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 alpha 2,3-sialylated human alpha(1)-acid glycoprotein (AGP) but not to alpha 2,6-sialylated AGP. Mutagenesis revealed that Y-152 of NKG2D and F-144 and N-160 of CD94 were critical for HepTF binding. This is the first report that NKG2D and CD94 bind to a2,3-sialylated but not to a2,6-sialylated multi-antennary N-glycans. (C) 2009 Elsevier Inc. All rights reserved.