RESEARCH ARTICLE


NKG2D: Binding Properties for Glycan Ligands, and Mutagenesis Analysis



Koji Higai*, 1, Sayo Matsumoto1, Megumi Kimura1, 2, Yuzo Imaizumi1, Kazuyuki Yanai2, Yutaro Azuma1, Kojiro Matsumoto1
1 Department of Clinical Chemistry, Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
2 Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, Japan


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© 2011 Higai et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Department of Clinical Chemistry, Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan; Tel: +81-474-72-1481; Fax: +81-474-1485; E-mail: koji@phar.toho-u.ac.jp


Abstract

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-Stransferase- 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 sulfatecontaining glycan-BSA with Kd values of 25 nM ±15 for λ-carrageenan-BSA, 66 ±23 nM for fucoidan-BSA, and 1.5±0.5 μM for heparan sulfate-BSA. Of the site-directed rGST-NKG2D mutants, Y152A, Q185A, K197A, Y199A, E201A, and N207A reduced binding to these glycans. These results indicate that NKG2D interacts with highly sulfated- and α2,3- NeuAc-containing glycans and suggest that the glycan-binding sites on NKG2D are shared between sulfate- and α2,3- NeuAc-containing glycans, and might overlap with protein ligand binding sites.

Keywords: NKG2D, Glycan ligands, glycosaminoglycan, sialyl lewis X, mutagenesis.