|Molecular Vision 2004;
Received 29 January 2004 | Accepted 16 April 2004 | Published 20 April 2004
Functional analyses of mutant recessive GUCY2D alleles identified in Leber congenital amaurosis patients: protein domain comparisons and dominant negative effects
Chandra L. Tucker,1 Visvanathan Ramamurthy,1
Ana-Luisa Pina,2 Magali Loyer,2 Sharola Dharmaraj,3
Yingying Li,3 Irene H. Maumenee,3
James B. Hurley,1
(The first two authors contributed equally to this publication)
1Department of Biochemistry, University of Washington, Seattle, WA; 2McGill Ocular Genetics Laboratory, Montreal Children's Hospital Research Institute, McGill University, Montreal, Canada; 3Johns Hopkins Center for Hereditary Eye Diseases, Johns Hopkins University, Baltimore, MD
Correspondence to: Robert K. Koenekoop, MD, PhD, Ophthalmology, Montreal Children's Hospital, 2300 Tupper, Montreal, PQ, Canada, H3H 1P3; Phone: (514) 412-4400, ext 22891; FAX: (514) 412-4443; email: firstname.lastname@example.org
Purpose: Recessive mutations in GUCY2D, the gene encoding the retinal guanylyl cyclase protein, RetGC-1, have been shown to cause Leber Congenital Amaurosis (LCA), a severe retinal dystrophy. The purpose of this study was to determine the functional consequences of selected mutations in GUCY2Dlinked to LCA. The mutations investigated in this study map to the catalytic domain (P858S, L954P) and the extracellular domain (C105Y, L325P) of RetGC-1.
Methods: All four mutations were introduced into the in vitro expression plasmid, pRC-CMV human RetGC-1, and expressed in HEK-293 cells. We assayed the abilities of the mutant cyclases to generate cGMP (basal activity), and to be activated by guanylyl cyclase activating proteins (GCAP-1 and GCAP-2). Additionally, we co-expressed the catalytic domain mutations (P858S and L954P) with a wild-type allele to test for dominant negative effects on wild-type RetGC-1.
Results: The P858S and L954P mutations, both in highly conserved residues of the catalytic domain of RetGC-1, severely impair basal, GCAP-1, and GCAP-2 stimulated catalytic activity of the enzyme. In addition, when co-expressed with the wild-type allele, both catalytic domain mutations act as dominant negative proteins and reduce the activity of wild-type RetGC-1. The basal activities of the C105Y and L325P mutants are unaltered, but GCAP-1 and GCAP-2 stimulated cyclase activities are reduced approximately 50%.
Conclusions: GUCY2D mutations from LCA patients have distinct functional consequences on RetGC-1 catalytic activity in vitro. Our analyses showed that the catalytic domain mutations cause a marked reduction in cyclase activity, while the extracellular domain mutations moderately reduce activity. The catalytic domain mutant alleles cause dominant negative effects, indicating that the functionality of RetGC-1 is compromised even in heterozygotes. This is consistent with abnormalities in cone electroretinograms (ERGs) detected in obligate heterozygous GUCY2D parents that carry the L954P mutation.
Leber congenital amaurosis (LCA; OMIM 204000) is an autosomal recessive group of diseases that represent the severest retinal dystrophies leading to congenital blindness in children . Although rare, with a worldwide prevalence of 3 in 100,000 births, it represents 5% of all retinal dystrophies . The great majority of LCA is transmitted in an autosomal recessive fashion , while dominant inheritance has rarely been reported [3,4]. LCA is genetically heterogeneous; since 1996, six genes with disparate retinal functions have been implicated, while three additional loci have been mapped to 1p36, 6q11-16, and 14q24.
The first LCA gene to be identified was GUCY2D, which encodes the photoreceptor membrane specific guanylyl cyclase RetGC-1 . Strong evidence that defects in GUCY2D cause LCA comes from linkage studies that used homozygosity mapping to map a gene for LCA to 17p13.1 , and studies reporting recessive frameshift mutations in GUCY2D in four unrelated LCA probands from families who showed linkage to the GUCY2D locus on 17p13.1 . In some populations, GUCY2D may be the most common LCA gene, with as many as 20% of LCA patients carrying GUCY2D mutations . Heterozygous defects in GUCY2D have been linked to human dominant cone-rod dystrophy (CORD6) [8,9] and a homozygous defective GUCY2D gene was found in the rd chicken .
RetGC-1, the protein encoded byGUCY2D, is expressed in photoreceptor outer segments . It shares structural similarities with other surface receptor guanylyl cyclases, containing an N-terminal extracellular domain (ECD), a single membrane-spanning domain, an intracellular kinase homology domain (KHD), and a catalytic domain (CD). While related membrane guanylyl cyclases have ligands that bind to the extracellular domain, the function of the extracellular domain of RetGC-1 is unknown, as it lacks a known ligand. The ECD of RetGC-1 is less conserved than the catalytic domain or the KHD, but does contain highly conserved cysteines, which are thought to be important for the structure of ECD and oligomerization of various membrane guanylyl cyclases .
RetGC-1 and a second photoreceptor guanylyl cyclase, RetGC-2, [13,14] function to replenish cGMP in the retina after it has been depleted by light-activated phosphodiesterase . Photoisomerization of rhodopsin initiates a cascade of events leading to depletion of cGMP in outer segments of the photoreceptor and closure of cGMP-gated Na+ / Ca2+ channels in the plasma membrane. This causes a drop in intracellular Ca2+ and Na+ concentrations, hyperpolarization of the cell, and reduction of neurotransmitter release. Restoration of cGMP in photoreceptors is initiated by this drop in intracellular Ca2+. Two Ca2+ binding proteins, GCAP-1 and GCAP-2, stimulate RetGC-1 at lowered Ca2+ concentrations to restore levels of cGMP [16-19]. This reopens the cGMP-gated channels, depolarizes the photoreceptor, and restores signaling.
Because of the severity of the mutations found in GUCY2D , it was hypothesized that the retinal blindness of LCA patients with GUCY2D mutations is caused by absent or severely reduced levels of cGMP. In agreement with this, the F514S point mutation from the kinase homology domain (KHD) of RetGC-1 associated with LCA was found to severely compromise the ability of the protein to generate cGMP and also damaged the response to GCAP-1 . In a separate study, a majority of missense mutations in the catalytic domain (CD) resulted in compromised cyclase activity. All ECD mutations characterized in that study had normal guanylyl cyclase activity .
We recently screened the GUCY2D coding region in LCA families and identified ten new mutations . These mutations were identified in small nuclear families with 1 sporadic case, which precluded linkage analysis, but allowed a mutation screening strategy of the entire coding sequence of GUCY2D. In the absence of linkage data, we used a set of well established criteria to establish the relation between the mutation and the disease. These included: 1. Exclusion of the mutation in 100 normal controls from the same ethnic background as the patient, 2. Conservation of the predicted mutant amino-acid across other species, 3. The biochemical significance of the predicted amino-acid change to the protein, 4. The presence of two mutations, one paternal, and one maternal, either in homozygous or compound heterozygous state, and 5. Co-segregation of the mutations in the small pedigree. We found four LCA patients with mutations that meet these criteria. The patients have the following genotypes: patient 1: L954P and S981 1 bp del (G); patient 2: C105Y and L325P; patient 3: -18 5' UTR 1 bp del (G) in homozygous state, and patient 4: with heterozygous P858S.
We examined the effects of these point mutations on RetGC-1 activity in vitro. In this paper, we show that they are deleterious to RetGC-1 activity and that the two mutations in the catalytic domain of RetGC-1 (L954P and P858S) act in a dominant-negative manner to reduce wild-type RetGC-1 activity. Furthermore, we show that the ECD mutation in the highly conserved cysteine, C105Y, and L325P reduce the cyclase activity by 50%.
GUCY2D mutations and definition of LCA
All GUCY2D mutations were initially identified in patients affected with LCA. Our definition of LCA is central visual acuity loss at birth or in the first 6 months of life, wandering nystagmus, amaurotic pupils, and severely attenuated or non-detectable rod and cone electroretinograms in the affected patients. The CD L954P missense (maternal) mutation was found in compound heterozygous state with a S981 1 bp del (G) frameshift (paternal) mutation (bp 2843 del G) also in exon 15 in the same LCA patient. From the extracellular domain, the missense mutations, C105Y and L325P, were found in compound heterozygous state in a second LCA patient. The ECD mutation 5' UTR -18 1 bp del (del G) was found in homozygous state in a third LCA patient. The P858S missense mutation was found in heterozygous state in a fourth LCA patient, in whom a second mutation has not yet been identified.
Generation of mutations
To generate catalytic domain mutations, signal overlap extension PCR  was used to introduce the desired point mutations (L954P, P858S) into RetGC-1. A 700 bp fragment containing the mutation flanked by Aat II and Nde I recognition cut sites was subcloned into pBluescript (Stratagene) containing the full-length RetGC-1 cDNA in which the wild-type sequence between these sites had been removed. Clones were sequenced in the replaced region, and the entire RetGC-1 cDNA (3.6 kb) was excised with Hind III/Xba I and subcloned into the eukaryotic expression plasmid pRC-CMV (Invitrogen). Extracellular Domain (ECD) mutations were created by single-stranded mutagenesis . In brief, single-stranded DNA was made from pBluescript containing a 1.35 Kb N-terminal RetGC-1 fragment. The second strand DNA was synthesized using a mutant oligonucleotide. The 677 bp HindIII/BstEII fragment containing the mutant fragment was used to replace the corresponding wild-type sequence in plasmid pRC-CMV RetGC-1. To avoid undesirable changes, all generated mutations were confirmed by uni-directional DNA sequencing of the entire exonic coding and flanking sequences, in which the mutation occurs, using the published intronic GUCY2D primers .
Expression in HEK 293 cells
Constructs were transiently transfected via a calcium phosphate method  into human embryonic kidney (HEK) 293 cells. Cells were harvested after 48 h, washed with PBS, and removed from dishes by agitation in PBS with 0.2% EGTA. Cells were pelleted gently by centrifugation and pellets were swollen in homogenization buffer (10 mM Tris pH 7.5, 5 mM MgCl2, and 5 mM 2-mercaptoethanol) for 10 min. Four strokes of a 26 gauge needle were used to lyse cells. Cell lysates were pelleted at 2000 rpm at 4 °C in a tabletop microcentrifuge to remove large debris. The supernatant from this spin was pelleted at 14000 rpm, resuspended in homogenization buffer, then frozen at -70 °C in aliquots for use during assays.
Total membrane proteins from transfected HEK 293 cells were electrophoresed on a 7.5% SDS-PAGE gel and transferred to nitrocellulose membranes. Westerns were performed using a polyclonal antibody that recognizes the KHD of RetGC-1 .
Measurement of guanylyl cyclase activity
Membranes from transiently transfected HEK 293 cells containing equal amounts of total protein were resuspended in GC buffer (100 mM KCl, 50 mM MOPS, 7 mM 2-mercaptoethanol, 10 mM MgCl2, 8 mM NaCl, 1 mM EGTA). All reactions also contained 0.5 mM ATP. Measurement of guanylyl cyclase activity was carried out at 30 °C for the indicated times essentially as previously described  In short, this method uses the radiolabeled substrate a-32P-GTP and then detects the amount of 32P-cGMP formed. Spots corresponding to cGMP are visualized on a short wavelength UV illuminator, and both 3H and 32P can be counted in a scintillation counter. Stimulated reactions contained recombinant myristoylated GCAP-1 or GCAP-2 . Measurement of Mn2+/Triton X-100 activity contained 1% Triton X-100 and 10 mM MnCl2 instead of MgCl2. All experiments shown were repeated three times with similar results.
All mutations in GUCY2D mentioned in this work have been previously reported , except S981 1 bp del (G), which was found in compound heterozygous state with the L954P mutation. The S981 1 bp del represents a deletion of the last base-pair of exon 15 (CACTCGGgtaatcc; bp 2943 del G) and is predicted to cause a frameshift. A recent study reported that the same change (bp 2943 del G) in RetGC-1 was found to be associated with LCA in a homozygous state .
Catalytic domain mutants
We examined the biochemical consequences of the catalytic domain mutations, P858S and L954P, on RetGC-1 activity in vitro. RetGC-1 is activated in the retina by GCAPs, EF-hand calcium binding proteins that stimulate RetGC-1 at calcium concentrations below 300 nM and inhibit RetGC-1 at micromolar concentrations . In addition, RetGC-1 and all other membrane guanylyl cyclases can be activated by Mn2+ and Triton X-100. In this study we used Mn2+/Triton activation as a direct measurement of competence of the catalytic domain . Figure 1 shows basal, GCAP-1, GCAP-2, and Mn2+/Triton stimulated activities of wild-type and mutant constructs. Basal, GCAP-stimulated, and Mn2+/Triton X-100 activities of P858S and L954P were all severely reduced, indicating that the catalytic abilities of the enzymes are greatly impaired, with the L954P mutation appearing more deleterious. With 17 μM GCAP-1 or GCAP-2, the proteins retain some responsiveness to GCAPs (10 fold stimulation over basal activity for P858S, four-fold for L954P), but this is very slight compared to wild-type stimulation (90-fold over basal activity). To determine expression levels, equal amounts of total membrane protein were probed with an antibody to the RetGC-1 kinase homology domain . Both mutants expressed equivalently to wild-type RetGC-1 (Figure 1, inset).
Membrane guanylyl cyclases are thought to exist in a dimeric state [32-36]. Recent modeling of the catalytic domain of RetGC-1 indicated an active catalytic site is formed from two head-to-tail oriented catalytic domain monomers [37,38] and the model has been confirmed by biochemical analysis . Mutations in one monomer can cause dominant negative effects on the other half .
We therefore tested whether the P858S and L954P mutants affect wild-type RetGC-1 activity when co-expressed (Figure 2). HEK 293 cells were co-transfected with 2.5 μg wild-type RetGC-1 and 2.5 or 5 μg of either pRC-CMV (as a control), P858S, or L954P. The total amount of DNA transfected was kept constant by the addition of pRC-CMV to a total of 15 μg. Western blots (Figure 2A) show that the total amount of wild-type and mutant protein transfected correlates with the amount of protein used in the assays. The addition of increasing amounts of either P858S or L954P reduced GCAP-2 stimulated activity by up to 55% (Figure 2B). P858S and L954P similarly reduced Mn2+/Triton X-100 stimulated activity of wild-type RetGC-1 by approximately 40% (Figure 2C), showing that this represents a reduction in general catalytic ability, not affinity for GCAP-2.
These results showing a significant decrease in wild-type RetGC-1 activity when L954P or P858S are coexpressed suggest that any heterodimers formed are inactive or poorly active. If the proteins were fully active as a heteromer, activity should be increased. If the mutants were not expressed or not dimerizing, total RetGC-1 activity would remain constant in these experiments, reflecting unhindered wild-type RetGC-1 activity.
Extracellular domain mutants
We tested the basal, Mn2+/Triton X-100 and GCAP-stimulated cyclase activities of versions of RetGC-1 containing the extracellular domain mutations, C105Y and L325P, and the 5' UTR mutation (Figure 3). Western blots with an antibody to the KHD showed that all three mutants expressed comparably to wild-type RetGC-1 (Figure 3, inset). Basal activities of C105Y, L325P, and 5' UTR -18 1 bp del were normal compared to wild-type RetGC-1, indicating that the catalytic abilities of the enzymes were intact. However, the C105Y and L325 mutants show reduced responsiveness compared to wild-type with Mn2+/Triton X-100 or with 17 μM GCAP-1 or GCAP-2. The C105Y mutation appears more deleterious than L325P in the presence of the GCAPs. Co-expression of L325P and C105Y reduced the levels of cyclase activity similar to L325P alone (Figure 3). The activity of the 5' UTR 1 bp del RetGC-1 mutant in the presence or absence of the activators was similar to that of wild-type RetGC-1.
In this study, we describe the biochemical consequences of four mutations found in the GUCY2D gene from three patients with typical features of LCA: poor fixation noted during the first six months of life, sensory nystagmus, poor pupillary light reflex, essentially normal retinal appearance, with mild vascular attenuation, hyperopic refractions, abolished ERGs (performed early in the disease process), stable visual evolution, and normal sighted parents. In the first patient, a missense mutation was identified that results in a substitution of L954P in the catalytic domain of the corresponding protein, RetGC-1. This was found in compound heterozygous state with a S981 1 bp del frameshift mutation, which was not tested in our experiments. The frameshift would result in a truncated version of RetGC-1, eliminating critical amino acid residues necessary for catalytic activity [37,38]. In a second patient, a missense mutation causing the substitution C105Y in the extracellular domain of RetGC-1 was found in compound heterozygous state with L325P, also in the extracellular domain. The last patient contained a mutation causing the substitution P858S in the catalytic domain, while a second mutation could not be identified.
The extracellular domain mutations, C105Y and L325P, result in a decrease of at least 50% of cGMP production by RetGC-1 when stimulated by GCAP-1, GCAP-2 and Mn2+/Triton X-100, although basal activity is unaffected. RetGC-1 is regulated by GCAP through the intracellular domain, and removal of the extracellular domain does not have a significant effect on regulation by GCAP, Ca2+, or ATP . Unlike previously characterized LCA-associated extracellular domain mutations that showed no effect on cyclase activity , L325P and C105Y reduced the cyclase activity by half. The exact cause for this decrease in activity is unknown to us at present. Cys105 is one of six highly conserved cysteine residues in the ECD of cyclases. These residues have been suggested to be involved in intramolecular disulfide bond formation and in oligomerization of the protein . Leu325 is another highly conserved position that is typically occupied by a hydrophobic residue. It is possible that the ECD mutations may cause alterations in structure or stability of the protein, such that the oligomerization of cyclase is affected.
The 5' UTR -18 1 bp del did not show any alteration in cyclase activity in our experimental system. This is not surprising, as this mutation was expected to reduce the levels of mRNA. However, our expression system uses a strong CMV promoter that would most likely mask this effect.
In contrast to the extracellular domain mutations, mutations from the catalytic domain (L954P and P858S) severely diminished basal catalytic enzyme function, and severely compromised the ability of GCAP to stimulate cGMP production. Pro858 and Leu954 are both absolutely conserved in all of the membrane and soluble forms of guanylyl cyclase catalytic domains. Leu954 or a hydrophobic counterpart (Met in AC V and Val in AC VI) is also conserved in the C1 domain of adenylate cyclases. In a catalytic domain model of RetGC-1 [37,38], Leu954 is positioned in the center of an alpha-helical region on the outer surface, which corresponds to the alpha3 loop of adenylyl cyclase (Figure 4). The insertion of a proline into this region would likely create a bend in this structure and be deleterious for the helical segment. Pro858 is the last amino acid at the end of a predicted amphipathic coil, which is thought to form a dimerization domain (Figure 4). Since Pro858 and its adjacent amino acid, Pro859, likely play roles in terminating the coil and turning the chain, the removal of a proline at this site could drastically change the orientation of the subsequent secondary structure.
The P858S and L954P RetGC-1 variants not only had severely reduced catalytic activities themselves, but they were able to act in a dominant negative fashion to reduce activity of wild-type RetGC-1 when coexpressed. Since previous experiments suggest that such dominant negative activity reflects formation of inactive mutant/wild-type dimers , P858S and L954P likely retain the abilities to dimerize. Due to this dominant negative effect, a heterozygote with one mutant and one wild-type allele would be predicted to have approximately 25% of wild-type RetGC-1 activity.
In light of these in vitro dominant negative findings, we found it curious that the heterozygous carrier parents appeared to tolerate a large reduction of RetGC-1 activity (perhaps a 75% reduction of normal) without developing overt disease. We thus tested the possibility that the carrier parents had in vivo dominant negative effects. ERGs were performed on the mother who carries the L954P substitution and the father who carries the 2843 1 bp del in GUCY2D . We found normal rod responses, but significant and repeatable cone ERG abnormalities in both parents . Our findings were most consistent with a mild cone dysfunction, which correlates well with the expression profile of GUCY2D, which is much higher in cones than in rods [11,16] and with the GUCY2D knockout mouse, which develops a cone dystrophy .
These results indicate that at both the biochemical and clinical level, the obligate carriers of LCA mutations have a phenotype, distinguishable from the wild-type. Other recent studies have also shown a phenotype in heterozygous carriers. Autosomal recessive mutations are well known to cause ataxia telangiectasia (AT). AT carriers have a significant decrease in the expression levels of many genes compared to normals, and the carriers indeed have an "expression phenotype" .
Based on the initial frameshift mutations in GUCY2D , and the deletion in the RetGC-1 gene of the rd/rd chicken , which is a model for human LCA, it was postulated that cGMP production is abolished in the retinas of LCA patients. This was supported by the absence of immunoreactive RetGC-1 in the retinas of the rd/rd chickens . Consistent with this, the patient with the heterozygous L954P substitution and the frameshift mutation is predicted to have essentially abolished or very limited RetGC-1 activity. Although we have been unable to identify a second mutation in the patient with the P858S substitution that would abolish cyclase activity, the severity of the P858S substitution in our biochemical studies (predicted to reduce RetGC-1 activity in this patient by about 75%) strongly supports a major role in LCA. Due to the severity of the P858S substitution, a second mutation, perhaps even a polymorphism that causes only a slight reduction in RetGC-1 activity, stability, expression, or regulation may be sufficient to cause the disease phenotype.
In contrast to the severe catalytic domain mutations, the mutations from the extracellular domain appear much milder. This is not reflected in the phenotype of the children, which is severe visual loss. Establishing the cause of LCA in these children will require further investigation.
We would like to thank all the patients and their families for their support and cooperation. This study was supported in part by a Montreal Children's Hospital Research Institute establishment fund, the E. Mildred Kanigsberg Estate Fund, a FRSQ Chercheur boursier Junior 1 award (970260-103), a Foundation Fighting Blindness of Canada grant and a CIHR operating grant to RKK. We are grateful for a Foundation Fighting Blindness Canada grant to ALP, and a National Institutes of Health grant (EY06641) to JBH, and we thank The James Adams Special Scholar Award of Research to Prevent Blindness to JBH. The Grousbeck Family Foundation, the Edel & Krieble Funds of the Johns Hopkins Center for Hereditary Eye Disease, and the Foundation for Retinal Research are acknowledged for their support of IHM.
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