Characterization of a discontinuous epitope of the HIV envelope protein gp120 recognized by a human monoclonal antibody using chemical modification and mass spectrometric analysis.

A subset of the neutralizing anti-HIV antibodies recognize epitopes on the envelope protein gp120 of the human immunodeficiency virus. These epitopes are exposed during conformational changes when gp120 binds to its primary receptor CD4. Based on chemical modification of lysine and arginine residues followed by mass spectrometric analysis, we determined the epitope on gp120 recognized by the human monoclonal antibody 559/64-D, which was previously found to be specific for the CD4 binding domain.

The HIV-neutralizing monoclonal antibody 4E10 recognizes N-terminal sequences on the native antigen.

Characterization of the epitope recognized by the broadly neutralizing anti-HIV Ab 4E10 has, heretofore, focused on a linear sequence from the gp41 pretransmembrane region (PTMR). Attempts to generate neutralizing Abs based on this linear epitope sequence have been unsuccessful. We have characterized the antigenic determinants on recombinant glycosylated full-length Ags, and nonglycosylated and truncated Ags recognized by 4E10 using epitope extraction and excision assays in conjunction with MALDI mass spectrometry.

Determination of protein-derived epitopes by mass spectrometry.

Mass spectrometry has evolved as a technique suitable for the characterization of peptides and proteins beyond their linear sequence. The advantages of mass spectrometric sample analysis are high sensitivity, high mass accuracy, rapid analysis time and low sample consumption. In epitope mapping, the molecular structure of an antigen (the epitope or antigenic determinant) that interacts with the paratope (recognition surface) of the antibody is identified.

Determination of epitopes by mass spectrometry.

As a response to an infection, the immune system produces antibodies. The determination of the antigenic structure recognized by the antibody through epitope mapping provides information about the interaction between antigen and antibody for the diagnosis of a disease on a molecular level, for characterizing the pathogenesis of the infectious material, and for the development of interfering drugs or preventative vaccines. Here we present the determination of the fine structure of the linear epitope located on the gp41 protein of the human immunodeficiency virus recognized by the monoclonal antibody 2F5.

Characterization of the tertiary structure of soluble CD4 bound to glycosylated full-length HIVgp120 by chemical modification of arginine residues and mass spectrometric analysis.

The initial step of infection of blood cells with the human immunodeficiency virus, HIV, is the formation of a complex of the viral envelope protein gp120 and its human receptor CD4. We have examined structural features of recombinant soluble CD4 (sCD4) by chemical modification of arginine residues with hydroxyphenylglyoxal and subsequent analysis by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. As R58, R59, R131, R134, R219, R240, R293, and R329 could be derivatized free in solution, these arginine residues were exposed on the surface of the protein.