Glycosylation Influences the Lectin Activities of the Macrophage Mannose Receptor

Yunpeng Su,Talitha Bakker,James Harris,Clarence Tsang,Gordon D Brown,Mark R Wormald,Siamon Gordon,Raymond A Dwek,Pauline M Rudd,Luisa Martinez-Pomares

doi:10.1074/jbc.m503457200

Abstract

The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognizes both mannosylated and sulfated ligands through its C-type lectin domains and cysteine-rich (CR) domain, respectively. Differential binding properties have been described for MR isolated from different sources, and we hypothesized that this could be due to altered glycosylation. Using MR transductants and purified MR, we demonstrate that glycosylation differentially affects both MR lectin activities. MR transductants generated in glycosylation mutant cell lines lacked most mannose internalization activity, but could internalize sulfated glycans. Accordingly, purified MR bearing truncated Man5-GlcNAc2 glycans (Man5 -MR) or non-sialylated complex glycans (SA0-MR) did not bind mannosylated glycans, but could recognize SO4-3-Gal in vitro. Additional studies showed that, although mannose recognition was largely independent of the oligomerization state of the protein, recognition of sulfated carbohydrates was mostly mediated by self-associated MR and that, in SA0-MR, there was a higher proportion of oligomeric MR. These results suggest that self-association could lead to multiple presentation of CR domains and enhanced avidity for sulfated sugars and that non-sialylated MR is predisposed to oligomerize. Therefore, the glycosylation of MR, terminal sialylation in particular, could influence its binding properties at two levels. (i) It is required for mannose recognition; and (ii) it modulates the tendency of MR to self-associate, effectively regulating the avidity of the CR domain for sulfated sugar ligands.

Highlights

Research Fund. 1 Present address: Whitehead Inst. for Medical Research, Nine Cambridge Centre, Cambridge, MA 02142. 2 Present address: Dept. of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131. 3 Present address: Inst. of Infectious Disease and Molecular Medicine, Faculty of Health
We have demonstrated binding of the mannose receptor (MR) CR domain to sulfated carbohydrate ligands expressed by subsets of macrophages and dendritic cells associated with B cell follicles in spleen and lymph nodes (20 –22)
The monoclonal antibody (mAb) internalization data seemed to give a good indication of the levels of MR expressed by the transductants, as they correlated with the results obtained by Western blot analysis

Summary

EXPERIMENTAL PROCEDURES

Cells—Chinese hamster ovary (CHO) cells were obtained from frozen stocks stored at the Sir William Dunn School of Pathology and cultured in Ham’s F-12K medium with 2 mM L-glutamine. Ligand Binding Assays—96-Well Maxisorp plates (Nalge Nunc International, Naperville, IL) were coated overnight at 37 °C with 50 ␮l of 100 ␮g/ml mannan, 10 ␮g/ml Gal-BSA, or 10 ␮g/ml SO4-3-Gal-PAA in PBS and washed twice with binding buffer (10 mM Tris-HCl (pH 7.5), 15 mM CaCl2, 150 mM NaCl, and 0.1% Tween 20) for 5 times; in the last wash, the plate was incubated at 25 °C for 5 min for blocking. MAb 5D3 (10 ␮g/ml) and alkaline phosphatase-conjugated goat anti-rat IgG, both diluted in binding buffer and incubated for 1 h at 25 °C, were subsequently used to detect MR binding. After washes with Tris-buffered saline, filters were incubated with an alkaline phosphatase-conjugated sheep anti-digoxigenin F(abЈ) fragment (0.75 unit/ml). Carboxypeptidase Y was used as a positive control for the lectins peanut agglutinin and Datura stramonium agglutinin

RESULTS

TABLE ONE

DISCUSSION

Tendency to multimerize