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.
Subject(s)
Antibodies, Anti-Idiotypic/metabolism , Antigens/metabolism , Subtilisins/immunology , Amino Acid Sequence , Animals , Antibodies, Anti-Idiotypic/chemistry , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Antigens/immunology , Base Sequence , Binding Sites , Catalysis , Hydrolysis , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Sequence Alignment , Subtilisins/genetics , Subtilisins/metabolismABSTRACT
An important challenge in the field of catalytic antibodies is the generation of antibodies with designed sequence-specific protease activities. Such catalysts would not only be recruited for diverse applications in basic biological science, but could also offer new approaches in biotechnology and medicine. We have previously used the "internal image" property of the idiotypic network to elicit antibodies with efficient esterase and amidase activities. In the present report, we present preliminary results for the production of anti-idiotypic antibodies mimicking subtilisin. A monoclonal inhibitory antibody of subtilisin was characterized and used to elicit anti-idiotypic antibodies. Some of these antibodies exhibit not only an amidase activity against synthetic substrates, but are also able to cleave a protein, bovine serum albumin (BSA).
