Functional characterization of a novel GUCA1A missense mutation (D144G) in autosomal dominant cone dystrophy: A novel pathogenic GUCA1A variant in COD

Participants and clinical examination

Four generations of a family with adCOD were recruited at theQingdao eye hospital of Shandong Eye Institute (Qingdao,China). There were 16 members in the family (7 affected, 9 unaffected; 7 males, 9 females). The 7 affected patients included (I:I, male, 82 years; II:2, female, 63 years; II:3, male, 61 years; III:2, female, 38 years;III:3, male, 37 years; IV:2, female, 8 years; IV:3, male, 15 years).The 9 unaffected patients included (I:2, male, 70years; II:1, male, 65 years; II:4, male, 61 years; III:1, male, 39 years; III:4, male, 37 years; III:5, male, 35 years; III:6, female, 33 years; IV:1, female, 10 years; IV:4, female, 9 years).They all had no other systemic abnormalities. Two hundred individuals with no medical history associated with any ophthalmic diseases were included in the control group. All participants received a comprehensive ophthalmic examination, including measurement of visual acuity and intraocular pressure, color vision testing, fundoscopy, multifocal electroretinography, and optical coherence tomography (OCT). The study was conducted according to the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Qingdao University with informed consent from all participants. Written informed consent were obtained from all participants. The study was conducted according to the principles of the Declaration of Helsinki and ARVO statement for research involving human subjects.

Genetic testing

Peripheral venous blood (5 ml) of the participating family members was sampled, and genomic DNA was extracted with a DNA isolation kit for mammalian blood (Tiangen, Beijing, China). Venous blood and genomic DNA samples were stored at −80 °C before useas described [1]. Peripheral venous blood of the participating family members was sampled, and genomic DNA was extracted with a DNA isolation kit for mammalian blood (Tiangen, Beijing, China). The proband (III:2) underwent next-generation sequencing captured with 532 inherited genes related to the genetic visual system. The exon regions of these genes were particularly enriched with a biotinylated capture probe (Joy Orient Translational Medicine Research, Beijing, China). The exon regions of these genes were particularly enriched with a biotinylated capture probe (Joy Orient Translational Medicine Research, Beijing, China)as described [2]. Burrows-Wheeler Aligner (BWA) software was used to perform short-read mapping and alignment, while SOAPsnp software and GATK Indel Genotype were used to test single nucleotide polymorphism (SNP) and insertion and deletion mutations, respectively. Identified mutants were filtered in databases. Sanger sequencing verified whether any remaining variants were cosegregated with the family disease phenotype. The novel mutations in GUCA1A were also genotyped with Sanger sequencing in the 200 normal control subjects. The possible pathogenicity of the mutations was predicted using the Sorting Intolerant from Tolerant (SIFT) algorithm, Polymorphism Phenotyping v2 (PolyPhen-2), Protein Variation Effect Analyzer (PROVEAN), and MutationTaster.

Sequence alignment and structure modeling of GCAP1

The human GCAP1 protein (NP_000400.2) sequence was aligned for analysis of the conservation of the mutated residues with the sequences of the following orthologous proteins: Bostaurus (NP_776971), Daniorerio (NP_571945), Gallusgallus (NP_989651), Mus musculus (NP_032215), Rattusnorvegicus (NP_001100357), Takifugurubripes (NP_001027790), and Xenopustropicalis (NP_001096291). Multiple alignments were made using ClustalX2 software. The SWISS-MODEL was used to model the homology structure of the GCAP1 mutant based on the chicken wild-type (WT) GCAP1 (Protein Data Bank identifier: 2r2i), and three-dimensional (3D) models of proteins were constructed via PyMol software.

Preparation of plasmids and cloning, protein expression, and purification

Glutathione-S-transferase (GST)-tagged GCAP1 (GST-GCAP1) was generated via PCR amplification of human GCAP1 cDNA and inserted into pGEX-4T-1. After an initial denaturation step at 94 °C for 3 min, 35 PCRcycles (denaturation: 94 °C, 40 s; annealing: 52 °C, 40 s; extension: 72 °C, 2 min) and a final extension step at 72°C for 10 min were performed. For FLAG-tagging, full-length GCAP1 was inserted into pcDNA3.1. For green fluorescent protein (GFP)-tagging, full-length retGC1 was inserted into the pEGFP-N1 vector to obtain the recombinant plasmid pEGFP-retGC1. GCAP1-D144G was created with PCR amplification and confirmed with sequencing. The methods for expressing fusion proteins were the same as previously described [19].

Fusion proteins GCAP1-WT and GCAP1-D144G were expressed from the PGEX-4T-1 vector in BL21 (DE3) competent E. coli cells. The overexpressed protein was subsequently purified as described previously [20] with some modifications. Cells was grown in standard Luria-Bertani (LB) medium (Solarbio, Beijing, China) containing 100 µg/ml ampicillin at 1.0 l, until they reached A₆₀₀ 0.6–0.7. After induction with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) for 2 h, bacterial precipitation obtained with centrifugation at 956 ×g for 30 min at 4 °C was resuspended, and lysozyme was added into the buffer. The cells were then thawed and disrupted with ultrasonication, before 10 mg of streptomycin sulfate was added after the supernatant was centrifuged. The supernatant was centrifuged again, and 60 mg of glutathione agarose (Solarbio, Beijing, China) was added and incubated end-over-end for 2 h at 4 °C. The precipitate was washed with 30 ml of harvest buffer (1 M HEPES, 1 M NaCl, 1 mM benzamidine, and water) after 956 ×g centrifugation at 4 °C. The purity of the final purified GCAP1 protein determined with sodium dodecyl sulfate (SDS) gel electrophoresis was greater than or equal to 95%.

Cell culture and transfection

Human embryonic kidney (HEK)-293 cells (Appendix 1) were grown at 37 °C in 5% CO₂, incubated, and maintained in complete, high-glucose Dulbecco’s modified Eagle’s medium (HyClone, Logan, UT), which was supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. According to the manufacturer’s instructions, the cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). During the detection of protein stability, pcDNA3.1-GUCA1A-WT and pcDNA3.1-GUCA1A-D144G were transfected into the HEK-293 cells, and the protein supernatants were extracted for western blotting at 24, 48, and 72 h after transfection. To express retGC1 for the functional assay in vitro, the HEK-293 cells were transiently transfected with a 30 μg of pEGFP-retGC1 plasmid using Lipofectamine 2000 in a 90-mm culture dish according to the manufacturer’s instructions.

Coimmunoprecipitation and western blotting

Coimmunoprecipitation (co-IP) and western blotting were performed as previously reported [19]. Briefly, HEK-293 cells were transfected with equal amounts of pcDNA3.1-GUCA1A-WT or pcDNA3.1-GUCA1A-D144G in the presence or absence of pEGFP-retGC1, followed by lysis in 1 ml of ice-cold lysis buffer containing 10 mM HEPES, 100 mM NaCl, 1 mM benzamidine, and 0.5% Triton X-100 (pH 7.4). After centrifugation, the lysates were incubated with anti-GFP antibody with protein-A/G agarose (Beyotime Biotechnology, Haimen, China) for 3 h. After being washed with cold wash buffer (10 mM HEPES, 100 mM NaCl, 0.1% Tween-20, benzamidine, and 3% bovine serum albumin [BSA]) three times, the immunoprecipitated proteins were eluted from the beads with SDS sample loading buffer. The co-IP assay was performed using anti-GFP affinity gel, while the bound proteins were detected with immunoblotting with anti-FLAG antibody. Then, the eluted samples were separated with SDS–polyacrylamide gel electrophoresis (PAGE), visualized with western blotting, and quantified by NIH Image 1.62. Each sample was normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primary antibodies included anti-FLAG antibody (Proteintech Group, Chicago, IL), anti-GFP antibody (Proteintech Group), and anti-GAPDH antibody (Kangchen, Shanghai, China).

Limited protein hydrolysis of GCAP1

Limited protein hydrolysis of purified GCAP1 and GCAP1-D144G was observed in SDS–PAGE in the presence or absence of 2.5 μM Ca²⁺ as previously described [21]. Purified recombinant GCAP1 or GCAP1-D144G was incubated with trypsin at 30 °C, and the digest was analyzed with SDS-PAGE at 0, 5, 10, and 20 min.

Immunofluorescence staining

The HEK-293 cells were cotransfected with pEGFP-retGC1 and pcDNA3.1-GUCA1A-WT/D144G plasmid as previously reported. The following is a supplement to the protocol’sdetails. PEGFP-retGC1 and pcDNA3.1-GUCA1A-WT / D144G plasmids were co-transfected into HEK-293 cells with a GCAP1: retGC1 plasmid ratio of 1: 0.5. According to the reagent manufacturer's instructions, the cells were transfected with Lipofectamine 2000 The slides of the cells cultured in the standard glass coverslip chambers (four 2 cm² chambers per slide) were washed twice with PBS (1X; 8 mM Na₂HPO₄, 137 mM NaCl, 2mM KH₂PO₄, 2.6 mM, KCL, pH 7.2), fixed with 4% paraformaldehyde for 15 min, and washed again following permeation with 0.2% Triton X-100. After being blocked overnight with 5% normal goat serum, the samples were incubated with anti-FLAG antibody at 37 °C for 1 h, washed, and stained with a fluorescein-conjugated second antibody (donkey anti-rabbit immunoglobulin (IgG; H⁺L; Life Technologies, Carlsbad, CA) at 37 °C for 30 min. The cell nucleus was counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) for 5 min and then washed as mentioned above. Cells were observed using a fluorescence microscope (CKX53, Olympus, Tokyo, Japan).

ELISA

pcDNA3.1-GUCA1A-WT and pcDNA3.1-GUCA1A-D144G were transfected into the HEK-293 cells. The cell pellets were divided into groups of EGTA (1 μM EGTA) and Ca²⁺ (2.5 μM Ca²⁺) after they were homogenized in 300 µl of lysis buffer with the added protease inhibitor cocktail.

Human retGC1 was expressed in the HEK-293 cells, and the washed cell membranes were prepared as previously described [22]. HEK-293 cells were transiently transfected with pEGFP-retGC1 plasmid using Lipofectamine 2000 according to the manufacturer and purification of membrane fractions containing expressed cyclase as previously described [3]. Briefly, the membranes containing equal amounts of total protein were resuspended in GC buffer (100 mM KCl, 50 mM Mops, 7 mM 2-mercaptoethanol, 8 mM NaCl, 10 mM MgCl2, 1 mM EGTA). Cell membranes were incubated with equal amounts of purified GCAP1-WT/D144G proteins, and the cGMP concentrations were measured at different free Ca²⁺ levels using a Ca²⁺-EGTA buffer system (1 μM EGTA or 2.5 μM Ca²⁺).

Protein concentrations were standardized by extracting the protein and using a bicinchoninic acid (BCA) protein assay kit. Human cGMP competition enzyme-linked immunosorbent assay (ELISA; human cGMP ELISA Kit; Mlbio, Shanghai, China) was used to determine the cGMP levels following the manufacturer’s instructions.

Statistical analysis

SPSS 19.0 was used for statistical analysis, and quantitative data were analyzed by using the Student t test to compare groups. The data are presented as the mean ± standard deviation (SD), and a p value of less than 0.05 was considered statistically significant.

Clinical assessment and findings

Ophthalmic examination confirmed that seven of 16 members across the four generations of the sampled family were affected by adCOD (Figure 1). All patients had similar features, including progressive vision loss, photophobia, nystagmus, color vision impairment, and visual field impairment. The proband (III:2) was a female patient aged 43 years at the time of observation. Her best corrected visual acuity was oculus dexter (OD) 0.1 and oculus sinister (OS) 0.05. The fundus appearance (Figure 2A,B) and macular OCT (Figure 2C) were consistent with the diagnosis. Patient IV:3 was first seen at age 16. His best corrected visual acuity was OD 0.5 and OS 0.4. His fundoscopy (Figure 3A,B) and macular OCT (Figure 3C) results were also in accordance with the diagnosis. For IV:3, the multifocal electroretinography examination showed that the amplitude density of the left eye’s macular area was significantly reduced (Figure 3D). Humphrey visual field examination showed that the visual fields of both eyes were defected to different degrees (Figure 3E).

Identification of GUCA1A as a candidate gene

The proband (III:2) was genetically tested with exome sequencing of a set of candidate genes. The average sequence depth was 58.34, and 72.21% of the exon sequences were sequenced at least ten times. Sixty-two non-synonymous SNPs, two splicing sites, and one indel were selected according to the recommended filtering criteria. In exon 5 of GUCA1A, only a novel missense mutation (c.431A > G, p.D144G) was cosegregated in the family (Figure 4A and Table 1). This mutation was not found in the 200 control individuals. PolyPhen-2 and SIFT predicted that the D144G missense mutation would lead to harmful changes in the function of GUCA1A (Table 1).

D144 located in the EF4 hand loop was involved in calcium binding (Figure 4B,C). Multiple alignments of D144 of the GCAP1 protein from different species revealed 100% identification, which suggested it was highly conserved during evolution (Figure 4B). The homology structure of GCAP1-D144G (Figure 4C) showed that the hydrogen bonds between residue 144 and residue Phe140 or Gly147 in GCAP1 were eliminated due to substitution from Aspartyl (Asp) to Glycine (Gly) (Figure 4D).

Regulation of retGC1 by GCAP1-WT and GCAP1-D144G

We assayed the ability of GCAP1-WT and GCAP1-D144G to regulate retGC1in vitro (Figure 5A). The concentration of cGMP in the Ca²⁺ group became lower than that in the EGTA group after induction by GCAP1-WT (p<0.05), whereas it was higher than that in the EGTA group due to induction by GCAP1-D144G (p<0.01). GCAP1-D144G caused a statistically significant increase in the cGMP concentration compared to GCAP1-WT (p<0.01) in the presence of Ca²⁺. The same experimental result was also found in vitro (Figure 5B). GCAP1-D144G led to a higher increase of cGMP synthesis than did GCAP1-WT (p<0.01; Figure 5B).

Biochemical analysis of GCAP1-D144G

To detect the effect of GCAP1-D144G on the protein structure, limited protein hydrolysis experiments were performed. In the absence of Ca²⁺, GCAP1-WT was readily susceptible to proteolysis after 5 min of exposure to trypsin, while GCAP1-D144G was completely digested (Figure 6A), indicating the open conformation of GCAP1-WT and D144G. Furthermore, the tight core of GCAP1-WT remained after 20 min of digestion in the presence of Ca²⁺. Under the same condition, GCAP1-D144G was more susceptible to hydrolysis after 20 min of exposure to trypsin (Figure 6A). It indicated that GCAP1-WT exhibited a tighter conformation than D144G in the presence of Ca²⁺.

The electrophoresis shift assay showed that GCAP1-WT exhibited a higher degree of electrophoretic mobility than GCAP1-D144G in the presence of Ca²⁺ (Figure 6B), which was typical for calmodulin-like Ca²⁺ binding proteins (the faster-moving, inactive form). However, the same experiment with GCAP1-D144G highlighted slower mobility during the electrophoresis shift assay in the presence of Ca²⁺, indicating that the Ca²⁺ induction of the mutant conformation was less pronounced, indicating a significantly decreased affinity for Ca²⁺.

The expression levels and stability of GCAP1 in HEK-293 cells were analyzed with western blotting. With the extension of the transfection time, the GCAP1-WT protein levels remained almost unchanged (Figure 6C). However, the levels of GCAP1-D144G decreased statistically significantly in a time-dependent manner (Figure 6C). Even after 72 h of transfection, the expression levels of GCAP1-D144G were statistically significantly lower than those of GCAP1-WT (p<0.01; Figure 6C).

Combination and colocalization of retGC1 and GCAP1-WT or GCAP1-D144G

To determine whether GCAP1-WT/D144G was colocalized with retGC1, the HEK-293 cells were cotransfected with FLAG-GCAP1-WT/D144G and GFP-retGC1. GCAP1-WT and GCAP1-D144G exhibited a similar membrane distribution, predominantly endoplasmic reticulum membranes, when they were coexpressed with retGC1 (Figure 7A).

To test the ability of GCAP1-WT/D144G to bind to retGC1, co-IP experiments were performed. As shown in Figure 7B, the FLAG-GCAP1 protein was not detectable in the immunoprecipitated complex in the cells transfected with FLAG-GCAP1-WT or FLAG-GCAP1-D144G alone. In the cells cotransfected with FLAG-GCAP1 and GFP-retGC1, however, the FLAG-GCAP1 protein could be detected in its immunoprecipitated complex. GCAP1-D144G enhanced the association of GCAP1 with retGC1 in HEK-293 cells.