Altered glycosylation of recombinant NKp30 hampers binding to heparan sulfate: a lesson for the use of recombinant immunoreceptors as an immunological tool
Altered glycosylation of recombinant NKp30 hampers binding to heparan sulfate: a lesson for the use of recombinant immunoreceptors as an immunological tool
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DOI：
10.1093/glycob/cwm125
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发表时间：
2008-01-01
期刊：
影响因子：
4.3
通讯作者：
Porgador, Angel
中科院分区：
文献类型：
--
作者：
Hershkovitz, Oren;Jarahian, Mostafa;Porgador, Angel
NKp 30是由人NK细胞表达并参与NK裂解活性的天然细胞毒性受体。我们以前发表的，膜硫酸乙酰肝素作为一个共配体的人NKp 30。在本研究中，我们补充我们的结果，显示直接结合的重组NKp 30固定肝素。靶肿瘤细胞上的硫酸乙酰肝素表位和NKp 30识别的肝素表位具有相似的特征。Warren及其同事（Warren HS，Jones AL，Freeman C，Bettadapura J，教区CR。2005.人NK细胞活化受体NKp 30的细胞配体不是硫酸乙酰肝素糖胺聚糖的证据。175：207-212）公开了NKp 30不与靶细胞上的膜硫酸乙酰肝素结合，并且硫酸乙酰肝素不参与NKp 30介导的裂解。在目前的研究中，我们研究了6种不同的重组NKp 30与膜硫酸乙酰肝素的结合，并得出结论，NKp 30确实与膜硫酸乙酰肝素相互作用。然而，六种重组NKp 30中的两种，包括可商购的重组NKp 30（由Warren等人采用），未显示硫酸乙酰肝素依赖性结合。我们证明，这是由于这两个重组NKp 30的糖基化改变。去除其N-连接聚糖后，硫酸乙酰肝素与肿瘤细胞的依赖性结合和与肝素的直接结合得以恢复。总体而言，我们的研究结果强调了适当的糖基化的重要性，分析NKp 30结合其配体和膜硫酸乙酰肝素可以作为共配体NKp 30。在细胞水平上，可溶性硫酸乙酰肝素增强了用抗NKp 30单克隆抗体激活的NK-92自然杀伤细胞分泌IFN γ。我们讨论了硫酸乙酰肝素与NKp 30结合参与NKp 30介导的NK细胞活化。
NKp30 is a natural cytotoxicity receptor expressed by human NK cells and involved in NK lytic activity. We previously published that membranal heparan sulfate serves as a coligand for human NKp30. In the present study, we complement our results by showing direct binding of recombinant NKp30 to immobilized heparin. The heparan sulfate epitope(s) on target tumor cells and the heparin epitope(s) recognized by NKp30 share similar characteristics. Warren and colleagues (Warren HS, Jones AL, Freeman C, Bettadapura J, Parish CR. 2005. Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan. J Immunol. 175:207-212) published that NKp30 does not bind to membranal heparan sulfate on target cells and that heparan sulfate is not involved in NKp30-mediated lysis. In the current study, we examine the binding of six different recombinant NKp30s to membranal heparan sulfate and conclude that NKp30 does interact with membranal heparan sulfate. Yet, two of the six recombinant NKp30s, including the commercially available recombinant NKp30 (employed by Warren et al.) did not show heparan sulfate-dependent binding. We demonstrate that this is due to an altered glycosylation of these two recombinant NKp30s. Upon removal of its N-linked glycans, heparan sulfate-dependent binding to tumor cells and direct binding to heparin were restored. Overall, our results emphasize the importance of proper glycosylation for analysis of NKp30 binding to its ligand and that membranal heparan sulfate could serve as a coligand for NKp30. At the cellular level, soluble heparan sulfate enhanced the secretion of IFN gamma by NK-92 natural killer cells activated with anti-NKp30 monoclonal antibody. We discuss the involvement of heparan sulfate binding to NKp30 in NKp30-mediated activation of NK cells.