Structure of the carbonic anhydrase VI (CA6) gene: evidence for two distinct groups within the α-CA gene family

The secreted carbonic anhydrase (CA VI) is believed to be one of the oldest mammalian CAs in evolutionary terms. To elucidate its gene structure and compare it with other members of the α-CA family, we cloned genomic fragments encoding the bovine CA6 gene and determined its exon/intron organization. The gene spans approx. 25 kb and consists of eight exons and seven introns. Exon 1 encodes the 5′ untranslated region, the signal peptide and the N-terminus of the mature enzyme. Exon 8 encodes the 3′ untranslated region and the C-terminal extension that is unique to CA VI. Exons 2-7 encode the CA domain, which shows significant sequence similarity to other CAs. Two distinct groups exist in the α-CA family on the basis of a comparison of the known gene structures. One group consists of the cytoplasmic (CA I, II, III and VII) and mitochondrial (CA V) members. The other group consists of the membrane-bound (CA IV and IX) and secreted (CA VI) members. In particular, the seven exon/intron boundaries in the CA domain of the CA6 gene are conserved in the CA9 gene, which encodes the multidomain protein CA IX that is overexpressed in tumours and has transforming potential.

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Expression of membrane-associated carbonic anhydrase isoforms IV, IX, XII, and XIV in the rabbit: induction of CA IV and IX during maturation

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00735.2004 ◽

2005 ◽

Vol 288 (5) ◽

pp. R1256-R1263 ◽

Cited By ~ 25

Author(s):

Jeffrey M. Purkerson ◽

George J. Schwartz

Keyword(s):

Skeletal Muscle ◽

Carbonic Anhydrase ◽

Gall Bladder ◽

Plasma Membranes ◽

Rabbit Kidney ◽

Α Subunit ◽

Anion Transporters ◽

Ca Ix ◽

Membrane Bound ◽

Physiological Demands

Several carbonic anhydrase (CA) isoforms are associated with plasma membranes. It is probable that these enzymes interact with anion transporters to facilitate the movement of HCO3− into or out of the cell. A better knowledge of CA isoform expression in a given tissue would facilitate a systematic examination of any associations with such transporters. We examined the expression of CAs IV, IX, XII, and XIV mRNAs in rabbit tissues, including kidney, heart, lung, skeletal muscle, liver, pancreas, gall bladder, stomach, small intestine, colon, and spleen, using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). CA IV mRNA was mainly in kidney, heart, lung, colon, and gall bladder. CA IX mRNA was restricted to stomach, gall bladder, duodenum, and early jejunum. CA XII mRNA was found in kidney and colon. CA XIV mRNA was localized to heart, lung, skeletal muscle, and liver. The data indicate that there are different patterns of CA expression in various tissues: CA IX was expressed in the proximal gastrointestinal tract, whereas CA XII and CA IV were more distal. CA IV and CA XII are important kidney isoforms. CA XIV was abundant in metabolically active tissues such as liver, heart, lung, and skeletal muscle. Some significant species differences were noted in the expression of some of these isoforms; for example, CA XIV is not expressed in rabbit kidney, despite being abundant in mouse kidney. Maturational studies showed that the expression of CA IX mRNA and protein increased markedly with weaning (∼3–4 postnatal wk) and was well correlated with the maturational expression of the α-subunit of the gastric H+,K+-ATPase, suggesting that function of CA IX and the gastric H+ pump might be linked in the digestion of adult foodstuffs. The unique pattern of membrane-bound CA isoforms suggests different functional associations with transporters, depending on the physiological demands on the tissue.

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Heterocyclic periphery in the design of carbonic anhydrase inhibitors: 1,2,4-Oxadiazol-5-yl benzenesulfonamides as potent and selective inhibitors of cytosolic h CA II and membrane-bound h CA IX isoforms

Bioorganic Chemistry ◽

10.1016/j.bioorg.2017.10.005 ◽

2018 ◽

Vol 76 ◽

pp. 88-97 ◽

Cited By ~ 30

Author(s):

Mikhail Krasavin ◽

Anton Shetnev ◽

Tatyana Sharonova ◽

Sergey Baykov ◽

Tiziano Tuccinardi ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Selective Inhibitors ◽

Carbonic Anhydrase Inhibitors ◽

Ca Ix ◽

Membrane Bound

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Immunochemical evidence for a unique GPI-anchored carbonic anhydrase isozyme in human cardiomyocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.278.4.h1335 ◽

2000 ◽

Vol 278 (4) ◽

pp. H1335-H1344 ◽

Cited By ~ 5

Author(s):

Anja Sylvia Knüppel-Ruppert ◽

Gerolf Gros ◽

Wolfgang Harringer ◽

Hans-Peter Kubis

Keyword(s):

Carbonic Anhydrase ◽

Microsomal Fraction ◽

Cell Types ◽

Human Cardiomyocytes ◽

Ca Ix ◽

Specific Distribution ◽

Carbonic Anhydrase Isozyme ◽

Membrane Bound ◽

Membrane Anchoring ◽

Triton X

To clarify the controversial question of cell-specific distribution of carbonic anhydrase (CA) in the heart, endothelial cells and cardiomyocytes were isolated from porcine and human hearts and were characterized with cell-specific markers. CA activity was found in the microsomal fraction of both cell types. It was shown by Triton X-114 phase separation that both cell types possess a membrane-bound form of CA. These CAs share the same mechanism of membrane-anchoring via glycosylphosphatidylinositol (GPI), which excludes identity with transmembrane isoforms CA IX or CA XII. Western blotting analysis of human microsomes with anti-human CA IV antibodies revealed a marked difference in immunoreactivity. Endothelial CA activity resulted in 11-fold stronger CA IV bands compared with identical amounts of myocytic CA activity, indicating that cardiac endothelium and cardiomyocytes possess immunologically distinct forms of CA. We conclude that in human hearts CA IV is associated with the endothelium, whereas most of the CA in myocytes is not identical with one of the known CA isozymes. This suggests that cardiomyocytic CA is a novel isozyme.

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Distinct Expression Patterns of Carbonic Anhydrase IX in Clear Cell, Microcystic, and Angiomatous Meningiomas

Journal of Neuropathology & Experimental Neurology ◽

10.1093/jnen/nlz091 ◽

2019 ◽

Vol 78 (12) ◽

pp. 1081-1088

Author(s):

Rati Chkheidze ◽

Patrick J Cimino ◽

Kimmo J Hatanpaa ◽

Charles L White ◽

Manuel Ferreira ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Clear Cell ◽

Expression Patterns ◽

Carbonic Anhydrase Ix ◽

World Health ◽

Loss Of Function ◽

Ca Ix ◽

Hypoxia Marker ◽

Other World ◽

Health Organization

Abstract Clear cell, microcytic, and angiomatous meningiomas are 3 vasculature-rich variants with overlapping morphological features but different prognostic and treatment implications. Distinction between them is not always straightforward. We compared the expression patterns of the hypoxia marker carbonic anhydrase IX (CA-IX) in meningiomas with predominant clear cell (n = 15), microcystic (n = 9), or angiomatous (n = 11) morphologies, as well as 117 cases of other World Health Organization recognized histological meningioma variants. Immunostaining for SMARCE1 protein, whose loss-of-function has been associated with clear cell meningiomas, was performed on all clear cell meningiomas, and selected variants of meningiomas as controls. All clear cell meningiomas showed absence of CA-IX expression and loss of nuclear SMARCE1 expression. All microcystic and angiomatous meningiomas showed diffuse CA-IX immunoreactivity and retained nuclear SMARCE1 expression. In other meningioma variants, CA-IX was expressed in a hypoxia-restricted pattern and was highly associated with atypical features such as necrosis, small cell change, and focal clear cell change. In conclusion, CA-IX may serve as a useful diagnostic marker in differentiating clear cell, microcystic, and angiomatous meningiomas.

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CHOP-Dependent Stress-Inducible Expression of a Novel Form of Carbonic Anhydrase VI

Molecular and Cellular Biology ◽

10.1128/mcb.19.1.495 ◽

1999 ◽

Vol 19 (1) ◽

pp. 495-504 ◽

Cited By ~ 102

Author(s):

John Sok ◽

Xiao-Zhong Wang ◽

Nikoleta Batchvarova ◽

Masahiko Kuroda ◽

Heather Harding ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Target Gene ◽

Target Genes ◽

Direct Evidence ◽

Nuclear Protein ◽

Intracellular Form ◽

Carbonic Anhydrase Vi ◽

Responsive Element

ABSTRACT CHOP (also called GADD153) is a stress-inducible nuclear protein that dimerizes with members of the C/EBP family of transcription factors and was initially identified as an inhibitor of C/EBP binding to classic C/EBP target genes. Subsequent experiments suggested a role for CHOP-C/EBP heterodimers in positively regulating gene expression; however, direct evidence that this is the case has so far not been uncovered. Here we describe the identification of a positively regulated direct CHOP-C/EBP target gene, that encoding murine carbonic anhydrase VI (CA-VI). The stress-inducible form of the gene is expressed from an internal promoter and encodes a novel intracellular form of what is normally a secreted protein. Stress-induced expression of CA-VI is both CHOP and C/EBPβ dependent in that it does not occur in cells deficient in either gene. A CHOP-responsive element was mapped to the inducibleCA-VI promoter, and in vitro footprinting revealed binding of CHOP-C/EBP heterodimers to that site. Rescue of CA-VIexpression in c/ebpβ−/− cells by exogenous C/EBPβ and a shorter, normally inhibitory isoform of the protein known as LIP suggests that the role of the C/EBP partner is limited to targeting the CHOP-containing heterodimer to the response element and points to a preeminent role for CHOP in CA-VI induction during stress.

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Inhibition of Carbonic Anhydrase IX Promotes Apoptosis through Intracellular pH Level Alterations in Cervical Cancer Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22116098 ◽

2021 ◽

Vol 22 (11) ◽

pp. 6098

Author(s):

Ebru Temiz ◽

Ismail Koyuncu ◽

Mustafa Durgun ◽

Murat Caglayan ◽

Ataman Gonel ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Intracellular Ph ◽

Hela Cells ◽

Protein Level ◽

Caspase 3 ◽

Solid Tumours ◽

Carbonic Anhydrase Ix ◽

Parp Activation ◽

Ca Ix ◽

Cervical Cancer Cells

Carbonic anhydrase IX (CAIX) is a hypoxia-related protein that plays a role in proliferation in solid tumours. However, how CAIX increases proliferation and metastasis in solid tumours is unclear. The objective of this study was to investigate how a synthetic CAIX inhibitor triggers apoptosis in the HeLa cell line. The intracellular effects of CAIX inhibition were determined with AO/EB, AnnexinV-PI, and γ-H2AX staining; measurements of intracellular pH (pHi), reactive oxygen species (ROS), and mitochondrial membrane potential (MMP); and analyses of cell cycle, apoptotic, and autophagic modulator gene expression (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin, and LC3), caspase protein level (pro-caspase 3 and cleaved caspase-3, -8, -9), cleaved PARP activation, and CAIX protein level. Sulphonamide CAIX inhibitor E showed the lowest IC50 and the highest selectivity index in CAIX-positive HeLa cells. CAIX inhibition changed the morphology of HeLa cells and increased the ratio of apoptotic cells, dramatically disturbing the homeostasis of intracellular pHi, MMP and ROS levels. All these phenomena consequent to CA IX inhibition triggered apoptosis and autophagy in HeLa cells. Taken together, these results further endorse the previous findings that CAIX inhibitors represent an important therapeutic strategy, which is worth pursuing in different cancer types, considering that presently only one sulphonamide inhibitor, SLC-0111, has arrived in Phase Ib/II clinical trials as an antitumour/antimetastatic drug.

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