Binding of natural cytotoxicity receptor NKp46 to sulfate- and α2,3-NeuAc-containing glycans and its mutagenesis

► NKp46 binds to heparin; however, other natural ligands for NKp46 have yet to be elucidated. ► We clarify the binding of NKp46-H6 to sulfate-containing glycans and sialyl Lewis X (sLeX). ► NKp46-H6 bound to multimeric sialyl Lewis X with a K d value of 530 nM. ► Moreover, we investigated the putative glycan binding sites on NKp46 using mutants. 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.