The human chorionic gonadotropin-beta arginine68 to glutamic acid substitution fixes the conformation of the C-terminal peptide.
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 cross-reactivity 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.
Charrel-Dennis
Marie
M
Department of Immunology and Molecular Pathology, University College London, 46 Cleveland Street, London W1T 4JF, United Kingdom.
Terrazzini
Nadia
N
McBride
Jeffrey D
JD
Kaye
Paul
P
Martensen
Pia M
PM
Justesen
Just
J
Berger
Peter
P
Lapthorn
Adrian
A
Kelly
Charles
C
Roitt
Ivan M
IM
Delves
Peter J
PJ
Lund
Torben
T
D019943:Amino Acid Substitution / Q000235:genetics / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000097:blood; D000911:Antibodies, Monoclonal / Q000276:immunology; D001120:Arginine / Q000235:genetics; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D003429:Cross Reactions / Q000276:immunology; D005260:Female; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D007114:Immunization; D007986:Luteinizing Hormone / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D010455:Peptides / Q000235:genetics / Q000276:immunology; D011487:Protein Conformation
D000906:Antibodies; D000911:Antibodies, Monoclonal; D018997:Chorionic Gonadotropin, beta Subunit, Human; D010455:Peptides; D018698:Glutamic Acid; D007986:Luteinizing Hormone; D001120:Arginine