Renata Štouračová1, Jan Závada1, Zuzana Závadová1, Silvia Pastoreková2, Jiří Brynda1, Milan Fábry1, Vlastimil Král1, Magda Hořejši1, and Juraj Sedláček1


1Institute of Molecular Genetics, Academy of Sciences, 166 37 Prague, Czech Republic

2Institute of Virology, Slovak Academy of Sciences, 842 46 Bratislava, Slovak Republic


Carbonic anhydrase IX (CA IX) is a cell surface protein, strongly associated with certain types of human carcinomas. The predicted protein of cloned CA IX cDNA consists of the signal peptide, proteoglycan-related sequence, carbonic anhydrase domain, trasmembrane segment and a short intracellular tail (1, 2). Until now, molecular basis of involvement of CA IX in carcinogenesis has remained unclear. CA IX is a cell adhesion molecule, its carbonic anhydrase (CA) is enzymaticaly active. Structural study of a CA IX-binding monoclonal antibody (mAb) M75, complexed with its epitope peptide may contribute toward elucidation of the role of CA IX. To achieve this goal, two parallel approaches were chosen: analysis of Fab fragment, or of a smaller scFv fragment, both containing the complete antigen binding site present in mAb M75.

Monoclonal antibody M75 was obtained (3) and proved to react excellently with native and denaturated CA IX. Using synthetic oligopeptides,  the epitope of mAb M75 was localized  in the proteoglycan domain of CA IX, in the region of a tandem repeat and identified  as amino acids PGEEDLP (4). The Fab fragment was obtained by papain cleavage. We obtained crystals of free Fab M75 and Fab M75 complexed with two different epitope peptides. The data set for Fab M75 was collected and the structure solving is underway.

Another approach is to prepare  sc Fv fragment of this antibody (described in the contribution of Vlastimil KRAL, Milan Fabry, Magda Horejsi, Jan Zavada, Juraj Sedlacek: Molecular cloning, E. coli expression and purification of scFv antibody fragments
 of diagnostic/therapeutic interest.



1. Pastorek J, Pastoreková S, Callebaut I, Mornon JP, Zelník V, Opavský R, Zat'ovicová M, Liao S, Portetelle D, Stanbridge EJ, et al. Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix  DNA binding segment. (1994) Oncogene 10:2877-88.

2. Opavský R, Pastoreková S, Zelník V, Gibadulinová A, Stanbridge EJ, Závada J, Kettmann R, Pastorek Human MN/CA9 gene, a novel member of the carbonic anhydrase family: structure and exon to protein domain relationships. (1996) Genomics. 33:480-7.

3. Pastoreková S, Závadová Z, Kostál M, Babusiková O, Závada J. A novel quasi-viral agent, MaTu, is a two-component system. (1992) Virology. 187:620-6.

4. Závada J, Závadová Z, Pastorek J, Biesová Z, Ježek J, Velek J.   Human tumour-associated cell adhesion protein MN/CA IX: identification of M75 epitope and of the region mediating cell adhesion. (2000) Br J Cancer. 82:1808-13