Applications of biotinylated V3 loop peptides of human immunodeficiency virus type 1 to flow cytometric analyses and affinity chromatographic techniques.
A principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) lies within the V3 loop of gp120, the external major envelope glycoprotein. V3 loop peptides derived from two HIV-1 strains, HTLV-III BH-10 (V3-BH10) and LAVELI (V3-ELI), were synthesized and biotinylated. The binding of both biotinylated V3-BH10 and V3-ELI to the surfaces of MOLT-4 clone 8 cells was demonstrated by flow cytometric analyses. Both the peptides (more than 2 microM) bound to the cells (2 x 10(5) in a dose-dependent manner. The binding of biotinylated V3-BH10 was specifically inhibited by a neutralizing monoclonal antibody (0.5 beta). The binding of both of the biotinylated V3 loop peptides was enhanced by the addition of unlabeled V3-BH10. In addition, the peptides were employed as ligands on affinity columns. A major V3 loop binding protein (V3BP) was purified from the membrane soluble fraction of MOLT-4 cells by successive application to two different V3 loop columns. V3BP consisted of two major polypeptides (32 and 33 kDa). The SDS-PAGE profile of V3BP did not change under non-reducing conditions, but only a single band was observed after analysis on native PAGE. The major peak of the eluate as determined by size exclusion chromatography was broad and the estimated relative molecular mass was much larger than 33 kDa, suggesting that V3BP comprises several subunits. Taken together, we confirmed that the V3 loop peptides are useful in the characterization of V3BP(s) of which they are conformational ligands.
Murakami
T
T
Laboratory for AIDS Immunology, Kyoto University, Japan.
Matsushita
S
S
Maeda
Y
Y
Takatsuki
K
K
Uchiyama
T
T
Hattori
T
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D015699:HIV Envelope Protein gp120 / Q000737:chemistry*; D015497:HIV-1 / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry*
D015699:HIV Envelope Protein gp120; D010446:Peptide Fragments; D001710:Biotin