Cloning, Expression, Post-Translational Modifications and Inhibition Studies on the Latest Mammalian Carbonic Anhydrase Isoform, CA XV Mika Hilvo, †,‡ Anna Maria Salzano, †,§ Alessio Innocenti, | Markku S. Kulomaa, ‡ Andrea Scozzafava, | Andrea Scaloni, § Seppo Parkkila, ‡,⊥ and Claudiu T. Supuran* ,| Institute of Medical Technology, UniVersity of Tampere and Tampere UniVersity Hospital, Biokatu 6, FI-33520, Tampere, Finland, Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, Via Argine 1085, 80147 Naples, Italy, Bioinorganic Chemistry Laboratory, UniVersity of Florence, Room 188, Via della Lastruccia 3, I-50019, Sesto Fiorentino (Florence), Italy, and School of Medicine, UniVersity of Tampere and Tampere UniVersity Hospital, Biokatu 6, FI-33520, Tampere, Finland ReceiVed October 7, 2008 We have cloned and purified to homogeneity the latest member of the mammalian R-carbonic anhydrase (CA, EC 4.2.1.1) family, the mouse CA XV (mCA XV) protein. An investigation on the post-translational modifications of the enzyme has also been performed. The enzyme shows a moderate catalytic activity for the physiologic reaction, similarly to the physiologically relevant isoforms CA I, IV, VI, XII, and XIV, and it is susceptible to inhibition by sulfonamides and sulfamates. Best mCA XV inhibitors were celecoxib, sulfanilyl-sulfonamides, methazolamide, ethoxzolamide, dorzolamide, brinzolamide, and sulthiame, with K I s in the range of 45-65 nM. Due to the presence of this enzyme in rather high amounts in the rodent kidneys, the contribution of this isoform to the overall drug response should be taken into account when animal models are used to investigate CA inhibitors. Introduction Carbonic anhydrases (CAs) are zinc-containing metalloen- zymes that catalyze the reversible hydration of carbon dioxide and water to bicarbonate and a proton (CO 2 + H 2 O S HCO 3 - + H + ). 1 The CA proteins can be found in organisms all over the phylogenetic tree, and the mammalian enzymes belong to the so-called R-CA family, as it was the first one to be described. 2 In mammals, the members of the R-CA family participate in several physiological processes, such as pH regulation, CO 2 and HCO 3 - transport, production of biological fluids, bone resorption, metabolic processes, as well as cell adhesion and cell proliferation. 3,4 The mammalian CA family consists of thirteen active members that differ in their kinetic and inhibitory properties, cell and tissue distribution, and function. The isozymes can be classified according to their subcellular localization: CAs I, II, III, VII, and XIII are cytosolic enzymes, 3,5,6 CAs VA and VB are located in the mitochondria, 7 CA VI is the only secreted isozyme, 8 CAs IX, XII, and XIV are transmembrane proteins, 9-11 and CAs IV and XV are GPI- anchored to the cell membrane (Table 1). 12,13 The latest member of the mammalian CA enzyme family was characterized in 2005, when the isozyme XV was reported for the first time. 13 CA XV appeared to be an exceptional member of this family because it is expressed in several species in different vertebrates, whereas in the primates, such as humans and chimpanzees, it has become a nonfunctional pseudogene. CA XV is a GPI-anchored enzyme like CA IV, and it is likely that CA IV or some other membrane-bound isozyme has taken the functional role of CA XV in primates. Nevertheless, CA XV has a high relevance for the biomedical research, because it is expressed 13 in the widely used model organisms, such as the rodents (mice and rats). This issue has to be taken into particular account when the results from these organisms are inferred to human physiology. In the first publication, the activity of mouse CA XV was measured for a recombinant protein form produced in the bacterial expression system, and it appeared to be very low. 13 Recently, we have obtained preliminary experi- mental indications suggesting that mouse CA XV possesses a catalytic activity comparable to those of the physiologically relevant isoforms CA XII and XIV, with k cat of 4.7 × 10 5 s -1 and k cat /K M of 3.3 × 10 7 M -1 s -1 (Table 1). 14 In the present study we describe in detail the cloning and protein production of CA XV in the baculovirus-insect cell expression system and use mass spectrometric analyses to characterize the obtained recombinant product, particularly its post-translational modifica- tions. CA XV is the only isozyme whose inhibition profile has not been published earlier (apart from acetazolamide, which showed K I of 72 nM), 14 and therefore, we have investigated * To whom correspondence should be addressed. Tel.: +39-055-4573005. Fax: +39-055-4573385. E-mail: claudiu.supuran@unifi.it. † These authors contributed equally to this work. ‡ Institute of Medical Technology, University of Tampere and Tampere University Hospital. § ISPAAM. | University of Florence. ⊥ School of Medicine, University of Tampere and Tampere University Hospital. Table 1. Kinetic Parameters for CO 2 Hydration Reaction Catalyzed by the 13 Vertebrate Catalytically Active R-CA Isozymes, at 20 °C and pH 7.5, and Their Subcellular Localization isozyme a k cat (s -1 ) K m (mM) k cat /K m (M -1 × s -1 ) subcellular localization hCA I 2.0 × 10 5 4.0 5.0 × 10 7 cytosol hCA II 1.4 × 10 6 9.3 1.5 × 10 8 cytosol hCA III 1.3 × 10 4 52.0 2.5 × 10 5 cytosol hCA IV 1.1 × 10 6 21.5 5.1 × 10 7 membrane-bound hCA VA 2.9 × 10 5 10.0 2.9 × 10 7 mitochondria hCA VB 9.5 × 10 5 9.7 9.8 × 10 7 mitochondria hCA VI 3.4 × 10 5 6.9 4.9 × 10 7 secreted into saliva/milk hCA VII 9.5 × 10 5 11.4 8.3 × 10 7 cytosol hCA IX b 1.1 × 10 6 7.5 1.5 × 10 8 transmembrane hCA XII 4.2 × 10 5 12.0 3.5 × 10 7 transmembrane hCA XIII 1.5 × 10 5 13.8 1.1 × 10 7 cytosol hCA XIV 3.1 × 10 5 7.9 3.9 × 10 7 transmembrane mCA XV 4.7 × 10 5 14.2 3.3 × 10 7 membrane-bound a h ) human; m ) mouse enzyme. b Proteoglycan and CA catalytic domains. 15 J. Med. Chem. 2009, 52, 646–654 646 10.1021/jm801267c CCC: $40.75  2009 American Chemical Society Published on Web 12/24/2008