Abstract
C-type lectin receptors expressed on the surface of dendritic cells and macrophages are able to bind glycoproteins of microbial pathogens via mannose, fucose, and N-acetylglucosamine. Langerin on Langerhans cells, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin on dendritic cells, and mannose receptor (MR) on dendritic cells and macrophages bind the human immunodeficiency virus (HIV) envelope protein gp120 principally via high mannose oligosaccharides. These C-type lectin receptors can also oligomerize to facilitate enhanced ligand binding. This study examined the effect of oligomerization of MR on its ability to bind to mannan, monomeric gp120, native trimeric gp140, and HIV type 1 BaL. Mass spectrometry analysis of cross-linked MR showed homodimerization on the surface of primary monocyte-derived dendritic cells and macrophages. Both monomeric and dimeric MR were precipitated by mannan, but only the dimeric form was co-immunoprecipitated by gp120. These results were confirmed independently by flow cytometry analysis of soluble monomeric and trimeric HIV envelope and a cellular HIV virion capture assay. As expected, mannan bound to the carbohydrate recognition domains of MR dimers mostly in a calcium-dependent fashion. Unexpectedly, gp120-mediated binding of HIV to dimers on MR-transfected Rat-6 cells and macrophages was not calcium-dependent, was only partially blocked by mannan, and was also partially inhibited by N-acetylgalactosamine 4-sulfate. Thus gp120-mediated HIV binding occurs via the calcium-dependent, non-calcium-dependent carbohydrate recognition domains and the cysteine-rich domain at the C terminus of MR dimers, presenting a much broader target for potential inhibitors of gp120-MR binding.
Highlights
The mannose receptor (MR)2 is a C-type lectin receptor that is expressed on the surface of a variety of cells, including immature monocyte-derived dendritic cells (MDDC), dermal dendritic cells, macrophages, and hepatic endothelial cells
The quaternary structure of MR has been defined for soluble recombinant molecules, it has not been shown that oligomerization occurs on the cell surface or whether it affects its function
This study showed that MR forms dimers on the surface of MDDCs, monocyte-derived macrophages (MDMs), and MR Rat-6 cells and investigated whether this enhances its ability to bind to oligosaccharide-expressing ligands such as yeast-derived mannan and human immunodeficiency virus (HIV)-1 gp120
Summary
Cells and Reagents—All reagents, unless otherwise stated, were purchased from Sigma. Monocytes were positively selected from whole peripheral blood mononuclear cells by magnetic cell sorting using CD14 microbeads (Miltenyi Biotec, Auburn, CA). Harvested cells were washed twice with cross-link buffer (CLB) (10 mM HEPES, pH 8.0, 140 mM NaCl, 1 mM MgCl2, 0.1 mM EGTA, 0.02% (w/v) NaN3) and resuspended at 5 ϫ 106 cells/ml. Proteins were eluted by boiling the beads for 5 min and separated on a 7.5% polyacrylamide gel (Bio-Rad), and MR was detected with a monoclonal ␣-MR antibody by Western blotting. Cells were cross-linked as described above and resuspended in binding buffer (RPMI, 1% bovine serum albumin, 10 mM HEPES, pH 7.5) at 1 ϫ 106 cells per 50 l. For inhibition of gp120 binding, 5 mg/ml mannan, 1 mg/ml D(ϩ)-mannose, 1 mg/ml quinic acid, 1 mg/ml shikimic acid, 10 mg/ml GalNAc-4-SO4, or 20 mM EGTA were added to the cell suspension and incubated for 30 min. Generating High Titer Purified HIV-1 BaL Stocks—To increase infectivity of initial seed stocks, vesicular stomatitis virus g protein (VSVg) envelope pseudotyped HIV-1 BaL was generated by co-transfecting HEK 293T (Invitrogen) with fullkDa
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