New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA.
Bruker, 40 Manning Road, Billerica, MA, 01821, USA.
Commun Biol. 2023 Jan 13;6(1):48. doi: 10.1038/s42003-023-04439-4.
N-glycosylation is implicated in cancers and aberrant N-glycosylation is recognized as a hallmark of cancer. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 displays hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells. This comparative site-specific N-glycoproteome analysis provides a pool of potential N-glycosylation-related cancer biomarkers, but also gives insights into the M6P pathway in cancer.
N-糖基化与癌症有关，异常的 N-糖基化被认为是癌症的一个标志。在这里，我们使用 Fbs1-GYR N-糖肽富集技术和俘获离子淌度谱法，对结肠癌 HCT116 细胞和同源非致瘤性 DNMT1/3b 双敲除（DKO1）细胞进行了定点 N-糖蛋白组学的作图和比较。揭示了许多定点 N-糖基化的显著变化，为进一步阐明 N-糖基化在蛋白质功能中的作用提供了分子基础。HCT116 细胞表现出过度唾液酸化，特别是在细胞膜蛋白中。HCT116 和 DKO1 都显示出低聚糖和 80%的低聚糖丰富蛋白丰富，这些蛋白被注释为位于外体中。最显著的 N-糖基化改变是甘露糖-6-磷酸（M6P）修饰的程度。N-糖蛋白组学分析显示，HCT116 表现出高 M6P 修饰，这通过 M6P 免疫检测得到了正交验证。在 HCT116 和 DKO1 中主要 M6P 受体 CI-MPR 的 N-糖基化模式存在显著差异，这可能导致 HCT116 细胞的高 M6P 表型。这项定点 N-糖蛋白组学比较分析提供了一组潜在的与 N-糖基化相关的癌症生物标志物，但也深入了解了癌症中的 M6P 途径。
