Martti Tolvanen

Characterization of CA XV, a new GPI-anchored form of carbonic anhydrase

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.

Cloning, Characterization, and Sulfonamide and Thiol Inhibition Studies of an α‑Carbonic Anhydrase from Trypanosoma cruzi, the Causative Agent of Chagas Disease

An α-carbonic anhydrase (CA, EC 4.2.1.1) has been identified, cloned, and characterized from the unicellular protozoan Trypanosoma cruzi, the causative agent of Chagas disease. The enzyme (TcCA) has a very high catalytic activity for the CO2 hydration reaction, being similar kinetically to the human (h) isoform hCA II, although it is devoid of the His64 proton shuttle. A large number of aromatic/heterocyclic sulfonamides and some 5-mercapto-1,3,4-thiadiazoles were investigated as TcCA inhibitors. The aromatic sulfonamides were weak inhibitors (K(I) values of 192 nM to 84 μM), whereas some heterocyclic compounds inhibited the enzyme with K(I) values in the range 61.6-93.6 nM. The thiols were the most potent in vitro inhibitors (K(I) values of 21.1-79.0 nM), and some of them also inhibited the epimastigotes growth of two T. cruzi strains in vivo.

Characterization of the first beta-class carbonic anhydrase from an arthropod (Drosophila melanogaster) and phylogenetic analysis of beta-class carbonic anhydrases in invertebrates

BackgroundThe β-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 β-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.ResultsThe novel β-CA gene, here named DmBCA, was identified from FlyBase, and its orthologs were searched and reconstructed from sequence databases, confirming the presence of β-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 × 105 s-1 and a kcat/KM of 1.1 × 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).ConclusionsThe Drosophila β-CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development.

Phylogeny and expression of carbonic anhydrase-related proteins.

BackgroundCarbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate α-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.ResultsWe 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.ConclusionsCARP 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.

Carbonic Anhydrase Related Proteins: Molecular Biology and Evolution

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.

Carbonic Anhydrase Related Protein VIII and its Role in Neurodegeneration and Cancer

Mammalian carbonic anhydrase (α-CA) gene family comprises sixteen isoforms, thirteen of which are active isozymes and three isoforms lack classical CA activity of reversible hydration of CO(2) due to absence of one or more histidine residues required for CA catalytic activity. The inactive isoforms are known as carbonic anhydrase related proteins (CARPs) VIII, X and XI. Among these three, CARP VIII was reported first in 1990 from a mouse brain cDNA library and is well studied structurally as well as functionally compared to CARP X and XI. CARP VIII is an intriguing protein and is widely distributed and evolutionarily well-conserved across the species. It is mainly expressed in the Purkinje cells of cerebellum and in wide variety of other tissues both in mouse and human. CARP VIII has been linked to development of colorectal and lung cancers in humans, and overexpression of CARP VIII has been observed in several other cancers. A mutation in the CA8 gene has been associated with ataxia, mild mental retardation and quadrupedal gait in humans and with lifelong gait disorder in mice, suggesting an important role for CARP VIII in the brain. However, the precise function of CARP VIII is still an enigma. The present review article describes the previous data on CARP VIII, including its structure, role in neurodegeneration and cancer; and bioinformatic and expression analyses recently performed in our laboratory.

Abnormal cerebellar development and ataxia in CARP VIII morphant zebrafish

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.

Bioinformatic analysis of beta carbonic anhydrase sequences from protozoans and metazoans

BackgroundDespite the high prevalence of parasitic infections, and their impact on global health and economy, the number of drugs available to treat them is extremely limited. As a result, the potential consequences of large-scale resistance to any existing drugs are a major concern. A number of recent investigations have focused on the effects of potential chemical inhibitors on bacterial and fungal carbonic anhydrases. Among the five classes of carbonic anhydrases (alpha, beta, gamma, delta and zeta), beta carbonic anhydrases have been reported in most species of bacteria, yeasts, algae, plants, and particular invertebrates (nematodes and insects). To date, there has been a lack of knowledge on the expression and molecular structure of beta carbonic anhydrases in metazoan (nematodes and arthropods) and protozoan species.MethodsHere, the identification of novel beta carbonic anhydrases was based on the presence of the highly-conserved amino acid sequence patterns of the active site. A phylogenetic tree was constructed based on codon-aligned DNA sequences. Subcellular localization prediction for each identified invertebrate beta carbonic anhydrase was performed using the TargetP webserver.ResultsWe verified a total of 75 beta carbonic anhydrase sequences in metazoan and protozoan species by proteome-wide searches and multiple sequence alignment. Of these, 52 were novel, and contained highly conserved amino acid residues, which are inferred to form the active site in beta carbonic anhydrases. Mitochondrial targeting peptide analysis revealed that 31 enzymes are predicted with mitochondrial localization; one was predicted to be a secretory enzyme, and the other 43 were predicted to have other undefined cellular localizations.ConclusionsThese investigations identified 75 beta carbonic anhydrases in metazoan and protozoan species, and among them there were 52 novel sequences that were not previously annotated as beta carbonic anhydrases. Our results will not only change the current information in proteomics and genomics databases, but will also suggest novel targets for drugs against parasites.

The β-carbonic anhydrase from the malaria mosquito Anopheles gambiae is highly inhibited by sulfonamides

A β-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 β-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 β-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.

Characterization of Non-Specific Cytotoxic Cell Receptor Protein 1: A New Member of the Lectin-Type Subfamily of F-Box Proteins

Our previous microarray study showed that the non-specific cytotoxic cell receptor protein 1 (Nccrp1) transcript is significantly upregulated in the gastric mucosa of carbonic anhydrase IX (CA IX)-deficient (Car9−/−) mice. In this paper, we aimed to characterize human NCCRP1 and to elucidate its relationship to CA IX. Recombinant NCCRP1 protein was expressed in Escherichia coli, and a novel polyclonal antiserum was raised against the purified full-length protein. Immunocytochemistry showed that NCCRP1 is expressed intracellularly, even though it has previously been described as a transmembrane protein. Using bioinformatic analyses, we identified orthologs of NCCRP1 in 35 vertebrate genomes, and up to five paralogs per genome. These paralogs are FBXO genes whose protein products are components of the E3 ubiquitin ligase complexes. NCCRP1 proteins have no signal peptides or transmembrane domains. NCCRP1 has mainly been studied in fish and was thought to be responsible for the cytolytic function of nonspecific cytotoxic cells (NCCs). Our analyses showed that in humans, NCCRP1 mRNA is expressed in tissues containing squamous epithelium, whereas it shows a more ubiquitous tissue expression pattern in mice. Neither human nor mouse NCCRP1 expression is specific to immune tissues. Silencing CA9 using siRNAs did not affect NCCRP1 levels, indicating that its expression is not directly regulated by CA9. Interestingly, silencing NCCRP1 caused a statistically significant decrease in the growth of HeLa cells. These studies provide ample evidence that the current name, “non-specific cytotoxic cell receptor protein 1,” is not appropriate. We therefore propose that the gene name be changed to FBXO50.