title: Anti-idiotypic antibody mimics proteolytic function of parent antigen.
abstract: 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.
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, 16/10, Miklukho-Maklaya str, Moscow, 117997, Russia.
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Ponomarenko
authors.forename: Dominique
authors.initials: D
authors.lastname: Pillet
authors.forename: Marjorie
authors.initials: M
authors.lastname: Paon
authors.forename: Ivan I
authors.initials: II
authors.lastname: Vorobiev
authors.forename: Ivan V
authors.initials: IV
authors.lastname: Smirnov
authors.forename: Hervé
authors.initials: H
authors.lastname: Adenier
authors.forename: Bérangère
authors.initials: B
authors.lastname: Avalle
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Kolesnikov
authors.forename: Arina V
authors.initials: AV
authors.lastname: Kozyr
authors.forename: Daniel
authors.initials: D
authors.lastname: Thomas
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Gabibov
authors.forename: Alain
authors.initials: A
authors.lastname: Friboulet
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D000941:Antigens / Q000276:immunology / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D006868:Hydrolysis; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013381:Subtilisins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000941:Antigens; D013381:Subtilisins