National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
Department of Molecular Microbiology, Washington University, St. Louis, MO, 63130, USA.
Biochem Biophys Res Commun. 2018 Nov 17;506(1):60-65. doi: 10.1016/j.bbrc.2018.08.131. Epub 2018 Oct 15.
Investigations on the structure and functional roles of glycosylation - an intricate, complex, and dynamic post translational modification on proteins - in biological processes has been a challenging task. Glycan modifications vary depending on the specific cell type, its developmental stage, and resting or activated state. In the present study, we aim to understand the differences between the mucin-type O-glycosylation (MTOG) of two functionally divergent human cell lines, K562 (chronic myeloid leukemia) and U937 (histiocytic lymphoma), having myeloid origins. MTOG is initiated by the addition of N-acetyl-α-d-galactosamine (GalNAc) to Ser/Thr of glycoproteins. We exploited the metabolic glycan engineering (MGE) strategy using the peracetyl N-thioglycolyl-d-galactosamine (AcGalNTGc), a synthetic GalNAc analogue, to engineer the glycoconjugates. AcGalNTGc was metabolized and incorporated as N-thioglycolyl-d-galactosamine (GalNTGc) in cell surface glycoproteins in both the cell lines with varying degrees of efficiency. Notably, metabolic incorporation of GalNTGc resulted in differential inhibition of MTOG. It was observed that endogenous glycosylation machinery of K562 is relatively more stringent for selecting GalNTGc whereas U937 is flexible towards this selection. Additionally, we studied how the glycan modifications vary on a given CD antigen in these cell lines. Particularly, MTOG on CD43 was differentially inhibited in K562 and U937 as revealed by glycan-dependent and glycan-independent antibodies. It was observed that the effect of MGE on CD43 was similar to global effects on both cell lines. Consequences of MGE using GalNAc analogues depend on the expression and activity of various glycosyl transferases which determine global glycosylation on cell surface as well as on specific glycoproteins.
研究糖基化的结构和功能作用——蛋白质上一种复杂、动态的翻译后修饰——一直是一项具有挑战性的任务。糖基化修饰因特定的细胞类型、其发育阶段、静止或激活状态而异。在本研究中，我们旨在了解具有髓系起源的两种功能不同的人细胞系（慢性髓系白血病的 K562 和组织细胞淋巴瘤的 U937）之间的粘蛋白型 O-糖基化（MTOG）的差异。MTOG 是通过在糖蛋白的 Ser/Thr 上添加 N-乙酰-α-d-半乳糖胺（GalNAc）而启动的。我们利用代谢糖基化工程（MGE）策略，使用乙酰基 N-硫代甘氨酰-d-半乳糖胺（AcGalNTGc），一种合成的 GalNAc 类似物，来设计糖缀合物。AcGalNTGc 在两种细胞系的细胞表面糖蛋白中被代谢并掺入，效率不同。值得注意的是，GalNTGc 的代谢掺入导致 MTOG 的差异抑制。观察到 K562 的内源性糖基化机制在选择 GalNTGc 时相对更为严格，而 U937 对此选择较为灵活。此外，我们研究了这些细胞系中给定 CD 抗原上的聚糖修饰如何变化。特别是，通过依赖聚糖和不依赖聚糖的抗体，观察到 K562 和 U937 中 CD43 的 MTOG 受到不同程度的抑制。观察到 MGE 对 CD43 的影响类似于对两种细胞系的全局影响。使用 GalNAc 类似物的 MGE 的后果取决于各种糖基转移酶的表达和活性，这些酶决定了细胞表面的全局糖基化以及特定糖蛋白的糖基化。
