ABSTRACT
The blood-brain barrier (BBB) presents a major challenge for delivering large molecules to study and treat the central nervous system. This is due in part to the scarcity of targets known to mediate BBB crossing. To identify novel targets, we leverage a panel of adeno-associated viruses (AAVs) previously identified through mechanism-agnostic directed evolution for improved BBB transcytosis. Screening potential cognate receptors for enhanced BBB crossing, we identify two targets: murine-restricted LY6C1 and widely conserved carbonic anhydrase IV (CA-IV). We apply AlphaFold-based in silico methods to generate capsid-receptor binding models to predict the affinity of AAVs for these identified receptors. Demonstrating how these tools can unlock target-focused engineering strategies, we create an enhanced LY6C1-binding vector, AAV-PHP.eC, that, unlike our prior PHP.eB, also works in Ly6a-deficient mouse strains such as BALB/cJ. Combined with structural insights from computational modeling, the identification of primate-conserved CA-IV enables the design of more specific and potent human brain-penetrant chemicals and biologicals, including gene delivery vectors.
Subject(s)
Blood-Brain Barrier , Carbonic Anhydrase IV , Mice , Humans , Animals , Blood-Brain Barrier/metabolism , Carbonic Anhydrase IV/genetics , Carbonic Anhydrase IV/metabolism , Brain/metabolism , Gene Transfer Techniques , Primates/genetics , Dependovirus/genetics , Dependovirus/metabolismABSTRACT
The functional role of membrane-bound carbonic anhydrases (CAs) has been of keen interest in the past decade, and in particular, studies have linked CA in red muscle, heart, and eye to enhanced tissue oxygen extraction in bony fishes (teleosts). However, the number of purported membrane-bound CA isoforms in teleosts, combined with the imperfect system of CA isoform nomenclature, present roadblocks for ascribing physiological functions to particular CA isoforms across different teleost lineages. Here we developed an organizational framework for membrane-bound CAs in teleosts, providing the latest phylogenetic analysis of extant CA4 and CA4-like isoforms. Our data confirm that there are three distinct isoforms of CA4 (a, b, and c) that are conserved across major teleost lineages, with the exception of CA4c gene being lost in salmonids. Tissue distribution analyses suggest CA4a functions in oxygen delivery across teleost lineages, while CA4b may be specialized for renal acid-base balance and ion regulation. This work provides an important foundation for researchers to elucidate the functional significance of CA4 isoforms in fishes.
Subject(s)
Carbonic Anhydrase IV , Carbonic Anhydrases , Animals , Carbonic Anhydrase IV/genetics , Phylogeny , Carbonic Anhydrases/genetics , Protein Isoforms/genetics , Fishes/genetics , OxygenABSTRACT
Pulmonary cartilage plays a crucial structural role determining the physiologic airway compressibility and distensibility, necessary for proper mechanical function. This functionality deteriorates with aging due to increased stiffness of both airway muscle and cartilage, as well as, decreased renewal capacity. Altered airway remodeling has been suggested as a pathogenic driver of chronic obstructive pulmonary disease (COPD) through mechanisms still incompletely understood. Using paraffin-embedded lung tissue sections from archived autopsy material from COPD with non-COPD age matched controls a histopathologic analysis focused on inflammation, fibrosis and calcification was performed with special stains (Masson's trichrome and Von Kossa) and immunohistochemistry for carbonic anhydrase IV (CA IV) and Ki-67. COPD lung tissues showed increased peribronchial inflammation compared to the non-COPD. Coarse amphophilic crystalline deposits in bronchial cartilage were more frequently observed in COPD sections, which were compatible with early dystrophic calcification of the extracellular matrix and chondrocytes. Moreover, Von Kossa staining revealed a significant calcium deposition in the cartilages from COPD in comparison to the controls. Interestingly, Ki-67 immunostains demonstrated a higher overall proliferative rate, including epithelial cells, in COPD. Furthermore, Masson's trichrome staining revealed relatively increased peribronchial collagen deposition associated with a fibrotic stromal response, which may be secondary to the inflammatory milieu in COPD. To further characterize the tissue microenvironment associated with dystrophic calcification, immunohistochemistry for CA IV was used, revealing significantly increased expression in chondrocytes and peribronchial tissue in COPD. Our findings demonstrate that dystrophic calcification of the extracellular matrix and chondrocytes can be linked to CA IV expression in COPD and suggest that pH changes in pulmonary tissue associated with inflammation and calcification may play an active role in COPD.
Subject(s)
Carbonic Anhydrase IV/genetics , Carbonic Anhydrases , Pulmonary Disease, Chronic Obstructive , Bronchi/metabolism , Carbonic Anhydrase IV/metabolism , Cartilage/metabolism , Cartilage/pathology , Fibrosis , Humans , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/pathologyABSTRACT
In most vertebrates, red blood cell carbonic anhydrase (RBC CA) plays a critical role in carbon dioxide (CO2) transport and excretion across epithelial tissues. Many early-diverging fishes (e.g., hagfish and chondrichthyans) are unique in possessing plasma-accessible membrane-bound CA-IV in the gills, allowing some CO2 excretion to occur without involvement from the RBCs. However, implications of this on RBC CA function are unclear. Through homology cloning techniques, we identified the putative protein sequences for RBC CA from nine early-diverging species. In all cases, these sequences contained a modification of the proton shuttle residue His-64, and activity measurements from three early-diverging fish demonstrated significantly reduced CA activity. Site-directed mutagenesis was used to restore the His-64 proton shuttle, which significantly increased RBC CA activity, clearly illustrating the functional significance of His-64 in fish red blood cell CA activity. Bayesian analyses of 55 vertebrate cytoplasmic CA isozymes suggested that independent evolutionary events led to the modification of His-64 and thus reduced CA activity in hagfish and chondrichthyans. Additionally, in early-diverging fish that possess branchial CA-IV, there is an absence of His-64 in RBC CAs and the absence of the Root effect [where a reduction in pH reduces hemoglobin's capacity to bind with oxygen (O2)]. Taken together, these data indicate that low-activity RBC CA may be present in all fish with branchial CA-IV, and that the high-activity RBC CA seen in most teleosts may have evolved in conjunction with enhanced hemoglobin pH sensitivity.
Subject(s)
Carbonic Anhydrase IV/blood , Carbonic Anhydrase IV/genetics , Erythrocytes/enzymology , Fish Proteins/blood , Fish Proteins/genetics , Fishes/blood , Fishes/genetics , Amino Acid Substitution , Animals , PhylogenyABSTRACT
Colon adenocarcinoma (COAD) is a common noncutaneous carcinoma worldwide with high morbidity and mortality. Effective prevention methods are far from being met. Both diagnostic and prognostic models that can precisely and accurately predict the status and survival time of COAD are urgently needed. In the field of COAD, there have been limited studies on molecular biomarkers that can predict disease status and prognosis. Hence, an important task is to identify these biomarkers. We aimed to identify important risk genes that have the ability not only to diagnose tumors but also to predict overall survival. A comprehensive analysis was performed in this study. Finally, carbonic anhydrase 1 (CA1) and CA4 were identified as potential biomarkers due to their predictive roles in diagnosis and prognosis, and the results were further confirmed by a series of analyses. Overall, these findings are of great importance and may facilitate individualized treatment in diagnosis and prognosis.
Subject(s)
Adenocarcinoma/genetics , Biomarkers, Tumor/genetics , Carbonic Anhydrase IV/genetics , Carbonic Anhydrase I/genetics , Colonic Neoplasms/genetics , Adenocarcinoma/diagnosis , Adenocarcinoma/pathology , Adult , Aged , Colon/pathology , Colonic Neoplasms/diagnosis , Colonic Neoplasms/pathology , Gene Expression Regulation, Neoplastic/genetics , Humans , PrognosisABSTRACT
The lung microvasculature is essential for gas exchange and commonly considered homogeneous. We show that VEGFA from the epithelium is required for a distinct endothelial cell (EC) population in the mouse lung. Vegfa is predominantly expressed by alveolar type 1 (AT1) cells and locally required to specify a subset of ECs. Single-cell RNA sequencing (scRNA-seq) reveals that â¼15% of lung ECs are transcriptionally distinct-marked by Carbonic anhydrase 4 (Car4)-and arise from bulk ECs, as suggested by trajectory analysis. Car4 ECs have extensive cellular projections and are separated from AT1 cells by a limited basement membrane without intervening pericytes. Car4 ECs are specifically lost upon epithelial Vegfa deletion; without Car4 ECs, the alveolar space is aberrantly enlarged despite the normal appearance of myofibroblasts. Lung Car4 ECs and retina tip ECs have common and distinct features. These findings support a signaling role of AT1 cells and shed light on alveologenesis.
Subject(s)
Alveolar Epithelial Cells/metabolism , Endothelial Cells/cytology , Endothelium, Vascular/cytology , Lung/metabolism , Vascular Endothelial Growth Factor A/metabolism , Alveolar Epithelial Cells/cytology , Animals , Carbonic Anhydrase IV/genetics , Carbonic Anhydrase IV/metabolism , Cells, Cultured , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Lung/cytology , Lung/growth & development , Mice , Morphogenesis , Myofibroblasts/cytology , Neovascularization, Physiologic , Vascular Endothelial Growth Factor A/geneticsABSTRACT
Giant clams represent symbiotic associations between a host clam and its extracellular zooxanthellae. They are able to grow in nutrient-deficient tropical marine environments and conduct light-enhanced shell formation (calcification) with the aid of photosynthates donated by the symbiotic zooxanthellae. In light, there is a high demand for inorganic carbon (Ci) to support photosynthesis in the symbionts and light-enhanced calcification in the host. In this study, we cloned and characterized a host Carbonic Anhydrase 4 homolog (CA4-like) from the whitish inner mantle of the giant clam Tridacna squamosa. The full cDNA coding sequence of CA4-like consisted of 1002â¯bp, encoding for 334 amino acids of 38.5â¯kDa. The host CA4-like was phenogramically distinct from algal CAs. The transcript level of CA4-like in the inner mantle was ~3-fold higher than those in the colorful outer mantle and the ctenidium. In the inner mantle, CA4-like was immunolocalized in the apical membrane of the seawater-facing epithelial cells, but absent from the shell-facing epithelium. Hence, CA4-like was positioned to catalyze the conversion of HCO3- to CO2 in the ambient seawater which would facilitate CO2 uptake. The absorbed CO2 could be converted back to HCO3- by the cytoplasmic CA2-like. As the protein abundance of CA4-like increased in the inner mantle after 6 or 12â¯h of light exposure, there could be an augmentation of the total CA4-like activity to increase Ci uptake in light. It is plausible that the absorbed Ci was allocated preferentially for shell formation due to the close proximity of the seawater-facing epithelium to the shell-facing epithelium in the inner mantle that contains only few zooxanthellae.
Subject(s)
Bivalvia/physiology , Carbonic Anhydrase IV/genetics , Cloning, Molecular/drug effects , Animal Shells/metabolism , Animal Shells/physiology , Animals , Bivalvia/genetics , Carbonic Anhydrase IV/metabolism , Open Reading Frames , Phylogeny , Sequence Analysis, DNA , Tissue Distribution , Up-RegulationABSTRACT
Monocarboxylate transporters (MCTs) mediate the proton-coupled transport of high-energy metabolites like lactate and pyruvate and are expressed in nearly every mammalian tissue. We have shown previously that transport activity of MCT4 is enhanced by carbonic anhydrase II (CAII), which has been suggested to function as a 'proton antenna' for the transporter. In the present study, we tested whether creation of an endogenous proton antenna by introduction of a cluster of histidine residues into the C-terminal tail of MCT4 (MCT4-6xHis) could facilitate MCT4 transport activity when heterologously expressed in Xenopus oocytes. Our results show that integration of six histidines into the C-terminal tail does indeed increase transport activity of MCT4 to the same extent as did coexpression of MCT4-WT with CAII. Transport activity of MCT4-6xHis could be further enhanced by coexpression with extracellular CAIV, but not with intracellular CAII. Injection of an antibody against the histidine cluster into MCT4-expressing oocytes decreased transport activity of MCT4-6xHis, while leaving activity of MCT4-WT unaltered. Taken together, these findings suggest that transport activity of the proton-coupled monocarboxylate transporter MCT4 can be facilitated by integration of an endogenous proton antenna into the transporter's C-terminal tail.
Subject(s)
Carbonic Anhydrase II/metabolism , Carbonic Anhydrase IV/metabolism , Histidine/metabolism , Monocarboxylic Acid Transporters/metabolism , Muscle Proteins/metabolism , Oligopeptides/metabolism , Protons , Recombinant Fusion Proteins/metabolism , Animals , Antibodies, Neutralizing/pharmacology , Biological Transport/drug effects , Carbonic Anhydrase II/genetics , Carbonic Anhydrase IV/genetics , Gene Expression , Histidine/antagonists & inhibitors , Histidine/genetics , Lactic Acid/metabolism , Microinjections , Monocarboxylic Acid Transporters/genetics , Muscle Proteins/genetics , Oligopeptides/antagonists & inhibitors , Oligopeptides/genetics , Oocytes/cytology , Oocytes/drug effects , Oocytes/metabolism , Protein Engineering , Pyruvic Acid/metabolism , Rats , Recombinant Fusion Proteins/genetics , Xenopus laevisABSTRACT
OBJECTIVE: We found that carbonic anhydrase IV (CA4), a member of the carbonic anhydrases, is silenced in colorectal cancer (CRC). We analysed its epigenetic inactivation, biological effects and prognostic significance in CRC. DESIGN: The biological functions of CA4 were determined by in vitro and in vivo tumorigenicity assays. The CA4 co-operator was identified by immunoprecipitation and mass spectrometry. CA4 downstream effectors and signalling pathways were elucidated by promoter luciferase assay, electrophoretic mobility shift assay and chromatin immunoprecipitation. The clinical impact of CA4 was assessed in 115 patients with CRC. RESULTS: CA4 was silenced in all nine CRC cell lines and 92.6% of CRC tumours. The promoter hypermethylation contributed to the inactivation of CA4, and it was detected in 75.7% of the patients with CRC. After a median follow-up of 49.3â months, multivariate analysis showed that the patients with CA4 hypermethylation had a recurrence of Stage II/III CRC. The re-expression of CA4 inhibited cell proliferation, induced apoptosis and cell cycle arrest in the G1 phase. CA4 inhibited the activity of the Wnt signalling pathway and mediated the degradation of ß-catenin. CA4 interacted with Wilms' tumour 1-associating protein (WTAP) and induced WTAP protein degradation through polyubiquitination. Moreover, CA4 promoted the transcriptional activity of Wilms' tumour 1 (WT1), an antagonist of the Wnt pathway, which resulted in the induction of transducin ß-like protein 1 (TBL1) and the degradation of ß-catenin. CONCLUSIONS: CA4 is a novel tumour suppressor in CRC through the inhibition of the Wnt signalling pathway by targeting the WTAP-WT1-TBL1 axis. CA4 methylation may serve as an independent biomarker for the recurrence of CRC.
Subject(s)
Carbonic Anhydrase IV/genetics , Colorectal Neoplasms , Neoplasm Recurrence, Local/genetics , Wnt Signaling Pathway/genetics , Cell Cycle Proteins , Cell Line, Tumor , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor/physiology , Humans , Neoplasm Staging , Nuclear Proteins/genetics , Prognosis , RNA Splicing Factors , Transducin/genetics , WT1 Proteins/genetics , beta Catenin/metabolismABSTRACT
BACKGROUND AND PURPOSE: Carbonic anhydrase IX (CAIX) plays a pivotal role in pH homeostasis, which is essential for tumor cell survival. We examined the effect of the CAIX inhibitor 4-(3'(3",5"-dimethylphenyl)-ureido)phenyl sulfamate (S4) on the tumor microenvironment in a laryngeal tumor model by analyzing proliferation, apoptosis, necrosis, hypoxia, metabolism and CAIX ectodomain shedding. METHODS: SCCNij202 tumor bearing-mice were treated with S4 for 1, 3 or 5 days. CAIX ectodomain shedding was measured in the serum after therapy. Effects on tumor cell proliferation, apoptosis, necrosis, hypoxia (pimonidazole) and CAIX were investigated with quantitative immunohistochemistry. Metabolic transporters and enzymes were quantified with qPCR. RESULTS: CAIX ectodomain shedding decreased after treatment with S4 (p<0.01). S4 therapy did neither influence tumor cell proliferation nor the amount of apoptosis and necrosis. Hypoxia (pimonidazole) and CAIX expression were also not affected by S4. CHOP and MMP9 mRNA as a reference of intracellular pH did not change upon treatment with S4. Compensatory mechanisms of pH homeostasis at the mRNA level were not observed. CONCLUSION: As the clinical and biological meaning of the decrease in CAIX ectodomain shedding after S4 therapy is not clear, studies are required to elucidate whether the CAIX ectodomain has a paracrine or autocrine signaling function in cancer biology. S4 did not influence the amount of proliferation, apoptosis, necrosis and hypoxia. Therefore, it is unlikely that S4 can be used as single agent to influence tumor cell kill and proliferation, and to target primary tumor growth.
Subject(s)
Carbonic Anhydrase IV/antagonists & inhibitors , Carbonic Anhydrase Inhibitors/pharmacology , Laryngeal Neoplasms/metabolism , Laryngeal Neoplasms/pathology , Tumor Microenvironment/drug effects , Animals , Apoptosis/drug effects , Carbonic Anhydrase IV/genetics , Carbonic Anhydrase IV/metabolism , Carbonic Anhydrase Inhibitors/administration & dosage , Cell Proliferation/drug effects , Disease Models, Animal , Female , Gene Expression , Gene Expression Regulation, Neoplastic/drug effects , Humans , Laryngeal Neoplasms/drug therapy , Laryngeal Neoplasms/genetics , Mice , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Eosinophilia and its cellular activation are hallmark features of asthma, as well as other allergic/Th2 disorders, yet there are few, if any, reliable surface markers of eosinophil activation. We have used a FACS-based genome-wide screening system to identify transcriptional alterations in murine lung eosinophils recruited and activated by pulmonary allergen exposure. Using a relatively stringent screen with false-positive correction, we identified 82 candidate genes that could serve as eosinophil activation markers and/or pathogenic effector markers in asthma. Carbonic anhydrase IV (Car4) was a top dysregulated gene with 36-fold induction in allergen-elicited pulmonary eosinophils, which was validated by quantitative PCR, immunohistochemistry, and flow cytometry. Eosinophil CAR4 expression was kinetically regulated by IL-5, but not IL-13. IL-5 was both necessary and sufficient for induction of eosinophil CAR4. Although CAR4-deficient mice did not have a defect in eosinophil recruitment to the lung, nor a change in eosinophil pH-buffering capacity, allergen-challenged chimeric mice that contained Car4(-/-) hematopoietic cells aberrantly expressed a series of genes enriched in biological processes involved in epithelial differentiation, keratinization, and anion exchange. In conclusion, we have determined that eosinophils express CAR4 following IL-5 or allergen exposure, and that CAR4 is involved in regulating the lung transcriptome associated with allergic airway inflammation; therefore, CAR4 has potential value for diagnosing and monitoring eosinophilic responses.
Subject(s)
Asthma/immunology , Carbonic Anhydrase IV/immunology , Eosinophils/immunology , Interleukin-5/immunology , Allergens/genetics , Allergens/immunology , Animals , Asthma/diagnosis , Asthma/genetics , Asthma/metabolism , Asthma/pathology , Carbonic Anhydrase IV/biosynthesis , Carbonic Anhydrase IV/genetics , Cell Differentiation/genetics , Cell Differentiation/immunology , Enzyme Induction/genetics , Enzyme Induction/immunology , Eosinophils/metabolism , Eosinophils/pathology , Hematopoietic Stem Cells , Interleukin-13/genetics , Interleukin-13/immunology , Interleukin-13/metabolism , Interleukin-5/genetics , Interleukin-5/metabolism , Lung/immunology , Lung/metabolism , Lung/pathology , Mice , Mice, KnockoutABSTRACT
Proton-coupled monocarboxylate transporters (MCTs) are carriers of high-energy metabolites such as lactate, pyruvate, and ketone bodies and are expressed in most tissues. It has previously been shown that transport activity of MCT1 and MCT4 is enhanced by the cytosolic carbonic anhydrase II (CAII) independent of its catalytic activity. We have now studied the influence of the extracellular, membrane-bound CAIV on transport activity of MCT1/4, heterologously expressed in Xenopus oocytes. Coexpression of CAIV with MCT1 and MCT4 resulted in a significant increase in MCT transport activity, even in the nominal absence of CO2/HCO3(-). CAIV-mediated augmentation of MCT activity was independent of the CAIV catalytic function, since application of the CA-inhibitor ethoxyzolamide or coexpression of the catalytically inactive mutant CAIV-V165Y did not suppress CAIV-mediated augmentation of MCT transport activity. The interaction required CAIV at the extracellular surface, since injection of CAIV protein into the oocyte cytosol did not augment MCT transport function. The effects of cytosolic CAII (injected as protein) and extracellular CAIV (expressed) on MCT transport activity, were additive. Our results suggest that intra- and extracellular carbonic anhydrases can work in concert to ensure rapid shuttling of metabolites across the cell membrane.
Subject(s)
Carbonic Anhydrase IV/metabolism , Ketone Bodies/metabolism , Lactic Acid/metabolism , Monocarboxylic Acid Transporters/metabolism , Muscle Proteins/metabolism , Pyruvic Acid/metabolism , Symporters/metabolism , Animals , Biological Transport/physiology , Carbonic Anhydrase II/metabolism , Carbonic Anhydrase IV/genetics , Cytosol/metabolism , Extracellular Space/metabolism , Humans , Hydrogen-Ion Concentration , Membrane Proteins/metabolism , Monocarboxylic Acid Transporters/genetics , Muscle Proteins/genetics , Oocytes/physiology , Oxygen Isotopes/pharmacokinetics , Rats , Symporters/genetics , XenopusABSTRACT
Carbonic anhydrase IV is one of 12 active human isozymes and one of four expressed on the extracellular surfaces of certain endothelial and epithelial cells. It is unique in being attached to the plasma membrane by a glycosyl-phosphatiydyl-inositol (GPI) anchor rather than by a membrane-spanning domain. It is also uniquely resistant to high concentrations of sodium dodecyl sulfate (SDS), which allows purification from tissues by inhibitor affinity chromatography without contamination by other isozymes. This unique resistance to SDS and recovery following denaturation is explained by the two disulfide bonds. The 35-kDa human CA IV is a "high activity" isozyme in CO2 hydration activity, like CA II, and has higher activity than other isozymes in catalyzing the dehydration of HCO3 (-). Human CA IV is also unique in that it contains no oligosaccharide chains, where all other mammalian CA IVs are glycoproteins with one to several oligosaccharide side chains.Although CA IV has been shown to be active in mediating CO2 and HCO3 (-) transport in many important tissues like kidney and lung, and in isolated cells from brain and muscle, the gene for CA IV appears not to be essential. The CA IV knockout mouse produced by targeted mutagenesis, though slightly smaller and produced in lower than expected numbers, is viable and has no obvious mutant phenotype. Conversely, several dominant negative mutations in humans are associated with one form of reitinitis pigmentosa (RP-17), which we attribute to unfolded protein accumulation in the choreocapillaris, leading to apoptosis of cells in the overlying retina.
Subject(s)
Carbonic Anhydrase IV/biosynthesis , Carbonic Anhydrase IV/chemistry , Carbonic Anhydrase IV/isolation & purification , Structure-Activity Relationship , Animals , Carbonic Anhydrase IV/genetics , Carbonic Anhydrase IV/metabolism , Catalysis , Enzyme Stability , Gene Expression Regulation, Enzymologic , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Mutation , Rats , Tissue DistributionABSTRACT
Soluble cytosolic carbonic anhydrases (CAs) are well known to participate in pH regulation of the cytoplasm of mammalian cells. Membrane-bound CA isoforms--such as isoforms IV, IX, XII, XIV, and XV--also catalyze the reversible conversion of carbon dioxide to protons and bicarbonate, but at the extracellular face of the cell membrane. When human CA isoform IV was heterologously expressed in Xenopus oocytes, we observed, by measuring H(+) at the outer face of the cell membrane and in the cytosol with ion-selective microelectrodes, not only extracellular catalytic CA activity but also robust intracellular activity. CA IV expression in oocytes was confirmed by immunocytochemistry, and CA IV activity measured by mass spectrometry. Extra- and intracellular catalytic activity of CA IV could be pharmacologically dissected using benzolamide, the CA inhibitor, which is relatively slowly membrane-permeable. In acute cerebellar slices of mutant mice lacking CA IV, cytosolic H(+) shifts of granule cells following CO(2) removal/addition were significantly slower than in wild-type mice. Our results suggest that membrane-associated CA IV contributes robust catalytic activity intracellularly, and that this activity participates in regulating H(+) dynamics in the cytosol, both in injected oocytes and in mouse neurons.
Subject(s)
Carbonic Anhydrase IV/metabolism , Animals , Benzolamide/pharmacology , Carbonic Anhydrase II/antagonists & inhibitors , Carbonic Anhydrase II/genetics , Carbonic Anhydrase II/metabolism , Carbonic Anhydrase IV/antagonists & inhibitors , Carbonic Anhydrase IV/deficiency , Carbonic Anhydrase IV/genetics , Carbonic Anhydrase Inhibitors/pharmacology , Cerebellum/cytology , Cerebellum/enzymology , Cytosol/enzymology , Extracellular Fluid/enzymology , Female , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Humans , Hydrogen-Ion Concentration , Intracellular Fluid/enzymology , Mice , Mice, Knockout , Neurons/enzymology , Oocytes/enzymology , RNA, Complementary/genetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Xenopus laevisABSTRACT
BACKGROUND: Carbonic anhydrase enzymes (CA) catalyze the reversible hydration of carbon dioxide to bicarbonate in mammalian cells. Trans-membrane transport of CA-produced bicarbonate contributes significantly to cellular pH regulation. A body of evidence implicates pH-regulatory processes in the hypertrophic growth pathway characteristic of hearts as they fail. In particular, Na+/H+ exchange (NHE) activation is pro-hypertrophic and CA activity activates NHE. Recently Cardrase (6-ethoxyzolamide), a CA inhibitor, was found to prevent and revert agonist-stimulated cardiac hypertrophy (CH) in cultured cardiomyocytes. Our goal thus was to determine whether hypertrophied human hearts have altered expression of CA isoforms. METHODS: We measured CA expression in hypertrophied human hearts to begin to examine the role of carbonic anhydrase in progression of human heart failure. Ventricular biopsies were obtained from patients undergoing cardiac surgery (CS, n = 14), or heart transplantation (HT, n = 13). CS patients presented mild/moderate concentric left ventricular hypertrophy and normal right ventricles, with preserved ventricular function; ejection fractions were ~60%. Conversely, HT patients with failing hearts presented CH or ventricular dilation accompanied by ventricular dysfunction and EF values of 20%. Non-hypertrophic, non-dilated ventricular samples served as controls. RESULTS: Expression of atrial and brain natriuretic peptide (ANP and BNP) were markers of CH. Hypertrophic ventricles presented increased expression of CAII, CAIV, ANP, and BNP, mRNA levels, which increased in failing hearts, measured by quantitative real-time PCR. CAII, CAIV, and ANP protein expression also increased approximately two-fold in hypertrophic/dilated ventricles. CONCLUSIONS: These results, combined with in vitro data that CA inhibition prevents and reverts CH, suggest that increased carbonic anhydrase expression is a prognostic molecular marker of cardiac hypertrophy.
Subject(s)
Carbonic Anhydrases/genetics , Heart Failure/genetics , Hypertrophy, Left Ventricular/genetics , Myocardium/enzymology , Aged , Animals , Atrial Natriuretic Factor/genetics , Biopsy , Carbonic Anhydrase II/genetics , Carbonic Anhydrase IV/genetics , Female , Gene Expression Regulation, Enzymologic , Genetic Markers , HEK293 Cells , Heart Failure/enzymology , Heart Failure/pathology , Heart Failure/physiopathology , Humans , Hypertrophy, Left Ventricular/enzymology , Hypertrophy, Left Ventricular/pathology , Hypertrophy, Left Ventricular/physiopathology , Isoenzymes , Male , Mice , Middle Aged , Myocardium/pathology , Natriuretic Peptide, Brain/genetics , RNA, Messenger/analysis , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Stroke Volume , Transfection , Ventricular Function, LeftABSTRACT
Carbonic anhydrase (CA) isozymes CA IV and CA XV are anchored on the extracellular cell surface via glycosylphosphatidylinositol (GPI) linkage. Analysis of evolution of these isozymes in vertebrates reveals an additional group of GPI-linked CAs, CA XVII, which has been lost in mammals. Our work resolves nomenclature issues in GPI-linked fish CAs. Review of expression data brings forth previously unreported tissue and cancer types in which human CA IV is expressed. Analysis of collective glycosylation patterns of GPI-linked CAs suggests functionally important regions on the protein surface.
Subject(s)
Biological Evolution , Animals , Carbonic Anhydrase IV/classification , Carbonic Anhydrase IV/genetics , Carbonic Anhydrase IV/metabolism , Carbonic Anhydrases/classification , Carbonic Anhydrases/genetics , Carbonic Anhydrases/metabolism , Gene Duplication , Gene Expression , Glycosylation , Glycosylphosphatidylinositols/chemistry , Humans , Isoenzymes/classification , Isoenzymes/genetics , Isoenzymes/metabolism , PhylogenyABSTRACT
AIM: The aim of this study was to investigate carbonic anhydrase (CA) VI exon 2 genetic polymorphism and its possible association with low caries experience in healthy young adults. PATIENTS AND METHODS: Healthy young adults with caries or who where caries-free were recruited and unstimulated whole saliva and blood samples were taken. The number of decayed, missing and filled teeth (DMFT) and oral hygiene parameters were examined. Single nucleotide polymorphism (SNP) of CA VI gene exon 2 was determined by PCR and DNA sequencing. Salivary CA activity, buffering capacity and pH were also determined. RESULTS: Two SNPs (dbSNP: 142460367 and 142460368), which are responsible for amino acid changes, were found. The frequencies of these SNPs were not significantly different between the caries-free group and the group with caries. There was no correlation between these SNPs and the salivary parameters. CONCLUSION: Two SNPs found in young Turkish adults have no correlation with low caries prevalence.
Subject(s)
Carbonic Anhydrase IV/genetics , Exons , Polymorphism, Genetic , Base Sequence , DNA Primers , Female , Humans , Male , Polymerase Chain Reaction , TurkeyABSTRACT
HCO(3) (-) is the signal for early activation of sperm motility. In vivo, this occurs when sperm come into contact with the HCO(3) (-) containing fluids in the reproductive tract. The activated motility enables sperm to travel the long distance to the ovum. In spermatozoa HCO(3) (-) stimulates the atypical sperm adenylyl cyclase (sAC) to promote the cAMP-mediated pathway that increases flagellar beat frequency. Stimulation of sAC may occur when HCO(3) (-) enters spermatozoa either directly by anion transport or indirectly via diffusion of CO(2) with subsequent hydration by intracellular carbonic anhydrase (CA). We here show that murine sperm possess extracellular CA IV that is transferred to the sperm surface as the sperm pass through the epididymis. Comparison of CA IV expression by qRT PCR analysis confirms that the transfer takes place in the corpus epididymidis. We demonstrate murine and human sperm respond to CO(2) with an increase in beat frequency, an effect that can be inhibited by ethoxyzolamide. Comparing CA activity in sperm from wild-type and CA IV(-/-) mice we found a 32.13% reduction in total CA activity in the latter. The CA IV(-/-) sperm also have a reduced response to CO(2). While the beat frequency of wild-type sperm increases from 2.86±0.12 Hz to 6.87±0.34 Hz after CO(2) application, beat frequency of CA IV(-/-) sperm only increases from 3.06±0.20 Hz to 5.29±0.47 Hz. We show, for the first time, a physiological role of CA IV that supplies sperm with HCO(3) (-), which is necessary for stimulation of sAC and hence early activation of spermatozoa.
Subject(s)
Bicarbonates/pharmacology , Carbonic Anhydrase IV/metabolism , Sperm Motility/drug effects , Spermatozoa/drug effects , Adenylyl Cyclases/metabolism , Animals , Bicarbonates/metabolism , Carbon Dioxide/pharmacology , Carbonic Anhydrase IV/genetics , Dose-Response Relationship, Drug , Epididymis/cytology , Epididymis/enzymology , Ethoxzolamide/pharmacology , Humans , Immunoblotting , Immunohistochemistry , Kidney/enzymology , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Reverse Transcriptase Polymerase Chain Reaction , Sperm Motility/physiology , Sperm Tail/drug effects , Sperm Tail/physiology , Spermatozoa/cytology , Spermatozoa/enzymology , Time FactorsABSTRACT
Retinitis pigmentosa is a highly heterogeneous form of inherited blindness which affects more than 1.3 million individuals worldwide. The RP17 form of the disease is caused by an arginine to tryptophan (R14W) mutation in the signal sequence of carbonic anhydrase IV (CAIV). While CAIV is expressed in the choriocapillaries of the eye and renal epithelium, the R14W mutation results in an exclusively ocular phenotype in affected individuals. In order to investigate the mechanism of disease in RP17 and the lack of kidney phenotype, we compared the subcellular localization and post-translational processing of wild-type (WT)- and mutant-CAIV in three cell types. We show using immunocytochemistry that unlike WT CAIV which is transported to the plasma membrane of transfected COS-7 and HT-1080 cells, the R14W mutant CAIV is retained in the endoplasmic reticulum. Western blot analyses further reveal that whereas the WT CAIV is processed to its mature form in both these cell lines, significant levels of the R14W mutant protein remain in its immature form. Importantly, flow cytometry experiments demonstrate that compared to WT CAIV protein, expression of specifically the R14W CAIV results in an S and G2/M cell-cycle block, followed by apoptosis. Interestingly, when the above experiments were repeated in the human embryonic kidney cell line, HEK-293, strikingly different results were obtained. These cells were unaffected by the expression of the R14W mutant CAIV and were able to process the mutant and WT protein equally effectively. This study has important implications for our understanding of the RP17 phenotype.