Characterization of CA XV, a new GPI-anchored form of carbonic anhydrase

Abstract

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.

Biochemical Characterization of CA IX, One of the Most Active Carbonic Anhydrase Isozymes

“…Their main function is to maintain an appropriate acid-base balance in organisms; thus, they participate in several physiological processes, such as CO 2 and HCO 3 Ϫ transport, bone resorption, production of body fluids, gluconeogenesis, ureagenesis, and lipogenesis (2). The CA family consists of 13 active isozymes in mammals, 12 of which are expressed and function in humans (3). The CA isozymes show various tissue expression patterns, unique kinetic and inhibitory properties, and different subcellular localizations.…”

“…tion, we have recently produced another recombinant CA domain of CA IX, which is otherwise identical to the CA form, but has an N-terminal His tag that can be cleaved off; after the His tag removal, this protein showed identical k cat and K m values with the CA form, thus confirming that the C-terminal His tag does not interfere with the catalytic activity. 3 Because it was not known whether the insect cell protein preparations contained enough Zn 2ϩ to saturate the active sites of all enzyme molecules, measurements were also performed in the presence of 50 M ZnCl 2 . Under this experimental condition, the catalytic efficiency of the CA form became 10 times higher (k cat /k M ϭ 5.6 ϫ 10 8 M Ϫ1 s…”

<i>In Vivo</i> Effects of Naringenin and Lead on Rat Erythrocyte Carbonic Anhydrase Enzyme

“…CA XV isoform is not expressed in humans and other anthropoids, while expression is very commonly found in rats and other higher vertebrates. 3,4 Many of this CA isozymes are therapeutic targets to fight some disorders like oedema, glaucoma, obesity, cancer, epilepsy and osteoporosis. Also, it is emphasized that CA inhibitors may be used for treatment of infections caused by protozoa, fungi and bacteria.…”

Synthesis and inhibitory properties of some carbamates on carbonic anhydrase and acetylcholine esterase

“…Human CAs (hCAs) all belongs to the a-family and so far 16 different CA isoforms discovered in this class [32][33][34] . In humans, CAs are dispersed in different tissues including the reproductive tract, the gastrointestinal tract, kidneys, the nervous system, skin, eyes, lungs, and among some others [35][36][37][38] . Carbonic anhydrase isoenzymes contain a zinc ion (Zn 2+ ) in their active site, coordinated by three His residues and a H 2 O molecule/hydroxide ion (ÀOH) in the a-and g-CAs or by two Cys and one His residues (in the b class), with the fourth ligand being a H 2 O molecule/ÀOH ion acting as nucleophile in the catalyzed reactions [39][40][41][42][43] .…”