Comprehensive N-glycosylation profiling of recombinant spike S1 protein from the wild-type SARS-CoV-2 and its variants
IntroductionBy 2024, COVID-19 has become endemic, with new variants contributing to its continued spread. The Spike protein forms trimers that bind to the ACE2 receptor on host cells, with the S1 subunit being a primary target for vaccines and antiviral treatments.MethodsHerein, we performed an in-d...
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Frontiers Media S.A.
2025-07-01
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| author | Yao Peng Yao Peng Yao Peng Tian-Tian Tong Qiu-Yu Deng Lee-Fong Yau Jia-Qi Qiu Qing Zhao Jia-Qi Wu Zhi-Qiang Xin Man-Ci Guan Yue Li Zhi-Hong Jiang Hu-Dan Pan Hu-Dan Pan Liang Liu Liang Liu Liang Liu Jing-Rong Wang Jing-Rong Wang |
| author_facet | Yao Peng Yao Peng Yao Peng Tian-Tian Tong Qiu-Yu Deng Lee-Fong Yau Jia-Qi Qiu Qing Zhao Jia-Qi Wu Zhi-Qiang Xin Man-Ci Guan Yue Li Zhi-Hong Jiang Hu-Dan Pan Hu-Dan Pan Liang Liu Liang Liu Liang Liu Jing-Rong Wang Jing-Rong Wang |
| author_sort | Yao Peng |
| collection | DOAJ |
| description | IntroductionBy 2024, COVID-19 has become endemic, with new variants contributing to its continued spread. The Spike protein forms trimers that bind to the ACE2 receptor on host cells, with the S1 subunit being a primary target for vaccines and antiviral treatments.MethodsHerein, we performed an in-depth analysis of the N-glycosylation of the recombinant Spike S1 protein (S1 protein) across the wild-type (WT) virus and its 5 variants, including Alpha, Beta, Gamma, Delta, and Lambda, by integrating ultrahigh-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF MS) and unique TiO₂-PGC chip-based LC/MS techniques.ResultsA total of 332 glycan structures arising from 180 compositions on the S1 and RBD regions were identified, revealing remarkable glycosylation diversity of the S1 protein. Complex glycan was shown to be the dominant structure across variants. Neutral N-glycans are mainly di-antennary with two fucosyl groups, while the majority of acidic N-glycans were multi-antennary with mono-fucosyl residues. In addition, sialic acid linkages of the N-glycans were extensively studied by utilizing ¹³C-labeled standards and specific enzymes for the first time, showing the existence of both α-2,3 and α-2,6 linkages across WT and five variants. It should be noted that the Lambda variant shows more complex α-2,3 and α-2,6-linked glycans in the RBD region, which may potentially enhance its glycan shield effect. Acetylated glycans, which were identified on S protein for the first time, were found to be fully fucosylated on the S1 region and sialylated on the RBD region across all variants. UHPLC-TOF MS analysis revealed unoccupied N-glycosylation sites in S1-Gamma (N657), S1-Delta (N61), and S1-Lambda (N17, N61, N657), with N17 and N61 showing low glycan occupancy (0%-3.4%), suggesting these sites may lack glycan shield protection.DiscussionThis study provides a comprehensive N-glycosylation profile of the S1 protein across different variants, offering an essential structural basis for future vaccine development and research on viral functions. |
| format | Article |
| id | doaj-art-54cfddb8c63d4fcfbb3a71522a875e2f |
| institution | Kabale University |
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| language | English |
| publishDate | 2025-07-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Immunology |
| spelling | doaj-art-54cfddb8c63d4fcfbb3a71522a875e2f2025-08-20T03:51:25ZengFrontiers Media S.A.Frontiers in Immunology1664-32242025-07-011610.3389/fimmu.2025.15921421592142Comprehensive N-glycosylation profiling of recombinant spike S1 protein from the wild-type SARS-CoV-2 and its variantsYao Peng0Yao Peng1Yao Peng2Tian-Tian Tong3Qiu-Yu Deng4Lee-Fong Yau5Jia-Qi Qiu6Qing Zhao7Jia-Qi Wu8Zhi-Qiang Xin9Man-Ci Guan10Yue Li11Zhi-Hong Jiang12Hu-Dan Pan13Hu-Dan Pan14Liang Liu15Liang Liu16Liang Liu17Jing-Rong Wang18Jing-Rong Wang19School of Pharmacy, Faculty of Medicine & Faculty of Chinese Medicine, Macau University of Science and Technology, Macao, Macao SAR, ChinaChinese Medicine Guangdong Laboratory, Guangdong-Macao In-Depth Cooperation Zone in Hengqin, Zhuhai, ChinaState Key Laboratory of Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, ChinaState Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macao, Macao SAR, ChinaState Key Laboratory of Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, ChinaState Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macao, Macao SAR, ChinaState Key Laboratory of Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, ChinaState Key Laboratory of Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, ChinaState Key Laboratory of Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, ChinaState Key Laboratory of Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, ChinaState Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macao, Macao SAR, ChinaState Key Laboratory of Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, ChinaState Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macao, Macao SAR, ChinaChinese Medicine Guangdong Laboratory, Guangdong-Macao In-Depth Cooperation Zone in Hengqin, Zhuhai, ChinaState Key Laboratory of Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, ChinaSchool of Pharmacy, Faculty of Medicine & Faculty of Chinese Medicine, Macau University of Science and Technology, Macao, Macao SAR, ChinaChinese Medicine Guangdong Laboratory, Guangdong-Macao In-Depth Cooperation Zone in Hengqin, Zhuhai, ChinaState Key Laboratory of Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, ChinaChinese Medicine Guangdong Laboratory, Guangdong-Macao In-Depth Cooperation Zone in Hengqin, Zhuhai, ChinaState Key Laboratory of Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, ChinaIntroductionBy 2024, COVID-19 has become endemic, with new variants contributing to its continued spread. The Spike protein forms trimers that bind to the ACE2 receptor on host cells, with the S1 subunit being a primary target for vaccines and antiviral treatments.MethodsHerein, we performed an in-depth analysis of the N-glycosylation of the recombinant Spike S1 protein (S1 protein) across the wild-type (WT) virus and its 5 variants, including Alpha, Beta, Gamma, Delta, and Lambda, by integrating ultrahigh-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF MS) and unique TiO₂-PGC chip-based LC/MS techniques.ResultsA total of 332 glycan structures arising from 180 compositions on the S1 and RBD regions were identified, revealing remarkable glycosylation diversity of the S1 protein. Complex glycan was shown to be the dominant structure across variants. Neutral N-glycans are mainly di-antennary with two fucosyl groups, while the majority of acidic N-glycans were multi-antennary with mono-fucosyl residues. In addition, sialic acid linkages of the N-glycans were extensively studied by utilizing ¹³C-labeled standards and specific enzymes for the first time, showing the existence of both α-2,3 and α-2,6 linkages across WT and five variants. It should be noted that the Lambda variant shows more complex α-2,3 and α-2,6-linked glycans in the RBD region, which may potentially enhance its glycan shield effect. Acetylated glycans, which were identified on S protein for the first time, were found to be fully fucosylated on the S1 region and sialylated on the RBD region across all variants. UHPLC-TOF MS analysis revealed unoccupied N-glycosylation sites in S1-Gamma (N657), S1-Delta (N61), and S1-Lambda (N17, N61, N657), with N17 and N61 showing low glycan occupancy (0%-3.4%), suggesting these sites may lack glycan shield protection.DiscussionThis study provides a comprehensive N-glycosylation profile of the S1 protein across different variants, offering an essential structural basis for future vaccine development and research on viral functions.https://www.frontiersin.org/articles/10.3389/fimmu.2025.1592142/fullSARS-CoV-2spike proteinvariantsN-glycanschip LC/MSN-glycopeptides |
| spellingShingle | Yao Peng Yao Peng Yao Peng Tian-Tian Tong Qiu-Yu Deng Lee-Fong Yau Jia-Qi Qiu Qing Zhao Jia-Qi Wu Zhi-Qiang Xin Man-Ci Guan Yue Li Zhi-Hong Jiang Hu-Dan Pan Hu-Dan Pan Liang Liu Liang Liu Liang Liu Jing-Rong Wang Jing-Rong Wang Comprehensive N-glycosylation profiling of recombinant spike S1 protein from the wild-type SARS-CoV-2 and its variants Frontiers in Immunology SARS-CoV-2 spike protein variants N-glycans chip LC/MS N-glycopeptides |
| title | Comprehensive N-glycosylation profiling of recombinant spike S1 protein from the wild-type SARS-CoV-2 and its variants |
| title_full | Comprehensive N-glycosylation profiling of recombinant spike S1 protein from the wild-type SARS-CoV-2 and its variants |
| title_fullStr | Comprehensive N-glycosylation profiling of recombinant spike S1 protein from the wild-type SARS-CoV-2 and its variants |
| title_full_unstemmed | Comprehensive N-glycosylation profiling of recombinant spike S1 protein from the wild-type SARS-CoV-2 and its variants |
| title_short | Comprehensive N-glycosylation profiling of recombinant spike S1 protein from the wild-type SARS-CoV-2 and its variants |
| title_sort | comprehensive n glycosylation profiling of recombinant spike s1 protein from the wild type sars cov 2 and its variants |
| topic | SARS-CoV-2 spike protein variants N-glycans chip LC/MS N-glycopeptides |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1592142/full |
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