Nucleotide sequence and structure of the mouse carbonic anhydrase III gene

At least 14 distinct isozymes of carbonic anhydrase have been identified in mammals. These enzymes catalyze the hydration of carbon dioxide and are essential for regulation of cellular pH and carbon dioxide transport. Carbonic anhydrase III is highly expressed in certain tissues, including muscle and fat where it constitutes up to 25% of the soluble protein. We cloned a cDNA encoding mouse carbonic anhydrase III. This cDNA contains 1653 bp, consisting of 79 bp in the 5' UTR, a 780 bp open reading frame, and 794 bp of the 3' UTR, including two potential polyadenylation signals. Fluorescent in situ hybridization confirmed the existence of a single copy of the gene on chromosome 3. We then isolated the genomic DNA for mouse carbonic anhydrase III and analyzed its structure. The gene consists of seven exons and six introns which span 10.5 kb. The 5' flanking region of the genomic DNA is notable for a pyrimidine rich region consisting of two dinucleotide repeats containing 23 and 20 TC pairs separated by the same 15 bp spacer.     

The nucleotide sequence and derived amino acid sequence of cDNA coding for mouse carbonic anhydrase II

The nucleotide sequence of a clone containing mouse carbonic anhydrase (CA) cDNA in pBR322 has been determined. The cloned cDNA contains all of the coding region except for nucleotides specifying the first eight amino acids, and all of the 3' noncoding region, which consists of 700 nucleotides. A cDNA clone was identified which contains an additional 54 bp at the 5' end, so that the complete amino acid sequence of mouse CA could be deduced. This sequence showed a 73-81% homology with other mammalian CA form II isozymes, 56-63% with form I isozymes, and 52-56% with form III isozymes. By examination of the amino acids which are unique and invariant for each isozyme, the mouse amino acid sequence was found to contain 16 of the 23 residues that are unique and invariant to mammalian CA form II isozymes, but only one or no residue for forms I and III, respectively.     

Cloning, expression, and sequence homologies of cDNA for human carbonic anhydrase II

A cDNA clone for human carbonic anhydrase (CA) II was isolated from a kidney lambda gt10 library. Expression of the cDNA insert in Cos-7 cells produced an immunoprecipitable product and enzymatically active carbonic anhydrase. The cDNA insert is 1551 bp in length and contains an open reading frame which encodes a 260-amino-acid polypeptide. The deduced amino acid sequence is identical to that reported for human CA II. The protein coding region of this cDNA for human CA II shows 81 and 70% nucleotide identity with cDNAs for CA II from mouse and chick, respectively. Even the long 3'-untranslated region of the cDNA for human CA II (703 bp) is 64 and 42% identical to those of CA II from mouse and chick, showing remarkable conservation of the CA II cDNAs in amniotes. The protein coding region of the human CA II cDNA is 64 and 65% identical with those of human CA I and CA III, which are thought to have arisen from a common precursor by gene duplication.     

Activation of mammalian skeletal-muscle carbonic anhydrase III by arginine modification

Purified carbonic anhydrase isozymes I, II, and III (CA I, CA II, CA III) from various sources were treated with 2,3-butanedione and their bicarbonate dehydration reactions followed. The specific activities of human and bovine CA I and CA II and chicken CA III were not affected by the butanedione treatment, whereas the activities of human, gorilla, and bovine CA III were rapidly activated. These findings suggest that one, or both, of the two arginyl residues which appear to be unique to the active sites of the mammalian CA III isozymes are modified by butanedione.     

The gene for human carbonic anhydrase II (CA2) is located at chromosome 8q22

The gene CA2 for the human carbonic anhydrase II isozyme is encoded in band q22 of chromosome 8. These data and supporting evidence predict that the genes for carbonic anhydrase I and III are also physically closely linked in this chromosomal region.     

Characterization of erythrocyte carbonic anhydrase in an ancient fish,the longnose gar ( Lepisosteus osseus)

This study investigates the evolutionary history of vertebrate red blood cell carbonic anhydrase (CA) by characterizing the isozyme properties and nucleotide sequence of an ancient fish, the longnose gar ( Lepisosteus osseus). The inhibitor sensitivities of gar rbc CA closely resembled those for mammalian CA II, as well as those for CAs from more recently evolved fishes. The kinetic properties of gar rbc CA were not closely aligned with either mammalian CA I and CA II, but fit well into an emerging phylogenetic pattern for early vertebrates. Gar rbc CA cDNA was also amplified from mRNA using 5' and 3'-RACE and the open reading frame consisted of 786 bp. This sequence shares approximately 65% identity with the nucleotide and amino acid sequences of both mammalian CA I and CA II. When the amino acid sequences within the active site are compared, gar rbc CA differs from mammalian CA I, CA II and CA VII by 9, 4 and 3 of the 36 amino acids, respectively. Phylogenetic analyses suggest that gar rbc CA diverged before the amniotic CAs (CA I, CA II and CA III), but after CA V and CA VII.     

DNA polymorphism in the 5′ flanking region of the human carbonic anhydrase II gene on chromosome 8

Summary A restriction-fragment-length polymorphism (RFLP) is described which is associated with the human carbonic anhydrase II gene (CA2) that codes for one of the three genetically distinct carbonic anhydrase isozymes, CA I, CA II, and CA III. The isolated DNA was cleaved with several restriction enzymes and subjected to Southern blot hybridization analysis using a DNA probe containing the 5 end of the human CA II gene. A two allele RFLP which was detected with the restriction endonuclease, Taq I, is expressed phenotypically on Southern blots as either a 5.4 kilobase (kb) fragment or as 4.0 and 1.4 kb fragments. These fragments result from the presence or absence of a Taq I recognition site in the 5 flanking region approximately 1.0kb from the initiation codon of the CA II gene. Segregation analysis showed that the alleles are inherited in a Mendelian fashion, with a frequency of 50%.     

4-Substituted-2,3,5,6-tetrafluorobenzenesulfonamides as inhibitors of carbonic anhydrases I,II, VII,XII, and XIII

A series of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides were synthesized and their binding potencies as inhibitors of recombinant human carbonic anhydrase isozymes I, II, VII, XII, and XIII were determined by the thermal shift assay, isothermal titration calorimetry, and stop-flow CO₂ hydration assay. All fluorinated benzenesulfonamides exhibited nanomolar binding potency toward tested CAs and fluorinated benzenesulfonamides posessed higher binding potency than non-fluorinated compounds. The crystal structures of 4-[(4,6-dimethylpyrimidin-2-yl)thio]-2,3,5,6-tetrafluorobenzenesulfonamide in complex with CA II and CA XII, and 2,3,5,6-tetrafluoro-4-[(2-hydroxyethyl)sulfonyl]benzenesulfonamide in complex with CA XIII were determined. The observed dissociation constants for several fluorinated compounds reached subnanomolar range for CA I isozyme. The affinity and the selectivity of the compounds towards tested isozymes are presented.     

4-[N-(substituted 4-pyrimidinyl)amino]benzenesulfonamides as inhibitors of carbonic anhydrase isozymes I, II, VII, and XIII

A series of 4-[N-(substituted 4-pyrimidinyl)amino]benzenesulfonamides were designed and synthesised. Their binding potencies as inhibitors of selected recombinant human carbonic anhydrase (hCA) isozymes I, II, VII, and XIII were measured using isothermal titration calorimetry and the thermal shift assay. To determine the structural features of inhibitor binding, the crystal structures of several compounds in complex with hCA II were determined. Several compounds exhibited selectivity towards isozymes I, II, and XIII, and some were potent inhibitors of hCA VII.     

Molecular analysis of G+C-rich upstream sequences regulating transcription of the human carbonic anhydrase II gene.

The upstream promoter sequences of the human carbonic anhydrase II (CA II) gene have been studied by 5' deletion analysis. Promoter activity was assayed by transfection and chloramphenicol acetyltransferase assay in both human HeLa cells and murine L cells. This investigation showed that the CA II promoter is comparable in activity to that of the simian virus 40 early-region promoter and enhancer and that the CA II upstream sequences exert a different pattern of control in the two cell lines.     

Carbonic anhydrase inhibitors. Interaction of 2-N,N-dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide with 12 mammalian isoforms: kinetic and X-ray crystallographic studies

2-N,N-Dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide was tested for its interaction with the 12 catalytically active mammalian carbonic anhydrase (CA, EC 4.2.1.1) isozymes, CA I-XIV. The compound is a potent inhibitor of CA IV, VII, IX, XII, and XIII (K(I)s of 0.61-39 nM), a medium potency inhibitor of CA II and VA (K(I)s of 121-438 nM), and a weak inhibitor against the other isoforms (CA III, VB, VI, and XIV), making it a very interesting candidate for situations in which a strong/selective inhibition of certain isozymes is needed. The crystal structure of the hCA II adduct of this sulfonamide revealed interesting interactions between the inhibitor and the enzyme which are quite different from those observed in the adducts of CA II with the structurally related aliphatic derivatives zonisamide, 2-amino-1,3,4-thiadiazolyl-5-difluoromethanesulfonamide, and 2-dimethylamino-5-[sulfonamido-(aminomethyl)]-1,3,4-thiadiazole reported earlier.     

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.     

First evaluation of organotellurium derivatives as carbonic anhydrase I,II, IV,VII and IX inhibitors

A series of tellurides was evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against the human (h) carbonic anhydrase isoforms hCA I, II, IV, VII and IX, involved in a variety of diseases, including glaucoma, retinitis pigmentosa, epilepsy, arthritis and tumors. These compounds, which are the first tellurium-containing derivatives acting as inhibitors of carbonic anhydrase enzymes, showed effective inhibition against all isoforms investigated and some of them were selective for inhibiting the cytosolic or the membrane-bound CAs. Thus, these carbonic anhydrase inhibitors are interesting leads for the development of isoform-selective inhibitors.     

Characterization of human carbonic anhydrase III from skeletal muscle

A third form of human carbonic anhydrase (CA III), found at high concentrations in skeletal muscle, has been purified and characterized. This isozyme shows relatively poor hydratase and esterase activities compared to the red cell isozymes, CA I and CA II, but is similar to these isozymes in subunit structure (monomer) and molecular size (28,000). CA III is liable to posttranslational modification by thiol group interaction. Monomeric secondary isozymes, sensitive to beta-mercaptoethanol, are found in both crude and purified material and can be generated in vitro by the addition of thiol reagents. Active dimeric isozymes, generated apparently by the formation of intermolecular disulfide bridges, also occur but account for only a small proportion of the total protein and appear only when the concentration of CA III is particularly high.     

Differential regulation of hepatic carbonic anhydrase isozymes in the streptozotocin-diabetic rat

Most work with the male rat liver carbonic anhydrase isozymes in the past decade has centered on the cytosolic CA III and the mitochondrial CA V. This paper reports that the relative activity of both isozymes is altered in streptozotocin-diabetes. Carbonic anhydrase activity of perfused liver homogenates and disrupted, isolated mitochondria was measured by the mass spectrometric 18O decay technique at 37 degrees C. The contributions of the different isozymes were determined based on intracellular location and sensitivity to acetazolamide inhibition. Diabetes resulted in a twofold increase in the activity of CA V but a halving in the activity of CA III. This is the first time that liver CA V has been shown to be altered by physiological stress. The total carbonic anhydrase activity in the diabetic rat liver was unaltered compared with control rats; however, CA III never accounted for more than 50% of this activity. Since CA isozymes I, II, and IV together account for 30% of the CA activity in control rats and 70% in diabetic rats it is concluded that one or more of these isozymes is subject to regulation in the diabetic male rat. The increase in CA V during diabetes is in accord with this isozyme having an important function in provision of substrate for hepatic gluconeogenesis and ureagenesis.     

Characterization of the rat carbonic anhydrase II gene structure: sequence analysis of the 5′ flanking region and 3′ UTR

The rat carbonic anhydrase II gene was characterized and found to be approximately 15.5 kb in length and to contain 7 exons and 6 introns. All intron/exon junction and branch point sequences conform to consensus sequences, and the overall rat CA II genomic structure appears to be conserved upon comparison with mouse, human, and chicken CA II genes. The putative cis-acting elements within the analyzed 1014 bp 5′ flanking region include: TATA box, 4 Sp1 binding sites, 2 AP2 sites and putative tissue-specific β-globin-like repeat elements. A CpG island of approximately 800 bp was identified that begins about 600 bp upstream of exon 1 and extends about 200 bp into intron 1. In the 3′ UTR, two polyadenylation signals (AATAAA) are present, the second of which is believed to be utilized. Northern blot analysis reveals that the 1.7 kb rat CA II mRNA is abundantly expressed in adult male brain and kidney, while negligible amounts are detected in heart and liver.     

Purification and characterization of human salivary carbonic anhydrase

A novel carbonic anhydrase was purified from human saliva with inhibitor affinity chromatography followed by ion-exchange chromatography. The molecular weight was determined to be 42,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the human salivary enzyme is larger than the cytosolic isoenzymes CA I, CA II, and CA III (Mr 29,000) from human tissue sources. Each molecule of the salivary enzyme had two N-linked oligosaccharide chains which were cleaved by endo-beta-N-acetylglucosaminidase F but not by endo-beta-N-acetylglucosaminidase H, indicating that the oligosaccharides are complex type. The isoelectric point was determined to be 6.4, but significant charge heterogeneity was found in different preparations. The human salivary isozyme has lower specific activity than the rat salivary isozyme and the human red blood cell isozyme II in the CO2 hydratase reaction. The inhibitory properties of the salivary isozyme resemble those of CA II with iodide, sulfanilamide, and bromopyruvic acid, but the salivary enzyme is less sensitive to acetazolamide and methazolamide than CA II. Antiserum raised in a rabbit against the salivary enzyme cross-reacted with CA II from human erythrocytes, indicating that human salivary carbonic anhydrase and CA II must share at least one antigenic site. CA I and CA III did not crossreact with this antiserum. The amount of salivary carbonic anhydrase in the saliva of the CA II-deficient patients was greatly reduced, indicating that the CA II deficiency mutation directly or indirectly affects the expression of the salivary carbonic anhydrase isozyme. From these results we conclude that the salivary carbonic anhydrase is immunologically and genetically related to CA II, but that it is a novel and distinct isozyme which we tentatively designate CA VI.     

Carbonic anhydrase inhibitors. Interaction of 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide with 12 mammalian isoforms: kinetic and X-ray crystallographic studies

2-(Hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide was tested for its interaction with 12 carbonic anhydrase (CA, EC 4.2.1.1) isoforms in the search of compounds with good inhibitory activity against isozymes with medicinal chemistry applications, such as CA I, II, VA, VB, VII, IX, and XII among others. This sulfonamide is a potent inhibitor of CA I and II (K(I)s of 7.2-7.5 nM), a medium potency inhibitor of CA VII, IX, XII, and XIV, and a weak inhibitor against the other ubiquitous isoforms, making it thus a very interesting clinical candidate for situations in which a strong inhibition of CA I and II is needed. The crystal structure of the hCA II adduct of this sulfonamide revealed many favorable interactions between the inhibitor and the enzyme which explain its strong low nanomolar affinity for this isoform but may also be exploited for the design of effective inhibitors incorporating bicyclic moieties.     

Genetic variation in the carbonic anhydrase isozymes of macaque monkeys. IV. Degradation by heat and proteolysis of normal and variant carbonic anhydrase isozymes of Macaca nemestrina

Studies were undertaken on the heat denaturation and proteolytic degradation by alpha-chymotrypsin of the normal red cell carbonic anhydrase isozyme, CA II, and two electrophoretic variants of carbonic anhydrase I, CA Ia and CA Ib, of the pigtail macaque. The heat degradation results showed a difference of about 40-fold in the rate constants between CA Ia and CA Ib, which is due to the marked thermostability of CA Ib compared to CA Ia. The enthalpies and entropies of activation were calculated from the heat denaturation constants. These values were compared, on enthalpy-entropy compensation plots, with those values previously determined for the human CA I and CA II isozymes. They were highly correlated and clearly fell into two distinct clusters, separated by about 200 kJ mol-1; one group comprising the macaque and human CA I isozymes and the other the CA II isozymes. The proteolytic degradation results showed that CA Ia is degraded about 2.5 times more rapidly than CA Ib by alpha-chymotrypsin. Thus, the characteristic 3/1 ratio of CA Ib/CA Ia in mature red cells could be accounted for by the greater susceptibility of CA Ia to degradation at some stage in red cell development.