Indentification of B7H3 WT/NQ N-Glycosites

Most patients with triple negative breast cancer (TNBC) fail to respond to anti-PD1/PDL1 immunotherapy, indicating the necessity to explore immune checkpoint targets. B7H3 is a highly glycosylated protein. However, the mechanisms of B7H3 glycosylation regulation and whether the sugar moiety contributes to immunosuppression remain elusive. Here, we identify aberrant B7H3 glycosylation and found N-glycosylation of B7H3 at NXT motif sites are responsible for its protein stability and immunosuppression in TNBC tumors. Mechanistically, fucosyltransferase FUT8 catalyzes B7H3 core fucosylation at N-glycans to maintain its high expression. Knockdown of FUT8 rescues glycosylated B7H3-mediated immunosuppressive function in TNBC cells. Abnormal B7H3 glycosylation mediated by FUT8 overexpression could be physiologically significant and clinically relevant in TNBC patients. Notably, combination of core fucosylation inhibitor 2F-Fuc and anti-PDL1 results in enhanced therapeutic efficacy in B7H3-positive TNBC tumors. These suggest targeting FUT8-B7H3 axis can be a promising strategy for improving anti-tumor immune responses in TNBC patients. To obtain the direct evidence that B7H3 is N-glycosylated in TNBC cells, we analysed the peptides of purified human B7H3 protein from B7H3-WT re-expressed and B7H3-8NQ re-expressed MDA-MB-231 cell lines by Nanoscale liquid chromatography coupled to tandem MS (nano LC-MS/MS). The result showed that there were eight N-glycosylation sites (Asn positions 91, 104, 189, 215, 309, 322, 407, and 433) in B7H3-WT cells, but not in B7H3-8NQ cells, as determined by Asn to Asp conversion after PNGase F treatment. As B7H3 contains a nearly exact tandem duplication of the IgV-IgC domain, there were four pairs of N-glycosylation sites identified through identical peptide sequence, including N91 and N309, N104 and N322, N189 and N407, and N215 and N433, in each of the IgV-IgC domains. Together, the results indicate that B7H3 is exclusively N-glycosylated at these four pairs of glycosylation sites in TNBC cells.

绝大多数三阴性乳腺癌（triple negative breast cancer, TNBC）患者无法对抗PD-1/PD-L1免疫治疗产生应答，凸显了探索新型免疫检查点靶点的迫切需求。B7H3是一种高度糖基化蛋白，然而其糖基化调控机制，以及糖基部分是否参与免疫抑制过程，目前仍不明确。本研究证实了B7H3存在异常糖基化修饰，并发现B7H3在NXT基序位点的N-糖基化修饰，是其在TNBC肿瘤中维持蛋白稳定性并发挥免疫抑制作用的关键机制。从机制层面来看，岩藻糖基转移酶FUT8（fucosyltransferase FUT8）可催化B7H3的N-聚糖核心岩藻糖基化修饰，从而维持其高表达水平；敲低FUT8可逆转糖基化B7H3介导的TNBC细胞免疫抑制功能。由FUT8过表达介导的B7H3异常糖基化修饰，在TNBC患者中具有重要的生理意义与临床相关性。值得注意的是，核心岩藻糖基化抑制剂2F-Fuc与抗PD-L1药物联合使用，可显著增强B7H3阳性TNBC肿瘤的治疗效果。上述结果表明，靶向FUT8-B7H3信号轴有望成为改善TNBC患者抗肿瘤免疫应答的有效策略。为直接证实TNBC细胞中B7H3存在N-糖基化修饰，本研究通过纳米液相色谱-串联质谱（nano LC-MS/MS）分析了分别重表达B7H3野生型（B7H3-WT）与B7H3-8NQ突变体的MDA-MB-231细胞中纯化的人源B7H3蛋白的肽段。经肽N-糖苷酶F（PNGase F）处理后通过天冬酰胺（Asn）向天冬氨酸（Asp）的转化实验验证，结果显示B7H3-WT组细胞中共存在8个N-糖基化位点（分别位于天冬酰胺残基91、104、189、215、309、322、407与433位），而B7H3-8NQ组细胞中未检测到相关位点。由于B7H3的IgV-IgC结构域存在近乎完全一致的串联重复序列，通过同源肽段序列鉴定，每个IgV-IgC结构域中均存在两对N-糖基化位点：分别为N91与N309、N104与N322、N189与N407以及N215与N433。综上，上述结果证实TNBC细胞中B7H3仅在这四对糖基化位点处发生N-糖基化修饰。