Anti-Idiotypic Antibody Mimics Proteolytic Function of Parent Antigen

• Published in: Biochemistry (Easton) Vol. 46; no. 50; pp. 14598 - 14609
• Main Authors: Ponomarenko, Natalia A, Pillet, Dominique, Paon, Marjorie, Vorobiev, Ivan I, Smirnov, Ivan V, Adenier, Herve, Avalle, Berangere, Kolesnikov, Alexer V, Kozyr, Arina V, Thomas, Daniel, Gabibov, Alexer G, Friboulet, Alain
• Format: Journal Article
• Language: English
• Published: 01-12-2007
• Subjects: Escherichia coli
• ISSN: 0006-2960
• DOI: 10.1021/bi7013954PII:S0006-2960(70)01395-8

Functional imaging of subtilisin Carlsberg active center by the idiotypic network yielded a catalytic anti-idiotypic antibody with endopeptidase, amidase, and esterase activities. A monoclonal antibody inhibitory to subtilisin (Ab1 5-H4) was employed as the template for guiding the idiotypic network to produce the catalytic anti-idiotypic Ab2 6B8-E12. Proteolytic activity of 6B8-E12 was demonstrated by zymography using self-quenched fluorescein-BSA conjugate and in a coupled assay detecting Ab2-dependent RNase A inactivation. Cleavage of peptide substrates by 6B8-E12 revealed distinct patterns of hydrolysis with high preference for aromatic residues before or after the scissile bond. Catalytic activity of Ab2 was inhibited by phenylmethylsulfonyl fluoride, a mechanism-based inhibitor of serine hydrolases. 5-H4 and 6B8-E12 were cloned, produced in Escherichia coli as single-chain variable fragments (scFvs), and purified. Kinetic parameters for amidolytic and esterolytic activities were similar in Ab2 and its scFv derivative. Although the antigen-specific portion of 6B8-E12 possesses no primary structure similarity to subtilisin, it mimics proteolytic and amidolytic functions of the parental antigen, albeit with 4 orders of magnitude slower acceleration rates. The lack of detectable endopeptidase activity of 6B8-E12 scFv raises interesting issues concerning general evolution of catalytic activity. The in silico 3D models of Ab1 and Ab2 revealed strong structural similarity to known anti-protease antibodies and to abzymes, respectively. These results indicate that the idiotypic network is capable, to a significant extent, of reproducing catalytic apparatus of serine proteases and further validate the use of imaging of enzyme active centers by the immune system for induction of abzymes accelerating energy- demanding amide bond hydrolysis.