Crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase IX

Carbonic anhydrase (CA) IX is a plasma membrane-associated member of the α-CA enzyme family, which is involved in solid tumor acidification. It is a marker of tumor hypoxia and a prognostic factor in several human cancers. An aberrant increase in CA IX expression in chronic hypoxia and during development of various carcinomas contributes to tumorigenesis through at least two mechanisms: pH regulation and cell adhesion control. Here we report the X-ray structure of the catalytic domain of CA IX in complex with a classical, clinically used sulfonamide inhibitor, acetazolamide. The structure reveals a typical α-CA fold, which significantly differs from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the PG domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. A correlation between the three-dimensional structure and the physiological role of the enzyme is here suggested, based on the measurement of the pH profile of the catalytic activity for the physiological reaction, CO2 hydration to bicarbonate and protons. On the basis of the structural differences observed between CA IX and the other membrane-associated α-CAs, further prospects for the rational drug design of isozyme-specific CA inhibitors are proposed, given that inhibition of this enzyme shows antitumor activity both in vitro and in vivo

Carbonic anhydrase isozyme-II-deficient mice lack the duodenal bicarbonate secretory response to prostaglandin E(2)

Duodenal bicarbonate secretion (DBS) is accepted as the primary mucosal defense against acid discharged from the stomach and is impaired in patients with duodenal ulcer disease. The secretory response to luminal acid is the main physiological stimulus for DBS and involves mediation by PGE(2) produced by mucosal cells. The aim of this investigation is to elucidate the role of carbonic anhydrases (CAs) II and IX in PGE(2)-mediated bicarbonate secretion in the murine duodenum. CA II- and IX-deficient mice and different combinations of their heterozygous and WT counterparts were studied. A 10-mm segment of the proximal duodenum with intact blood supply was isolated, and DBS was titrated by pH-stat (TitroLine-easy, Schott, Mainz, Germany). Mean arterial blood pressure (MAP) was continuously recorded, and blood acid/base balance and gastrointestinal morphology were analyzed. The duodenal segment spontaneously secreted HCO(3)(-) at a steady basal rate of 5.3 ± 0.6 μmol·cm(-1)·h(-1). Perfusing the duodenal lumen for 20 min with 47 μM PGE(2) caused a significant increase in DBS to 13.0 ± 2.9 μmol·cm(-1)·h(-1), P < 0.0001. The DBS response to PGE(2) was completely absent in Car2(-/-) mice, whereas basal DBS was normal. The CA IX-deficient mice with normal Car2 alleles showed a slight increase in DBS. Histological abnormalities were observed in the gastroduodenal epithelium in both CA II- and IX-deficient mice. Our data demonstrate a gastrointestinal phenotypic abnormality associated with CA II deficiency. The results show that the stimulatory effect of the duodenal secretagogue PGE(2) completely depends on CA II