Natural killer group 2A (NKG2A) and natural killer group 2C (NKG2C) bind to sulfated glycans and α2,3-NeuAc-containing glycoproteins.

Killer lectin-like receptors on natural killer (NK) cells mediate cytotoxicity through glycans on target cells. We prepared recombinant glutathione S-transferase-fused extracellular lectin-like domains (AA 94-231) of natural killer group 2A (NKG2A) (rGST-NKG2A) and NKG2C (rGST-NKG2C) and determined the binding of these receptors to plates coated with heparin-conjugated bovine serum albumin (heparin-BSA) and glycoproteins. rGST-NKG2A and rGST-NKG2C directly bound to heparin-BSA with K(d) values of 20 and 40 nM, respectively. Binding of rGST-NKG2A and rGST-NKG2C to heparin-BSA was suppressed in the presence of soluble heparin, heparan sulfate, fucoidan, λ-carrageenan, and dextran sulfate. 2-O-Sulfate residues in heparin were essential for the binding of rGST-NKG2A and rGST-NKG2C. Moreover, rGST-NKG2A and rGST-NKG2C bound to multimeric sialyl Lewis X expressing transferrin secreted by HepG2 cells with K(d) values of 80 and 114 nM, respectively. This is the first report showing that NKG2A and NKG2C bind to heparin and α2,3-NeuAc-containing glycoproteins.

Binding affinities of NKG2D and CD94 to sialyl Lewis X-expressing N-glycans and heparin.

Lectin-like receptors natural killer group 2D (NKG2D) and CD94 on natural killer (NK) cells bind to α2,3-NeuAc-containing N-glycans and heparin/heparan sulfate (HS). Using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rGST-NKG2Dlec) and CD94 (rGST-CD94lec), we evaluated their binding affinities (K(d)) to high sialyl Lewis X (sLeX)-expressing transferrin secreted by HepG2 cells (HepTf) and heparin-conjugated bovine serum albumin (Heparin-BSA), using quartz crystal microbalance (QCM) and enzyme immunoassay (EIA) microplate methods. K(d) values obtained by linear reciprocal plots revealed good coincidence between the two methods. K(d) values of rGST-NKG2Dlec obtained by QCM and EIA, respectively, were 1.19 and 1.11 µM for heparin-BSA >0.30 and 0.20 µM for HepTf, while those of rGST-CD94lec were 1.31 and 1.45 µM for HepTf >0.37 and 0.36 µM for heparin-BSA. These results suggested that these glycans can interact with NKG2D and CD94 to modulate NK cell-dependent cytotoxicity.

NKG2D and CD94 bind to multimeric alpha2,3-linked N-acetylneuraminic acid.

Killer lectin-like receptors on natural killer cells mediate cytotoxicity through glycans on target cells including the sialyl Lewis X antigen (sLeX). We investigated whether NK group 2D (NKG2D) and CD94 can bind to sialylated N-linked glycans, using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rNKG2Dlec) and CD94 (rCD94lec). Both rNKG2Dlec and rCD94lec bound to plates coated with high-sLeX-expressing transferrin secreted by HepG2 cells (HepTF). The binding of rNKG2Dlec and rCD94lec to HepTF was markedly suppressed by treatment of HepTF with neuraminidase and in the presence of N-acetylneuraminic acid. Moreover, rNKG2Dlec and rCD94lec bound to alpha2,3-sialylated human alpha(1)-acid glycoprotein (AGP) but not to alpha2,6-sialylated AGP. Mutagenesis revealed that (152)Y of NKG2D and (144)F and (160)N of CD94 were critical for HepTF binding. This is the first report that NKG2D and CD94 bind to alpha2,3-sialylated but not to alpha2,6-sialylated multi-antennary N-glycans.