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 (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.
Hilvo
Mika
M
Institute of Medical Technology, FI-33014 Tampere, Finland. Electronic address: Mika.Hilvo@uta.fi.
Baranauskiene
Lina
L
Laboratory of Biothermodynamics and Drug Design, Institute of Biotechnology, LT-02241 Vilnius, Lithuania.
Salzano
Anna Maria
AM
Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147 Naples, Italy.
Scaloni
Andrea
A
Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147 Naples, Italy.
Matulis
Daumantas
D
Laboratory of Biothermodynamics and Drug Design, Institute of Biotechnology, LT-02241 Vilnius, Lithuania.
Innocenti
Alessio
A
Bioinorganic Chemistry Laboratory, University of Florence, 50019 Sesto Fiorentino (Florence), Italy.
Scozzafava
Andrea
A
Bioinorganic Chemistry Laboratory, University of Florence, 50019 Sesto Fiorentino (Florence), Italy.
Monti
Simona Maria
SM
Institute of Biostructures and Bioimages, National Research Council, 80134 Naples, Italy.
Di Fiore
Anna
A
Institute of Biostructures and Bioimages, National Research Council, 80134 Naples, Italy.
De Simone
Giuseppina
G
Institute of Biostructures and Bioimages, National Research Council, 80134 Naples, Italy.
Lindfors
Mikaela
M
Institute of Medical Technology, FI-33014 Tampere, Finland.
Jänis
Janne
J
Department of Chemistry, University of Joensuu, FI-80101 Joensuu, Finland.
Valjakka
Jarkko
J
Institute of Medical Technology, FI-33014 Tampere, Finland.
Pastoreková
Silvia
S
Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovak Republic.
Pastorek
Jaromir
J
Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovak Republic.
Kulomaa
Markku S
MS
Institute of Medical Technology, FI-33014 Tampere, Finland.
Nordlund
Henri R
HR
Institute of Medical Technology, FI-33014 Tampere, Finland.
Supuran
Claudiu T
CT
Bioinorganic Chemistry Laboratory, University of Florence, 50019 Sesto Fiorentino (Florence), Italy.
Parkkila
Seppo
S
Institute of Medical Technology, FI-33014 Tampere, Finland; School of Medicine, University of Tampere and Tampere University Hospital, FI-33014 Tampere, Finland.
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
D000951:Antigens, Neoplasm; D004220:Disulfides; D007527:Isoenzymes; D011134:Polysaccharides; D011994:Recombinant Proteins; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases