Structural Studies of Carbonic Anhydrase IX

 

Pavel Mader, Renata Štouračová, Jiří Brynda, Milan Fábry, Magdalena Hořejší,
Vlastimil Kr
ál, Juraj Sedláček

 

Department of Recombinant Expression and Structural Biology, Institute of Molecular Genetics, Flemingovo nám. 2, CZ - 166 37 Praha 6, mader@img.cas.cz

 

Carbonic anhydrase IX (CA IX) belongs to a family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide: CO2 + H2O → H+ + HCO3-. These enzymes play a role in pH regulation, CO2 and HCO3- transport, bone resorption, production of biological fluids, ureagenesis, gluconeogenesis, and lipogenesis [[1]]. CA IX is a unique member of the CA protein family. In contrast to the other isozymes, it has been implicated to play a role in regulation of cell proliferation, adhesion, and malignant cell invasion [[2], [3]]. This integral membrane protein was shown to function as a cell adhesion molecule with the binding site localized in the extracellular proteoglycan-like (PG) domain; the same site is recognized by the monoclonal antibody M75 thus abrogating cell adhesion in in vitro test [2]. Interestingly, CA IX is overexpressed in human epithelial tumours derived from tissues that normally do not express this isozyme, including carcinomas of cervix, lung, kidney, and breast [4]. In contrast, tumours originating from CA IX-positive tissues, such as stomach, tend to have lowered expression of CA IX [[4]]. CA IX is also overexpressed in von Hippel-Lindau (VHL)-defective tumors and under hypoxic conditions [[5]]. There are several reasons to consider CA IX as a suitable target molecule for cancer therapy: (i) it is expressed ectopically in various commonly occurring carcinomas, which are rather resistant to conventional therapy; (ii) the antigen is exposed on the cell surface; (iii) normal expression of CA IX is restricted to the luminal epithelia of the alimentary tract, with limited accessibility to immune cells, antibodies and many drugs [[6]].

The aim of our research is to help elucidate the molecular basis of CA IX involvement in cell proliferation by solving the structure of recombinant forms of extracellular domain of CA IX. Further, we plan to solve structure of CA IX complexes with specific sulfonamide inhibitors, and recombinant scFv fragment derived from monoclonal antibody M75, and finally to identify the putative CA IX binding partner.

To obtain recombinant extracellular CA IX domain, various expression systems were tested, however only Drosophila Schneider 2 Cells yielded sufficient amount (60 mg.l-1) of native protein in serum free medium. The protein was purified in 3 chromatographic steps: affinity chromatography on p-aminobenzene sulfonamide agarose, anion exchange chromatography, and gel filtration. The first crystals of CA IX have been obtained using vapour diffusion hanging drop method [Figure 1].

 

Structure of M75 Fab fragment in complex with its epitope peptide [Figure 2] derived from PG domain of CA IX, which will also be presented, should be helpful in rational drug design of compounds suitable for use in human oncology.

Text Box: Figure 2:
Fab M75 in complex with the epitope peptide PGEEDLPGEEDL
 



[[1]] W.S. Sly, P.Y.Hu, Annu. Rev. Biochem., 64 (1995) 375–401

[[2]] J. Závada, Z. Závadová, J. Pastorek, Z. Biesova, J. Ježek, J. Velek, Br. J. Cancer 82 (2000) 1808-13

[[3]] M. Hilvo, M. Rafajová, S. Pastoreková, J. Pastorek, S. Parkkila,  J. Histochem. Cytochem. 52(10) (2004) 1313-22

[[4]] M. Leppilampi, J. Saarnio, T. J. Karttunen, J. Kivelä, S. Pastoreková, J. Pastorek, A. Waheed, et al, World. J. Gastroenterol. 9 (2003) 1398–1403

[[5]] C. C. Wykoff, N. J. Beasley, P. H. Watson, K. J. Turner, J. Pastorek, A. Sibtain, G. D. Wilson, et al, Cancer Res. 60 (2000) 7075–7083

[[6]] S. Pastoreková, J. Závada, Cancer Therapy 2 (2004) 245-262