2. Academic Validation
  3. Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes

Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes

  • J Biol Chem. 2008 Oct 10;283(41):27799-27809. doi: 10.1074/jbc.M800938200.
Mika Hilvo 1,  Lina Baranauskiene 2,  Anna Maria Salzano 3,  Andrea Scaloni 3,  Daumantas Matulis 2,  Alessio Innocenti 4,  Andrea Scozzafava 4,  Simona Maria Monti 5,  Anna Di Fiore 5,  Giuseppina De Simone 5,  Mikaela Lindfors 6,  Janne Jänis 7,  Jarkko Valjakka 6,  Silvia Pastoreková 8,  Jaromir Pastorek 8,  Markku S Kulomaa 6,  Henri R Nordlund 6,  Claudiu T Supuran 4,  Seppo Parkkila 9
Affiliations

Affiliations

  • 1 Institute of Medical Technology, FI-33014 Tampere, Finland. Electronic address: [email protected].
  • 2 Laboratory of Biothermodynamics and Drug Design, Institute of Biotechnology, LT-02241 Vilnius, Lithuania.
  • 3 Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147 Naples, Italy.
  • 4 Bioinorganic Chemistry Laboratory, University of Florence, 50019 Sesto Fiorentino (Florence), Italy.
  • 5 Institute of Biostructures and Bioimages, National Research Council, 80134 Naples, Italy.
  • 6 Institute of Medical Technology, FI-33014 Tampere, Finland.
  • 7 Department of Chemistry, University of Joensuu, FI-80101 Joensuu, Finland.
  • 8 Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovak Republic.
  • 9 Institute of Medical Technology, FI-33014 Tampere, Finland; School of Medicine, University of Tampere and Tampere University Hospital, FI-33014 Tampere, Finland.
PMID: 18703501 DOI: 10.1074/jbc.M800938200
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.

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