RESUMO
During the past three decades, mice, zebrafish, fruit flies, and Caenorhabditis elegans have been the primary model organisms used for the study of various biological phenomena. These models have also been adopted and developed to investigate the physiological roles of carbonic anhydrases (CAs) and carbonic anhydrase-related proteins (CARPs). These proteins belong to eight CA families and are identified by Greek letters: α, ß, γ, δ, ζ, η, θ, and ι. Studies using model organisms have focused on two CA families, α-CAs and ß-CAs, which are expressed in both prokaryotic and eukaryotic organisms with species-specific distribution patterns and unique functions. This review covers the biological roles of CAs and CARPs in light of investigations performed in model organisms. Functional studies demonstrate that CAs are not only linked to the regulation of pH homeostasis, the classical role of CAs, but also contribute to a plethora of previously undescribed functions.
Assuntos
Anidrases Carbônicas , Equilíbrio Ácido-Base , Animais , Humanos , Camundongos , Especificidade da Espécie , Peixe-ZebraRESUMO
Schistosoma mansoni is an intestinal parasite with one ß-class carbonic anhydrase, SmaBCA. We report the sequence enhancing, production, catalytic activity, and inhibition results of the recombinant SmaBCA. It showed significant catalytic activity on CO2 hydration in vitro with kcat 1.38 × 105 s-1 and kcat/Km 2.33 × 107 M-1 s-1. Several sulphonamide inhibitors, from which many are clinically used, showed submicromolar or nanomolar inhibitory effects on SmaBCA. The most efficient inhibitor with a KI of 43.8 nM was 4-(2-amino-pyrimidine-4-yl)-benzenesulfonamide. Other effective inhibitors with KIs in the range of 79.4-95.9 nM were benzolamide, brinzolamide, topiramate, dorzolamide, saccharin, epacadostat, celecoxib, and famotidine. The other tested compounds showed at least micromolar range inhibition against SmaBCA. Our results introduce SmaBCA as a novel target for drug development against schistosomiasis, a highly prevalent parasitic disease.
Assuntos
Anidrases Carbônicas , Parasitos , Animais , Schistosoma mansoni , Benzolamida , Clonagem MolecularRESUMO
A ß-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned from the genome of the Monogenean platyhelminth Gyrodactylus salaris, a parasite of Atlantic salmon. The new enzyme, GsaCAß has a significant catalytic activity for the physiological reaction, CO2 + H2O â HCO3- + H+ with a kcat of 1.1 × 105 s-1 and a kcat/Km of 7.58 × 106 M-1 × s-1. This activity was inhibited by acetazolamide (KI of 0.46 µM), a sulphonamide in clinical use, as well as by selected inorganic anions and small molecules. Most tested anions inhibited GsaCAß at millimolar concentrations, but sulfamide (KI of 81 µM), N,N-diethyldithiocarbamate (KI of 67 µM) and sulphamic acid (KI of 6.2 µM) showed a rather efficient inhibitory action. There are currently very few non-toxic agents effective in combating this parasite. GsaCAß is subsequently proposed as a new drug target for which effective inhibitors can be designed.
Assuntos
Anidrases Carbônicas , Parasitos , Platelmintos , Salmo salar , Animais , Ânions/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/genética , Clonagem Molecular , Parasitos/genética , Platelmintos/genética , Salmo salar/genéticaRESUMO
Congenital ataxia and mental retardation are mainly caused by variations in the genes that affect brain development. Recent reports have shown that mutations in the CA8 gene are associated with mental retardation and ataxia in humans and ataxia in mice. The gene product, carbonic anhydrase-related protein VIII (CARP VIII), is predominantly present in cerebellar Purkinje cells, where it interacts with the inositol 1,4,5-trisphosphate receptor type 1, a calcium channel. In this study, we investigated the effects of the loss of function of CARP VIII during embryonic development in zebrafish using antisense morpholino oligonucleotides against the CA8 gene. Knockdown of CA8 in zebrafish larvae resulted in a curved body axis, pericardial edema and abnormal movement patterns. Histologic examination revealed gross morphologic defects in the cerebellar region and in the muscle. Electron microscopy studies showed increased neuronal cell death in developing larvae injected with CA8 antisense morpholinos. These data suggest a pivotal role for CARP VIII during embryonic development. Furthermore, suppression of CA8 expression leads to defects in motor and coordination functions, mimicking the ataxic human phenotype. This work reveals an evolutionarily conserved function of CARP VIII in brain development and introduces a novel zebrafish model in which to investigate the mechanisms of CARP VIII-related ataxia and mental retardation in humans.
Assuntos
Ataxia/genética , Anidrases Carbônicas/genética , Cerebelo/anormalidades , Proteínas do Tecido Nervoso/genética , Peixe-Zebra/embriologia , Animais , Ataxia/fisiopatologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Anidrases Carbônicas/metabolismo , Morte Celular/genética , Clonagem Molecular , Biologia Computacional , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Marcação In Situ das Extremidades Cortadas , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Deficiência Intelectual/genética , Deficiência Intelectual/fisiopatologia , Larva/genética , Larva/crescimento & desenvolvimento , Masculino , Camundongos , Microscopia Eletrônica , Mutação , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fenótipo , Células de Purkinje/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
A ß-carbonic anhydrase (CA, EC 4.2.1.1) was cloned, purified and characterized from Anopheles gambiae, the mosquito species mainly involved in the transmission of malaria. The new enzyme, AgaCA, showed a significant catalytic activity for the physiologic reaction, CO2 hydration to bicarbonate and protons, with a kcat of 7.2×10(5)s(-1) and kcat/Km of 5.6×10(7)M(-1)s(-1), being thus similar to parasite ß-CAs which were discovered earlier as drug targets for antifungal or anti-protozoan agents. An inhibition study of AgaCA with a panel of aromatic, aliphatic and heterocyclic sulfonamides allowed us to identify several low nanomolar inhibitors of the enzyme. Benzolamide and aminobenzolamide showed inhibition constants of 6.8-9.8nM, whereas a structurally related aromatic derivative, 4-(2-hydroxymethyl-4-nitrophenyl-sulfonamidoethyl)-benzenesulfonamide was the strongest inhibitor with a KI of 6.1nM. As ß-CAs are not present in mammals, including humans, finding effective and selective A. gambiae CA inhibitors may lead to alternative procedures for controlling malaria by impairing the growth of its transmission vector, the mosquito.
Assuntos
Anopheles/química , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/química , Proteínas de Insetos/antagonistas & inibidores , Inseticidas/química , Prótons , Sulfanilamidas/química , Sequência de Aminoácidos , Animais , Anopheles/classificação , Anopheles/enzimologia , Baculoviridae/genética , Bicarbonatos/química , Dióxido de Carbono/química , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Clonagem Molecular , Expressão Gênica , Ensaios de Triagem em Larga Escala , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Cinética , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Células Sf9 , SpodopteraRESUMO
The catalytically inactive isoforms of α-carbonic anhydrases are known as carbonic anhydrase related proteins (CARPs). The CARPs occur independently or as domains of other proteins in animals (both vertebrates and invertebrates) and viruses. The catalytic inactivity of CARPs is due to the lack of histidine residues required for the coordination of the zinc atom. The phylogenetic analysis shows that these proteins are highly conserved across the species. The three CARPs in vertebrates are known as CARP VIII, X and XI. CARPs orthologous to CARP VIII are found in deuterostome invertebrates, whereas protostomes only possess orthologs of CARP X. The CA-like domains of receptor-type protein tyrosine phosphatases (PTPR) are found only in PTPRG and PTPRZ. Most of these CARPs are predominantly expressed in central nervous system. Among the three vertebrate CA isoforms, CARP VIII is functionally associated with motor coordination in human, mouse and zebrafish and certain types of cancers in humans. Vertebrate expression studies show that CARP X is exclusively expressed in the brain. CARP XI is only found in tetrapods and is highly expressed in the central nervous system (CNS) of humans and mice and is also associated with several cancers. CARP VIII, PTPRZ and PTPRG have been shown to coordinate the function of other proteins by protein-protein interaction, and viral CARPs participate in attachment to host cells, but the precise biological function of CARPs X and XI is still unknown. The findings so far suggest many novel functions for the CARP subfamily, most likely related to binding to other proteins.
Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Neoplasias/enzimologia , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Sequência de Aminoácidos , Animais , Anidrases Carbônicas/genética , Cristalografia por Raios X , Histidina/genética , Humanos , Camundongos , Filogenia , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Vertebrados/genéticaRESUMO
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.
Assuntos
Evolução Biológica , Animais , Anidrase Carbônica IV/classificação , Anidrase Carbônica IV/genética , Anidrase Carbônica IV/metabolismo , Anidrases Carbônicas/classificação , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Duplicação Gênica , Expressão Gênica , Glicosilação , Glicosilfosfatidilinositóis/química , Humanos , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , FilogeniaRESUMO
Carbonic anhydrases (CAs) are essential and ubiquitous enzymes. Thus far, there are no articles on characterization of Drosophila melanogaster α-CAs. Data from invertebrate CA studies may provide opportunities for anti-parasitic drug development because α-CAs are found in many parasite or parasite vector invertebrates. We have expressed and purified D. melanogaster CAH1 and CAH2 as proteins of molecular weights 30kDa and 28kDa. CAH1 is cytoplasmic whereas CAH2 is a membrane-attached protein. Both are highly active enzymes for the CO2 hydration reaction, being efficiently inhibited by acetazolamide. CAH2 in the eye of D. melanogaster may provide a new animal model for CA-related eye diseases. A series of dithiocarbamates were also screened as inhibitors of these enzymes, with some representatives showing inhibition in the low nanomolar range.
Assuntos
Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica I/antagonistas & inibidores , Inibidores da Anidrase Carbônica/química , Drosophila melanogaster/enzimologia , Tiocarbamatos/química , Animais , Anidrase Carbônica I/classificação , Anidrase Carbônica I/metabolismo , Anidrase Carbônica II/classificação , Anidrase Carbônica II/metabolismo , Inibidores da Anidrase Carbônica/metabolismo , Biologia Computacional , Cinética , Filogenia , Ligação Proteica , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Tiocarbamatos/metabolismoRESUMO
The catalytically inactive isoforms of carbonic anhydrase (CAs) are known as CA-related proteins (CARPs) VIII, X, and XI. They have highly conserved amino acid sequences. These proteins are predominantly expressed in human and mouse brain, however, their precise roles are poorly known. CARP VIII is functionally associated with motor coordination in human and mouse. CARP X is more highly expressed in the pineal gland during night compared to the day time, suggesting a function for wake/sleep patterns. Phylogeny shows that CARP XI has emerged from CARP X. It is only found in tetrapods and is highly expressed in the central nervous system (CNS) of humans and is also associated with several cancers. Detailed analysis of CARPs is in progress in our laboratory to understand their role in normal physiology. We present a review of literature on CARPs and present some novel data on CARPs obtained in our laboratory.
Assuntos
Anidrases Carbônicas/metabolismo , Animais , Biocatálise , Biotransformação , Anidrases Carbônicas/genética , Ativação Enzimática , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Over the past decade, the demand for automated protein function prediction has increased due to the volume of newly sequenced proteins. In this paper, we address the function prediction task by developing an ensemble system automatically assigning Gene Ontology (GO) terms to the given input protein sequence. We develop an ensemble system which combines the GO predictions made by random forest (RF) and neural network (NN) classifiers. Both RF and NN models rely on features derived from BLAST sequence alignments, taxonomy and protein signature analysis tools. In addition, we report on experiments with a NN model that directly analyzes the amino acid sequence as its sole input, using a convolutional layer. The Swiss-Prot database is used as the training and evaluation data. In the CAFA3 evaluation, which relies on experimental verification of the functional predictions, our submitted ensemble model demonstrates competitive performance ranking among top-10 best-performing systems out of over 100 submitted systems. In this paper, we evaluate and further improve the CAFA3-submitted system. Our machine learning models together with the data pre-processing and feature generation tools are publicly available as an open source software at https://github.com/TurkuNLP/CAFA3.
Assuntos
Redes Neurais de Computação , Proteínas , Bases de Dados de Proteínas , Proteínas/química , Alinhamento de Sequência , SoftwareRESUMO
Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the hydration of carbon dioxide and take part in many essential physiological processes. In humans, 15 CAs are characterized, including the only secreted isoenzyme CA VI. CA VI has been linked to specific processes in the mouth, namely bitter taste perception, dental caries, and maintenance of enamel pellicle, and implicated in several immunity-related phenomena. However, little is known of the mechanisms of the above. In this study, we characterized human CA VI purified from saliva and milk with biophysical methods and measured their enzyme activities and acetazolamide inhibition. Size-exclusion chromatography showed peaks of salivary and milk CA VI corresponding to hexameric state or larger at pH 7.5. At pH 5.0 the hexamer peaks dominated. SDS- PAGE of milk CA VI protein treated with a bifunctional crosslinker further confirmed that a majority of CA VI is oligomers of similar sizes in solution. Mass spectrometry experiments confirmed that both of the two putative N-glycosylation sites, Asn67 and Asn256, are heterogeneously glycosylated. The attached glycans in milk CA VI were di- and triantennary complex-type glycans, carrying both a core fucose and 1 to 2 additional fucose units, whereas the glycans in salivary CA VI were smaller, seemingly degraded forms of core fucosylated complex- or hybrid-type glycans. Mass spectrometry also verified the predicted signal peptide cleavage site and the terminal residue, Gln 18, being in pyroglutamate form. Thorough characterization of CA VI paves way to better understanding of the biological function of the protein.
Assuntos
Anidrases Carbônicas , Leite Humano , Saliva , Anidrases Carbônicas/análise , Fucose , Humanos , Leite Humano/enzimologia , Saliva/enzimologiaRESUMO
BACKGROUND: Carbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate alpha-CA family consists of 16 subfamilies, three of which (VIII, X and XI) consist of acatalytic proteins. These are named carbonic anhydrase related proteins (CARPs), and their inactivity is due to absence of one or more Zn-binding histidine residues. In this study, we analyzed and evaluated the distribution of genes encoding CARPs in different organisms using bioinformatic methods, and studied their expression in mouse tissues using immunohistochemistry and real-time quantitative PCR. RESULTS: We collected 84 sequences, of which 22 came from novel or improved gene models which we created from genome data. The distribution of CARP VIII covers vertebrates and deuterostomes, and CARP X appears to be universal in the animal kingdom. CA10-like genes have had a separate history of duplications in the tetrapod and fish lineages. Our phylogenetic analysis showed that duplication of CA10 into CA11 has occurred only in tetrapods (found in mammals, frogs, and lizards), whereas an independent duplication of CA10 was found in fishes. We suggest the name CA10b for the second fish isoform. Immunohistochemical analysis showed a high expression level of CARP VIII in the mouse cerebellum, cerebrum, and also moderate expression in the lung, liver, salivary gland, and stomach. These results also demonstrated low expression in the colon, kidney, and Langerhans islets. CARP X was moderately expressed in the cerebral capillaries and the lung and very weakly in the stomach and heart. Positive signals for CARP XI were observed in the cerebellum, cerebrum, liver, stomach, small intestine, colon, kidney, and testis. In addition, the results of real-time quantitative PCR confirmed a wide distribution for the Car8 and Car11 mRNAs, whereas the expression of the Car10 mRNA was restricted to the frontal cortex, parietal cortex, cerebellum, midbrain, and eye. CONCLUSIONS: CARP sequences have been strongly conserved between different species, and all three CARPs show high expression in the mouse brain and CARP VIII is also expressed in several other tissues. These findings suggest an important functional role for these proteins in mammals.
Assuntos
Anidrases Carbônicas/genética , Sequência de Aminoácidos , Animais , Anidrases Carbônicas/química , Expressão Gênica , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Alinhamento de SequênciaRESUMO
BACKGROUND: The beta-carbonic anhydrase (CA, EC 4.2.1.1) enzymes have been reported in a variety of organisms, but their existence in animals has been unclear. The purpose of the present study was to perform extensive sequence analysis to show that the beta-CAs are present in invertebrates and to clone and characterize a member of this enzyme family from a representative model organism of the animal kingdom, e.g., Drosophila melanogaster. RESULTS: The novel beta-CA gene, here named DmBCA, was identified from FlyBase, and its orthologs were searched and reconstructed from sequence databases, confirming the presence of beta-CA sequences in 55 metazoan species. The corresponding recombinant enzyme was produced in Sf9 insect cells, purified, kinetically characterized, and its inhibition was investigated with a series of simple, inorganic anions. Holoenzyme molecular mass was defined by dynamic light scattering analysis and gel filtration, and the results suggested that the holoenzyme is a dimer. Double immunostaining confirmed predictions based on sequence analysis and localized DmBCA protein to mitochondria. The enzyme showed high CO2 hydratase activity, with a kcat of 9.5 x 105 s-1 and a kcat/KM of 1.1 x 108 M-1s-1. DmBCA was appreciably inhibited by the clinically-used sulfonamide acetazolamide, with an inhibition constant of 49 nM. It was moderately inhibited by halides, pseudohalides, hydrogen sulfide, bisulfite and sulfate (KI values of 0.67 - 1.36 mM) and more potently by sulfamide (KI of 0.15 mM). Bicarbonate, nitrate, nitrite and phenylarsonic/boronic acids were much weaker inhibitors (KIs of 26.9 - 43.7 mM). CONCLUSIONS: The Drosophila beta-CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development.
Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/classificação , Proteínas de Drosophila/química , Proteínas de Drosophila/classificação , Drosophila melanogaster/enzimologia , Sequência de Aminoácidos , Animais , Anidrases Carbônicas/genética , Bases de Dados de Proteínas , Dimerização , Proteínas de Drosophila/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Cinésica , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Over half of the DNA of mammalian genomes is transcribed, and one of the emerging enigmas in the field of RNA research is intergenic splicing or transcription induced chimerism. We argue that fused low-copy-number transcripts constitute neglected pathological mechanism akin to copy number variation, due to loss of stoichiometric subunit ratios in protein complexes. An obstacle for transcriptomics meta-analysis of published microarrays is the traditional nomenclature of merged transcript neighbors under same accession codes. Tandem transcripts cover 4-20% of genomes but are only loosely overlapping in population. They were most enriched in systems medicine annotations concerning neurology, thalassemia and genital disorders in the GeneGo Inc. MetaCore-MetaDrug(TM) knowledgebase, evaluated with external randomizations here. Clinical transcriptomics is good news since new disease etiologies offer new remedies. We identified homeotic HOX-transfactors centered around BMI-1, the Grb2 adaptor network, the kallikrein system, and thalassemia RNA surveillance as vulnerable hotspot chimeras. As a cure, RNA interference would require verification of chimerism from symptomatic tissue contra healthy control tissue from the same patient.
Assuntos
Bases de Dados de Ácidos Nucleicos , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Proteínas Mutantes Quiméricas/genética , Algoritmos , Dosagem de Genes , Redes Reguladoras de Genes , Humanos , Interferência de RNA , Splicing de RNARESUMO
INTRODUCTION: Nigeria has the largest burden of Sickle Cell Disease (SCD) with estimated 100,000 new born affected annually. SCD is a Hemoglobin (Hb) disorder with the major form resulting from the substitution of a polar glutamate (Glu) by non-polar Valine (Val) in an invariant region of Hbß chain-subunit. Species of Hb found in the sickle cell trait are HbA and HbS in a 60:40 proportion, in SCD only HbS, in the HbC disease only HbC, and in the SC disease it's HbS and HbC in a 50:50 equal proportion. OBJECTIVE: This paper reviews herbal medicines usage in sub-Saharan Africa (sSA) to ameliorate the crisis associated with SCD. The model Hb tetramer suggests a higher membrane affinity of HbS and HbC, promoting dehydration of RBCs, with concomitant in vivo crystallization. Some drawbacks using these herbal drugs include; poor bioavailability and the lack of proper pharmacovigilance monitoring procedures arising from weak governance structure combined with under reporting of herbal usage to physicians were discussed. Probable epigenetic loci that could be targeted using phytomedicines for effective SCD management were also discussed. METHODS: Using search engines, several databases including Google scholar, PubMed, Academic Resource Index were utilized as a source for relevant publications/ literature. The protein coordinates for the Hb tetramer were obtained from the Protein Data Bank (PDB). CONCLUSION: Manipulation of epigenetics to achieve better SCD management involves careful thinking. Herein, we discuss some epigenetic interactions that could be putatively tweaked with a view of enhancing soluble bioactive small molecular components with the potential to reactivate γ -globin genes, thereby boosting immune response in patient with SCD.
Assuntos
Anemia Falciforme/tratamento farmacológico , Fitoterapia , África Subsaariana , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Animais , Composição de Medicamentos , Epigenômica , Humanos , Legislação de Medicamentos , Compostos Fitoquímicos/uso terapêutico , Compostos Fitoquímicos/toxicidadeRESUMO
Carbonic anhydrases (CA) catalyze the reversible hydration of CO2 to protons and bicarbonate and thereby play a fundamental role in the epithelial acid/base transport mechanisms serving fluid secretion and absorption for whole-body acid/base regulation. The three carbonic anhydrase-related proteins (CARPs) VIII, X, and XI, however, are catalytically inactive. Previous work has shown that some CA isoforms noncatalytically enhance lactate transport through various monocarboxylate transporters (MCT). Therefore, we examined whether the catalytically inactive CARPs play a role in lactate transport. Here, we report that CARP VIII, X, and XI enhance transport activity of the MCT MCT1 when coexpressed in Xenopus oocytes, as evidenced by the rate of rise in intracellular H+ concentration detected using ion-sensitive microelectrodes. Based on previous studies, we suggest that CARPs may function as a 'proton antenna' for MCT1, to drive proton-coupled lactate transport across the cell membrane.
Assuntos
Anidrases Carbônicas/metabolismo , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/metabolismo , Animais , Animais Geneticamente Modificados , Bicarbonatos/metabolismo , Transporte Biológico/fisiologia , Transporte Biológico Ativo , Biomarcadores Tumorais/metabolismo , Catálise , Humanos , Concentração de Íons de Hidrogênio , Transportadores de Ácidos Monocarboxílicos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Oócitos/metabolismo , Prótons , Simportadores/fisiologia , Xenopus laevis/metabolismoRESUMO
Secretory human carbonic anhydrase VI (CA VI) has emerged as a potential drug target due to its role in pathological states, such as excess acidity-caused dental caries and injuries of gastric epithelium. Currently, there are no available CA VI-selective inhibitors or crystallographic structures of inhibitors bound to CA VI. The present study focuses on the site-directed CA II mutant mimicking the active site of CA VI for inhibitor screening. The interactions between CA VI-mimic and a series of benzenesulfonamides were evaluated by fluorescent thermal shift assay, stopped-flow CO2 hydration assay, isothermal titration calorimetry, and X-ray crystallography. Kinetic parameters showed that A65T, N67Q, F130Y, V134Q, L203T mutations did not influence catalytic properties of CA II, but inhibitor affinities resembled CA VI, exhibiting up to 0.16 nM intrinsic affinity for CA VI-mimic. Structurally, binding site of CA VI-mimic was found to be similar to CA VI. The ligand interactions with mutated side chains observed in three crystallographic structures allowed to rationalize observed variation of binding modes and experimental binding affinities to CA VI. This integrative set of kinetic, thermodynamic, and structural data revealed CA VI-mimic as a useful model to design CA VI-specific inhibitors which could be beneficial for novel therapeutic applications.
Assuntos
Substituição de Aminoácidos , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas , Modelos Químicos , Modelos Moleculares , Dióxido de Carbono/química , Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Cristalografia por Raios X , Humanos , Mutação de Sentido Incorreto , Domínios ProteicosRESUMO
BACKGROUND: Carbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15-21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX) domain, a novel combination for both CAs and PTXs. METHODS: We isolated and sequenced zebrafish (Danio rerio) CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI-PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI-PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf). The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish. RESULTS: The recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org). The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is consistent with the modifications observed in mass spectrometry. Phylogenetics and sequence analyses provide a consistent hypothesis of the evolutionary history of domains associated with CA VI in mammals and non-mammals. Briefly, the evidence suggests that ancestral CA VI was a transmembrane protein, the exon coding for the cytoplasmic domain was replaced by one coding for PTX domain, and finally, in the therian lineage, the PTX-coding exon was lost. We knocked down CA VI expression in zebrafish embryos with antisense morpholino oligonucleotides, resulting in phenotype features of decreased buoyancy and swim bladder deflation in 4 dpf larvae. DISCUSSION: These findings provide novel insights into the evolution, structure, and function of this unique CA form.
RESUMO
The main function of CAs (carbonic anhydrases) is to participate in the regulation of acid-base balance. Although 12 active isoenzymes of this family had already been described, analyses of genomic databases suggested that there still exists another isoenzyme, CA XV. Sequence analyses were performed to identify those species that are likely to have an active form of this enzyme. Eight species had genomic sequences encoding CA XV, in which all the amino acid residues critical for CA activity are present. However, based on the sequence data, it was apparent that CA XV has become a non-processed pseudogene in humans and chimpanzees. RT-PCR (reverse transcriptase PCR) confirmed that humans do not express CA XV. In contrast, RT-PCR and in situ hybridization performed in mice showed positive expression in the kidney, brain and testis. A prediction of the mouse CA XV structure was performed. Phylogenetic analysis showed that mouse CA XV is related to CA IV. Therefore both of these enzymes were expressed in COS-7 cells and studied in parallel experiments. The results showed that CA XV shares several properties with CA IV, i.e. it is a glycosylated glycosylphosphatidylinositol-anchored membrane protein, and it binds CA inhibitor. The catalytic activity of CA XV is low, and the correct formation of disulphide bridges is important for the activity. Both specific and non-specific chaperones increase the production of active enzyme. The results suggest that CA XV is the first member of the alpha-CA gene family that is expressed in several species, but not in humans and chimpanzees.
Assuntos
Anidrases Carbônicas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Anidrases Carbônicas/genética , Anidrases Carbônicas/fisiologia , Chlorocebus aethiops , Biologia Computacional , Dissulfetos/metabolismo , Regulação Enzimológica da Expressão Gênica , Glicosilação , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Pan troglodytes/genética , Filogenia , Ligação Proteica , Estrutura Quaternária de Proteína , Pseudogenes/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
BACKGROUND: Horizontal gene transfer (HGT) is a movement of genetic information occurring outside of normal mating activities. It is especially common between prokaryotic endosymbionts and their protozoan, insect, and nematode hosts. Although beta carbonic anhydrase (ß-CA) plays a crucial role in metabolic functions of many living organisms, the origin of ß-CA genes in eukaryotic species remains unclear. METHODS: This study was conducted using phylogenetics, prediction of subcellular localization, and identification of ß-CA, transposase, integrase, and resolvase genes on the MGEs of bacteria. We also structurally analyzed ß-CAs from protozoans, insects, and nematodes and their putative prokaryotic common ancestors, by homology modelling. RESULTS: Our investigations of a number of target genomes revealed that genes coding for transposase, integrase, resolvase, and conjugation complex proteins have been integrated with ß-CA gene sequences on mobile genetic elements (MGEs) which have facilitated the mobility of ß-CA genes from bacteria to protozoan, insect, and nematode species. The prokaryotic origin of protozoan, insect, and nematode ß-CA enzymes is supported by phylogenetic analyses, prediction of subcellular localization, and homology modelling. CONCLUSION: MGEs form a complete set of enzymatic tools, which are relevant to HGT of ß-CA gene sequences from prokaryotes to protozoans, insects, and nematodes.