Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA.
Department of Chemistry, University of Alberta, Edmonton, Canada.
Nat Commun. 2024 Nov 18;15(1):9970. doi: 10.1038/s41467-024-53225-1.
Glycans play critical roles in cellular signaling and function. Unlike proteins, glycan structures are not templated from genetic sequences but synthesized by the concerted activity of many genes, making them historically challenging to study. Here, we present a strategy that utilizes CRISPR screens and lectin microarrays to uncover and characterize regulators of glycosylation. We applied this approach to study the regulation of high mannose glycans - the starting structure of all asparagine(N)-linked-glycans. We used CRISPR screens to uncover the expanded network of genes controlling high mannose levels, followed by lectin microarrays to fully measure the complex effect of select regulators on glycosylation globally. Through this, we elucidated how two high mannose regulators - TM9SF3 and the CCC complex - control complex N-glycosylation via regulating Golgi morphology and function. Notably, this allows us to interrogate Golgi function in-depth and reveals that similar disruption to Golgi morphology can lead to drastically different glycosylation outcomes. Collectively, this work demonstrates a generalizable approach for systematically dissecting the regulatory network underlying glycosylation.
糖链在细胞信号转导和功能中起着至关重要的作用。与蛋白质不同，糖链结构不是由遗传序列模板化合成的，而是由许多基因的协同活性合成的，这使得它们在历史上难以研究。在这里，我们提出了一种利用 CRISPR 筛选和凝集素微阵列来揭示和表征糖基化调控因子的策略。我们将这种方法应用于研究高甘露糖聚糖的调控，即所有天冬酰胺（N）连接聚糖的起始结构。我们使用 CRISPR 筛选来揭示控制高甘露糖水平的基因扩展网络，然后使用凝集素微阵列全面测量选定调节剂对糖基化的复杂影响。通过这种方法，我们阐明了两个高甘露糖调节剂——TM9SF3 和 CCC 复合物——如何通过调节高尔基体形态和功能来控制复杂的 N-糖基化。值得注意的是，这使我们能够深入研究高尔基体的功能，并揭示出对高尔基体形态的类似破坏可能导致截然不同的糖基化结果。总的来说，这项工作展示了一种可用于系统剖析糖基化调控网络的通用方法。
