Carbonic anhydrase 9

Carbonic anhydrases (CAs) are a large family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide. They participate in a variety of biological processes, including respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. They show extensive diversity in tissue distribution and in their subcellular localization.[7]

CA IX is mainly expressed in the gastrointestinal tract where it facilitates acid secretion.[11] The CA IX enzyme, along with the CA II enzyme, binds to Anion Exchanger 2 (AE2) which increases bicarbonate transport and maximizes the rate of acid secretion by gastric parietal cells.[8]

CA IX is a transmembrane glycoprotein with an extracellular active site.[9] The cytoplasmic tail of the enzyme contains three residues that may be phosphorylated (Thr-443, Ser-448, and Tyr-449) and participate in signal transduction.[9][12] Phosphorylated tyrosine 449 can interact with PI3K which activates protein kinase B to affect cellular glucose metabolism.[13]

Under physiological conditions, the enzyme exists as two nearly identical dimers.[14] Both dimers are stabilized by two hydrogen bonds between Arg-137 and the Ala-127 carbonyl oxygen as well as many Van der Waals interactions.[14] One dimer, however, has additional stabilization due to a disulfide bridge formed by two cysteine residues.[14]

Two hydrogen bonds between Arg-137 and the Ala-127 carbonyl oxygen stabilize the CA IX dimer.

One face of the dimer contains proteoglycan (PG) domains-a feature that is unique from other CA enzymes- and the opposite face contains the C-termini which help the enzyme attach to the cell membrane.[15] CA IX contains an N-linked glycosylation site bearing mannose-type glycan structures on Asn-309 as well as an O-linked glycosylation site on Thr-78.[16]

CA IX dimer with its four distinct pairs of domains labeled: the proteoglycan domain (PG), catalytic domain (blue), transmembrane segment (TM) and the intracellular tail (IC).

Expression of CA IX is primarily regulated at the transcriptional level.[17] The promoter region of the CA9 gene contains an HRE (hypoxia responsive element) where HIF-1 can bind, which allows hypoxic conditions to increase CA IX expression.[17] Expression can also be regulated post-translationally by metalloproteinases which cause shedding of the enzyme's ectodomain.[18] Unlike other CA isozymes, CA IX is not inhibited by high lactate concentrations.[19] However, it is inhibited by bicarbonate.[19]

CA IX is a transmembrane protein and is a tumor-associated carbonic anhydrase isoenzyme. It is over-expressed in VHL mutated clear cell renal cell carcinoma (ccRCC) and hypoxic solid tumors, but is low-expressed in normal kidney and most other normal tissues. It may be involved in cell proliferation and transformation. This gene is mapped to 9p13-p12.[7]

CA IX is a cellular biomarker of hypoxia. Furthermore, recent studies examining the association between CA IX levels and various clinicopathological outcomes suggest that CA IX expression may also be a valuable prognostic indicator for overall survival[20] although this association has been questioned.[21]

CA IX shows high expression in carcinomas of the uterine cervix, kidney, oesophagus, lung, breast, colon, brain, and vulva compared to expression in few noncancerous tissues.[22][11] Its overexpression in cancerous tissues compared to normal ones is due to hypoxic conditions in the tumor microenvironment caused by abnormal vasculature and subsequent transcriptional activation by HIF-1 binding.[17] In clear cell renal carcinomas, CA IX shows high expression under normoxia due to a mutation in the VHL gene that normally negatively regulates HIF-1.[22] Because of its overexpression in many types of cancer and low expression in normal tissues, CAIX has become a useful target for clear cell RCC and breast cancer tumor imaging in mice.[23][24]

CA IX plays a very significant role in tumor acidification as it has very high catalytic activity with the highest rate of proton transfer of the known CAs.[16] The enzyme converts carbon dioxide outside of the tumor into bicarbonate and protons, contributing to extracellular acidosis and promoting tumor growth by regulating the pH of the cytosol.[10]

Because of its low expression in normal tissues and overexpression in many cancer tissues, CA IX has also become a desirable drug target. Girentuximab, an antibody that binds to CA IX, failed to improve disease-free as well as overall survival of patients with clear cell RCC in Phase III clinical trials.[25]

However, a number of small molecules have been used to inhibit CA IX. The main classes of these inhibitors are inorganic anions, sulfonamides, phenols, and coumarins.[15] Anions and sulfonamides inhibit CA IX by coordinating the zinc ion within CA IX while phenols bind to the zinc-coordinated water molecule.[15] Coumarins serve as mechanism-based inhibitors that are hydrolyzed by the enzyme to form a cis-2-hydroxy-cinnamic acid derivative that then binds to the active site.[26]