Antigen-specific proteolysis by hybrid antibodies containing promiscuous proteolytic light chains paired with an antigen-binding heavy chain.
Antigen-specific proteolysis by hybrid antibodies containing promiscuous proteolytic light chains paired with an antigen-binding heavy chain.
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通过含有与抗原结合重链配对的混杂蛋白水解轻链的混合抗体进行抗原特异性蛋白水解。
DOI：
10.1074/jbc.m109.011858
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Paul,Sudhir
中科院分区：
文献类型：
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作者：
Sapparapu,Gopal;Planque,StephanieA;Nishiyama,Yasuhiro;Foung,StevenK;Paul,Sudhir
The antigen recognition site of antibodies consists of the heavy and light chain variable domains (VLand VHdomains). VLdomains catalyze peptide bond hydrolysis independent of VHdomains (Mei, S., Mody, B., Eklund, S. H., and Paul, S. (1991)J. Biol. Chem.266, 15571–15574). VHdomains bind antigens noncovalently independent of VLdomains (Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989)Nature341, 544–546). We describe specific hydrolysis of fusion proteins of the hepatitis C virus E2 protein with glutathioneS-transferase (GST-E2) or FLAG peptide (FLAG-E2) by antibodies containing the VHdomain of an anti-E2 IgG paired with promiscuously catalytic VLdomains. 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 ∼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.
