Robustness in glycosylation systems: effect of modified monosaccharides, acceptor decoys and azido sugars on cellular nucleotide-sugar levels and pattern of N-linked glycosylation.,Molecular Omics

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Robustness in glycosylation systems: effect of modified monosaccharides, acceptor decoys and azido sugars on cellular nucleotide-sugar levels and pattern of N-linked glycosylation.
Molecular Omics ( IF 3.0 ) Pub Date : 2020-04-22 , DOI: 10.1039/d0mo00023j
Virginia Del Solar ₁ , Rohitesh Gupta , Yusen Zhou , Gabrielle Pawlowski , Khushi L Matta , Sriram Neelamegham

Affiliation

Small molecule monosaccharide analogs (e.g. 4F-GlcNAc, 4F-GalNAc) and acceptor decoys (e.g. ONAP, SNAP) are commonly used as metabolic glycoengineering tools to perturb molecular and cellular recognition processes. Azido-derivatized sugars (e.g. ManNAz, GlcNAz, GalNAz) are also used as bioorthogonal probes to assay the glycosylation status of cells and tissue. With the goal of obtaining a systems-level understanding of how these compounds work, we cultured cells with these molecules and systematically evaluated their impact on: (i) cellular nucleotide-sugar levels, and (ii) N-linked glycosylation. To this end, we developed a streamlined, simple workflow to quantify nucleotide-sugar levels using amide-based hydrophilic interaction liquid chromatography (HILIC) separation followed by negative-mode electrospray ionization mass spectrometry (ESI-MS/MS) using an Orbitrap detector. N-Glycans released from cells were also procainamide functionalized and quantified using positive-mode ESI-MS/MS. Results show that all tested compounds changed the baseline nucleotide-sugar levels, with the effect being most pronounced for the fluoro-HexNAc compounds. These molecules depressed UDP-HexNAc levels in cells by up to 80%, while concomitantly elevating UDP-4F-GalNAc and UDP-4F-GlcNAc. While the measured changes in nucleotide-sugar concentration were substantial in many cases, their impact on N-linked glycosylation was relatively small. This may be due to the high nucleotide-sugar concentrations in the Golgi, which far exceed the K_M values of the glycosylating enzymes. Thus, the glycosylation system output exhibits ‘robustness’ even in the face of significant changes in cellular nucleotide-sugar concentrations.

中文翻译：

糖基化系统的稳健性：修饰的单糖、受体诱饵和叠氮基糖对细胞核苷酸-糖水平和 N-连接糖基化模式的影响。

小分子单糖类似物（例如4F-GlcNAc、4F-GalNAc）和受体诱饵（例如ONAP、SNAP）通常用作代谢糖工程工具来干扰分子和细胞识别过程。叠氮衍生的糖类（例如ManNAz、GlcNAz、GalNAz) 也用作生物正交探针来检测细胞和组织的糖基化状态。为了获得对这些化合物如何工作的系统级理解，我们用这些分子培养细胞并系统地评估它们对以下方面的影响：（i）细胞核苷酸 - 糖水平，和（ii）N-连接糖基化。为此，我们开发了一种简化的工作流程，使用基于酰胺的亲水相互作用液相色谱 (HILIC) 分离和使用 Orbitrap 检测器的负模式电喷雾电离质谱 (ESI-MS/MS) 来量化核苷酸 - 糖水平。从细胞中释放的 N-聚糖也被普鲁卡因胺功能化并使用正模式 ESI-MS/MS 进行定量。结果表明，所有测试的化合物都改变了基线核苷酸 - 糖水平，氟-HexNAc 化合物的效果最为显着。这些分子将细胞中的 UDP-HexNAc 水平降低了 80%，同时提高了 UDP-4F-GalNAc 和 UDP-4F-GlcNAc。虽然在许多情况下测得的核苷酸-糖浓度变化很大，但它们对 N-连接糖基化的影响相对较小。这可能是由于高尔基体中的高核苷酸糖浓度，远远超过糖基化酶的K _M值。因此，即使面对细胞核苷酸-糖浓度的显着变化，糖基化系统输出也表现出“稳健性”。

更新日期：2020-04-22

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