The human chorionic gonadotropin-beta arginine(68) to glutamic acid substitution fixes the conformation of the C-terminal peptide

TY - JOUR

T1 - The human chorionic gonadotropin-beta arginine(68) to glutamic acid substitution fixes the conformation of the C-terminal peptide

AU - Charrel-Dennis, M

AU - Terrazzini, N

AU - McBride, J D

AU - Kaye, P

AU - Martensen, P M

AU - Justesen, J

AU - Berger, P

AU - Lapthorn, A

AU - Kelly, C

AU - Roitt, I M

AU - Delves, P J

AU - Lund, T

PY - 2005/7

Y1 - 2005/7

N2 - Wild-type human chorionic gonadotropin (hCG) has been used as a contraceptive vaccine. However, extensive sequence homology with LH elicits production of cross-reactive antibodies. Substitution of arginine(68) of the beta-subunit (hCG beta) with glutamic acid (R68E) profoundly reduces the crossreactivity while refocusing the immune response to the hCG beta-specific C-terminal peptide (CTP). To investigate the molecular basis for this change in epitope usage, we immunized mice with a plasmid encoding a truncated hCG beta-R68E chain lacking the CTP. The animals produced LH-cross-reactive antibodies, suggesting that the refocused immunogenicity of R68E is a consequence of epitope masking by a novel disposition of the CTP in the mutant rather than a structural change in the cross-reactive epitope region. This explanation was strongly supported by surface plasmon resonance analysis using a panel of anti-hCG beta-specific and anti-hCG beta/LH cross-reactive monoclonal antibodies (mAbs). Whereas the binding of the LH cross-reactive mAbs to hCG beta-R68E was eliminated, mAbs reacting with hCG beta-specific epitopes bound to hCG beta and hCG beta-R68E with identical affinities. In a separate series of experiments, we observed that LH cross-reactive epitopes were silent after immunization with a plasmid encoding a membrane form of hCG beta-R68E, as previously observed with the soluble mutant protein itself. In contrast, the plasmid encoding the soluble secreted form of hCG beta-R68E evoked LH cross-reactive antibodies, albeit of relatively low titer, suggesting that the handling and processing of the proteins produced by the two constructs differed

AB - Wild-type human chorionic gonadotropin (hCG) has been used as a contraceptive vaccine. However, extensive sequence homology with LH elicits production of cross-reactive antibodies. Substitution of arginine(68) of the beta-subunit (hCG beta) with glutamic acid (R68E) profoundly reduces the crossreactivity while refocusing the immune response to the hCG beta-specific C-terminal peptide (CTP). To investigate the molecular basis for this change in epitope usage, we immunized mice with a plasmid encoding a truncated hCG beta-R68E chain lacking the CTP. The animals produced LH-cross-reactive antibodies, suggesting that the refocused immunogenicity of R68E is a consequence of epitope masking by a novel disposition of the CTP in the mutant rather than a structural change in the cross-reactive epitope region. This explanation was strongly supported by surface plasmon resonance analysis using a panel of anti-hCG beta-specific and anti-hCG beta/LH cross-reactive monoclonal antibodies (mAbs). Whereas the binding of the LH cross-reactive mAbs to hCG beta-R68E was eliminated, mAbs reacting with hCG beta-specific epitopes bound to hCG beta and hCG beta-R68E with identical affinities. In a separate series of experiments, we observed that LH cross-reactive epitopes were silent after immunization with a plasmid encoding a membrane form of hCG beta-R68E, as previously observed with the soluble mutant protein itself. In contrast, the plasmid encoding the soluble secreted form of hCG beta-R68E evoked LH cross-reactive antibodies, albeit of relatively low titer, suggesting that the handling and processing of the proteins produced by the two constructs differed

U2 - 10.1210/me.2004-0109

DO - 10.1210/me.2004-0109

M3 - Article

VL - 19

SP - 1803

EP - 1811

JO - Molecular Endocrinology

JF - Molecular Endocrinology

IS - 7

ER -