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

被引:238
作者
Hilvo, Mika [1 ]
Baranauskiene, Lina [4 ]
Salzano, Anna Maria [5 ]
Scaloni, Andrea [5 ]
Matulis, Daumantas [4 ]
Innocenti, Alessio [6 ]
Scozzafava, Andrea [6 ]
Monti, Simona Maria [7 ]
Di Fiore, Anna [7 ]
De Simone, Giuseppina [7 ]
Lindfors, Mikaela [1 ]
Janis, Janne [8 ]
Valjakka, Jarkko [1 ]
Pastorekova, Silvia [9 ]
Pastorek, Jaromir [9 ]
Kulomaa, Markku S. [1 ]
Nordlund, Henri R. [1 ]
Supuran, Claudiu T. [6 ]
Parkkila, Seppo [1 ,2 ,3 ]
机构
[1] Univ Tampere, Inst Med Technol, FI-33014 Tampere, Finland
[2] Univ Tampere, Sch Med, FI-33014 Tampere, Finland
[3] Tampere Univ Hosp, FI-33014 Tampere, Finland
[4] Inst Biotechnol, Lab Biothermodynam & Drug Design, LT-02241 Vilnius, Lithuania
[5] CNR, ISPAAM, Proteom & Mass Spectrometry Lab, I-80147 Naples, Italy
[6] Univ Florence, Bioinorgan Chem Lab, I-50019 Florence, Italy
[7] CNR, Inst Biostruct & Bioimages, I-80134 Naples, Italy
[8] Univ Joensuu, Dept Chem, FI-80101 Joensuu, Finland
[9] Slovak Acad Sci, Ctr Mol Med, Inst Virol, Bratislava 84505, Slovakia
关键词
D O I
10.1074/jbc.M800938200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 mannosetype 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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收藏
页码:27799 / 27809
页数:11
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