Das Tuhin, Luo Shuhong, Wang Panning, Fang Jianmin, Shajahan Asif, Peppi Lauren, Dash Sabyasachi, Maravillas Kino, Wickramasekara Rochelle N, Azadi Parastoo, Huang Ruo-Pan
RayBiotech Inc, 3607 Parkway Lane, Peachtree Corners, Atlanta, GA 30092, USA.
RayBiotech Guangzhou Co. Ltd. Guangzhou, Guangzhou 510000, China.
Biochim Biophys Acta Proteins Proteom. 2025 Sep 1;1873(5):141089. doi: 10.1016/j.bbapap.2025.141089. Epub 2025 Jul 14.
The binding of the SARS-CoV-2 spike (S) glycoprotein to human host receptors, including ACE2, NRP1, and AXL, is essential for viral entry. Glycosylation of both the spike protein and its host receptors can significantly influence these interactions. While NRP1 is recognized as a key host receptor, the role of its glycosylation in spike binding and viral infectivity has not been fully elucidated. In this study, we developed a spike-NRP1 binding assay using recombinant proteins in HEK293 cells and performed LC-MS-based glycoproteomic analysis to characterize NRP1 glycosylation. We identified three N-linked glycosylation sites (N150, N261, N522) and four O-linked glycosylation sites (S612, S637, T638, S641) on NRP1. Mutational analysis revealed that glycosylation at these specific sites is critical for spike binding, as single-site mutants showed significantly reduced interaction. Further characterization of terminal sialic acid linkages, bisecting GlcNAc, and fucosylation patterns provided insight into the complexity of NRP1 glycosylation. Functional assays demonstrated that loss of glycosylation impaired SARS-CoV-2 pseudovirus entry and altered IL-6 secretion, indicating a broader role in host immune modulation. These findings demonstrate that site-specific NRP1 glycosylation modulates spike binding and viral entry in a cell-based model, providing a foundation for future studies to explore the potential of targeting glycosylation-dependent mechanisms in SARS-CoV-2 infection.
严重急性呼吸综合征冠状病毒2(SARS-CoV-2)刺突(S)糖蛋白与包括血管紧张素转换酶2(ACE2)、神经纤毛蛋白1(NRP1)和AXL在内的人类宿主受体的结合对于病毒进入至关重要。刺突蛋白及其宿主受体的糖基化可显著影响这些相互作用。虽然NRP1被认为是一种关键的宿主受体,但其糖基化在刺突结合和病毒感染性中的作用尚未完全阐明。在本研究中,我们利用重组蛋白在人胚肾293(HEK293)细胞中开发了一种刺突-NRP1结合试验,并进行了基于液相色谱-质谱联用(LC-MS)的糖蛋白组学分析,以表征NRP1的糖基化。我们在NRP1上鉴定出三个N-连接糖基化位点(N150、N261、N522)和四个O-连接糖基化位点(S612、S637、T638、S641)。突变分析表明,这些特定位点的糖基化对于刺突结合至关重要,因为单点突变体显示相互作用显著降低。对末端唾液酸连接、平分型N-乙酰葡糖胺和岩藻糖基化模式的进一步表征揭示了NRP1糖基化的复杂性。功能试验表明,糖基化缺失会损害SARS-CoV-2假病毒进入并改变白细胞介素-6(IL-6)分泌,表明其在宿主免疫调节中具有更广泛的作用。这些发现表明,在基于细胞的模型中,位点特异性NRP1糖基化调节刺突结合和病毒进入,为未来研究探索靶向SARS-CoV-2感染中糖基化依赖性机制的潜力奠定了基础。