免疫疗法彻底改变了癌症治疗，但缺乏可靠的治疗反应预测生物标志物仍然是一个挑战。 Alpha-1,6-甘露糖基糖蛋白 6-β-N-乙酰氨基葡萄糖转移酶 5 (MGAT5) 是复杂 N-聚糖合成的关键调节因子，其失调与癌症进展相关。凝集素菜豆白细胞凝集素 (PHA-L) 特异性结合成熟的 MGAT5 产物。先前的研究表明头颈鳞状细胞癌 (HNSCC) 中 PHA-L 染色升高，这意味着 MGAT5 活性增加。然而，MGAT5 在 HNSCC 中的具体作用仍不清楚。在这项研究中，我们发现与邻近的非肿瘤组织相比，HNSCC 肿瘤中的 PHA-L 染色和 MGAT5 表达显着更高。使用基于质谱 (MS) 的糖蛋白组学方法，我们鉴定了 MGAT5 的 163 种潜在蛋白质底物。功能分析表明 MGAT5 的蛋白质底物调节与 T 细胞增殖和激活相关的途径。我们进一步发现PD-L1是MGAT5的蛋白质​​底物之一，MGAT5的表达可以保护肿瘤细胞免受细胞毒性T淋巴细胞（CTL）的杀伤。纳武单抗治疗减轻了 MGAT5 对 CTL 活性的保护作用。一致的是，与 MGAT5 阴性肿瘤患者相比，MGAT5 阳性肿瘤患者对免疫治疗的反应有所改善。使用从 HNSCC 细胞中纯化的 PD-L1 和糖蛋白组学方法，我们进一步破译了 N35 和 N200 位点携带 PD-L1 上的大部分复杂 N-聚糖。我们的研究结果强调了 MGAT5 介导的 PD-L1 分支 N-聚糖在调节与免疫检查点受体 PD-1 相互作用方面的关键作用。因此，我们建议 MGAT5 可以作为生物标志物来预测患者对抗 PD-1 治疗的反应。此外，针对 PD-L1 N35 和 N200 处的分支 N-聚糖可能会导致新的诊断和治疗方法的开发。© 2024。作者。

Immunotherapy has revolutionized cancer treatment, but the lack of a reliable predictive biomarker for treatment response remains a challenge. Alpha-1,6-Mannosylglycoprotein 6-β-N-Acetylglucosaminyltransferase 5 (MGAT5) is a key regulator of complex N-glycan synthesis, and its dysregulation is associated with cancer progression. The lectin Phaseolus vulgaris leukoagglutinin (PHA-L) specifically binds to mature MGAT5 products. Previous studies have indicated elevated PHA-L staining in head and neck squamous cell carcinoma (HNSCC), which implies increased activity of MGAT5. However, the specific role of MGAT5 in HNSCC remains unclear. In this study, we found significantly higher PHA-L staining and MGAT5 expression in HNSCC tumors compared to adjacent non-tumor tissues. Using a mass spectrometry (MS)-based glycoproteomic approach, we identified 163 potential protein substrates of MGAT5. Functional analysis revealed that protein substrates of MGAT5 regulated pathways related to T cell proliferation and activation. We further discovered that PD-L1 was among the protein substrates of MGAT5, and the expression of MGAT5 protected tumor cells from cytotoxic T lymphocyte (CTL) killing. Treatment of nivolumab alleviated the protective effects of MGAT5 on CTL activity. Consistently, patients with MGAT5-positive tumors showed improved responses to immunotherapy compared to those with MGAT5-negative tumors. Using purified PD-L1 from HNSCC cells and a glycoproteomic approach, we further deciphered that the N35 and N200 sites carry the majority of complex N-glycans on PD-L1. Our findings highlight the critical role of MGAT5-mediated branched N-glycans on PD-L1 in modulating the interaction with the immune checkpoint receptor PD-1. Consequently, we propose that MGAT5 could serve as a biomarker to predict patients' responses to anti-PD-1 therapy. Furthermore, targeting the branched N-glycans at N35 and N200 of PD-L1 may lead to the development of novel diagnostic and therapeutic approaches.© 2024. The Author(s).