Inhibition of Mucin-Type O-Glycosylation through Metabolic Processing and Incorporation of N-Thioglycolyl-D-galactosamine Peracetate (Ac5GalNTGc)
Inhibition of Mucin-Type O-Glycosylation through Metabolic Processing and Incorporation of N-Thioglycolyl-D-galactosamine Peracetate (Ac5GalNTGc)
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DOI：
10.1021/ja405189k
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发表时间：
2013-09-25
影响因子：
15
通讯作者：
Sampathkumar, Srinivasa-Gopalan
中科院分区：
文献类型：
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作者：
Agarwal, Kavita;Kaul, Rachna;Sampathkumar, Srinivasa-Gopalan
粘蛋白型o -聚糖在细胞表面蛋白上形成最丰富和最复杂的翻译后修饰（PTM）之一，控制造血细胞的粘附、迁移和运输。针对o -聚糖功能的探测方法尚处于早期阶段。在几种方法中，具有独特化学官能团的小分子可以调节聚糖的生物合成，这是一种关键的工具。本研究表明，在Jurkat细胞中，携带n -巯基（Ac(5)GalNTGc, 1）片段的过乙酰n -酰基- d -半乳糖胺衍生物通过n -乙酰- d -半乳糖胺（GalNAc）挽救途径代谢，而不携带n -巯基（Ac(5)GalNGc, 2）和n -乙酰基（Ac(4)GalNAc, 3）片段，诱导MAL-II和PNA表位的消除。来自Jurkat细胞的过甲基化o -聚糖质谱分析证实了大量精化o -聚糖（唾液酰t和双尿酰t）的存在，它们在1处理后被抑制。富含o -聚糖的细胞表面抗原CD43的o -糖基化以巯基依赖的方式急剧减少1。相比之下，CD45糖型仅观察到轻微的影响。通过免疫沉淀CD43-myc/FLAG的巯基选择性Michael加成反应证实了1的直接代谢掺入。从机制上看，CD43糖型不受过乙酰化的N-（3-乙酰基硫代丙炔基）(4)、N-（4-乙酰基硫代丁烷基）(5)、N-甲基硫代乙酰基(6)半乳糖胺衍生物、N-巯基氨基-o-葡萄糖糖苷（7，C-4外聚体1）和α -o-苄基2-乙酰氨基-2-脱氧-o-半乳糖苷(8)的干扰，证实了游离巯基和半乳糖胺部分对抑制黏液型o-聚糖的关键要求。在多种造血细胞中观察到1对CD43糖型的类似但不同的影响。以抗原选择和细胞类型选择的方式改变聚糖模式的小分子的发展可能为了解聚糖的生物学功能提供途径。
Mucin-type O-glycans form one of the most abundant and complex post-translational modifications (PTM) on cell surface proteins that govern adhesion, migration, and trafficking of hematopoietic cells. Development of targeted approaches to probe functions of O-glycans is at an early stage. Among several approaches, small molecules with unique chemical functional groups that could modulate glycan biosynthesis form a critical tool. Herein, we show that metabolism of peracetyl N-acyl-D-galactosamine derivatives carrying an N-thioglycolyl (Ac(5)GalNTGc, 1) moiety but not N-glycolyl (Ac(5)GalNGc, 2) and N-acetyl (Ac(4)GalNAc, 3) through the N-acetyl-D-galactosamine (GalNAc) salvage pathway induced abrogation of MAL-II and PNA epitopes in Jurkat cells. Mass spectrometry of permethylated O-glycans from Jurkat cells confirmed the presence of significant amounts of elaborated O-glycans (sialyl-T and disialyl-T) which were inhibited upon treatment with 1. O-Glycosylation of CD43, a cell surface antigen rich in O-glycans, was drastically reduced by 1 in a thiol-dependent manner. By contrast, only mild effects were observed for CD45 glycoforms. Direct metabolic incorporation of 1 was confirmed by thiol-selective Michael addition reaction of immunoprecipitated CD43-myc/FLAG. Mechanistically, CD43 glycoforms were unperturbed by peracetylated N-(3-acetylthiopropanoyl) (4), N-(4-acetylthiobutanoyl) (5), and N-methylthioacetyl (6) galactosamine derivatives, N-thioglycolyl-o-glucosarnine (7, C-4 epimer of 1), and alpha-O-benzyl 2-acetamido-2-deoxy-o-galactopyranoside (8), confirming the critical requirement of both free sulthydryl and galactosamine moieties for inhibition of mucin-type O-glycans. Similar, yet differential, effects of 1 were observed for CD43 glycoforms in multiple hematopoietic cells. Development of small molecules that could alter glycan patterns in an antigen-selective and cell-type selective manner might provide avenues for understanding biological functions of glycans.