Antigen-specific proteolysis by hybrid antibodies containing promiscuous proteolytic light chains paired with an antigen-binding heavy chain.
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.
Sapparapu
Gopal
G
Chemical Immunology Research Center, Department of Pathology and Laboratory Medicine, University of Texas Houston Medical School, Houston, Texas 77030, USA.
Planque
Stephanie A
SA
Nishiyama
Yasuhiro
Y
Foung
Steven K
SK
Paul
Sudhir
S
D000818:Animals; D017151:Antibodies, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D002384:Catalysis; D018937:Hepatitis C Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D018936:Hepatitis C Antigens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
D017151:Antibodies, Catalytic; D018937:Hepatitis C Antibodies; D018936:Hepatitis C Antigens; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus