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Human alpha1-acid glycoprotein binds to CCR5 expressed on the plasma membrane of human primary macrophages
A Atemezem1, E Mbemba, R Vassy
1Laboratoire de Biologie Cellulaire, JE 2138, Faculté de Médecine Léonard de Vinci, Université Paris XIII, Bobigny 93017, France.
Abstract:
We have reported previously that human alpha(1)-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 K(d) values of 16 nM and 4.9 microM 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 ( approximately 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-1beta (MIP-1beta) 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 33-36 kDa electroblotted AGP-bound MDM membrane ligands, characterized as CCR5 by their interactions with anti-CCR5 antibodies and with MIP-1beta. 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.
Insights
Human alpha(1)-acid glycoprotein (AGP) binds to macrophages via both its glycans and CCR5, inhibiting R5 HIV-1 infection. This dual binding mechanism involves interactions with macrophage lectins and the CCR5 co-receptor.
Area of Science:
- Immunology
- Virology
- Glycobiology
Background:
- Human alpha(1)-acid glycoprotein (AGP) previously shown to inhibit R5 HIV-1 infection of macrophages.
- R5 HIV-1 utilizes CCR5 as a co-receptor on human monocyte-derived macrophages (MDM).
Purpose of the Study:
- To elucidate the binding mechanism of AGP to MDM.
- To investigate the role of AGP glycans and CCR5 in AGP-MDM interactions.
- To understand how AGP binding relates to its HIV-1 inhibitory effect.
Main Methods:
- AGP binding assays to MDM with varying AGP treatments (heat denaturation, exoglycosidase).
- Inhibition studies using mannan, dextran, and anti-CCR5 antibodies.
- Identification of AGP-bound MDM membrane ligands via electroblotting and characterization with antibodies and MIP-1beta.
Main Results:
- AGP binds to MDM at high- and low-affinity sites, involving both protein-protein interactions and heat-resistant glycans (likely mannose residues).
- Mannan significantly inhibits AGP binding, while exoglycosidase treatment enhances it.
- AGP binding to MDM is partially inhibited by an anti-CCR5 antibody targeting the second extracellular loop, and AGP binds to CCR5 on MDM.
- AGP inhibits the binding of V3Cs and MIP-1beta to MDM.
Conclusions:
- AGP binds to MDM through interactions with both its glycans and the CCR5 co-receptor.
- This specific binding to MDM, involving potential lectin-like interactions and CCR5, likely underlies AGP's inhibitory effect on R5 HIV-1 infection.