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