Laboratory of Chemical Glycobiology (CGB), National Institute of Immunology (NII) , Aruna Asaf Ali Marg, New Delhi 110067, India.
J Am Chem Soc. 2013 Sep 25;135(38):14189-97. doi: 10.1021/ja405189k. Epub 2013 Sep 16.
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 (Ac5GalNTGc, 1) moiety-but not N-glycolyl (Ac5GalNGc, 2) and N-acetyl (Ac4GalNAc, 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-D-glucosamine (7, C-4 epimer of 1), and α-O-benzyl 2-acetamido-2-deoxy-D-galactopyranoside (8), confirming the critical requirement of both free sulfhydryl 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.
粘蛋白型 O-聚糖是细胞表面蛋白上最丰富和最复杂的翻译后修饰（PTM）之一，控制着造血细胞的黏附、迁移和运输。目前，开发针对 O-聚糖功能的靶向方法仍处于早期阶段。在几种方法中，带有 N-硫代糖基（Ac5GalNTGc，1）部分的乙酰基 N-酰基-D-半乳糖胺衍生物等具有独特化学官能团的小分子成为了关键工具。本文中，我们发现通过 N-乙酰-D-半乳糖胺（GalNAc）补救途径代谢携带 N-硫代糖基（Ac5GalNTGc，1）部分的乙酰基 N-酰基-D-半乳糖胺衍生物，但不能代谢 N-糖基（Ac5GalNGc，2）和 N-乙酰基（Ac4GalNAc，3），可导致 Jurkat 细胞 MAL-II 和 PNA 表位缺失。对 Jurkat 细胞中甲基化 O-聚糖的质谱分析证实，存在大量复杂的 O-聚糖（唾液酸-T 和二唾液酸-T），而这些 O-聚糖在 1 处理后受到抑制。1 以依赖巯基的方式显著降低富含 O-聚糖的细胞表面抗原 CD43 的 O-糖基化。相比之下，CD45 糖型的影响则较为轻微。通过免疫沉淀的 CD43-myc/FLAG 的硫醇选择性迈克尔加成反应证实了 1 的直接代谢掺入。在机制上，未乙酰化的 N-（3-乙酰硫代丙酰基）（4）、N-（4-乙酰硫代丁酰基）（5）和 N-甲基硫代乙酰基（6）半乳糖胺衍生物、N-硫代糖基-D-葡萄糖胺（7，1 的 C-4 差向异构体）和α-O-苄基 2-乙酰氨基-2-脱氧-D-半乳糖吡喃糖苷（8）对 CD43 糖型无影响，这证实了游离巯基和半乳糖胺部分对于抑制粘蛋白型 O-聚糖的关键要求。在多种造血细胞中，1 对 CD43 糖型也表现出类似但又不同的作用。开发能够以抗原选择性和细胞类型选择性方式改变聚糖模式的小分子可能为理解聚糖的生物学功能提供途径。
