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.
Highlights
Carbonic anhydrase IX (CA IX) is an exceptional member of the Carbonic anhydrases (CAs) protein family; in addition to its classical role in pH regulation, it has been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes
The CA form included only the CA domain, encompassing residues 101–354 in the mature sequence (Fig. 1). Both recombinant proteins contained the CA IX signal sequence, which targets the protein to the secretory pathway within the cells, and the proteins were secreted into the medium due to the designated truncation at the C terminus
A series of novel biochemical features, which differentiate human CA IX from the other members of the CA family, were evident following characterization of two recombinant protein forms that were produced by the baculovirus-insect cell expression system
Summary
Construction of Recombinant Baculoviruses—Two cDNA constructs were designed to produce the recombinant CA IX forms, both of which lack the small transmembrane and intracytoplasmic domains. Because the PG domain was unstable in Tris buffer (data not shown), the purified PG ϩ CA form was changed to a buffer of 50 mM NaH2PO4, 100 mM NaCl, pH 8.0 To remove the His tag, recombinant proteins were treated with 30 l of resin-coupled thrombin (Thrombin CleanCleave KIT, Sigma) per 1 mg of protein with gentle shaking at 25 °C for 3 h, according to the manufacturer’s instructions. Purified recombinant CA IX proteins were analyzed by 10% SDS-PAGE under reducing conditions [21] followed either by treatment with the Colloidal Blue Staining Kit (Invitrogen) or Western blotting, which was performed as described previously [22].
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