Zilka Alon, Landau Guy, Hershkovitz Oren, Bloushtain Noga, Bar-Ilan Ahuva, Benchetrit Fabrice, Fima Eyal, van Kuppevelt Toin H, Gallagher John T, Elgavish Sharona, Porgador Angel
Department of Microbiology and Immunology, Faculty of Health Sciences, and Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.
Biochemistry. 2005 Nov 8;44(44):14477-85. doi: 10.1021/bi051241s.
NKp46 is a member of a group of receptors collectively termed natural cytotoxicity receptors (NCRs) that are expressed by natural killer (NK) cells. NCRs are capable of mediating direct killing of tumor and virus-infected cells by NK cells. We have recently shown that NKp46 recognizes the heparan sulfate moieties of membranal heparan sulfate proteoglycans (HSPGs), thus enabling lysis of tumor cells by NK cells. In the current study, we further examined the residues in NKp46 that may be involved in heparan sulfate binding on tumor cells. On the basis of both the electrostatic potential map and comparison to the heparin binding site on human fibronectin, we predicted a continuous region containing the basic amino acids K133, R136, H139, R142, and K146 to be involved in NKp46 binding to heparan sulfate. Mutating these amino acids on NKp46D2 to noncharged amino acids retained its virus binding capacity but reduced its binding to tumor cells with a 10-100 fold lower K(D) when tested for direct binding to heparin. The minimal length of the heparin/heparan sulfate epitope recognized by NKp46 was eight saccharides as predicted from the structure and proven by testing heparin oligomers. Testing selectively monodesulfated heparin oligomers emphasized the specific contributions of O-sulfation, N-sulfation, and N-acetylation to epitope recognition by NKp46. The characterization of heparan sulfate binding region in NKp46 offers further insight into the identity of the ligands for NKp46 and the interaction of NK and cancers.
NKp46是一组统称为自然细胞毒性受体（NCRs）的受体成员，由自然杀伤（NK）细胞表达。NCRs能够介导NK细胞直接杀伤肿瘤细胞和病毒感染细胞。我们最近发现NKp46识别膜硫酸乙酰肝素蛋白聚糖（HSPGs）的硫酸乙酰肝素部分，从而使NK细胞能够裂解肿瘤细胞。在本研究中，我们进一步研究了NKp46中可能参与与肿瘤细胞硫酸乙酰肝素结合的残基。基于静电势图以及与人纤连蛋白上肝素结合位点的比较，我们预测包含碱性氨基酸K133、R136、H139、R142和K146的连续区域参与NKp46与硫酸乙酰肝素的结合。将NKp46D2上的这些氨基酸突变为不带电荷的氨基酸，保留了其病毒结合能力，但在测试与肝素的直接结合时，其与肿瘤细胞的结合能力降低，解离常数（K(D)）低10 - 100倍。如从结构预测并通过测试肝素寡聚物所证实，NKp46识别的肝素/硫酸乙酰肝素表位的最小长度为八个糖。测试选择性单硫酸化肝素寡聚物强调了O-硫酸化、N-硫酸化和N-乙酰化对NKp46表位识别的特定贡献。NKp46中硫酸乙酰肝素结合区域的表征为NKp46配体的身份以及NK与癌症的相互作用提供了进一步的见解。
