Mutations In Carbonic Anhydrase Research Articles

Molecular characterization of a carbon dioxide-dependent Proteus mirabilis small-colony variant isolated from a clinical specimen

IntroductionCarbon dioxide-dependent Proteus mirabilis has been isolated from clinical specimens. It is not clear whether mutations in carbonic anhydrase are responsible for the carbon dioxide dependence of P. mirabilis. The pathogenicity of carbon dioxide-dependent P. mirabilis also remains unclear. The purpose of this study was to determine the cause carbon dioxide dependence of P. mirabilis and its pathogenicity. MethodsThe DNA sequence of can encoding carbonic anhydrase of a carbon dioxide-dependent P. mirabilis small colony variant (SCV) isolate was analyzed. To confirm that impaired carbonic anhydrase activity is responsible for the formation of the carbon dioxide-dependent SCV phenotype of P. mirabilis, we performed complementation experiments using plasmids with intact can. Additionally, mouse infection experiments were performed to confirm the change in virulence due to the mutation of carbonic anhydrase. ResultsWe found that the can gene of the carbon dioxide-dependent P. mirabilis SCV isolate showed had a frameshift mutation with a deletion of 1 bp (c. 173delC). The can of P. mirabilis encodes carbonic anhydrase was also found to function in Escherichia coli. The cause of the carbon dioxide-dependent SCV phenotype of P. mirabilis was an abnormality in carbonic anhydrase. Nevertheless, no changes were observed in virulence due to the mutation of carbonic anhydrase in mouse infection experiments. ConclusionsThe can gene is essential for the growth of P. mirabilis in ambient air. The mechanisms underlying this fitness advantage in terms of infection warrant further investigation.

Natural History and Clinical Manifestations of Hyponatremia and Hyperchlorhidrosis due to Carbonic Anhydrase XII Deficiency

Introduction: We identified patients of Bedouin origin with a mutation in carbonic anhydrase XII (CA XII) leading to hyponatremia due to excessive salt loss via sweat. Methods: The medical records of patients were reviewed for clinical and laboratory data. Results: A total of 11 subjects were identified; 7 symptomatic patients presented with hyponatremic dehydration in infancy. Screening of the entire kindred identified 4 asymptomatic individuals with elevated sweat chloride. All symptomatic patients had failure to thrive and moderate-severe hyponatremia (106-124 mmol·l^-1); 6 had hypochloremia (79-94 mmol·l^-1). All asymptomatic subjects had normal or near-normal serum sodium and chloride concentrations. Both symptomatic and asymptomatic subjects had normal renal functions and normal cortisol response on low-dose ACTH test. All symptomatic patients were treated by dietary salt, which prevents episodes of hyponatremic dehydration and promotes growth. At follow-up, the chief complaints remained heat intolerance, accumulation of salt precipitates on the face and hyperhidrosis. No evidence for chronic renal, respiratory, gastrointestinal or fertility abnormalities was found. Conclusion: Recognizing this newly described entity and differentiating it from cystic fibrosis and pseudohypoaldosteronism are important. Patients with CA XII mutations should be followed even after early childhood, especially in hot temperatures and intense physical activity.

Autosomal recessive hyponatremia due to isolated salt wasting in sweat associated with a mutation in the active site of Carbonic Anhydrase 12

Genetic disorders of excessive salt loss from sweat glands have been observed in pseudohypoaldosteronism type I (PHA) and cystic fibrosis that result from mutations in genes encoding epithelial Na+ channel (ENaC) subunits and the transmembrane conductance regulator (CFTR), respectively. We identified a novel autosomal recessive form of isolated salt wasting in sweat, which leads to severe infantile hyponatremic dehydration. Three affected individuals from a small Bedouin clan presented with failure to thrive, hyponatremic dehydration and hyperkalemia with isolated sweat salt wasting. Using positional cloning, we identified the association of a Glu143Lys mutation in carbonic anhydrase 12 (CA12) with the disease. Carbonic anhydrase is a zinc metalloenzyme that catalyzes the reversible hydration of carbon dioxide to form a bicarbonate anion and a proton. Glu143 in CA12 is essential for zinc coordination in this metalloenzyme and lowering of the protein-metal affinity reduces its catalytic activity. This is the first presentation of an isolated loss of salt from sweat gland mimicking PHA, associated with a mutation in the CA12 gene not previously implicated in human disorders. Our data demonstrate the importance of bicarbonate anion and proton production on salt concentration in sweat and its significance for sodium homeostasis.

Pathogenesis of retinitis pigmentosa associated with apoptosis-inducing mutations in carbonic anhydrase IV

Missense mutations in the carbonic anhydrase IV (CA IV) gene have been identified in patients with an autosomal dominant form of retinitis pigmentosa (RP17). We used two transient expression systems to investigate the molecular mechanism by which the newly identified CA IV mutations, R69H and R219S, contribute to retinal pathogenesis. Although the R219S mutation drastically reduced the activity of the enzyme, the R69H mutation had a minimal effect, suggesting that loss of CA activity is not the molecular basis for their pathogenesis. Defective processing was apparent for both mutant proteins. Cell surface-labeling techniques showed that the R69H and R219S mutations both impaired the trafficking of CA IV to the cell surface, resulting in their abnormal intracellular retention. Expression of both CA IV mutants induced elevated levels of the endoplasmic reticulum (ER) stress markers, BiP and CHOP, and led to cell death by apoptosis. They also had a dominant-negative effect on the secretory function of the ER. These properties are similar to those of R14W CA IV, the signal sequence variant found in the original patients with RP17. These findings suggest that toxic gain of function involving ER stress-induced apoptosis is the common mechanism for pathogenesis of this autosomal-dominant disease. Apoptosis induced by the CA IV mutants could be prevented, at least partially, by treating the cells with dorzolamide, a CA inhibitor. Thus, the use of a CA inhibitor as a chemical chaperone to reduce ER stress may delay or prevent the onset of blindness in RP17.

Inhibition of Nuclear Translocation of Apoptosis-Inducing Factor Is an Essential Mechanism of the Neuroprotective Activity of Pigment Epithelium-Derived Factor in a Rat Model of Retinal Degeneration

Photoreceptor apoptosis is a critical process of retinal degeneration in retinitis pigmentosa (RP), a group of retinal degenerative diseases that result from rod and cone photoreceptor cell death and represent a major cause of adult blindness. We previously demonstrated the efficient prevention of photoreceptor apoptosis by intraocular gene transfer of pigment epithelium-derived factor (PEDF) in animal models of RP; however, the underlying mechanism of the neuroprotective activity of PEDF remains elusive. In this study, we show that an apoptosis-inducing factor (AIF)-related pathway is an essential target of PEDF-mediated neuroprotection. PEDF rescued serum starvation-induced apoptosis, which is mediated by AIF but not by caspases, of R28 cells derived from the rat retina by preventing translocation of AIF into the nucleus. Nuclear translocation of AIF was also observed in the apoptotic photoreceptors of Royal College of Surgeons rats, a well-known animal model of RP that carries a mutation of the Mertk gene. Lentivirus-mediated retinal gene transfer of PEDF prevented the nuclear translocation of AIF in vivo, resulting in the inhibition of the apoptotic loss of their photoreceptors in association with up-regulated Bcl-2 expression, which mediates the mitochondrial release of AIF. These findings clearly demonstrate that AIF is an essential executioner of photoreceptor apoptosis in inherited retinal degeneration and provide a therapeutic rationale for PEDF-mediated neuroprotective gene therapy for individuals with RP.

ER stress and apoptosis‐inducing mutations in carbonic anhydrase IV associated with retinitis pigmentosa (RP17) respond to chemical chaperones

We examined two human mutations in the carbonic anhydrase IV (CA IV) gene, which are associated with RP17, an autosomal dominant form of retinitis pigmentosa. CA IV is highly expressed in the choriocapillaris, but not expressed in the retina. The R14W CA IV mutation (Rebello et al., Proc. Natl. Acad. Sci. USA 101:6617–6622, 2004) is located in the signal sequence. The subsequently discovered R219S CA IV mutation is located in the sequence of the mature protein. The biological effects of these mutations were studied in transfected COS cells. Although the signal sequence mutation had only a modest effect on total enzyme production (70% of normal), the R219S mutant protein was completely inactive. Both mutations induced upregulation of BiP, PERK, and CHOP, markers of ER stress. Both suppressed levels of production of other secretory proteins. Both dramatically induced apoptosis as measured by Annexin V binding and TUNEL staining. The markers of ER stress and the number of cells undergoing apoptosis were both greatly reduced by growth in 4-phenylbutyric acid or CA inhibitors, which appeared to act as nonspecific and specific chemical chaperones, respectively. We propose that autosomal dominant RP17 associated with both CA IV mutations is a protein folding disease produced by apoptosis in capillary endothelial cells that nourish the overlying retina. These results suggest that patients with RP17 may respond clinically to treatment with chemical chaperones. This research is supported by NIH grant DK40163 to W.S.S.

A Single Copy of Carbonic anhydrase 2 Restores Wild-type Circadian Period to Carbonic Anhydrase II-Deficient Mice

Carbonic anhydrase II (CA-II)-deficient mice have long circadian periods compared to their siblings with normal CA-II levels. The CA-II-deficient mice differ genetically from their siblings at proximal chromosome three, where the mutated carbonic anhydrase 2 gene sits on a small insert of DNA from the DBA/2J strain. The rest of the genome is that of the C57BL/6J strain. The goal of this study was to test the hypothesis that the null mutation in carbonic anhydrase 2 and the long circadian period phenotype were linked. In order to separate the effect of the null mutation in carbonic anhydrase 2 from the effect of DBA/2J alleles of other genes on the insert, two new lines of mice were studied. The first line, Kar, was developed from a CA-II-deficient mouse that had a fortuitous recombination restoring functional CA-II without affecting the rest of the DBA/2J insert. The second line was generated by breeding DBA/2J mice and C57BL/6J mice until they had the genomic composition of CA-II-deficient mice without the null mutation. Both lines of mice had circadian periods not different from C57BL/6J mice and shorter than CA-II-deficient mice. The phenotype of the new lines showed that the long circadian period characteristic of the CA-II-deficient mice arises when functional CA-II is absent, not when DBA/2J alleles are present on proximal chromosome three.

Chemical chaperones protect from effects of apoptosis-inducing mutation in carbonic anhydrase IV identified in retinitis pigmentosa 17.

Carbonic anhydrase (CA) IV is a glycosylphosphotidylinositol-anchored enzyme highly expressed on the plasma face of microcapillaries and especially strongly expressed in the choriocapillaris of the human eye. In collaboration with scientists at the University of Cape Town (Rondebosch, South Africa), we recently showed that the R14W mutation in the signal sequence of CA IV, which they identified in patients with the retinitis pigmentosa (RP) 17 form of autosomal dominant RP, results in accumulation of unfolded protein in the endoplasmic reticulum (ER), leading to ER stress, the unfolded protein response, and apoptosis in a large fraction of transfected COS-7 cells expressing mutant, but not wild-type, CA IV. Here we present experiments showing that several well characterized CA inhibitors largely prevent the adverse effects of expressing R14W CA IV in transfected COS-7 cells. Specifically, CA inhibitors prevent the accelerated turnover of the mutant protein, the up-regulation of Ig-binding protein, double-stranded RNA-regulated protein kinase-like ER kinase, and CCAAT/enhancer-binding protein homologous protein (markers of the unfolded protein response and ER stress), the inhibition of production of other secretory proteins expressed from COS-7-transfecting plasmids, and the induction of apoptosis, all characteristics of transfected cells expressing R14W CA IV. Furthermore, treatment with 4-phenylbutyric acid, a nonspecific chemical chaperone used in other protein-folding disorders, also dramatically reduces the apoptosis-inducing effect of expressing R14W CA IV cDNA in transfected COS-7 cells. These experiments suggest a promising approach to treatment of RP17 that might delay the onset or possibly prevent this autosomal dominant form of RP.

Apoptosis-inducing signal sequence mutation in carbonic anhydrase IV identified in patients with the RP17 form of retinitis pigmentosa.

Genetic and physical mapping of the RP17 locus on 17q identified a 3.6-megabase candidate region that includes the gene encoding carbonic anhydrase IV (CA4), a glycosylphosphatidylinositol-anchored protein that is highly expressed in the choriocapillaris of the human eye. By sequencing candidate genes in this region, we identified a mutation that causes replacement of an arginine with a tryptophan (R14W) in the signal sequence of the CA4 gene at position -5 relative to the signal sequence cleavage site. This mutation was found to cosegregate with the disease phenotype in two large families and was not found in 36 unaffected family members or 100 controls. Expression of the mutant cDNA in COS-7 cells produced several findings, suggesting a mechanism by which the mutation can explain the autosomal dominant disease. In transfected COS-7 cells, the R14W mutation (i) reduced the steady-state level of carbonic anhydrase IV activity expressed by 28% due to a combination of decreased synthesis and accelerated turnover; (ii) led to up-regulation of immunoglobulin-binding protein, double-stranded RNA-regulated protein kinase-like ER kinase, and CCAAT/enhancer-binding protein homologous protein, markers of the unfolded protein response and endoplasmic reticulum stress; and (iii) induced apoptosis, as evidenced by annexin V binding and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining, in most cells expressing the mutant, but not the WT, protein. We suggest that a high level of expression of the mutant allele in the endothelial cells of the choriocapillaris leads to apoptosis, leading in turn to ischemia in the overlying retina and producing autosomal dominant retinitis pigmentosa.

Seven novel mutations in carbonic anhydrase II deficiency syndrome identified by SSCP and direct sequencing analysis

Human MutationVolume 9, Issue 5 p. 383-387 Mutation Update Seven novel mutations in carbonic anhydrase II deficiency syndrome identified by SSCP and direct sequencing analysis Peiyi Y. Hu, Peiyi Y. Hu Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri; Fax: 314776-1183Search for more papers by this authorE. Justin Lim, E. Justin Lim Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri; Fax: 314776-1183Search for more papers by this authorJessica Ciccolella, Jessica Ciccolella Department of Pediatrics, University of Reggio Calabria, Catanzaro, ItalySearch for more papers by this authorPietro Strisciuglio, Pietro Strisciuglio Department of Pediatrics, University of Reggio Calabria, Catanzaro, ItalySearch for more papers by this authorWilliam S. Sly, Corresponding Author William S. Sly Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri; Fax: 314776-1183Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri; Fax: 314776-1183Search for more papers by this author Peiyi Y. Hu, Peiyi Y. Hu Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri; Fax: 314776-1183Search for more papers by this authorE. Justin Lim, E. Justin Lim Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri; Fax: 314776-1183Search for more papers by this authorJessica Ciccolella, Jessica Ciccolella Department of Pediatrics, University of Reggio Calabria, Catanzaro, ItalySearch for more papers by this authorPietro Strisciuglio, Pietro Strisciuglio Department of Pediatrics, University of Reggio Calabria, Catanzaro, ItalySearch for more papers by this authorWilliam S. Sly, Corresponding Author William S. Sly Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri; Fax: 314776-1183Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri; Fax: 314776-1183Search for more papers by this author First published: 07 January 1999 https://doi.org/10.1002/(SICI)1098-1004(1997)9:5<383::AID-HUMU1>3.0.CO;2-5Citations: 21AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume9, Issue51997Pages 383-387 RelatedInformation

Seven novel mutations in carbonic anhydrase II deficiency syndrome identified by SSCP and direct sequencing analysis.

Seven novel mutations in carbonic anhydrase II deficiency syndrome identified by SSCP and direct sequencing analysis.