Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1784, Issue 3, Pages 530-542Publisher
ELSEVIER
DOI: 10.1016/j.bbapap.2007.12.015
Keywords
hepatitis C E2 glycoprotein; human neutralizing and non-neutralizing antibodies; limited proteolysis; differential chemical modification; mass spectrometry; alanine scanning mutagenesis
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Funding
- Intramural NIH HHS [Z01 ES050127-15] Funding Source: Medline
- NHLBI NIH HHS [R01 HL079381, HL079381, R01 HL079381-04] Funding Source: Medline
- NIAID NIH HHS [R01 AI047355-05, AI047355, R01 AI047355] Funding Source: Medline
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Human monoclonal antibodies derived from B cells of HCV-infected individuals provide information on the immune response to native HCV envelope proteins as they are recognized during infection. Monoclonal antibodies have been useful in the determination of the function and structure of specific immunogenic domains of proteins and should also be useful for the structure/function characterization of HCV E1 and E2 envelope glycoproteins. The HCV E2 envelope glycoprotein has at least three immunodistinctive conformation domains, designated A, B, and C. Conformational epitopes within domain B and C are neutralizing antibody targets on HCV pseudoparticles as well as from infectious cell culture virus. In this study, a combination of differential surface modification and mass spectrometric limited proteolysis followed by alanine mutagenesis was used to provide insight into potential conformational changes within the E2 protein upon antibody binding. The arginine guanidine groups in the E2 protein were modified with CHD in both the affinity bound and free states followed by mass spectrometric analysis, and the regions showing protection upon antibody binding were identified. This protection can arise by direct contact between the residues and the monoclonal antibody, or by antibody-induced conformational changes. Based on the mass spectrometric data, site-directed mutagenesis experiments were performed which clearly identified additional amino acid residues on E2 distant from the site of antibody interaction, whose change to alanine inhibited antibody recognition by inducing conformational changes within the E2 protein. (C) 2008 Elsevier B.V. All rights reserved.
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