RESUMO
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.
Assuntos
Encéfalo/efeitos dos fármacos , Anidrases Carbônicas/metabolismo , Ácido Caínico/toxicidade , Estado Epiléptico/induzido quimicamente , Animais , Sequência de Bases , Northern Blotting , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Anidrases Carbônicas/genética , Primers do DNA , Hibridização In Situ , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Anidrases Carbônicas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Anidrases Carbônicas/genética , Anidrases Carbônicas/fisiologia , Chlorocebus aethiops , Biologia Computacional , Dissulfetos/metabolismo , Regulação Enzimológica da Expressão Gênica , Glicosilação , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Pan troglodytes/genética , Filogenia , Ligação Proteica , Estrutura Quaternária de Proteína , Pseudogenes/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Carbonic anhydrase (CA) XII is a membrane-associated enzyme that has been demonstrated to be normally expressed in some human tissues, to be upregulated in some cancers, and to be a hypoxia-inducible gene product. In mouse, CA XII has been recently localized in the kidney. In the present study, we investigated CA XII gene and protein expression in other mouse tissues, with the kidney serving as a positive control for the reagents. The expression of CA XII mRNA was examined using polymerase chain reaction (PCR) amplification of commercial cDNAs produced from selected mouse tissues. A strong positive signal for CA XII mRNA was detected in the kidney, and weak signals were obtained in the testis and lung. Heart, spleen, liver, and skeletal muscle were negative. Immunohistochemical staining was performed using a mouse CA XII-specific antibody and biotin-streptavidin complex method. The results showed high expression of CA XII in the kidney, as expected. It was also highly expressed in the surface epithelial cells of the colon, whereas it was absent in the stomach, proximal small intestine, pancreas, liver, heart, and skeletal muscle. The maturing sperm cells showed a weak staining in a pattern that most probably indicates expression in the developing acrosomal membrane. The high expression in the kidney and colon suggests a role for CA XII in the maintenance of body ion and pH homeostasis in the mouse. However, the present findings demonstrated that CA XII has a very limited distribution in mouse tissues outside these two organs.
