Mass spectrometric characterization of a discontinuous epitope of the HIV envelope protein HIV-gp120 recognized by the human monoclonal antibody 1331A.
The characterization of a discontinuous epitope in the C5 region of the HIV envelope protein HIV-gp120, recognized by 1331A, a human mAb, is reported. Regions involved in affinity binding in the HIV-gp120 molecule were identified by epitope excision/extraction methods followed by matrix assisted laser desorption-time of flight mass spectrometry. In epitope excision, the protein is bound in its native conformation to an immobilized Ab and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the Ab. A series of proteolytic digestions of the 1331A/HIV-gp120 complex allowed the identification of protected amino acids in two noncontinuous regions of the C5 region of HIV-gp120. Interaction of the Ab with amino acids I487 and E507 of HIV-gp120 is essential for efficient binding. This is the first application of this approach for the identification and characterization of a discontinuous epitope. The results are consistent with molecular modeling results, indicating that these amino acids are located on opposite sides of a hydrophobic pocket. This pocket is thought to be of importance for the interaction of HIV-gp120 with the transmembrane protein HIV-gp41.
Hochleitner
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EO
National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Gorny
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MK
Zolla-Pazner
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Tomer
K B
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D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D017951:Antigen Presentation; D001666:Binding Sites, Antibody; D018826:CD13 Antigens / Q000378:metabolism; D003488:Cyanogen Bromide; D015699:HIV Envelope Protein gp120 / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D016056:Immunodominant Epitopes / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D012685:Sepharose; D012697:Serine Endopeptidases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000911:Antibodies, Monoclonal; D015699:HIV Envelope Protein gp120; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D012685:Sepharose; D018826:CD13 Antigens; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D003488:Cyanogen Bromide