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

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys119-Cys299) and a unique N-linked glycosylation site (Asn309) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr78) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved. Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys119-Cys299) and a unique N-linked glycosylation site (Asn309) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr78) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved. Carbonic anhydrases (CAs) 2The abbreviations used are: CA, carbonic anhydrase; DTT, dithiotreitol; LS, light scattering; MALDI-TOF MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; NeuGc, N-glycolylneuraminic acid; PSD, post-source decay; SEC, size exclusion chromatography; SES, stepwise elongation of sequence; HPLC, high pressure liquid chromatography; PNGase, peptide:N-glycosidase; ah, anti-human. form a family of zinc-containing metalloenzymes that catalyze the reversible hydration of carbon dioxide according to the following reaction: CO2 + H2O ↔ H+ + HCO3− (1Supuran C.T. Supuran C.T. Scozzafava A. Conway J. Carbonic Anhydrase: Its Inhibitors and Activators. CRC Press, Boca Raton, FL2004: 1-23Crossref Google Scholar). Their main function is to maintain an appropriate acid-base balance in organisms; thus, they participate in several physiological processes, such as CO2 and HCO3− transport, bone resorption, production of body fluids, gluconeogenesis, ureagenesis, and lipogenesis (2Sly W.S. Hu P.Y. Annu. Rev. Biochem. 1995; 64: 375-401Crossref PubMed Scopus (735) Google Scholar). The CA family consists of 13 active isozymes in mammals, 12 of which are expressed and function in humans (3Hilvo M. Tolvanen M. Clark A. Shen B. Shah G.N. Waheed A. Halmi P. Hänninen M. Hämäläinen J.M. Vihinen M. Sly W.S. Parkkila S. Biochem. J. 2005; 392: 83-92Crossref PubMed Scopus (146) Google Scholar). The CA isozymes show various tissue expression patterns, unique kinetic and inhibitory properties, and different subcellular localizations. CAs I, II, III, VII, and XIII reside in the cytosol (2Sly W.S. Hu P.Y. Annu. Rev. Biochem. 1995; 64: 375-401Crossref PubMed Scopus (735) Google Scholar, 4Montgomery J.C. Venta P.J. Eddy R.L. Fukushima Y.S. Shows T.B. Tashian R.E. Genomics. 1991; 11: 835-848Crossref PubMed Scopus (59) Google Scholar, 5Lehtonen J. Shen B. Vihinen M. Casini A. Scozzafava A. Supuran C.T. Parkkila A.K. Saarnio J. Kivelä A.J. Waheed A. Sly W.S. Parkkila S. J. Biol. Chem. 2004; 279: 2719-2727Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), CAs IV, IX, XII, XIV and XV (XV is not expressed in humans) are associated with the cell membrane (3Hilvo M. Tolvanen M. Clark A. Shen B. Shah G.N. Waheed A. Halmi P. Hänninen M. Hämäläinen J.M. Vihinen M. Sly W.S. Parkkila S. Biochem. J. 2005; 392: 83-92Crossref PubMed Scopus (146) Google Scholar, 6Zhu X.L. Sly W.S. J. Biol. Chem. 1990; 265: 8795-8801Abstract Full Text PDF PubMed Google Scholar, 7Pastoreková S. Parkkila S. Parkkila A.K. Opavský R. Zelník V. Saarnio J. Pastorek J. Gastroenterology. 1997; 112: 398-408Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 8Türeci Ö. Sahin U. Vollmar E. Siemer S. Göttert E. Seitz G. Parkkila A.K. Shah G.N. Grubb J.H. Pfreundschuh M. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7608-7613Crossref PubMed Scopus (320) Google Scholar, 9Fujikawa-Adachi K. Nishimori I. Taguchi T. Onishi S. Genomics. 1999; 61: 74-81Crossref PubMed Scopus (112) Google Scholar, 10Whittington D.A. Grubb J.H. Waheed A. Shah G.N. Sly W.S. Christianson D.W. J. Biol. Chem. 2004; 279: 7223-7228Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar); CA VA and VB localize to mitochondria (11Fujikawa-Adachi K. Nishimori I. Taguchi T. Onishi S. J. Biol. Chem. 1999; 274: 21228-21233Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar); and CA VI is secreted (12Murakami H. Sly W.S. J. Biol. Chem. 1987; 262: 1382-1388Abstract Full Text PDF PubMed Google Scholar). In general, CAs exhibit a very high catalytic efficiency that approaches the diffusion-controlled limit (1Supuran C.T. Supuran C.T. Scozzafava A. Conway J. Carbonic Anhydrase: Its Inhibitors and Activators. CRC Press, Boca Raton, FL2004: 1-23Crossref Google Scholar). To date, CA II has been reported to represent the isozyme with the highest catalytic efficiency, with a J. Biol. Chem. Full Text PDF PubMed Google Scholar, I. A. Scozzafava A. Supuran C.T. Chem. PubMed Scopus Google Scholar). CA IX and are of the CA and expression is by CA IX is the exceptional it has been proposed to to cell proliferation, cell adhesion, and cell S. Pastorek J. Supuran C.T. Scozzafava A. Conway J. Carbonic Anhydrase: Its Inhibitors and Activators. CRC Press, Boca Raton, FL2004: Google Scholar, Parkkila S. E. J. M. Pastorek J. T. A. B. J. I. S. Gastroenterology. Full Text Full Text PDF PubMed Scopus (102) Google Scholar). is expressed in a very of in the it is expressed in several that CA such as and S. J. 2004; Scholar). for the CA IX protein and by Pastorek J. S. I. Zelník V. Opavský R. M. S. J. A. R. Google Scholar), and the characterized in by the R. S. V. A. J. R. Pastorek J. Genomics. PubMed Scopus Google Scholar). this study that CA IX with the domain of a R. S. V. A. J. R. Pastorek J. Genomics. PubMed Scopus Google Scholar). CA IX consists of the following an domain a CA catalytic domain a transmembrane and a intracytoplasmic the occurrence of on the PG CA IX has been to represent a in and is an IX that is in a to cell the information on CA IX that has the the biochemical of this enzyme has The main of the study to this in by information on the catalytic properties, and of CA IX. it is reported that membrane proteins for biochemical soluble CA IX forms were produced using the baculovirus-insect cell expression which has not been used to CA recombinant proteins were characterized by different and biochemical of were to the recombinant CA IX both of which the small transmembrane and intracytoplasmic recombinant proteins a CA IX for for protein and a site for The PG + CA form for a protein of both the PG and CA of the CA IX of the it the site The CA form only for the CA domain, of the the and a the were by the stepwise elongation of K. PubMed Scopus Google Scholar). The CA IX recombinant proteins were according to the on the and the of the and and of CA were in cell in an The were and the with the with and according to the The of cell as of and of the were to the cell and the protein to to the on a for The with of and the protein with pH and CA form to a of pH using an the PG domain in not the PG + CA form to a of pH To the recombinant proteins were with of of protein with for according to the The the protein by The the protein of the PG + CA form by the protein using an The for the PG + CA form for the CA using different and a enzyme excess and measured different by both the and the and the of used as the of and CA IX by in the CA form of CA IX. recombinant CA IX proteins were by PubMed Scopus Google either by with the or which as M. M. S. Pastorek J. Parkkila S. J. 2004; PubMed Google Scholar). In both the S. M. J. PubMed Scopus Google and the were used to CA IX recombinant the were and The were for and for the IX and they were both in study the oligomerization of CA IX, of both recombinant protein were for with different of were by The were using according to the by light were using a size exclusion to high liquid with an The by a a or and a laser light The system in pH the of were used for and the the and the the the the light and the the The the were also for and CA IX in the and recombinant of the CA IX protein used in the CA activity and has been M. K. E. A. V. M. M. Parkkila S. Parkkila A.K. Waheed A. Sly W.S. I. Pastorek J. S. J. PubMed Scopus Google Scholar). The production and of this protein in has been Ö. G. Scozzafava A. Supuran C.T. Chem. 2005; PubMed Scopus Google Scholar). of recombinant CA IX protein produced in cell and used in the mass CA used to CO2 hydration activity J. Biol. Chem. Full Text PDF PubMed Google Scholar). measured using as an in pH for a of To the kinetic and the CO2 to the of the were used to the of in used to to To form the and enzyme were for to were obtained by using kinetic were obtained as reported and represent the different were on the PG + CA and CA forms the baculovirus-insect cell expression system and on the CA domain of CA IX also the E. expression system. The for the proteins produced in the were also in the of or In the of the CO2 hydration activity measured also for CA I, II, IV, XII, and and the for production in M. A. G. Supuran C.T. Parkkila S. PubMed Scopus Google for disulfide were with in and pH for in the were excess and by the a as reported V. R. A. M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were and and were the and with as reported S. G. R. M. G. G. A. PubMed Scopus Google Scholar). were with by and were were either using MALDI-TOF or on a using a to of in a of for were and by MALDI-TOF and were by with of in pH for 12 disulfide were by with in pH for were by with of in pH for of to the and they were on MALDI-TOF recombinant proteins were with a system with an mass with an a of and a of were using a with a of in to in that to a of Mass were the in were using the in the by the Mass by using as an protein or were on the with in or in as using the were with a with a laser S. G. R. M. G. G. A. PubMed Scopus Google Scholar). Mass were in and using the either in or mass with or were of the appropriate as reported G. G. M. R. S. S. G. P. M. A. G. A. 2004; PubMed Scopus Google Scholar). In both were using the MALDI-TOF mass were to or using the This a on protein nature of the to and the mass of the Mass were to protein regions using a mass were by additional experiments on as are reported as to CA IX. of CA IX soluble recombinant forms of CA IX, both of which were the small transmembrane and intracytoplasmic domains the were produced using the baculovirus-insect cell expression system. The PG + CA form consisted of both the PG and CA catalytic which in the CA IX The CA form included only the CA domain, in the recombinant proteins the CA IX which the protein to the the and the proteins were secreted the to the the forms were to with a as by a or CA IX were on the site included in the recombinant proteins to the active in the for the PG + CA form not for the CA form not the the PG + CA form for the in the CA The nature of both CA IX by using two CA only the PG and a novel CA IX, that only the CA the PG + CA form by both and the CA form only by of the PG + CA a small of protein an mass in the protein The showed that the mass protein the CA domain, which the PG domain This may a site the PG and CA site the is to the of the PG + CA the only for this protein in the mass an N-linked which not to the as with in a of both in the of the oligomerization of the recombinant CA IX proteins by and SEC, and with laser light recombinant proteins were with various and to that both CA IX recombinant proteins as in addition to The PG + CA form consisted of and the CA form consisted of and by the with of the of the with a of the that the are stabilized by intermolecular disulfide bond(s). the CA the that the protein is of and the PG + CA the size of the and the size of the To the oligomerization of proteins and were In the experiments the CA form showed two mass a with protein demonstrated the of a protein and not in with the the PG + CA the also showed the occurrence of two and in an and were that obtained mass spectrometry for the an of this on the of the PG domain with a This by of the which E. J. 1999; Google Scholar). In this and showed an mass of and with in also that the PG + CA form as a of a protein and of the and demonstrated that showed to the the of the and were also in the this for the also In the experiments showed that both CA IX forms as a of and In the of CA IX the for a of the CA date, the catalytic activity of CA IX has been measured only for the expressed recombinant protein of the CA domain T. P.J. Biochem. PubMed Scopus Google Scholar). In the the CO2 hydration of the recombinant CA IX forms the baculovirus-insect cell expression system were measured and with the reported the measured kinetic of this with for mammalian the activity and inhibitory measured for the CA form expressed in either the or E. the were with reported T. P.J. Biochem. PubMed Scopus Google Scholar). This that the which not the CA not any on the function of the protein in the protein E. which the showed In produced recombinant CA domain of CA IX, which is to the CA has an that the this protein showed and with the CA that the not with the catalytic A. M. A. A. A. S. M. A. G. M. M. J. J. S. J. M. S. H. R. T. and S. it not the cell protein to the active of enzyme were also in the of this the catalytic efficiency of the CA form and that reported for CA II J. Biol. Chem. Full Text PDF PubMed Google Scholar, I. A. Scozzafava A. Supuran C.T. Chem. PubMed Scopus Google Scholar). addition of the CO2 hydration activity of the PG + CA form that measured for the CA form the In this addition of the activity to a that has been measured for any member of this enzyme this addition also a in the of for the PG + CA form to and inhibitory properties of CA IX to the mammalian CA IX E. + CA, + CA, + CA, + CA, + CA, in a To the of in the cell expression CO2 hydration activity measured in the of for CA which produced the used for CA IX M. A. G. Supuran C.T. Parkkila S. PubMed Scopus Google Scholar). The experiments demonstrated that the addition of to CA XV the enzyme catalytic activity In experiments with isozymes I, II, IV, XII, and the addition of to the also a in the CO2 hydration activity In that the cell expression system to the active site of the CA and that the addition of not an in the CO2 hydration measured by the CA activity that the of the catalytic activity the addition of to the enzyme is a unique for isozyme of on the CO2 hydration activity of various CA different PG + CA form produced in different The PG + CA form produced in in a both the catalytic activity and were following addition of the of metal ions on the PG + CA form of and also an in the with the measured for to that for In to the metal ions not exhibit an for the of the and not any on the CA form not CA IX the of the recombinant CA IX both were to showed the of that by mass to different the PG + CA the mass were and the CA the were and and the of were with protein by for the PG + CA and CA which and were with and with were either by MALDI-TOF in or by to which by MALDI-TOF of the in the mass obtained for both recombinant enzyme the mass measured for the PG + CA and an obtained for the CA form not the of measured and of were to a N-linked to the and and In both to a unique which showed a and were for the of the and the in the not associated with the were in any both that both of proteins were measured the of and of the for the PG + CA and CA and the occurrence of In the proteins to the PG + CA form and the CA form which the been by the with both high mannose-type N-linked to mass and (PG + CA mass and (CA CA IX in Shows and in glycosylation of CA IX produced in either or mammalian also a of recombinant enzyme produced in the cell The protein by a unique that and to MALDI-TOF mass experiments in as in this a the of measured and of were to high and N-linked to and in this to a unique with a to that reported the nature of by or as as in the not MALDI-TOF in additional were also in the and and occurrence by by and and the of measured mass were to and different and O-linked to a for and for and and N-glycolylneuraminic were not the of measured a or the of MALDI-TOF the of and 2004; PubMed Scopus Google Scholar). were also in the and and were associated to additional O-linked a they not following with for and for and in for the with and the nature by of ions either glycan or the site to the of a to the the occurrence of a In a of MALDI-TOF mass in and for recombinant CA IX of the protein CA IX a the of in recombinant CA IX obtained the baculovirus-insect cell expression a of the CA form with with and to MALDI-TOF in as reported In this a showed a of that by mass with and the of the glycosylation reported were to high mannose-type N-linked to and to a unique with a with both the of the and the of with and which were to and In the MALDI-TOF demonstrated the occurrence of the as a with a to that reported the of the PG + CA form showed the occurrence of with and which were to N-linked high mannose-type to a unique following demonstrated that both recombinant CA IX forms exhibited a bridge to of novel biochemical which CA IX the of the CA were following of two recombinant protein forms that were produced by the baculovirus-insect cell expression system. The cell expression system has been reported for the production of recombinant CA and it to enzymes with a to the mammalian that produced in The oligomerization of the recombinant enzymes by several size exclusion size exclusion with laser light and and experiments demonstrated that both recombinant CA IX forms consisted of a of and The were for the CA in the of a laser light E. J. 1999; Google Scholar), as for the of the size of a protein and its to the of the PG + CA of the CA IX extracellular to the and both an of the recombinant of the occurrence of the PG domain, which experiments an to of the of CA IX, the protein as to form a S. M. J. PubMed Scopus Google Scholar, J. S. Pastorek J. V. J. PubMed Scopus Google Scholar). experiments demonstrated that both recombinant CA IX forms an intramolecular bridge to which is also in CA that the CA form may only to form the the the of two and in the PG + CA of the intermolecular disulfide the on the and nature of the intermolecular in the not in an not the of of CA IX, a that CA IX may the transmembrane the intracytoplasmic to form the with the protein are to the of this protein may the CA IX oligomerization CA IX were by the the CO2 hydration activity of the recombinant protein The revealed a catalytic activity that that reported for this an the obtained for the catalytic domain produced in both the baculovirus-insect cell and the expression addition of to the enzyme its catalytic efficiency by of The PG + CA form showed metal it showed the as that measured for CA II, which has been to the active CA isozyme The only the PG + CA and CA forms the of the PG domain in the PG + CA that this domain to an in the CO2 hydration activity of the CA domain by an of the catalytic activity of the PG + CA form by a measured for any member of the CA isozyme also the for experiments with isozymes revealed that the catalytic activity in the of not to of in the cell expression system an of the CO2 hydration activity in the experiments in the of demonstrated a of on the catalytic activity of the PG + CA In to the addition of and showed on the and of the CA and the of only a in the of for the PG + CA the that the metal ions may either on the proteoglycan or catalytic to the the catalytic domain addition of to the protein the catalytic activity of both recombinant protein the the metal ions may the PG domain, which contains were only with the PG + CA The metal ions catalytic activity by the that in the PG domain, the in the of the PG + CA the by the may also to a PG or of the for which in the is to that both the catalytic and PG domains of CA IX reside in the which contains a of in the of Chem. 1995; PubMed Scopus Google Scholar). The PG domain of the PG + CA form of CA IX has been to in a which A. M. A. A. A. S. M. A. G. M. M. J. J. S. J. M. S. H. R. T. and S. experiments in that CA IX protein on the cell is M. M. R. Pastorek J. S. J. 2004; Google Scholar). The in the study to the that the ions in the may in the very high stability and catalytic activity of CA IX in Mass revealed information on the in CA IX. N-linked glycosylation site (Asn309) the CA domain, which by high mannose-type in both recombinant CA IX forms the baculovirus-insect cell expression system as as in that the In the N-linked were also of the high mannose-type is in with on glycosylation of the protein S. M. J. PubMed Scopus Google Scholar). In the of O-linked glycosylation also demonstrated in the recombinant mammalian This is a that has with the domain of a R. S. V. A. J. R. Pastorek J. Genomics. PubMed Scopus Google Scholar). and or O-linked and a were of the that has been in the proteoglycan domain of proteins in cell processes and PubMed Scopus Google Scholar). In a protein in cell and to the extracellular K. I. M. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (84) Google Scholar). N-linked and of the with to in and A. G. P. 1998; PubMed Scopus Google Scholar, M. R. P. PubMed Scopus Google Scholar, A. A. PubMed Scopus Google Scholar, K. PubMed Scopus Google Scholar). in the glycosylation of membrane been in by both the and of the and the of and expressed S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, I. PubMed Scopus Google Scholar). In and cell a has been in protein the addition of I. PubMed Scopus Google Scholar, I. J.M. J. J. J. Biochem. 1995; PubMed Scopus Google Scholar, J. V. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). with the of expression of the that are for the of and the of R. M. M. S. H. T. J. H. J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a is not may also the addition of to form K. Rev. PubMed Scopus Google Scholar, I. Biochem. PubMed Google Scholar), and several of enzymes different expression and activity in and J. S. PubMed Scopus Google Scholar). this that a of the processes protein is to the occurrence of in CA IX and to the of and O-linked and the of CA IX an the novel biochemical properties of CA IX, which information on its a of the enzyme in a by high mass or as as on cellular and using recombinant CA IX the of this protein to on the function of different domains CA IX, to the of the various pathophysiological processes in which this unique enzyme is involved. M. for the and and for with