Comparative physiology and molecular analysis of carbonic anhydrase from the red blood cells of teleost fish

This study investigates the early evolution of vertebrate red blood cell (rbc) carbonic anhydrase (CA) by examining the physiological and molecular properties of rbc CA in teleost fish. When representatives of four different families of teleosts were compared, it was found that differences in overall rbc CA activity were due to different concentrations of CA, rather than differences in the enzymes kinetic properties. Additional molecular analysis of CA from the rbcs of rainbow trout provided further evidence that critical elements of the enzyme, such as the active site, have been highly conserved during vertebrate evolution. The active site of the trout CA differed from that of gar rbc CA at only two amino acid positions. The rainbow trout rbc CA sequence also showed high sequence homology with CA sequences from other fish tissues, and fits into an emerging group of fish CAs that are basal to mammalian CA I, II and III. Northern blot analysis of the tissue expression of the sequenced CA indicated that it is primarily found in the rbcs, but high amounts of cytosolic CA activity were also found in the gill, suggesting the presence of other cytosolic CA isozymes in this species.Abbreviations Az acetazolamide - CA carbonic anhydrase - MP maximum parsimony - NJ neighbour joining - RACE rapid amplification of cDNA ends - rbc red blood cellCommunicated by L.C.-H. Wang     

Comparative physiology and molecular evolution of carbonic anhydrase in the erythrocytes of early vertebrates

The different isozymes of carbonic anhydrase (CA) have been the subject of intensive study in mammals, but there is still much to be learned about the early evolution of this enzyme in vertebrates. Erythrocyte CA plays an essential role in the respiratory processes of most vertebrates and is probably the most well studied CA isozyme. The available evidence indicates that there has been a progressive increase in the efficiency of erythrocyte CA during the early evolution of vertebrates. There also appears to be a substantial increase in erythrocyte CA activity during development in some species. At the present time, however, the selective pressures that may be influencing the properties of erythrocyte CA during vertebrate evolution and development have not been clearly determined. When the available molecular sequence information is examined, it is evident that the erythrocyte CAs of early vertebrates have active sites that are more similar to those of mammalian CA VII and II, rather than CA I. We can now also begin to examine the phylogenetic relationships between the different rbc CAs in vertebrates, but more CA sequence information is clearly required from different groups of vertebrates before we have a complete picture of the molecular evolution of erythrocyte CA.     

Sulfonamides incorporating 1,3,5-triazine moieties selectively and potently inhibit carbonic anhydrase transmembrane isoforms IX, XII and XIV over cytosolic isoforms I and II: Solution and X-ray crystallographic studies

Reaction of cyanuryl chloride with d,l-amino acids and amino alcohols afforded a new series of triazinyl-substituted benzenesulfonamides incorporating amino acyl/hydroxyalkyl-amino moieties. Inhibition studies of physiologically relevant human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, such as CA I, II, IX, XII and XIV with these compounds are reported. They showed moderate-weak inhibition of the cytosolic, offtarget isozymes CA I and II, but many of them were low nanomolar inhibitors of the transmembrane, tumor-associated CA IX and XII (and also of CA XIV). The X-ray crystal structure of two of these compounds in adduct with CA II allowed us to understand the features associated with this strong inhibitory properties and possibly also their selectivity. Two of these compounds were also investigated for the inhibition of other human isoforms, that is, hCA IV, VA, VB, VI, VII and XIII, as well as inhibitors of the fungal pathogenic CAs Nce103 (Candida albicans) and Can2 (Cryptococcus neoformans), showing interesting activity. The 1,3,5-triazinyl-substituted benzenesulfonamides constitute thus a class of compounds with great potential for obtaining inhibitors targeting both α-class mammalian, tumor-associated, and β-class from pathogenic organisms CAs.     

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.     

Expression, purification, kinetic, and structural characterization of an alpha-class carbonic anhydrase from Aedes aegypti (AaCA1)

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. The alpha-class CAs are found predominantly in vertebrates, but they are also expressed in insects like mosquitoes. Recently, an alpha-CA from the midgut of Aedes aegypti larvae (AaCA1) was identified, cloned, and subsequently shown to share high sequence homologous to human CA I (HCA I). This paper presents the bacterial expression, purification, and kinetic characterization of the soluble CA domain of AaCA1. The data show AaCA1 is a highly active CA that displays inhibition by methazolamide and ethoxzolamide with nM affinity. Additionally, a homology model of AaCA1, based on the crystal structure of HCA I, is presented and the overall structure, active site, and surface charge properties are compared to those of HCA I and II. Measurements of catalysis show that AaCA1 is more like HCA II in terms of proton transfer, but more similar to HCA I in terms of conversion of carbon dioxide to bicarbonate, and these differences are rationalized in terms of structure. These results also indicate that amino acid differences in the active site of AaCA1 compared to human CAs could be used to design specific CA inhibitors for the management of mosquito populations.     

Molecular evolution of the carbonic anhydrase genes: Calculation of divergence time for mouse carbonic anhydrase I and II

Summary A cDNA clone in pBR322 that cross-hybridizes with a mouse carbonic anhydrase form II (CAII) probe has been sequenced and identified as mouse carbonic anhydrase form I (CAI). The 1224-base-pair clone encodes the entire 260-amino-acid protein and appears to contain an Alu-like element in the 3 untranslated region. The deduced amino acid sequence exhibits 77% homology to human CAI and contains 17 of the 20 residues that are considered unique to and invariant for all mammalian CAI isozymes. The results of a detailed comparison of the nucleic acid sequences spanning the coding regions of mouse CAI and rabbit CAI have been used to calibrate an evolutionary clock for the carbonic anhydrases (CAs). These data have been applied to a comparison of the mouse CAI and CAII nucleic acid sequences to calculate the divergence time between the two genes. The divergence-time calculation provides the first estimation of the evolutionary relationship between CAs based entirely on nucleotide sequence comparison.     

Changes in ascorbic acid content and ascorbate peroxidase activity during the development of Acetabularia mediterranea

We cloned ras-related sequences from goldfish genomic libraries constructed as recombinants using the lambda phage. Restriction enzyme mapping of the clones obtained revealed three kinds of ras-related sequences among approximately 350,000 genomic clones. One of these clones was partially sequenced. Comparison with the nucleotide sequences of mammalian ras genes showed that the determined sequences covered the predicted amino acid coding regions and parts of the intervening regions. The predicted amino acid sequences of the cloned ras-related goldfish gene suggested that the coding region is localized separately in DNA, and that its exon-intron boundaries are exactly the same as those of corresponding mammalian genes. The nucleotide and amino acid sequences of the goldfish ras-related gene may have extensive homologies to mammalian p 21 protein. Among the three mammalian ras proteins, the predicted amino acid sequence of the sequenced ras-related goldfish clone is most closely homologous (96%) to the Kirsten ras protein. Differences in the predicted amino acid sequence were greatest in the sequence predicted from the fourth exon; fewer differences were found in the sequence from the third exon, and only slight or no differences were found in the sequence predicted for the first and second exons. The 12th and 61st amino acids from the N-terminal of the protein, which are thought to be critical positions for GTP binding and catalysis, are both conserved in the goldfish protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of two distinct rat pancreatic preproelastases determined by sequence analysis of the complete cloned messenger ribonucleic acid sequences

The mRNA sequences for two rat pancreatic elastolytic enzymes have been cloned by recombinant DNA technology and their nucleotide sequences determined. Rat elastase I mRNA is 1113 nucleotides in length, plus a poly(A) tail, and encodes a preproelastase of 266 amino acids. The amino acid sequence of the predicted active form of rat elastase I is 84% homologous to porcine elastase 1. Key amino acid residues involved in determining substrate specificity of porcine elastase 1 are retained in the rat enzyme. The activation peptide of the zymogen does not appear related to that of other mammalian pancreatic serine proteases. The mRNA for elastase I is localized in the rough endoplasmic reticulum of acinar cells, as expected for the site of synthesis of an exocrine secretory enzyme. Rat elastase II mRNA is 910 nucleotides in length, plus a poly(A) tail, and encodes a preproenzyme of 271 amino acids. The amino acid sequence is more closely related to porcine elastase 1 (58% sequence identity) than to the other pancreatic serine proteases (33-39% sequence identity). Predictions of substrate preference based upon key amino acid residues that define the substrate binding cleft are consistent with the broad specificity observed for mammalian pancreatic elastase 2. The activation peptide is similar to that of the chymotrypsinogens and retains an N-terminal cysteine available to form a disulfide link to an internal conserved cysteine residue.     

Conformational variability of different sulfonamide inhibitors with thienyl-acetamido moieties attributes to differential binding in the active site of cytosolic human carbonic anhydrase isoforms

The X-ray crystal structures of the adducts of human carbonic anhydrase (hCA, EC 4.2.1.1) II complexed with two aromatic sulfonamides incorporating 2-thienylacetamido moieties are reported here. Although, the two inhibitors only differ by the presence of an additional 3-fluoro substituent on the 4-amino-benzenesulfonamide scaffold, their inhibition profiles against the cytosolic isoforms hCA I, II, III, VII and XIII are quite different. These differences were rationalized based on the obtained X-ray crystal structures, and their comparison with other sulfonamide CA inhibitors with clinical applications, such as acetazolamide, methazolamide and dichlorophenamide. The conformations of the 2-thienylacetamido tails in the hCA II adducts of the two sulfonamides were highly different, although the benzenesulfonamide parts were superimposable. Specific interactions between structurally different inhibitors and amino acid residues present only in some considered isoforms have thus been evidenced. These findings can explain the high affinity of the 2-thienylacetamido benzenesulfonamides for some pharmacologically relevant CAs (i.e., isoforms II and VII) being also useful to design high affinity, more selective sulfonamide inhibitors of various CAs.     

Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes.

Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.     

Flavones and structurally related 4-chromenones inhibit carbonic anhydrases by a different mechanism of action compared to coumarins

An inhibition study of several carbonic anhydrase (CA, EC 4.2.1.1) isoforms with flavones and aminoflavones, compounds possessing a rather similar scaffold with the coumarins, recently discovered inhibitors of this enzyme, is reported. The natural product flavone and some of its hydroxylated derivatives did not show time-dependent inhibition of the CAs, sign that they are not hydrolyzed within the enzyme active site as the (thio)coumarins and lactones. These compounds were low micromolar inhibitors of hCA I, II, IX and XII, with K(I)s in the range of 1.88-9.07 μM. A series of substituted 2-amino-3-phenyl-4H-chromen-4-ones, incorporating chloro- and methoxy substituents in various positions of the heterocycle, were then prepared and assayed as hCA I and II inhibitors, showing activity in the micromolar range. Some of these derivatives, as well as cis+trans resveratrol, were then assayed for the inhibition of all catalytically active mammalian CA isoforms, hCA I, II, III, IV, VA, VB, VI, VII, IX, XII, XIII, XIV and mCA XV (h=human, m=murine enzyme). These derivatives inhibited these CAs in the submicromolar-low micromolar range. Flavones, although not as active as the coumarins, may be considered as interesting leads for the design of non-sulfonamide CA inhibitors.     

Expression of carbonic anhydrases II, IV, VII, VIII and XII in rat brain after kainic acid induced status epilepticus

Carbonic anhydrases (CAs) are important enzymes in the central nervous system (CNS), where they participate in regulating cerebrospinal fluid (CSF) secretion, blood-brain barrier and glial cell function. Using RT-PCR we found CA XII mRNA in rat and mouse brain. Cloning of rat CA XII revealed 94% homology with the mouse CA XII. To map the putative functional roles of different CAs, we studied the expression and localization of CA II, CA IV, CA VII, CA-related protein (CA-RP) VIII and CA XII mRNAs in rat brain after kainic acid induced epileptic seizures using Northern blot analysis and in situ hybridization. The expression of CA IV, CA VII and CA-RP VIII was somewhat similar: they were expressed in the cortex, hippocampus and midbrain structures and their expression did not change after the kainic acid treatment. The expression of CA II was concentrated in the white matter structures, which is in line with the preferential expression of CA II in the oligodendrocytes. High levels of CA II mRNA were also detected in the choroid plexus. Surprisingly, CA II was induced 3-12 h after seizures in the vulnerable CA1 region. CA XII was expressed in dentate granule cells, cortex and choroid plexus. Kainic acid stimulated CA XII expression throughout the cortical layer I. The observed hippocampal induction of CA II may indicate a pro-apoptotic and/or epileptogenic role of CA II after prolonged seizures. The physiological significance of the observed cortical induction of CA XII remains obscure. Cytosolic CA II is known to participate in CSF secretion, and the high expression of CA XII in the choroid plexus suggests an analogous role for this membrane-bound isozyme.     

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.     

The evolution of gonadotropins: some molecular data concerning a non-mammalian pituitary gonadotropin, the hormone from a teleost fish (Cyprinus carpio L.)

The amino acid and sugar compositions as well as long N-terminal sequences and the C-terminal amino acids of the two subunits of carp gonadotropin, SU I and SU II, were determined. An important homology was demonstrated between SU I and alpha-subunits and between SU II and beta-subunits of mammalian gonadotropins. Moreover SU II was more closely related to the beta-subunit of LH than to the beta-subunit of FSH.     

Primary structure of two mRNAs encoding putative salmon alpha-subunits of pituitary glycoprotein hormone

1. By random sequence analyses, we isolated from the cDNA library of salmon pituitary glands two clones, the deduced amino acid sequences corresponding to the C-terminal region of which are almost the same as those of the alpha subunits of mammalian glycoprotein hormones. 2. Comparison of the nucleotide sequences and deduced amino acid sequences from these two clones with those of mammalian species revealed that the two newly-isolated cDNAs corresponded to mRNAs encoding the putative salmon pre-alpha subunit of glycoprotein hormones. 3. Homology in the nucleotide sequences of these two clones suggested that corresponding mRNAs may be encoded by separate genes which probably evolved from a common ancestral gene.     

New chemotypes acting as isozyme-selective carbonic anhydrase inhibitors with low affinity for the offtarget cytosolic isoform II

Considering phenols and coumarins as lead molecules for obtaining non-sulfonamide inhibitors of carbonic anhydrases (CAs, EC 4.2.1.1), we screened a large number of compounds possessing diverse chemotypes, but structural features which resemble the two chemical classes. Here we report an investigation of such derivatives which do not significantly inhibit CA II, but show interesting inhibition profiles against other isozymes. Pyridine-N-oxide-2-thiophenol, thiobenzoic acid, thimerosal, two oximes derived from a six-membered-ring lactone and from coumarin; 2-hydroxyquinoline and coumaphos, were investigated as inhibitors of CA I-XIV. All these compounds did not inhibit CA II, whereas the two oximes and 2-hydroxyquinoline were low nanomolar inhibitors of CA I, IX, XII, XIII and XIV, showing a very different inhibition profile compared to sulfonamides and sulfamates. Some other compounds showed low micromolar inhibition of other isoforms of interest, such as CA VA/VB, CA VI and VII. This study demonstrates that a rather wide range of structures show low nanomolar-micromolar inhibitory activity against many CA isozymes, without inhibiting significantly the offtarget isoform CA II.