title: Binding of natural cytotoxicity receptor NKp46 to sulfate- and α2,3-NeuAc-containing glycans and its mutagenesis.
abstract: Natural cytotoxicity receptor 1 (NCR1, NKp46) binds to heparin and heparan sulfate; however, other natural ligands for NKp46 have yet to be elucidated. Using the recombinant extracellular region (coding for AA 22-258) of NKp46 tagged with 6× His (NKp46-H6), and mutants K136Q, R139Q, H142Q, R145Q, and K149Q, we determined their binding affinities to sulfate- and NeuAc-containing glycans-coated plates. NKp46-H6 directly bound to plates coated with heparin- and heparan sulfate-conjugated bovine serum albumin with K(d) values of 770 and 850 nM, respectively. The binding of NKp46-H6 to heparin-BSA was suppressed by soluble heparin, herparan sulfate, fucoidan, λ-carrageenan, and dextran sulfate, but not by 2-O-, 6-O-, and N-desulfated heparin. NKp46-H6 also bound to multimeric sialyl Lewis X expressing transferrin secreted by human hepatoma HepG2 cells (HepTF) with a K(d) value of 530 nM, but not to desialylated HepTF, commercially available TF, or 1-acid glycoprotein. Moreover, mutants R139Q, R145Q, and K149Q had significantly reduced binding to these sulfate-containing glycans, and K136Q and K149Q to HepTF, indicating that NKp46 binds to sulfate- and 2,3-NeuAc-containing glycans mainly via ionic interactions. However, the binding sites of NKp46 were different.
authors.affiliation: Department of Clinical Chemistry, Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 247-8510, Japan.
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Ito
authors.forename: Koji
authors.initials: K
authors.lastname: Higai
authors.forename: Mizuha
authors.initials: M
authors.lastname: Sakurai
authors.forename: Chihiro
authors.initials: C
authors.lastname: Shinoda
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Yanai
authors.forename: Yutaro
authors.initials: Y
authors.lastname: Azuma
authors.forename: Kojiro
authors.initials: K
authors.lastname: Matsumoto
mesh_terms: D006493:Heparin / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D055627:Natural Cytotoxicity Triggering Receptor 1 / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics
chemical_list: D006493:Heparin; D006497:Heparan Sulfate; D055627:Natural Cytotoxicity Triggering Receptor 1; C112260:NCR1 protein, human