Chemical modification of recombinant HIV-1 capsid protein p24 leads to the release of a hidden epitope prior to changes of the overall folding of the protein.
It was found that the affinity of a monoclonal antibody directed against a recombinantly expressed HIV-1 capsid protein p24 (rp24) strongly increased after chemical modification of the Iysine residues of rp24 with different amounts of maleic anhydride. The extent and the sites of modification were analyzed by MALDI-TOF mass spectrometry. Unmodified rp24 and the differently modified rp24 samples were tested for binding the murine monoclonal antibody CB4-1 which recognizes the epitope GATPQDLNTML comprising residues 46-56 of rp24. An increase in the number of modified lysine residues led to enhanced binding affinity of CB4-1. Most pronounced effects were observed after substitution of the first amino groups: an average number of three modified residues per protein molecule increases the binding affinity by a factor of 23, but the substitution of the remaining nine residues increases the binding affinity only by a factor of 11. Fully modified rp24 variant proteins were bound by CB4-1 with Kd values comparable to that of the peptide epitope. Conformation and stability of the unmodified rp24, highly (rp24F, 9 residues; rp24G, 11 residues) modified, and fully modified protein (rp24I, 11 lysine residues and N-terminus) were analyzed by circular dichroism (CD) and fluorescence spectroscopy under different solvent conditions. Little difference in conformation and unfolding behavior was observed between the unmodified and highly modified rp24, which differ drastically in the antibody binding behavior. The fully modified sample, however, displayed a significant decrease in alpha-helical content. Thus, the epitope seems to be hidden (cryptotope) in the unmodified rp24 in a low-affinity binding conformation and becomes displayed at low levels of chemical modification which obviously induce subtle structural changes prior to changes of the overall folding observable by spectroscopic means.
Ehrhard
B
B
Institut für Medizinische Immunologie and Institut für Biochemie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Germany.
Misselwitz
R
R
Welfle
K
K
Hausdorf
G
G
Glaser
R W
RW
Schneider-Mergener
J
J
Welfle
H
H
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D002942:Circular Dichroism; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D019791:Guanidine; D006146:Guanidines; D015483:HIV Antibodies / Q000276:immunology*; D016655:HIV Core Protein p24 / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry; D008299:Maleic Anhydrides / Q000494:pharmacology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D000911:Antibodies, Monoclonal; D000939:Epitopes; D006146:Guanidines; D015483:HIV Antibodies; D016655:HIV Core Protein p24; D008299:Maleic Anhydrides; D011994:Recombinant Proteins; C027132:1-anilino-8-naphthalenesulfonate; D019791:Guanidine; D008239:Lysine