High-Throughput Mass Spectrometry Analysis of <i>N</i>‑Glycans and Protein Markers after <i>FUT8</i> Knockdown in the Syngeneic SW480/SW620 Colorectal Cancer Cell Model

Disruption of the glycosylation machinery is a common feature in many types of cancer, and colorectal cancer (CRC) is no exception. Core fucosylation is mediated by the enzyme fucosyltransferase 8 (FucT-8), which catalyzes the addition of α1,6-l-fucose to the innermost GlcNAc residue of N-glycans. We and others have documented the involvement of FucT-8 and core-fucosylated proteins in CRC progression, in which we addressed core fucosylation in the syngeneic CRC model formed by SW480 and SW620 tumor cell lines from the perspective of alterations in their N-glycosylation profile and protein expression as an effect of the knockdown of the FUT8 gene that encodes FucT-8. Using label-free, semiquantitative mass spectrometry (MS) analysis, we found noticeable differences in N-glycosylation patterns in FUT8-knockdown cells, affecting core fucosylation and sialylation, the Hex/HexNAc ratio, and antennarity. Furthermore, stable isotopic labeling of amino acids in cell culture (SILAC)-based proteomic screening detected the alteration of species involved in protein folding, endoplasmic reticulum (ER) and Golgi post-translational stabilization, epithelial polarity, and cellular response to damage and therapy. This data is available via ProteomeXchange with identifier PXD050012. Overall, the results obtained merit further investigation to validate their feasibility as biomarkers of progression and malignization in CRC, as well as their potential usefulness in clinical practice.糖基化机制的异常是多数癌症的共同表型，结直肠癌（colorectal cancer, CRC）亦不例外。核心岩藻糖基化由岩藻糖基转移酶8（fucosyltransferase 8, FucT-8）介导，该酶可催化α1,6-L-岩藻糖连接至N-糖链最内侧的N-乙酰葡糖胺（GlcNAc）残基。本团队与其他研究者均已证实，FucT-8及核心岩藻糖基化蛋白参与结直肠癌的进展过程；本研究以编码FucT-8的FUT8基因敲低为干预手段，从N-糖基化谱与蛋白表达变化的角度，对SW480与SW620肿瘤细胞系构建的同基因结直肠癌模型中的核心岩藻糖基化进行了探究。通过无标记半定量质谱（mass spectrometry, MS）分析，本团队在FUT8敲低细胞中观察到N-糖基化模式的显著差异，该差异影响核心岩藻糖基化与唾液酸化、己糖/己糖胺比值以及糖链天线数。此外，基于细胞培养氨基酸稳定同位素标记（stable isotopic labeling of amino acids in cell culture, SILAC）的蛋白质组筛选发现，参与蛋白质折叠、内质网（endoplasmic reticulum, ER）与高尔基体翻译后修饰稳定、上皮极性维持以及细胞损伤与治疗应答的蛋白物种发生了表达改变。本研究数据可通过ProteomeXchange获取，登录编号为PXD050012。总体而言，本研究所得结果有待进一步验证，以确认其作为结直肠癌进展与恶性化生物标志物的可行性，以及其在临床实践中的应用潜力。