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Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes
Authors:Hilvo Mika  Baranauskiene Lina  Salzano Anna Maria  Scaloni Andrea  Matulis Daumantas  Innocenti Alessio  Scozzafava Andrea  Monti Simona Maria  Di Fiore Anna  De Simone Giuseppina  Lindfors Mikaela  Jänis Janne  Valjakka Jarkko  Pastoreková Silvia  Pastorek Jaromir  Kulomaa Markku S  Nordlund Henri R  Supuran Claudiu T  Parkkila Seppo
Institution:Institute of Medical Technology, University of Tampere and Tampere University Hospital, FI-33014 Tampere, Finland. Mika.Hilvo@uta.fi
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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