The production and biochemical characterization of α-carbonic anhydrase from Lactobacillus rhamnosus GG.

We report the production and biochemical characterization of an α-carbonic anhydrase (LrhCA) from gram-positive probiotic bacteria Lactobacillus rhamnosus GG. CAs form a family of metalloenzymes that catalyze hydration of CO2/interconversion between CO2 and water to bicarbonate ions and protons. They are divided into eight independent gene families (α, ß, γ, δ, ζ, η, θ, and ι). Interestingly, many pathogens have been identified with only ß- and/or γ-CAs, which can be targeted with CA-specific inhibitors (CAIs) acting as anti-pathogen drugs. Since it is important to study the potential off-target effects of CAIs for both the human body and its commensal bacteria, we took L. rhamnosus GG as our study subject. To date, only a single α-CA has been identified in L. rhamnosus GG, which was successfully produced and biochemically characterized. LrhCA showed moderate catalytic activity with the following kinetic parameters: kcat of 9.86 × 105 s-1 and kcat/KM of 1.41 × 107 s-1 M-1. Moderate inhibition was established with 11 of the 39 studied sulfonamides. The best inhibitors were 5-((4-aminophenyl)sulfonamido)-1,3,4-thiadiazole-2-sulfonamide, 4-(2-hydroxymethyl-4-nitrophenyl-sulfonamidoethyl)-benzenesulfonamide, and benzolamide with Ki values of 319 nM, 378 nM, and 387 nM, respectively. The other compounds showed weaker inhibitory effects. The Ki of acetazolamide, a classical CAI, was 733 nM. In vitro experiments with acetazolamide showed that it had no significant effect on cell growth in L. rhamnosus GG culture. Several sulfonamides, including acetazolamide, are in use as clinical drugs, making their inhibition data highly relevant to avoid any adverse off-target effects towards the human body and its probiotic organisms. KEY POINTS: • The α-carbonic anhydrase from Lactobacillus rhamnosus GG (LrhCA) is 24.3 kDa. • LrhCA has significant catalytic activity with a kcat of 9.9 × 105 s-1. • Acetazolamide resulted in a marginal inhibitory effect on cell growth.

Cloning, purification, kinetic and anion inhibition studies of a recombinant ß-carbonic anhydrase from the Atlantic salmon parasite platyhelminth Gyrodactylus salaris.

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 ⇋ 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.

Assessment of databases to determine the validity of ß- and γ-carbonic anhydrase sequences from vertebrates.

BACKGROUND: The inaccuracy of DNA sequence data is becoming a serious problem, as the amount of molecular data is multiplying rapidly and expectations are high for big data to revolutionize life sciences and health care. In this study, we investigated the accuracy of DNA sequence data from commonly used databases using carbonic anhydrase (CA) gene sequences as generic targets. CAs are ancient metalloenzymes that are present in all unicellular and multicellular living organisms. Among the eight distinct families of CAs, including α, ß, γ, δ, ζ, η, θ, and ι, only α-CAs have been reported in vertebrates. RESULTS: By an in silico analysis performed on the NCBI and Ensembl databases, we identified several ß- and γ-CA sequences in vertebrates, including Homo sapiens, Mus musculus, Felis catus, Lipotes vexillifer, Pantholops hodgsonii, Hippocampus comes, Hucho hucho, Oncorhynchus tshawytscha, Xenopus tropicalis, and Rhinolophus sinicus. Polymerase chain reaction (PCR) analysis of genomic DNA persistently failed to amplify positive ß- or γ-CA gene sequences when Mus musculus and Felis catus DNA samples were used as templates. Further BLAST homology searches of the database-derived "vertebrate" ß- and γ-CA sequences revealed that the identified sequences were presumably derived from gut microbiota, environmental microbiomes, or grassland ecosystems. CONCLUSIONS: Our results highlight the need for more accurate and fast curation systems for DNA databases. The mined data must be carefully reconciled with our best knowledge of sequences to improve the accuracy of DNA data for publication.

Expression of LHCG receptors in the human penis.

The aim of this study is to investigate the expression of the luteinizing hormone/choriogonadotropin (LHCG) receptor in the human penis to see, if the luteinizing hormone (LH) effects are possible in the spongious and cavernous tissue of the penis. The number of men with erection disturbances increases significantly simultaneously with the elevated LH concentrations between 40 and 70 years. It is possible that the elevated LH concentrations may influence locally the erectile mechanisms. The precondition for this is the expression of LHCG receptors in the penis. Penile tissue was obtained from three patients undergoing total or partial penectomy due to a rectal cancer with secondary penile metastasis or squamous cell carcinoma of the penis. Immunohistochemistry was used for the detection of the LHCG receptor. Positive immunoreaction for LHCG receptors was discovered in the endothelial cells of cavernous spaces in the corpus cavernosum and corpus spongiosum penis, also in the endothelial cells of the capillary walls in all patients. Our results show that LHCG receptor is expressed in the spongious and cavernous tissue of the human penis. This finding suggests that LH can affect the spongious and cavernous tissue in human and play a significant role in the development of erectile dysfunction among the aging men.

Sulphonamide inhibition profile of Staphylococcus aureus ß-carbonic anhydrase.

This paper presents the production and kinetic and inhibitory characterisation of ß-carbonic anhydrase from the opportunistic bacterium Staphylococcus aureus (SauBCA). From the eight different carbonic anhydrase (CA) families known to date, humans have only the α-form, whereas many clinically relevant pathogens have ß- and/or γ-form(s). Based on this discovery, ß- and γ-CAs have been introduced as promising new anti-infective targets. The results of this study revealed that recombinant SauBCA possesses significant CO2 hydration activity with a kcat of 1.46 × 105 s-1 and a kcat/KM of 2.56 × 107 s- 1M-1. Its enzymatic function was inhibited by various sulphonamides in the nanomolar - micromolar range, and the Ki of acetazolamide was 628 nM. The best inhibitor was the clinically used sulfamide agent famotidine (Ki of 71 nM). The least efficient inhibitors were zonisamide and dorzolamide. Our work encourages further investigations of SauBCA in an attempt to discover novel drugs against staphylococcal infections.

Biochemical and structural characterisation of a protozoan beta-carbonic anhydrase from Trichomonas vaginalis.

We report the biochemical and structural characterisation of a beta-carbonic anhydrase (ß-CA) from Trichomonas vaginalis, a unicellular parasite responsible for one of the world's leading sexually transmitted infections, trichomoniasis. CAs are ubiquitous metalloenzymes belonging to eight evolutionarily divergent groups (α, ß, γ, δ, ζ, η, θ, and ι); humans express only α-CAs, whereas many clinically significant pathogens express only ß- and/or γ-CAs. For this reason, the latter two groups of CAs are promising biomedical targets for novel antiinfective agents. The ß-CA from T. vaginalis (TvaCA1) was recombinantly produced and biochemically characterised. The crystal structure was determined, revealing the canonical dimeric fold of ß-CAs and the main features of the enzyme active site. The comparison with the active site of human CA enzymes revealed significant differences that can be exploited for the design of inhibitors selective for the protozoan enzyme with respect to the human ones.

Activation studies with amines and amino acids of the α-carbonic anhydrase from the pathogenic protozoan Trypanosoma cruzi.

The activation of a α-class carbonic anhydrase (CAs, EC 4.2.1.1) from Trypanosoma cruzi (TcCA) was investigated with the best known classes of activators, the amino acids and aromatic/heterocyclic amines. The best TcCA activators were l-/d-DOPA and 4-amino-l-phenylalanine, which had activation constants in the range of 0.38-0.83 µM. Low micromolar activators were also l-/d-Trp, l-/d-Tyr, l-Gln, histamine and serotonin (KAs of 1.79-4.92 µM), whereas l-/d-His, l-/d-Phe and l-Asp were less effective activators (KAs of 6.39-18.7 µM). Amines such as dopamine, pyridyl-alkylamines, aminoethyl-piperazine or l-adrenaline, were devoid of activating effects on TcCA. Since the role of autacoids as many of these compounds investigated here is not known for the life cycle of T. cruzi, our work provides new tools for further investigations of factors connected with this protozoan pathogen infection.

Anion inhibition studies of a beta carbonic anhydrase from the malaria mosquito Anopheles gambiae.

An anion inhibition study of the ß-class carbonic anhydrase, AgaCA, from the malaria mosquito Anopheles gambiae is reported. A series of simple as well as complex inorganic anions, and small molecules known to interact with CAs were included in the study. Bromide, iodide, bisulphite, perchlorate, perrhenate, perruthenate, and peroxydisulphate were ineffective AgaCA inhibitors, with KIs > 200 mM. Fluoride, chloride, cyanate, thiocyanate, cyanide, bicarbonate, carbonate, nitrite, nitrate, sulphate, stannate, selenate, tellurate, diphosphate, divanadate, tetraborate, selenocyanide, and trithiocarbonate showed KIs in the range of 1.80-9.46 mM, whereas N,N-diethyldithiocarbamate was a submillimolar AgaCA inhibitor (KI of 0.65 mM). The most effective AgaCA inhibitors were sulphamide, sulphamic acid, phenylboronic acid and phenylarsonic acid, with inhibition constants in the range of 21-84 µM. The control of insect vectors responsible of the transmission of many protozoan diseases is rather difficult nowadays, and finding agents which can interfere with these processes, as the enzyme inhibitors investigated here, may arrest the spread of these diseases worldwide.

Sulfonamide Inhibition Studies of a New ß-Carbonic Anhydrase from the Pathogenic Protozoan Entamoeba histolytica.

A newly described ß-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic protozoan Entamoeba histolytica, EhiCA, was recently shown to possess a significant catalytic activity for the physiologic CO2 hydration reaction (kcat of 6.7 × 105 s-1 and a kcat/Km of 8.9 × 107 M-1 s-1). A panel of sulphonamides and one sulfamate, some of which are clinically used drugs, were investigated for their inhibitory properties against EhiCA. The best inhibitors detected in the study were 4-hydroxymethyl/ethyl-benzenesulfonamide (KIs of 36⁻89 nM), whereas some sulfanilyl-sulfonamides showed activities in the range of 285⁻331 nM. Acetazolamide, methazolamide, ethoxzolamide, and dichlorophenamide were less effective inhibitors (KIs of 509⁻845 nM) compared to other sulfonamides investigated here. As ß-CAs are not present in vertebrates, the present study may be useful for detecting lead compounds for the design of more effective inhibitors with potential to develop anti-infectives with alternative mechanisms of action.

Cloning, Characterization and Anion Inhibition Studies of a ß-Carbonic Anhydrase from the Pathogenic Protozoan Entamoeba histolytica.

We report the cloning and catalytic activity of a ß-carbonic anhydrase (CA, EC 4.2.1.1), isolated from the pathogenic protozoan Entamoeba histolytica, EhiCA. This enzyme has a high catalytic activity for the physiologic CO2 hydration reaction, with a kcat of 6.7 × 105 s-1 and a kcat/Km of 8.9 × 107 M-1 × s-1. An anion inhibition study of EhiCA with inorganic/organic anions and small molecules revealed that fluoride, chloride, cyanide, azide, pyrodiphosphate, perchlorate, tetrafluoroborate and sulfamic acid did not inhibit the enzyme activity, whereas pseudohalides (cyanate and thiocyanate), bicarbonate, nitrate, nitrite, diethyldithiocarbamate, and many complex inorganic anions showed inhibition in the millimolar range (KIs of 0.51⁻8.4 mM). The best EhiCA inhibitors were fluorosulfonate, sulfamide, phenylboronic acid and phenylarsonic acid (KIs in the range of 28⁻86 µM). Since ß-CAs are not present in vertebrates, the present study may be useful for detecting lead compounds for the design of effective enzyme inhibitors, with potential to develop anti-infectives with alternative mechanisms of action.

ß carbonic anhydrase is required for female fertility in Drosophila melanogaster.

BACKGROUND: Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous enzymes that catalyze the reversible hydration reaction of carbon dioxide. CAs are present as six structurally divergent enzyme families: α, ß, γ, δ, ζ and η. ß-CAs have a wide distribution across different species including invertebrates. Previously, we showed that Drosophila melanogaster ß-CA is a highly active mitochondrial enzyme. In this study, we investigated the function of Drosophila ß-CA by silencing the expression of the ß-CA gene using UAS/GAL4-based RNA interference (RNAi) in Drosophila in vivo. RESULTS: Crossing ß-CA RNAi lines over ubiquitous Actin driver flies did not produce any viable progeny, indicating that ß-CA expression is required for fly development. RNAi silencing of ß-CA ubiquitously in adult flies did not affect their survival rate or function of mitochondrial electron transport chain. Importantly, ß-CA RNAi led to impaired reproduction. All ß-CA knockdown females were sterile, and produced few or no eggs. Whole ovaries of knockdown females looked normal but upon cadherin staining, there was an apparent functional defect in migration of border cells, which are considered essential for normal fertilization. CONCLUSIONS: These results indicate that although Drosophila ß-CA is dispensable for survival of adult flies, it is essential for female fertility.

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

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.

Carbonic anhydrase III: a neglected isozyme is stepping into the limelight.

CONTEXT: Carbonic anhydrase III (CA III) is a cytosolic enzyme which is known to be highly expressed in the skeletal muscle and has been recently linked to important roles in several physiological processes. OBJECTIVE: This review is focused on properties of CA III, including its distribution, function, structure and modulation of enzymatic activity by activators or inhibitors. We also provide some novel data on its expression in murine tissues. METHODS: In this article, the most relevant literature on CA III has been covered. New information on the distribution has been obtained by immunohistochemical staining and western blotting. RESULTS AND CONCLUSION: CA III shows the highest expression in the skeletal muscle and liver. Several other tissues contain lower levels of the enzyme. Activation or inhibition of CA III may offer a novel opportunity to treat some of the diseases linked to the defective expression or function of this enzyme.

Biochemical and structural characterization of beta-carbonic anhydrase from the parasite Trichomonas vaginalis.

Trichomonas vaginalis is a unicellular parasite and responsible for one of the most common sexually transmittable infections worldwide, trichomoniasis. Carbonic anhydrases (CAs) are enzymes found in all lifeforms and are known to play a vital role in many biochemical processes in organisms including the maintenance of acid-base homeostasis. To date, eight evolutionarily divergent but functionally convergent forms of CAs (α, ß, γ, δ, ζ, η, θ, and ι) have been discovered. The human genome contains only α-CAs, whereas many clinically significant pathogens express only ß-CAs and/or γ-CAs. The characterization of pathogenic ß- and γ-CAs provides important knowledge for targeting these biomolecules to develop novel anti-invectives against trichomoniasis. Here, we report the recombinant production and characterization of the second ß-CA of T. vaginalis (TvaCA2). Light scattering analysis revealed that TvaCA2 is a dimeric protein, which was further supported with in silico modeling, suggesting similar structures between TvaCA2 and the first ß-CA of T. vaginalis (TvaCA1). TvaCA2 exhibited moderate catalytic activity with the following kinetic parameters: kcat of 3.8 × 105 s-1 and kcat/KM of 4.4 × 107 M-1 s-1. Enzyme activity inhibition was studied with a set of clinically used sulfonamides and sulfonamide derivates. Twenty-seven out of the 39 compounds resulted in inhibition with a nanomolar range. These initial results encourage for future work entailing the design of more potent inhibitors against TvaCA2, which may provide new assets to fight trichomoniasis. KEY MESSAGES: • Protozoan parasite Trichomonas vaginalis has two ß-carbonic anhydrases (TvaCA1/2). • TvaCA1/TvaCA2 represents promising targets for antitrichomonal drug development. • TvaCA2 is a dimer of 20.3 kDa and possesses moderate catalytic activity. • The most efficient inhibitor was clinical drug acetazolamide with KI of 222.9 nM. • The 39 tested sulfonamides form the basis for the design of more potent inhibitors.

Novel carbonic anhydrase autoantibodies and renal manifestations in patients with primary Sjogren's syndrome.

OBJECTIVE: Anti-carbonic anhydrase II (anti-CA II) antibodies have been related to renal manifestations of primary SS (pSS), and animal studies have even suggested a pathogenic role for them. However, not all pSS patients with renal tubular acidosis (RTA) present with anti-CA II antibodies. Recently, several novel CA isoenzymes have been recognized and we aimed to investigate whether antibodies to these are associated with renal manifestations of pSS. METHODS: We examined anti-CA II antibodies as well as anti-CA I, VI, VII and XIII antibodies by ELISA tests in 74 pSS patients on whom detailed nephrological examinations had been performed and, as controls, in 56 subjects with sicca symptoms, but no pSS. RESULTS: The levels of anti-CA I, II, VI and VII antibodies were significantly higher in patients with pSS compared with subjects with sicca symptoms but no pSS. None of the anti-CA antibodies was associated with the presence of complete or incomplete RTA or proteinuria or urinary α1m excretion in patients with pSS. However, levels of anti-CA II, VI and XIII antibodies correlated significantly with urinary pH, and inversely with serum sodium concentrations. The degree of 24-h urinary protein excretion correlated weakly with levels of anti-CA VII antibodies. CONCLUSION: Not only antibodies to CA II, but also anti-CA VI and XIII antibodies seem to be associated with renal acidification capacity in patients with pSS.

Acetaldehyde-derived modifications on cytosolic human carbonic anhydrases.

Acetaldehyde can generate modifications in several proteins, such as carbonic anhydrase (CA) II. In this study, we extended in vitro investigations on acetaldehyde adduct formation by focusing on the other human cytosolic CA enzymes I, III, VII, and XIII. High-resolution mass spectrometric analysis indicated that acetaldehyde most efficiently formed covalent adducts with CA II and XIII. The binding of up to 19 acetaldehydes in CA II is probably attributable to the high number of lysine residues (n = 24) located mainly on the surface of the enzyme molecule. CA XIII formed more adducts (up to 25) than it contains lysine residues (n = 16) in its primary structure. Acetaldehyde treatment induced only minor changes in CA catalytic activity in most cases. The present study provides the first evidence that acetaldehyde can bind to several cytosolic CA isozymes. The functional consequences of such modifications will be further investigated in vivo by using animal models.

Activation Studies of the ß-Carbonic Anhydrase from the Pathogenic Protozoan Entamoeba histolytica with Amino Acids and Amines.

The ß-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic protozoan Entamoeba histolytica, EhiCA, was investigated for its activation with a panel of natural and non-natural amino acids and amines. EhiCA was potently activated by D-His, D-Phe, D-DOPA, L- and D-Trp, L- and D-Tyr, 4-amino-L-Tyr, histamine and serotonin, with KAs ranging between 1.07 and 10.1 M. The best activator was D-Tyr (KA of 1.07 µM). L-Phe, L-DOPA, L-adrenaline, L-Asn, L-Asp, L-Glu and L-Gln showed medium potency activation, with KAs of 16.5⁻25.6 µM. Some heterocyclic- alkyl amines, such as 2-pyridyl-methyl/ethyl-amine and 4-(2-aminoethyl)-morpholine, were devoid of EhiCA activating properties with KAs > 100 µM. As CA activators have poorly been investigated for their interaction with protozoan CAs, our study may be relevant for an improved understanding of the role of this enzyme in the life cycle of E. histolytica.

Thalamic calcification in vitamin D receptor knockout mice.

Vitamin D is a steroid hormone with many important functions in the brain, mediated through the nuclear vitamin D receptor. Here, we report that aging nuclear vitamin D receptor knockout mice demonstrate a symmetric thalamic calcification with numerous Ca/P-containing laminated bodies. These results are consistent with clinical findings showing brain calcification in patients with vitamin D deficiency. Our results suggest that nuclear vitamin D receptor deficiency leads to brain mineralization in vitamin D receptor knockout mice, which may represent an experimental model of intracranial calcification.

Behavioural anomalies in mice evoked by "Tokyo" disruption of the Vitamin D receptor gene.

Vitamin D is a steroid hormone with many important functions in the brain, mediated through the nuclear Vitamin D receptor (VDR). Mounting clinical data link VDR mutations to various psychiatric phenotypes. We have reported previously that mutant mice lacking functional VDR ("Tokyo" VDR mutant mice) display several behavioural anomalies, including high anxiety and aberrant grooming. Given the important role of Vitamin D and VDR in brain development and functioning, we hypothesized that several other important behavioural domains may be affected by disruption of the VDR gene in mice. Here we report that VDR mutants display unaffected depressive-like behaviour, but show abnormal social behaviours, reduced social barbering and aggressiveness, impaired nest building and aberrant maternal (pup neglect, cannibalism) behaviours. Taken together, these findings confirm the important role postulated for the VDR in the regulation of behaviour, and suggest the mice lacking functional VDR may be a useful tool to model different brain disorders.

Increased severity of chemically induced seizures in mice with partially deleted Vitamin D receptor gene.

Vitamin D is a neuroactive steroid hormone with multiple functions in the brain. Numerous clinical and experimental data link various Vitamin D-related dysfunctions to epilepsy. Here, we study the role of Vitamin D receptors (VDRs) in experimental epilepsy in mice. To examine this problem, we assessed the seizure profiles in VDR knockout mice following a systemic injection of pentylenetetrazole (70 mg/kg). Overall, compared to the wild-type (WT) 129S1 mice (n=10 in each group), the VDR knockout group significantly demonstrated shorter latencies to the onset, higher Racine scores and increased mortality rates. Our findings suggest that VDRs modulate seizure susceptibility in mice, and that the Vitamin D/VDR endocrine system may be involved in the pathogenesis of epilepsy.