Abstract
The antigen recognition site of antibodies consists of the heavy and light chain variable domains (V(L) and V(H) domains). V(L) domains catalyze peptide bond hydrolysis independent of V(H) domains (Mei, S., Mody, B., Eklund, S. H., and Paul, S. (1991) J. Biol. Chem. 266, 15571-15574). V(H) domains bind antigens noncovalently independent of V(L) domains (Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989) Nature 341, 544-546). We describe specific hydrolysis of fusion proteins of the hepatitis C virus E2 protein with glutathione S-transferase (GST-E2) or FLAG peptide (FLAG-E2) by antibodies containing the V(H) domain of an anti-E2 IgG paired with promiscuously catalytic V(L) domains. The hybrid IgG hydrolyzed the E2 fusion proteins more rapidly than the unpaired light chain. An active site-directed inhibitor of serine proteases inhibited the proteolytic activity of the hybrid IgG, indicating a serine protease mechanism. The hybrid IgG displayed noncovalent E2 binding in enzyme-linked immunosorbent assay tests. Immunoblotting studies suggested hydrolysis of FLAG-E2 at a bond within E2 located approximately 11 kDa from the N terminus. GST-E2 was hydrolyzed by the hybrid IgG at bonds in the GST tag. The differing cleavage pattern of FLAG-E2 and GST-E2 can be explained by the split-site model of catalysis, in which conformational differences in the E2 fusion protein substrates position alternate peptide bonds in register with the antibody catalytic subsite despite a common noncovalent binding mechanism. These studies provide proof-of-principle that the catalytic activity of a light chain can be rendered antigen-specific by pairing with a noncovalently binding heavy chain subunit.
Highlights
Grant R01 HL079381. □S The on-line version of this article contains supplemental Table S1. 1 To whom correspondence should be addressed: 6431 Fannin, MSB 2.230A, VH domains)
Disruption of the serine protease-like catalytic triad in an Ab light chain by site-directed mutagenesis was without effect on its ability to bind the polypeptide antigen by noncovalent means [13], and a discrete peptide epitope remote from the bond hydrolyzed by a proteolytic Ab preparation has been identified [25]
Light chains HK14 and HK13 hydrolyze vasoactive intestinal polypeptide [21] and amyloid- peptide [38], substrates unrelated in sequence to the E2 fusion proteins. This catalyst engineering strategy is predicated on the hypothesis that the specificity of hybrid IgGs can be derived from the noncovalent antigen binding activity contributed by the VH domain
Summary
Recombinant Abs—VH and VL cDNA of the anti-E2-IgG1 CBH-7 were prepared by reverse transcriptase-PCR using as template total RNA from hybridoma cells [29]. Recombinant E2 Fusion Proteins—A stable cell line coexpressing E1 and FLAG-E2 (residues 406 –746 of E2 HCV H77 polyprotein with an N-terminal FLAG peptide tag) was prepared by transfection with the plasmid pCDNA3/E1FLAGE2. This plasmid was constructed from vectors kindly provided by Dr L. E2, corresponding to full-length E2 (residues 384 –746 of HCV polyprotein) with an N-terminal glutathione S-transferase tag, produced in a baculovirus insect cell expression system was purchased from Immunodiagnostics Inc. Hydrolysis Assays—The enriched FLAG-E2 preparation (70 ng of FLAG-E2/ml; 1 nM) was incubated with electrophoretically homogenous IgG in 20 l of 100 mM glycine-HCl, 50 mM Tris, 0.1 mM CHAPS at 37 °C. Kinetic parameters were obtained by fitting the rate data obtained at increasing concentrations of peptide-AMC substrates to the MichaelisMenten-Henri equation: V ϭ kcat1⁄7[Ab] 1⁄7 [S]/(Km ϩ [S])
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