Natural killer (NK) cell cytotoxicity is regulated by the action of a large number of distinct inhibitory and activating receptors that bind to the major histocompatibility complex (MHC) on the surface of target cells. Two major types of MHC class I-binding receptors are expressed on human NK cells: immunoglobulin (Ig) superfamily molecules, including killer cell Ig-like receptors (KIRs) and leukocyte Ig-like receptors (LIRs), and heterodimers of the C-type lectin-like molecules CD94 and natural killer group 2 (NKG2), including NKG2A/B, NKG2C, and NKG2E/H. 1,2) NKG2B and NKG2H are alternatively spliced variants of NKG2A and NKG2E, respectively. NKG2F has no C-type lectin-like domain and does not associate with CD94.3) NKG2D, which is distantly related to other NKG2 proteins, forms a homodimer and associates with the DNAX-activating proteins DAP10 or DAP12 to induce NK cell cytotoxicity.The NKG2 receptors are closely related, as evidenced by the 81-94% identity in their C-type lectin-like extracellular domains, and form disulfide-linked heterodimers with CD94.4) The CD94/NKG2A heterodimer is an inhibitory receptor due to the two immunoreceptor tyrosine-based inhibitory motifs (ITIM) in the cytoplasmic tail of NKG2A.5-7) In contrast, CD94/NKG2C and CD94/NKG2E heterodimers function as activating receptors due to the association of positively charged Lys residues in the transmembrane regions of NKG2C and NKG2E with Asp residues of the immunoreceptor tyrosine-based activating motif (ITAM) of adaptor molecule DAP12. 8)Human leukocyte antigen (HLA)-E serves as the ligand for CD94/NKG2A, CD94/NKG2C, and CD94/NKG2E. HLA-E is a non-classical MHC class Ib molecule loaded with nonamer leader peptides derived from the signal sequences of MHC class Ia, HLA-A, -B, -C, or -G proteins. [9][10][11][12] Both the inhibitory CD94/NKG2A and activating CD94/NKG2C recognize the same nonamer peptide/HLA-E complex, although CD94/NKG2A has a higher binding affinity than CD94/ NKG2C. 13)These C-type lectin-like receptors lack most of the conserved Ca 2ϩ -binding region, and their glycan ligands have yet to be elucidated.14) However, of the C-type lectin-like receptors on NK cells, several have been reported to recognize sulfated glycans: mouse Ly-49A binds to fucoidan [15][16][17][18] and osteoclast inhibitory lectin (OCIL, lectin-like transcript 1: LLT1) binds to fucoidan, l-carrageenan, and dextran sulfate. 19) Preclinical and clinical studies suggest that heparin and low molecular weight heparins are effective in preventing tumor growth and metastasis. The molecular mechanisms of the anti-tumor effects of heparin are complicated and largely unclarified. Hypotheses include competition with the binding of growth factors to heparan sulfate proteoglycans, inhibition of heparanase, and inhibition of interaction with Land P-selectins on vascular endothelial cells and platelets. 20,21) We demonstrated that K562 cells transfected with the fucosyltransferase 3 gene that highly express the sialyl Lewis X (sLeX) antigen, NeuAca2,3Galb1,4(Fuca1,3)G...

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Killer lectin-like receptor CD94 on natural killer cells can bind to HLA-E, 2,3-NeuAc-containing glycoproteins, and heparin. Using the glutathione-S-transferase-fused CD94 (rGST-CD94), we investigated its binding to plates coated with 2,3-NeuAc-containing transferrin secreted by HepG2 cells (HepTF) and sulfate-containing glycanconjugated BSA. rGST-CD94 bound directly to several glycans with similar high affinities: rGST-CD94 bound to heparin-BSA, heparan sulfate-BSA, fucoidan-BSA, -carrageenan-BSA, and HepTF with Kd values of 36 nM, 141 nM, 36 nM, 92 nM and 84 nM, respectively. Different mutants of rGST-CD94 were generated by site-directed mutagenesis. Mutants N160A and C166G had reduced binding to sulfated glycans and HepTF, also F114A, L162A, D163A, and E164A showed reduced binding to sulfated glycans with some variability. These results indicated that CD94 interacts with sulfated glycans and 2,3-NeuAc-containing glycoproteins suggesting that glycan binding sites in CD94 partially overlap with the HLA-E binding sites to modulate natural killer cell-dependent cytotoxicity.

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