Bertelli, Matteo

AU - Dell'Orco, Daniele

PY - 2018/12/15

Y1 - 2018/12/15

N2 - 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.

AB - 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.

U2 - 10.1093/hmg/ddy311

DO - 10.1093/hmg/ddy311

M3 - Article

C2 - 30184081

VL - 27

SP - 4204

EP - 4217

JO - Human Molecular Genetics

JF - Human Molecular Genetics

SN - 0964-6906

IS - 24

ER -