Nkg2d: Binding Properties for Glycan Ligands, and Mutagenesis Analysis

Killer lectin-like receptor NKG2D, which is found on natural killer cells, recognizes MHC class 1-related ligands and also interacts with glycan ligands, heparin-conjugated bovine serum albumin (heparin-BSA) and sialyl Lewis X (sLeX) on multi-antennary N-glycans on transferrin secreted by HepG2 cells (HepTF). Using the glutathione-S-transferase-fused extracellular domain (AA 73-216) of NKG2D (rGST-NKG2D) and seven site-directed mutants, we explored in detail the binding of NKG2D to sulfate-containing glycan-BSA and HepTF. rGST-NKG2D binds to sulfate-containing glycan-BSA with K d values of 25 nM ±15 for-carrageenan-BSA, 66 ±23 nM for fucoidan-BSA, and 1.5±0.5 μM for heparan sulfate-BSA. Of the site-directed rGST-N207A reduced binding to these glycans. These results indicate that NKG2D interacts with highly sulfated-and 2,3-NeuAc-containing glycans and suggest that the glycan-binding sites on NKG2D are shared between sulfate-and 2,3-NeuAc-containing glycans, and might overlap with protein ligand binding sites.


INTRODUCTION
Natural killer (NK) cells are important in innate immunity and, the cytotoxic activity of NK cells is regulated by the integration of signals produced by missing self and induced self interactions and a shift of the balance in activating and inhibiting cell surface receptors of the immunoglobulinlike and C-type lectin-like superfamilies [1].
Of the C-type lectin-like receptors, natural killer group 2D (NKG2D) is one of the most important activating receptors [2][3][4][5].NKG2D is distantly related to other members of the NKG2 family, NKG2A, B, C, E, and H, which form heterodimers with CD94 and bind to the non-classical major histocompatibility complex (MHC) class 1 molecules HLA-E in human and Qa-1 in mouse.Activating receptor NKG2D is expressed on all NK cells and most T-cell receptor ( TCR)-positive T cells [6] and is a homodimeric type 2 transmembrane glycoprotein that forms a salt-bridged hexamer with two homodimers of the immunoreceptor tyrosinebased activation motif (ITAM) YINM-containing adaptor molecule DNAX-activating protein of 10 kDa (DAP10) in human [7].
Although the ligands for NKG2D are identified MHC class I related proteins: MHC class I related chain family protein (MIC) A and B [6] and UL 16-binding protein (ULBP) 1-5 [8], its glycan ligands are yet unidentified in human.
Here, we report that we have further characterized the binding of rGST-NKG2D and its mutants to sulfatecontaining glycans and HepTF.We found that rGST-NKG2D binds to glycans with K d values.Moreover, mutagenesis suggested that the glycan-binding sites on NKG2D may overlap with protein ligand binding sites.
The recombinant plasmids were re-transformed into Chaperone competent cells pTf16/BL21 (Takara, Otsu, Japan) to express the recombinant protein.
After induction with 1 mM isopropyl--D-thiogalactopyranoside (Promega Co., Madison, WI), the cells were harvested and sonicated on ice for 10 s 6.The recombinant proteins were purified on a GSTrap TM FF column (1 ml packed volume; GE Healthcare) according to the manufacturer's instructions and dialyzed against 1 mM dithiothreitol (DTT) in phosphate-buffered saline (PBS).The proteins were subjected to 10% SDS-PAGE with and without pretreatment in 1% 2-mercaptoethanol (2-ME) at 95°C for 3 min, and stained with Coomassie brilliant blue.Protein concentrations were determined using advanced protein assay reagent (Bio-Rad, Hercules, CA) with bovine serum albumin (BSA) as a standard.

Binding of Site-Directed Mutant rGST-NKG2D to Glycans
The concentrations of mutant used to determine their binding to glycans were based on the K d values of rGST-NKG2Dwild for each glycan: 20 nM for fucoidan-BSA and -carrageenan-BSA, and 230 nM for HepTF, heparin-BSA and heparan sulfate-BSA, respectively.The binding was measured as described above and compared with that of rGST-NKG2Dwild.

Binding of rGST-NKG2D to Sulfate-Containing Glycans
We prepared rGST-NKG2D and its mutants using Chaperon competent cells pTf16/BL21, purifying on a glutathione column.To evaluate the molecular forms of rGST-NKG2D and its mutants, these recombinant proteins were treated with or without 1% 2-ME and subjected to 10% SDS-PAGE.They separated at around 42 kDa under both reducing and non-reducing conditions, indicating that the forms mainly obtained were monomeric (Fig. 1).

Fig. (1). SDS-PAGE for rGST-NKG2D and its mutants.
Recombinant proteins purified on a glutathione column were separated on 10% SDS-PAGE with (R) and without (NR) pretreatment with 1% 2-ME at 95°C for 3 min.
These results indicate that interactions between NKG2D and glycans are mainly hydrophobic and/or hydrogenbonding interactions, while formation of ionic salt bridges is less important.The binding sites on NKG2D for sulfate-and 2,3-NeuAc-containing glycans seem to overlap with protein ligand binding sites.
Crystal structures of representative NK receptors have been determined in isolated and ligand-bound forms [22].The crystal structures of NKG2D alone [23] and in complex with MICA [14], MICB [17], ULBP3 [15], and RAE-1 [18]   have revealed that the NKG2D homodimer forms a concave surface to interact with the convex monomeric ligand via a network of hydrophobic, hydrogen-bonding, and saltbridging interactions and that each NKG2D monomer predominantly interacts with either the 1 or the 2 platform domain of these divergent ligands.This indicates that two symmetric binding sites on the NKG2D homodimer bind to different epitopes on the asymmetric ligand monomer [24,25].Each NKG2D subunit forms a hydrophobic patch with MICA [14] and ULBP3 [15], using the same receptor residues (Y152, I182, M184, and Y199) but different ligand residues.Y152, I181, E183, Q185, K186, S195, Y199, T205, and N207 in NKG2D are hydrogen bonded and K197 and E201 or K150 in NKG2D are salt bridged with MICA and/or ULBP3, respectively.At the center of the NKG2D binding sites lie two conserved residues, Y152 and Y199, that constitute the dominant binding-energy hotspots [25].Two mechanisms have been considered to explain NKG2D's multispecific ligand recognition: an induced-fit model [15,26] or a rigid adaptation mechanism [19,27].
We have revealed the glycan-ligand binding sites on NKG2D using single amino acid-substituted mutants: Y152, Q185, K197, Y199, E201, and N207 are essential for interactions with sulfate-and 2,3-NeuAc-containing glycans, with some variation between glycans.Interestingly, the dominant binding-energy hotspots Y152 and Y199 interact with both sulfate-and 2,3-NeuAc-containing glycans, suggesting that glycan-binding sites may overlap with protein ligand binding sites to physiologically modulate NKG2Dmediated NK cell cytotoxicity.
We have reported that increased cytotoxicity of IL2activated KHYG cells against K562/FUT cells is suppressed by pretreatment with anti-sLeX and KHYG cells with anti- CD94 and anti-NKG2D antibodies [9] and also that the other C-type lectin-like receptors CD94, NKG2A, and NKG2C interact with sulfate-and 2,3-NeuAc-containing glycans with high but different affinities [11][12][13]28].Heparin and heparan sulfate interact with the natural cytotoxicity receptors NKp46, NKp44, and NKp30 to induce NK cell cytotoxicity [29][30][31].Soluble forms of heparin compete with the sLeX-dependent interactions of tumor cells with L-and Pselectin and inhibit platelet aggregation and vascular-cell adhesion with tumor cells to prevent extravasation and migration of tumor cells [32][33][34].NK cell-dependent cytotoxicity is modulated through sulfation and sialylation status of target cells.Further studies are needed to clarify the role of sulfate-and sLeX-containing glycans in tumor surveillance via NK cells.