Binding Properties of CD94 to Sulfated Glycans and 2,3-NeuAc- Containing Glycoproteins and Mutagenesis Analysis

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.