Mutational and inhibitory analysis of a catalytic antibody. Implication for drug discovery.
Data on catalytic antibodies (abzymes) having critical roles in pathologies, for example in some auto-immune diseases accumulate at overwhelming pace. Nevertheless, the misunderstanding of how antibodies can mimic a catalytic activity may hamper the development of therapeutic tools. We thus investigated the structure function roles of residues of a catalytic antibody endowed with a beta-lactamase activity. The catalytic antibody 9G4H9 was generated using the internal image properties of anti-idiotypic antibodies. The single-chain fragment was cloned and produced in Escherichia coli. Based on the structure function knowledge of beta-lactamases pattern and on the tridimensional model of the scFv, five residues were selected for mutagenic analysis to learn about the contribution of putative residues in catalysis. Light chain mutants R24A, S26A, S28A, and E98A lost catalytic activity significantly. Mutant K27A retained catalytic activity but the estimated K(M) was affected. Kinetic outcomes support the argument that S26 and S28 function as nucleophile and E98 as general acid/base catalyst. We have selected a peptide Pep90 able to inhibit 9G4H9 catalytic activity. We also demonstrate the tryptophan and proline residues of Pep90 (YHFLWGP) are responsible for binding and inhibitory properties of Pep90 on 9G4H9. A three-dimensional model docked with Pep90 is therefore built in which critical residues of Pep90 are buried at the interface of CDR-L1 and CDR-L3 loops whereas other are exposed to the solvent.
Phichith
Denis
D
Université de Technologie de Compiègne-CNRS, UMR 6022 Génie Enzymatique et Cellulaire, BP 20529, 60205 Compiègne Cedex, France.
Bun
Sylvie
S
Padiolleau-Lefèvre
Séverine
S
Banh
Soun
S
Thomas
Daniel
D
Friboulet
Alain
A
Avalle
Bérangère
B
D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D055162:Biocatalysis; D004252:DNA Mutational Analysis; D055808:Drug Discovery; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000276:immunology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D020349:Surface Plasmon Resonance; D044366:Transition Temperature
D017151:Antibodies, Catalytic; D007147:Immunoglobulin Light Chains; D050505:Mutant Proteins; D010455:Peptides