title: Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes.
abstract: 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 (Cys(119)-Cys(299)) and a unique N-linked glycosylation site (Asn(309)) 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 (Thr(78)) 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.
authors.affiliation: Institute of Medical Technology, FI-33014 Tampere, Finland. Electronic address: Mika.Hilvo@uta.fi.
authors.forename: Mika
authors.initials: M
authors.lastname: Hilvo
authors.affiliation: Laboratory of Biothermodynamics and Drug Design, Institute of Biotechnology, LT-02241 Vilnius, Lithuania.
authors.forename: Lina
authors.initials: L
authors.lastname: Baranauskiene
authors.affiliation: Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147 Naples, Italy.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Salzano
authors.affiliation: Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147 Naples, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Scaloni
authors.affiliation: Laboratory of Biothermodynamics and Drug Design, Institute of Biotechnology, LT-02241 Vilnius, Lithuania.
authors.forename: Daumantas
authors.initials: D
authors.lastname: Matulis
authors.affiliation: Bioinorganic Chemistry Laboratory, University of Florence, 50019 Sesto Fiorentino (Florence), Italy.
authors.forename: Alessio
authors.initials: A
authors.lastname: Innocenti
authors.affiliation: Bioinorganic Chemistry Laboratory, University of Florence, 50019 Sesto Fiorentino (Florence), Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Scozzafava
authors.affiliation: Institute of Biostructures and Bioimages, National Research Council, 80134 Naples, Italy.
authors.forename: Simona Maria
authors.initials: SM
authors.lastname: Monti
authors.affiliation: Institute of Biostructures and Bioimages, National Research Council, 80134 Naples, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Di Fiore
authors.affiliation: Institute of Biostructures and Bioimages, National Research Council, 80134 Naples, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: De Simone
authors.affiliation: Institute of Medical Technology, FI-33014 Tampere, Finland.
authors.forename: Mikaela
authors.initials: M
authors.lastname: Lindfors
authors.affiliation: Department of Chemistry, University of Joensuu, FI-80101 Joensuu, Finland.
authors.forename: Janne
authors.initials: J
authors.lastname: Jänis
authors.affiliation: Institute of Medical Technology, FI-33014 Tampere, Finland.
authors.forename: Jarkko
authors.initials: J
authors.lastname: Valjakka
authors.affiliation: Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovak Republic.
authors.forename: Silvia
authors.initials: S
authors.lastname: Pastoreková
authors.affiliation: Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovak Republic.
authors.forename: Jaromir
authors.initials: J
authors.lastname: Pastorek
authors.affiliation: Institute of Medical Technology, FI-33014 Tampere, Finland.
authors.forename: Markku S
authors.initials: MS
authors.lastname: Kulomaa
authors.affiliation: Institute of Medical Technology, FI-33014 Tampere, Finland.
authors.forename: Henri R
authors.initials: HR
authors.lastname: Nordlund
authors.affiliation: Bioinorganic Chemistry Laboratory, University of Florence, 50019 Sesto Fiorentino (Florence), Italy.
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
authors.affiliation: Institute of Medical Technology, FI-33014 Tampere, Finland; School of Medicine, University of Tampere and Tampere University Hospital, FI-33014 Tampere, Finland.
authors.forename: Seppo
authors.initials: S
authors.lastname: Parkkila
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016367:Baculoviridae; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002448:Cell Adhesion / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000201:enzymology / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
chemical_list: D000951:Antigens, Neoplasm; D004220:Disulfides; D007527:Isoenzymes; D011134:Polysaccharides; D011994:Recombinant Proteins; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases