ABSTRACT
A cyclic closed-chain dodecapeptide (cDDR5) mimicking the conformation-specific domain of CCR5 was prepared in which Gly-Asp, as a dipeptide forming a spacer arm, links the amino and carboxyl termini of the decapeptidyl linear chain (Arg168 to Thr177) derived from the undecapeptidyl arch (UPA; Arg168 to Cys178) of extracellular loop 2 (ECL2) in CCR5. Novel monoclonal antibodies were raised against cDDR5 conjugated with a multiple-antigen peptide (cDDR5-MAP), and the purified antibody [KB8C12, immunoglobulin M(κ)] reacted with cDDR5, but not with linear DDR5, in real-time biomolecular interaction analysis using surface plasmon resonance. The antibody also reacted with cells expressing CCR5, but not with cells expressing CXCR4, and the immunoreaction was competed by cDDR5-MAP. The antibody significantly interfered with chemotaxis induced by macrophage inflammatory protein, 1β, and at a concentration of 1.67 nM it almost completely inhibited infection by human immunodeficiency virus type 1 (HIV-1) R5, but not by HIV-1 X4, as observed by use of a new phenotypic assay for drug susceptibility of HIV-1 using the CCR5-expressing HeLa CD4+ cell clone 1-10 (MAGIC-5). Furthermore, cDDR5-MAP suppressed infection by HIV-1 R5 at relatively high concentrations (50 to 400 μM) in a dose-dependent manner but did not suppress infection by HIV-1 X4. Taken together, these results indicate that the antibody is conformation specific and recognizes the conformation-specific domain of the UPA of ECL2. Moreover, both the antibody and its immunogen, the cDDR5-MAP conjugate, may be useful in developing a new candidate vaccine for HIV therapy.
Role of the second extracellular loop of adenosine receptors in agonist and antagonist binding. Analysis of chimeric A1/A3 adenosine receptors.
