Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, CZ - 166 37 Prague 6, Czech Republic
CA IX (carbonic anhydrase IX) is a cell surface protein, associated with several types of human carcinomas. It is almost 100 % associated with cervical carcinomas, carcinomas of oesophagus and with renal clear cell carcinomas, while it is absent in normal tissues of corresponding organs. This suggests that CA IX gene represents a novel oncogene . CA IX exerts capacity of binding to cell surface receptors . It is a member of the alpha class of carbonic anhydrases - zinc metalloenzymes that catalyze the reversible conversion between CO2 and bicarbonate.
Monoclonal antibody M75  kindly provided by Dr. Závada reacts excellently with CA IX. Using synthetic oligopeptides, the epitope of mAb M75 was localized in the proteoglycan‑like domain of CA IX and identified as six times repeating amino acid sequence PGEEDLP . Structural studies of the M75 Fab fragment in complex with its epitope peptides should be helpful in rational drug design of compounds suitable for use in human oncology.
M75 mAb was isolated from TC medium of hybridoma cells M75 using Protein A Sepharose. Fab fragment of M75 antibody was obtained by controlled papain digestion of IgG M75 and further purification on DEAE Sephacel. Purity of the product was assessed using gel filtration, SDS PAGE and isoelectric focusing.
Using vapor diffusion hanging drop technique we obtained crystals of free Fab M75 and crystals of Fab M75 in complex with epitope peptide PGEEDLPGEEDL. Datasets for both crystals were collected at 150 K using in-house diffractometer (Nonius FR 591 generator, 345mm MarResearch Image Plate detector). Crystals belong to triclinic spacegroup P1. The Fab M75 structure was solved by molecular replacement, using Fab Bv04-01 (PDB code 1NBV) as search model. Probably due to high mobility of the variable loops the structure of free Fab M75 could not be refined. However structure of Fab M75 in complex with the epitope peptide was successfully refined at 2.0 Å resolution (the final R- and free R factors are 0.194 and 0.260 respectively).
Only the first eight N-terminal residues of the peptide (PGEEDLPG) are visible in electron density maps. The C–terminal residues extend out from the antigen-binding site and are most probably flexible. The interactions between Fab M75 and epitope peptide include extensive van der Waals contacts and several hydrogen bonds. All six CDRs of Fab M75 are involved in contacts with antigen. With the exception of the third peptide residue (E), which points towards the exterior of the antigen binding groove the remaining residues from the peptide establish several contacts. Due to shift of the variable H3 loop in direction to the light chain CDRs the structure displays an interesting and unusual mode of epitope binding which involves van der Waals interactions between epitope peptide and the N terminal end of the Fab heavy chain.
Acknowledgment. The research was supported by the Grant Agency of the Czech Republic (project 203/02/0405).
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