Structural elucidation of critical residues involved in binding of human monoclonal antibodies to hepatitis C virus E2 envelope glycoprotein

Abstract

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 acids 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.

Identification of New Functional Regions in Hepatitis C Virus Envelope Glycoprotein E2

“…It is worth noting that the chimeric E2 protein containing DI domain from genotype 1a was still less efficiently pulled down by CD81, suggesting that in addition to the major CD81 determinants identified in DI (13,56), other residues within DIII might also modulate CD81 binding, as suggested previously (34,62). However, we cannot exclude the possibility that the involvement of domain DIII is indirect since mutations in this domain, which affect CD81 binding, can affect DIII folding (31).…”

Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Proteins Incorporated into Infectious Virions

“…It has been suggested that additional residues in the DIII domain can also affect CD81 binding (40). However, mutation of these residues might affect E2 folding, suggesting an indirect role for these residues (27). Glycans affecting CD81 binding are highlighted by light green circles.…”

“…Alternatively, we cannot exclude that the E2N11 mutation affects the DI-DIII interaction, which indirectly leads to better accessibility of the CD81 binding region to neutralizing antibodies. Interestingly, mutation of residues in DIII can affect CD81 binding (40); however, these mutations might affect E2 folding, suggesting an indirect role for these residues (27). Besides modulating accessibility to the CD81 binding site, the glycans associated with HCV envelope proteins reduce the accessibility of other regions of the protein moiety.…”

Functional mapping of the anti-idiotypic antibody anti-TS1 scFv using site-directed mutagenesis and kinetic analysis

“…A detailed description of the interactions between an Id mAb paratope, its antigen and an anti-Id mAb may enable more effective engineering since the effect of mutations intended to improve the properties and attributes of mAbs may be more reliably predicted. [10][11][12][13][14] Many research groups have analyzed the structural basis of antibody-antigen recognition. 11,[15][16][17] It has been demonstrated that amino acid residues involved in establishing the complementary determining region (CDR) conformations are less frequently involved in antigenic interactions compared to residues in other positions in the CDR.…”

“…[10][11][12][13][14] Many research groups have analyzed the structural basis of antibody-antigen recognition. 11,[15][16][17] It has been demonstrated that amino acid residues involved in establishing the complementary determining region (CDR) conformations are less frequently involved in antigenic interactions compared to residues in other positions in the CDR. Correlations between antigen type and the and, if the desired result of a mutation is to change the association rate, residues affecting the electrostatic interactions outside the paratope should most likely be altered.…”