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. Epub 2008 Aug 13.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Neoplasm / chemistry*
  • Antigens, Neoplasm / genetics
  • Antigens, Neoplasm / metabolism
  • Baculoviridae
  • Carbonic Anhydrase IX
  • Carbonic Anhydrases / chemistry*
  • Carbonic Anhydrases / genetics
  • Carbonic Anhydrases / metabolism
  • Catalysis
  • Cell Adhesion / genetics
  • Cell Line
  • Cell Proliferation
  • Dimerization
  • Disulfides / chemistry
  • Disulfides / metabolism
  • Glycosylation
  • Humans
  • Hydrogen-Ion Concentration
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Neoplasms / enzymology
  • Neoplasms / genetics
  • Polysaccharides / chemistry
  • Polysaccharides / genetics
  • Polysaccharides / metabolism
  • Protein Structure, Tertiary / genetics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spodoptera


  • Antigens, Neoplasm
  • Disulfides
  • Isoenzymes
  • Polysaccharides
  • Recombinant Proteins
  • CA9 protein, human
  • Carbonic Anhydrase IX
  • Carbonic Anhydrases