Tyrosine sulfation on a PSGL-1 glycopeptide influences the reactivity of glycosyltransferases responsible for synthesis of the attached O-glycan
Tyrosine sulfation on a PSGL-1 glycopeptide influences the reactivity of glycosyltransferases responsible for synthesis of the attached O-glycan
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DOI：
10.1021/ja993820o
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发表时间：
2000-02-02
影响因子：
15
通讯作者：
Wong, CH
中科院分区：
文献类型：
--
作者：
Koeller, KM;Smith, MEB;Wong, CH
P-选择素糖蛋白配体-1 (PSGL-1) 是炎症反应中白细胞外渗过程中 P-选择素的主要反受体。 1 此前已确定，P-选择素对 PSGL-1 的最佳识别需要连接至苏氨酸 16 的 O-聚糖和 N 端 19 个氨基酸内的至少一个酪氨酸硫酸化位点。 2 事实上，N 端糖磺肽与 P-选择素的结合效率几乎与全长二聚体 PSGL-1 一样有效，并且硫酸化糖肽与受体的结合比未硫酸化糖肽配体的结合紧密程度大约为 105 倍（图 1a）。 3 PSGL-1 的 O 连接聚糖结构包括从核心 2 聚糖延伸的末端唾液酸路易斯 x (sLex) 四糖（图 1b）。 4为了进一步检查 PSGL-1/P-选择素识别以及研究 PSGL-1 生物合成，对 PSGL-1 的结合决定簇进行了化学酶合成。目标结构代表含有硫酸酪氨酸和糖基化苏氨酸残基的最小序列，对应于成熟PSGL-1蛋白的氨基酸残基10-17。该合成结合了溶液和固相方法，得到了硫酸化和非硫酸化形式的二糖连接八肽。然后研究了糖基转移酶催化的聚糖的加工。 5 结果表明，酪氨酸的硫酸化会影响糖基转移酶的反应性，这些糖基转移酶负责在附着的 O-聚糖上合成 sLex。合成策略涉及将受保护的二糖连接的苏氨酸结构单元纳入固相肽合成中（方案 1）。二糖-苏氨酸缀合物3是通过16与糖基供体2a7在BF3-OEt2催化下反应或与供体2b8与DMTST作为活化剂反应获得的。 9 3 至 5 的转化是通过标准合成操作完成的。使用 Rink Amide 改性树脂作为固相，将结构单元 5 掺入糖肽 6（方案 2）。 N 末端乙酰化后，用 95% TFA、H2O 和乙二硫醇作为清除剂处理序列 6。这些条件导致序列从树脂中以C-末端酰胺的形式同时释放，并除去tBu酯和醚保护基团。获得的粗肽最初通过乙醚沉淀和小部分纯化，然后使用RP-HPLC进一步纯化，得到序列7。 10 然后用三氧化硫-吡啶络合物完成糖肽7的酪氨酸残基的硫酸化。涉及甲醇淬灭和立即硅胶色谱11的后处理方案允许以比之前报道的更高的产率分离硫酸化序列。 12 醋酸酯皂化，得到脱保护的糖肽 8a，用于随后的糖基转移酶催化
P-Selectin glycoprotein ligand-1 (PSGL-1) is the primary counter-receptor for P-selectin during leukocyte extravasation in the inflammatory response. 1 Previously, it has been determined that the O-glycan attached to threonine 16 and at least one site of tyrosine sulfation within the N-terminal 19 amino acids are required for optimal recognition of PSGL-1 by P-selectin. 2 In fact, the N-terminal glycosulfopeptide binds to P-selectin nearly as efficiently as the full-length dimeric PSGL-1, and the sulfated glycopeptide binds to the receptor approximately 105 times more tightly than does the unsulfated glycopeptide ligand (Figure 1a). 3 The O-linked glycan structure from PSGL-1 includes a terminal sialyl Lewis x (sLex) tetrasaccharide extended from a core 2 glycan (Figure 1b). 4To further examine PSGL-1/P-selectin recognition, as well as to investigate PSGL-1 biosynthesis, the chemoenzymatic synthesis of a binding determinant of PSGL-1 was undertaken. The target structure represents a minimal sequence containing a tyrosine sulfate and the glycosylated threonine residue, corresponding to amino acid residues 10-17 of the mature PSGL-1 protein. The synthesis combined solution-and solid-phase methods to arrive at a disaccharide-linked octapeptide in both sulfated and unsulfated forms. Glycosyltransferase-catalyzed elaboration of the glycan was then studied. 5 Results indicate that sulfation on tyrosine influences the reactivity of the glycosyltransferases responsible for the synthesis of sLex on the attached O-glycan. The synthetic strategy involved incorporation of a protected disaccharide-linked threonine building block into solid-phase peptide synthesis (Scheme 1). Disaccharide-threonine conjugate 3 was obtained by reaction of 16 with either glycosyl donor 2a7 under BF3-OEt2 catalysis or donor 2b8 with DMTST as the activating agent. 9 Conversion of 3 to 5 was accomplished by standard synthetic manipulations. Building block 5 was incorporated into glycopeptide 6 utilizing a Rink Amide modified resin as the solid phase (Scheme 2). Following N-terminal acetylation, sequence 6 was treated with 95% TFA, H2O, and ethane dithiol as a scavenger. These conditions caused simultaneous liberation of the sequence from the resin as the C-terminal amide and removal of the tBu ester and ether protecting groups. The crude peptide obtained was initially purified by ether precipitation and small portions then further purified using RP-HPLC to give sequence 7. 10 Sulfation on the tyrosine residue of glycopeptide 7 was then accomplished with sulfur trioxide-pyridine complex. A workup protocol involving a methanol quench and immediate silica gel chromatography11 allowed the sulfated sequence to be isolated in much higher yields than has been previously reported. 12 Saponification of the acetate esters then gave deprotected glycopeptide 8a for the subsequent glycosyltransferase-catalyzed