Tyrosine sulfation of the amino terminus of PSGL-1 is critical for enterovirus 71 infection.
Tyrosine sulfation of the amino terminus of PSGL-1 is critical for enterovirus 71 infection.
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DOI：
10.1371/journal.ppat.1001174
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发表时间：
2010-11-04
期刊：
影响因子：
6.7
通讯作者：
Shimizu H
中科院分区：
文献类型：
--
作者：
Nishimura Y;Wakita T;Shimizu H
肠道病毒71型(EV71)是手足口病的主要病原体之一，手足口病是一种常见的儿童发热性疾病；然而，EV71还与各种神经系统疾病有关，包括在亚太地区爆发的EV71大爆发中的死亡病例。最近，我们发现人P-选择素糖蛋白配体-1(PSGL-1)是EV71进入白细胞并在白细胞中复制的细胞受体。PSGL-1是一种表达于白细胞表面的唾液粘蛋白，作为选择素的高亲和力拮抗受体，介导白细胞在内皮细胞上滚动。PSGL-1-P-选择素的相互作用需要在PSGL-1的N-末端区域至少将三个簇状酪氨酸中的一个和邻近的表达唾液酸路易斯x的O-糖链硫化。为了阐明PSGL-1与EV71相互作用的分子基础，我们产生了一系列PSGL-1突变体，并鉴定了对PSGL-1与EV71结合至关重要的翻译后修饰。我们在293T细胞中表达了PSGL-1突变体，并用流式细胞仪检测了其与EV71的结合能力。我们发现T57上的O-糖基化是PSGL-1-选择素相互作用的关键，而不是PSGL-1与EV71结合所必需的。另一方面，PSGL-1 N-末端一个或多个潜在酪氨酸硫化位点的定点突变显著削弱了PSGL-1与EV71的结合。此外，硫酸盐抑制剂氯酸钠以剂量依赖的方式阻断PSGL-1与EV71的相互作用，并抑制PSGL-1介导的EV71在Jurkat T细胞中的复制。因此，这项研究的结果表明，PSGL-1 N-末端区域的酪氨酸硫酸基化而不是O-糖基化可能促进病毒进入和EV71在白细胞中的复制。肠道病毒71型(EV71)是儿童手足口病和包括致命性脑炎在内的多种神经系统疾病的主要病原体。EV71越来越多地引起手足口病的大规模暴发，特别是在亚太地区。最近，我们发现人P-选择素糖蛋白配体-1(PSGL-1)是EV71的功能性受体。免疫细胞上的PSGL-1是参与早期炎症事件的关键分子，PSGL-1与选择素的相互作用受PSGL-1翻译后修饰的调节。在这里，我们发现PSGL-1 N-末端的翻译后修饰，酪氨酸硫化，对于它与EV71结合和随后的淋巴细胞病毒复制是至关重要的。酪氨酸硫化在蛋白质-蛋白质相互作用中的重要作用已被广泛接受；然而，只有在HIV-1中报道了受体的酪氨酸硫化参与病毒-受体相互作用。因此，这是第二个也是唯一一个酪氨酸硫化参与特定病毒-受体相互作用的例子。我们的结果为酪氨酸硫化蛋白在细胞趋向性和EV71致病机制中的生物学作用提供了新的线索。
Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease, a common febrile disease in children; however, EV71 has been also associated with various neurological diseases including fatal cases in large EV71 outbreaks particularly in the Asia Pacific region. Recently we identified human P-selectin glycoprotein ligand-1 (PSGL-1) as a cellular receptor for entry and replication of EV71 in leukocytes. PSGL-1 is a sialomucin expressed on the surface of leukocytes, serves as a high affinity counterreceptor for selectins, and mediates leukocyte rolling on the endothelium. The PSGL-1–P-selectin interaction requires sulfation of at least one of three clustered tyrosines and an adjacent O-glycan expressing sialyl Lewis x in an N-terminal region of PSGL-1. To elucidate the molecular basis of the PSGL-1–EV71 interaction, we generated a series of PSGL-1 mutants and identified the post-translational modifications that are critical for binding of PSGL-1 to EV71. We expressed the PSGL-1 mutants in 293T cells and the transfected cells were assayed for their abilities to bind to EV71 by flow cytometry. We found that O-glycosylation on T57, which is critical for PSGL-1–selectin interaction, is not necessary for PSGL-1 binding to EV71. On the other hand, site-directed mutagenesis at one or more potential tyrosine sulfation sites in the N-terminal region of PSGL-1 significantly impaired PSGL-1 binding to EV71. Furthermore, an inhibitor of sulfation, sodium chlorate, blocked the PSGL-1–EV71 interaction and inhibited PSGL-1-mediated viral replication of EV71 in Jurkat T cells in a dose-dependent manner. Thus, the results presented in this study reveal that tyrosine sulfation, but not O-glycosylation, in the N-terminal region of PSGL-1 may facilitate virus entry and replication of EV71 in leukocytes. Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease and a diverse array of neurological diseases, including fatal encephalitis, in children. EV71 has increasingly caused large outbreaks of hand, foot, and mouth disease particularly in the Asia-Pacific region. Recently, we identified human P-selectin glycoprotein ligand-1 (PSGL-1) as a functional receptor for EV71. PSGL-1 on immune cells is a key molecule involved in early inflammatory events and the PSGL-1–selectin interaction is regulated by post-translational modifications of PSGL-1. Here, we found that a post-translational modification, tyrosine sulfation, at the N-terminal region of PSGL-1 is critical for its binding to EV71 and subsequent viral replication in lymphocytes. Important roles for tyrosine sulfation in protein-protein interactions have been widely accepted; however, involvement of tyrosine sulfation of the receptor in the virus-receptor interaction has been reported only for HIV-1. Therefore, this is the second and unique example of the involvement of tyrosine sulfation in specific virus-receptor interactions. Our results shed new light on biological roles for tyrosine-sulfated proteins in cell tropism and the pathogenesis of EV71.
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