Polysialic Acid on Neuropilin-2 Is Exclusively Synthesized by the Polysialyltransferase ST8SiaIV and Attached to Mucin-type O-Glycans Located between the b2 and c Domain
Polysialic Acid on Neuropilin-2 Is Exclusively Synthesized by the Polysialyltransferase ST8SiaIV and Attached to Mucin-type O-Glycans Located between the b2 and c Domain
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DOI：
10.1074/jbc.m113.463927
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发表时间：
2013-08-09
影响因子：
4.8
通讯作者：
Muehlenhoff, Martina
中科院分区：
文献类型：
--
作者：
Rollenhagen, Manuela;Buettner, Falk F. R.;Muehlenhoff, Martina
神经纤毛蛋白-2（NRP 2）是3类脑信号蛋白和血管内皮生长因子的共同受体，参与轴突导向和血管生成。此外，NRP 2显示出促进人单核细胞衍生的树突状细胞（DC）向趋化因子CCL 21的趋化性迁移，该功能依赖于聚唾液酸（polySia）的存在。在脊椎动物中，这种翻译后修饰主要存在于神经细胞粘附分子（NCAM）上，它通过两种聚唾液酸转移酶ST 8 SiaII或ST 8 SiaIV中的任一种在N-聚糖上合成。与NCAM相反，对NRP 2上polySia的生物合成知之甚少。在这里，我们确定了polySia附着位点，并证明NRP 2仅被ST 8 SiaIV识别。尽管在野生型和St 8 sia 2（-/-）小鼠的骨髓来源的DC上发现了polySia-NRP 2，但是在St 8 sia 4（-/-）小鼠的DC中polySia完全丧失，尽管NRP 2表达正常。在COS-7细胞中，NRP 2与ST 8 SiaIV而非ST 8 SiaII的共表达导致聚Sia-NRP 2的形成，突出了两种聚唾液酸转移酶的不同受体特异性。值得注意的是，ST 8 SiaIV在NRP 2糖型上选择性地合成polySia，所述NRP 2糖型的特征在于存在唾液酸化的核心1和核心20-聚糖。基于全面的定点突变研究，我们将polySia附着位点定位于位于b2和c结构域之间的接头区域中的O-聚糖簇。Thr-607、-613、-614、-615、-619和-624的组合丙氨酸交换有效地阻断聚唾液酸化。恢复单个站点仅部分挽救聚唾液酸化，这表明在该簇内，polySia连接到一个以上的站点。
Neuropilin-2 (NRP2) is well known as a co-receptor for class 3 semaphorins and vascular endothelial growth factors, involved in axon guidance and angiogenesis. Moreover, NRP2 was shown to promote chemotactic migration of human monocyte-derived dendritic cells (DCs) toward the chemokine CCL21, a function that relies on the presence of polysialic acid (polySia). In vertebrates, this posttranslational modification is predominantly found on the neural cell adhesion molecule (NCAM), where it is synthesized on N-glycans by either of the two polysialyltransferases, ST8SiaII or ST8SiaIV. In contrast to NCAM, little is known on the biosynthesis of polySia on NRP2. Here we identified the polySia attachment sites and demonstrate that NRP2 is recognized only by ST8SiaIV. Although polySia-NRP2 was found on bone marrow-derived DCs from wild-type and St8sia2(-/-) mice, polySia was completely lost in DCs from St8sia4(-/-) mice despite normal NRP2 expression. In COS-7 cells, co-expression of NRP2 with ST8SiaIV but not ST8SiaII resulted in the formation of polySia-NRP2, highlighting distinct acceptor specificities of the two polysialyltransferases. Notably, ST8SiaIV synthesized polySia selectively on a NRP2 glycoform that was characterized by the presence of sialylated core 1 and core 2 O-glycans. Based on a comprehensive site-directed mutagenesis study, we localized the polySia attachment sites to an O-glycan cluster located in the linker region between b2 and c domain. Combined alanine exchange of Thr-607,-613,-614, -615,-619, and -624 efficiently blocked polysialylation. Restoration of single sites only partially rescued polysialylation, suggesting that within this cluster, polySia is attached to more than one site.