We have reported previously that human α1-acid glycoprotein (AGP) inhibits the infection of human monocyte-derived macrophages (MDM) by R5 HIV-1, and that a disulphide-bridged peptide mimicking the clade B HIV-1 gp120 consensus V3 domain (V3Cs) binds specifically to CCR5 (the major co-receptor of R5 HIV strains) on these cells [Seddiki, Rabehi, Benjouad, Saffar, Ferriere, Gluckman and Gattegno (1997) Glycobiology 7, 1229–1236]. The present study demonstrates that AGP binds specifically to MDM at high- and low-affinity binding sites with Kd values of 16nM and 4.9μM respectively. The fact that heat denaturation of AGP only partly inhibited this binding (43%) suggests that protein–protein interactions are involved, as well as AGP glycans which are resistant to heat denaturation. Mannan, but not dextran, is a significant inhibitor (52%) of this binding, and sequential exoglycosidase treatment of AGP, which exposes penultimate mannose residues, has a strong stimulatory effect (∼ 2.8-fold). Therefore AGP glycans (probably mannose residues) are involved, at least partly, in the binding of AGP to MDM. In addition, AGP inhibits the binding of V3Cs and macrophage inflammatory protein-1β (MIP-1β) to MDM. The anti-CCR5 monoclonal antibody 2D7, specific for the second extracellular loop of CCR5, also inhibited AGP binding (67%), whereas anti-CCR5 antibodies specific for the C-terminus of CCR5 region had no effect. Native AGP, like V3Cs (but not heat-denatured AGP), binds to 46 and 33–36kDa electroblotted AGP-bound MDM membrane ligands, characterized as CCR5 by their interactions with anti-CCR5 antibodies and with MIP-1β. Therefore both AGP glycans and MDM CCR5 are involved in the binding of AGP to MDM. This suggests that the inhibitory effect of AGP on the infection of human primary macrophages by R5 HIV-1 may be related to specific binding of AGP to a macrophage membrane lectin or lectin-like component and to CCR5.
Abbreviations used: AGP, α1-acid glycoprotein; ECL2, second extracellular loop of CCR5; mAb, monoclonal antibody; MDM, monocyte-derived macrophages; MIP-1β, macrophage inflammatory protein-1β; SDF-1α, stromal-cell-derived factor-1α; V3Cs, disulphide-bridged peptide mimicking the clade B HIV-1 gp120 consensus V3 domain.