Carbonic Anhydrase-related Protein Research Articles

Inherited deficiencies and activity variants of the mammalian carbonic anhydrases.

The carbonic anhydrases are involved in a variety of physiological functions that are important for the survival of nearly all species. The most important biochemical function of these enzymes is the interconversion of carbon dioxide and water into bicarbonate ions and protons. These metabolites are used in a wide variety of metabolic pathways (Tashian, 1989). Some of the enzymes have other catalytic activities, although their physiological importance is not understood at this time (Verpoorte et al., 1967; Cabiscol and Levine, 1996). In humans, twelve carbonic anhydrases or carbonic anhydrase-related proteins are known to exist that belong to the alpha family of CAs (Hewett-Emmett and Tashian, 1996).

The membrane carbonic anhydrases: from CO2 transport to tumor markers.

The growing carbonic anhydrase family includes at least 11 enzymatically active isozymes (CAs) and several carbonic anhydrase-related proteins (CARPs) (Sly and Hu, 1995; Hewett-Emmett and Tashian, 1996). CAs I, II III, and VII are cytosolic isozymes covered extensively elsewhere in this volume. CA V is a mitochondrial isozyme expressed in the mitochondrial matrix (Dodgson et al., 1980It is thought to play a role in ureagenesis, providing HCO – 3 for carbamoyl phosphate synthase, and in gluconeogenesis, providing HCO –3 for pyruvate carboxylase (Dodgson and Cherian, 1989; Dodgson et al., 1993). There is emerging evidence for a second mitochondrial enzyme which has not yet been characterized. CA VI is the isozyme expressed in saliva (Murakami and Sly, 1987; Feldstein and Silverman, 1984; Fernley et al., 1989) that has been suggested to protect gastroesophageal mucosa from acid injury (Parkkila et al., 1997). The three known non-enzymatic CA-RPs CA VII, CA X, and CA XI are covered elsewhere in this volume. The focus of this report is on CA IV, CA IX, and CA XII, the three membrane CAs which were discovered in that order.KeywordsCarbonic AnhydraseHuman Carbonic AnhydraseCrab GillHuman Male Reproductive TractThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

CDNA sequence, mRNA expression, and chromosomal localization of human carbonic anhydrase-related protein, CA-RP XI

A full-length cDNA clone of a human carbonic anhydrase-related protein, CA-RP XI encoded by CA11, was obtained and sequenced. The cDNA sequence was 1475 bp long and predicted to encode a 328-amino acid polypeptide with a molecular mass of 36 200 Da. The deduced amino acid sequence of CA-RP XI showed an overall similarity of 42–53% to the active site residues of other active CA isozymes; however, it lacked three zinc-binding histidine residues, raising questions regarding its CA catalytic activity. Northern blot analysis demonstrated strong expression of an approx. 1.5 kb transcript in the human brain, particularly in the cerebellum, cerebral cortex, and putamen. A single copy of the CA11 gene was localized to the human chromosome 19q13.2-3. These results suggest that CA-RP XI plays a general role in the human central nervous system.

Sequence and Tissue Expression of a Novel Human Carbonic Anhydrase-Related Protein, CARP-2, Mapping to Chromosome 19q13.3

In this study, we report the identification and characterisation of a novel carbonic anhydrase related-protein. We have determined that the full length coding sequence of an anonymous expressed sequenced tag, D19S799E, encodes a novel carbonic anhydrase related-protein (CARP-2) that is 328 amino acids in length. This peptide exhibits between 23.1-28.8% amino acid identity with the seven active human carbonic anhydrase (CA) isozymes. Four substitutions of key amino acids in the catalytic domain of CAs (equivalent to His94Arg, His96Leu, His119Gln, and Thr199Ser) are likely to render CARP-2 inactive as a carbonic anhydrase. Northern blot analysis of 23 human tissues indicates that CARP2 is expressed abundantly in the brain with moderate expression also present in spinal cord and thyroid. D19S799E (and thus CARP2) has previously been localised close to the polymorphic marker D19S412 and the genes DBP and FUT1/FUT2 on 19q13.3.

Evolutionarily Conserved, “Acatalytic” Carbonic Anhydrase-Related Protein XI Contains a Sequence Motif Present in the Neuropeptide Sauvagine: The Human CA-RP XI Gene (CA11) Is Embedded between theSecretorGene Cluster and theDBPGene at 19q13.3

Conserved amino acid motifs are found in numerous expressed genes. Proteins and peptides with functional relationships may be identified using probes designed to hybridize with these motifs. An oligonucleotide probe was prepared to match the sequence of the expected active region of a frog corticotropin-releasing factor-like peptide sauvagine and used to screen a sheep brain cDNA library. A novel 1331-bp cDNA encoding a putative 328-residue protein with a theoretical mass of 36 kDa was identified. The presence of a strong signal sequence indicates that it is a secreted protein. The amino- and carboxy-terminal regions are characterized by several potential phosphorylation sites and binding motifs, suggesting a role in intracellular signal transduction. Although the protein possesses a 7-residue sequence identical to that found in sauvagine, its overall primary structure most closely resembles those of the α-carbonic anhydrases (α-CAs). Moreover, the detection of the human and mouse orthologues in the EST databases, together with an evolutionary analysis, indicates that the protein represents a new member of the α-CA gene family, which we designate carbonic anhydrase-related protein XI (CA-RP XI), encoded byCA11(human) andCar11(mouse, rat). The humanCA11gene appears to be located between the secretor type α(1,2)-fucosyltransferase gene cluster (FUT1–FUT2–FUT2P) and the D-site binding protein gene (DBP) on chromosome 19q13.3. Despite potentially inactivating changes in the active-site residues, CA-RP XI is evolving very slowly in mammals, a property indicative of an important function, which has also been observed in the two other “acatalytic” CA isoforms, CA-RP VIII and CA-RP X, whose functions are unknown.

Molecular characterization of the human carbonic anhydrase-related protein (HCA-RP VIII)

The very evolutionarily conserved human carbonic anhydrase-related polypeptide (CA-RP VIII) lacks the carbon-dioxide hydration-activity, characteristic of the enzymatically active carbonic anhydrases. We have expressed HCA-RP VIII as a glutathione- S-transferase fusion protein (GST-HCA-RP VIII). The purified HCA-RP VIII showed a substantially higher apparent molecular weight by gel-filtration compared to the molecular weight calculated from the amino acid sequence, indicating a larger than expected Stoke's radius. Like other studied CA's, the protein unfolds through two transitions at increasing concentrations of guanidine hydrochloride. The far-UV CD spectra of HCA-RP VIII indicates a secondary structure similar to that of the catalytically active HCA II. The very high sequence identity between human and mouse CA-RP VIII (98%), might indicate that the function of the protein involves binding of another protein. However, an attempt to use the GST-HCA-RP VIII fusion protein to affinity purify a ligand was unsuccessful.

Carbonic anhydrase II and carbonic anhydrase-related protein in the cerebellar cortex of normal and lurcher mice

The developmental profiles of carbonic anhydrase II (CA-II) and a carbonic anhydrase related protein (CARP) were studied in rat and mouse cerebella. Enzyme histochemistry, immunohistochemistry, in situ hybridisation and Western blotting were used to study the synthesis and expression of these enzymes in cerebellar sections from age matched control, CA-II deficient and lurcher mice, the latter being characterised by Purkinje cell degeneration. Both CA-II and CARP were first found to be expressed in the Purkinje cells in the 9 day old mouse, and the immunoreactivity of both peptides increased with time. Immunohistochemistry showed more intense staining of CARP than of CA-II in Purkinje cells throughout the developmental profile of the mouse, and this was mirrored by the mRNA levels determined by in situ hybridisation. Immunohistochemistry of CA-II and CARP also demonstrated the progressive dendritic growth of the mouse and rat Purkinje cells. CA-II and CARP immunoreactivity ceased by the end of cerebellar maturation. The onset of Purkinje cell degeneration was detected at day 10 in the lurcher mouse, with concomitant marked decrease in CA-II level: however CARP expression was found to be unchanged. By postnatal day 16 neither CA-II mRNA, protein, nor activity was detectable in contrast to CARP which remained at a decreased level until the Purkinje cell population had completely degenerated. Our findings suggest a role of CA-II in the degenerative processes of the lurcher Purkinje cells, with CARP playing an important role in the development and maturation of the cerebellar cortex.

Two point mutations convert a catalytically inactive carbonic anhydrase-related protein (CARP) to an active enzyme

A murine carbonic anhydrase-related protein (CARP) has been expressed in Escherichia coli and purified to near homogeneity. The polypeptide chain consists of 290 amino acid residues and has a calculated molecular mass of 32 950 Da. By introducing two mutations, Arg 117 → His and Glu 115 → Gln, we created a metal-binding center homologous to that in the carbonic anhydrases from the animal kingdom. In contrast to unmodified CARP, this double mutant was isolated as a 1 : 1 zinc-protein complex. While unmodified CARP is catalytically inactive, the mutant catalyzes CO 2 hydration with a significantly higher efficiency than the mammalian low-activity carbonic anhydrase isozyme III. The activity is strongly inhibited by the powerful and selective carbonic anhydrase inhibitor, acetazolamide.

The deduced amino acid sequence of human carbonic anhydrase-related protein (CARP) is 98% identical to the mouse homologue

A recently reported mRNA, encoding ‘carbonic anhydrase-related polypeptide’ (CARP) from the Purkinje cells of mouse cerebellum, was shown to have a 30–40% deduced amino acid sequence identity with the carbonic anhydrases (CA) of mammals. In order to compare the mouse and human CARP sequences, we used the polymerase chain reaction (PCR) to amplify human CARP sequences from several cDNA libraries (salivary gland, testis and placenta). The sequence has an 89.3% sequence identity with mouse CARP at the nucleotide level and 97.9% at the amino acid level. This extremely high evolutionary conservation suggests an important function for the CARP gene product.

Genetics of the Mammalian Carbonic Anhydrases

This chapter discusses the genetics of the mammalian carbonic anhydrases. The chapter provides information about various evolutionarily genes coding for enzymes that can surpass the carbonic anhydrase (CA) multigene family with respect to the distributional and functional diversity of their gene products. The CA gene family in mammals consists of eight genes coding for seven CA isozymes (CA I–CA VII) and a CA related protein (CARP). These genes may be expressed in certain cells of virtually all tissues or limited in expression to a single tissue, with the other CA genes ranging in their expression between these two extremes. Considerable chemical and biological information is now accumulated on the CAs regarding their primary and tertiary structures, enzyme kinetics, active site mechanisms, physiological roles, cellular and sub cellular locations, genetics, and evolution. Therefore, the related CA genes appear to be an attractive model for the study of the molecular genetics and evolution of a multigene family. It is possible that both types of carbonic anhydrase may be present in bacteria. The CA found in Neisseria sicca—and probably other bacteria—may be related to the algal and animal CAs, whereas the CA products of the cynT gene of E. coli and the IcfA gene of the cyanobacterium, Synechoccus sp. belong to the β-CA family.