Abstract
Macrophage galactose-type C-type lectins 1 and 2 (MGL1/2) are expressed on the surfaces of macrophages and immature dendritic cells. Despite the high similarity between the primary sequences of MGL1 and MGL2, they display different ligand specificities. MGL1 shows high affinity for the LewisX trisaccharide, whereas MGL2 shows affinity for N-acetylgalactosamine. To elucidate the structural basis for the ligand specificities of the MGLs, we performed NMR analyses of the MGL1-LewisX complex. To identify the LewisX binding site on MGL1, a saturation transfer experiment for the MGL1-LewisX complex where sugar-CH/CH2-selective saturation was applied was carried out. To obtain sugar moiety-specific information on the interface between MGL1 and the LewisX trisaccharide, saturation transfer experiments where each of galactose-H5-, fucose-CH3-, and N-acetylglucosamine-CH3-selective saturations was applied to the MGL1-LewisX complex were performed. Based on these results, we present a LewisX binding mode on MGL1 where the galactose moiety is bound to the primary sugar binding site, including Asp-94, Trp-96, and Asp-118, and the fucose moiety interacts with the secondary sugar binding site, including Ala-89 and Thr-111. Ala-89 and Thr-111 in MGL1 are replaced with arginine and serine in MGL2, respectively. The hydrophobic environment formed by a small side chain of Ala-89 and a methyl group of Thr-111 is a requisite for the accommodation of the fucose moiety of the LewisX trisaccharide within the sugar binding site of MGL1.
Highlights
The MGL molecules are ϳ42-kDa type II transmembrane glycoproteins, which possess a cytoplasmic domain, a transmembrane domain, a neck domain, and a carbohydrate recognition domain (CRD) within each molecule [1, 3]
We present a LewisX binding mode on MGL1 where the galactose moiety is bound to the primary sugar binding site, including Asp-94, Trp-96, and Asp-118, and the fucose moiety interacts with the secondary sugar binding site, including Ala-89 and Thr-111
This is because mouse scavenger receptor C-type lectin (mSRCL) lacks the glycine-rich loop, which is a characteristic structural component of Gal-type lectins, including MGLs and asialoglycoprotein receptor (ASGPR), and plays a crucial role in fixing the correct position of the indole ring of the tryptophan residue to interact with the galactose [17]
Summary
Sample Preparation—Recombinant MGL1 was expressed in Escherichia coli BL21-Codon Plus (DE3)-RP cells (Stratagene) using the pET-21a plasmid containing the CRD of MGL1 [3]. Isothermal Titration Calorimetry—Binding of the carbohydrates (the LewisX trisaccharide and -methylgalactose) to MGL1 (wild type, A89L mutant, A89R mutant, and T111S mutant) was measured by isothermal titration calorimetry (ITC) using a MicroCal VP-ITC MicroCalorimeter (MicroCal Inc.). The sample cell was loaded with the MGL1 solution, and the protein was titrated with the sugar solution. NMR Experiments—Uniformly 13C- and 15N-labeled MGL1 was complexed with the LewisX trisaccharide and dissolved in 95% H2O, 5% 2H2O containing 10 mM HEPES (pH 7.4), 2 mM CaCl2, and 50 mM NaCl. Sets of triple resonance spectra (HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, and HBHA(CO)NH (24 –27)) were collected for the sample at 25 °C on a Bruker Avance 500 spectrometer.
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