Binding of natural cytotoxicity receptor NKp46 to sulfate-and α2,3-NeuAc-containing glycans and its mutagenesis
Binding of natural cytotoxicity receptor NKp46 to sulfate-and α2,3-NeuAc-containing glycans and its mutagenesis
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DOI：
10.1016/j.bbrc.2011.02.050
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发表时间：
2011-03-18
影响因子：
3.1
通讯作者：
Matsumoto, Kojiro
中科院分区：
文献类型：
--
作者：
Ito, Kenichiro;Higai, Koji;Matsumoto, Kojiro
天然细胞毒性受体1（NCR 1，NKp 46）结合肝素和硫酸乙酰肝素;然而，NKp 46的其他天然配体尚未阐明。使用用6x His（NKp 46-H6）标记的NKp 46的重组胞外区（编码AA 22-258）以及突变体K136 Q、R139 Q、H142 Q、R145 Q和K149 Q，我们测定了它们与含硫酸酯和NeuAc的聚糖包被的平板的结合亲和力。NKp 46-H6直接结合到用肝素和硫酸乙酰肝素缀合的牛血清白蛋白包被的平板上，Kd值分别为770和850 nM。NKp 46-H6与肝素-BSA的结合被可溶性肝素、硫酸乙酰肝素、岩藻依聚糖、葡聚糖-角叉菜胶和硫酸葡聚糖抑制，但不被2-O-、6-O-和N-乙酰肝素化。NKp 46-H6也与人肝癌HepG 2细胞（HepTF）分泌的表达转铁蛋白的多聚体唾液酸刘易斯X结合，Kd值为530 nM，但不与去唾液酸化的HepTF、市售TF或1-酸性糖蛋白结合。此外，突变体R139 Q、R145 Q和K149 Q与这些含硫酸根的聚糖的结合显著降低，K136 Q和K149 Q与HepTF的结合显著降低，表明NKp 46主要通过离子相互作用与含硫酸根和2，3-NeuAc的聚糖结合。然而，NKp 46的结合位点不同。(C)2011 Elsevier Inc. All rights reserved.
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 6x 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, lambda-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. (C) 2011 Elsevier Inc. All rights reserved.