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 identi fied by epitope excision/extraction methods followed by matrix assisted las er desorption-time of flight mass spectrometry. In epitope excision, the pr otein is bound in its native conformation to an immobilized Ab and then dig ested with proteolytic enzymes, In epitope extraction, the protein is first digested and subsequently allowed to react with the Ab, A series of proteo lytic digestions of the 1331A/HIV-gp120 complex allowed the identification of protected amino acids in two noncontinuous regions of the C5 region of H IV-gpl20. 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 epi tope, The results are consistent with molecular modeling results, indicatin g that these amino acids are located on opposite sides of a hydrophobic poc ket, This pocket is thought to be of importance for the interaction of HIV- gpl20 with the transmembrane protein HIV-gp41.