Proximity Labeling-Based Identification of MGAT3 Substrates and Revelation of the Tumor-Suppressive Role of Bisecting GlcNAc in Breast Cancer via GLA Degradation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plasmid Construction
2.2. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)
2.3. Cell Lines and Cell Culture
2.4. Construction of Gene Knockin Cell Lines
2.5. Lentivirus-Mediated Stable Knockdown and Overexpression of GLA
2.6. Western Blot and Lectin Blot
2.7. CCK-8 Assay
2.8. Transwell Assay
2.9. Flow Cytometry
2.10. On-Beads Digestion
2.11. Intact Glycopeptide Enrichment
2.12. Immunoprecipitation-Mass Spectrometry
2.13. Liquid Chromatography Coupled to Tandem Mass Spectrometry
2.14. Data Analysis
3. Results
3.1. Validation of Proximity Labeling Tools in Living Cells
3.2. Profiling of the MGAT3 Proximitome Labeled by BASU and TurboID
3.3. Identification of Novel MGAT3 Substrates in the MGAT3 Proximitome
3.4. Bisecting GlcNAc-Facilitated GLA Degradation in Breast Cancer Cells
3.5. Bisecting GlcNAc Inhibited the Tumor-Promoting Effect of GLA in Breast Cancer Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Schjoldager, K.T.; Narimatsu, Y.; Joshi, H.J.; Clausen, H. Global view of human protein glycosylation pathways and functions. Nat. Rev. Mol. Cell Biol. 2020, 21, 729–749. [Google Scholar] [CrossRef] [PubMed]
- Chu, Y.-D.; Fan, T.-C.; Lai, M.-W.; Yeh, C.-T. GALNT14-mediated O-glycosylation on PHB2 serine-161 enhances cell growth, migration and drug resistance by activating IGF1R cascade in hepatoma cells. Cell Death Dis. 2022, 13, 956. [Google Scholar] [CrossRef]
- Clausen, T.M.; Sandoval, D.R.; Spliid, C.B.; Pihl, J.; Perrett, H.R.; Painter, C.D.; Narayanan, A.; Majowicz, S.A.; Kwong, E.M.; McVicar, R.N.; et al. SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2. Cell 2020, 183, 1043–1057.e15. [Google Scholar] [CrossRef] [PubMed]
- Sharma, P.; Zhang, X.; Ly, K.; Kim, J.H.; Wan, Q.; Kim, J.; Lou, M.; Kain, L.; Teyton, L.; Winau, F. Hyperglycosylation of prosaposin in tumor dendritic cells drives immune escape. Science 2024, 383, 190–200. [Google Scholar] [CrossRef]
- Li, J.; Zhang, J.; Xu, M.; Yang, Z.; Yue, S.; Zhou, W.; Gui, C.; Zhang, H.; Li, S.; Wang, P.G.; et al. Advances in glycopeptide enrichment methods for the analysis of protein glycosylation over the past decade. J. Sep. Sci. 2022, 45, 3169–3186. [Google Scholar] [CrossRef]
- Zhou, J.; Yang, W.; Hu, Y.; Höti, N.; Liu, Y.; Shah, P.; Sun, S.; Clark, D.; Thomas, S.; Zhang, H. Site-Specific Fucosylation Analysis Identifying Glycoproteins Associated with Aggressive Prostate Cancer Cell Lines Using Tandem Affinity Enrichments of Intact Glycopeptides Followed by Mass Spectrometry. Anal. Chem. 2017, 89, 7623–7630. [Google Scholar] [CrossRef]
- Ohkawa, Y.; Kizuka, Y.; Takata, M.; Nakano, M.; Ito, E.; Mishra, S.; Akatsuka, H.; Harada, Y.; Taniguchi, N. Peptide Sequence Mapping around Bisecting GlcNAc-Bearing N-Glycans in Mouse Brain. Int. J. Mol. Sci. 2021, 22, 8579. [Google Scholar] [CrossRef] [PubMed]
- Gao, L.; Song, Q.; Liang, H.; Zhu, Y.; Wei, T.; Dong, N.; Xiao, J.; Shao, F.; Lai, L.; Chen, X. Legionella effector SetA as a general O-glucosyltransferase for eukaryotic proteins. Nat. Chem. Biol. 2019, 15, 213–216. [Google Scholar] [CrossRef]
- Xu, S.; Sun, F.; Wu, R. A Chemoenzymatic Method Based on Easily Accessible Enzymes for Profiling Protein O-GlcNAcylation. Anal. Chem. 2020, 92, 9807–9814. [Google Scholar] [CrossRef]
- Zhu, Q.; Chaubard, J.-L.; Geng, D.; Shen, J.; Ban, L.; Cheung, S.T.; Wei, F.; Liu, Y.; Sun, H.; Calderon, A.; et al. Chemoenzymatic Labeling, Detection and Profiling of Core Fucosylation in Live Cells. J. Am. Chem. Soc. 2024, 146, 26408–26415. [Google Scholar] [CrossRef]
- Cummings, R.D.; Kornfeld, S. Characterization of the structural determinants required for the high affinity interaction of asparagine-linked oligosaccharides with immobilized Phaseolus vulgaris leukoagglutinating and erythroagglutinating lectins. J. Biol. Chem. 1982, 257, 11230–11234. [Google Scholar] [CrossRef]
- Nagae, M.; Soga, K.; Morita-Matsumoto, K.; Hanashima, S.; Ikeda, A.; Yamamoto, K.; Yamaguchi, Y. Phytohemagglutinin from Phaseolus vulgaris (PHA-E) displays a novel glycan recognition mode using a common legume lectin fold. Glycobiology 2014, 24, 368–378. [Google Scholar] [CrossRef] [PubMed]
- Gaunitz, S.; Nagy, G.; Pohl, N.L.B.; Novotny, M.V. Recent Advances in the Analysis of Complex Glycoproteins. Anal. Chem. 2016, 89, 389–413. [Google Scholar] [CrossRef] [PubMed]
- Cao, W.; Liu, M.; Kong, S.; Wu, M.; Zhang, Y.; Yang, P. Recent Advances in Software Tools for More Generic and Precise Intact Glycopeptide Analysis. Mol. Cell. Proteom. 2021, 20, 100060. [Google Scholar] [CrossRef] [PubMed]
- Santos-Barriopedro, I.; van Mierlo, G.; Vermeulen, M. Off-the-shelf proximity biotinylation for interaction proteomics. Nat. Commun. 2021, 12, 5015. [Google Scholar] [CrossRef]
- Villaseñor, R.; Pfaendler, R.; Ambrosi, C.; Butz, S.; Giuliani, S.; Bryan, E.; Sheahan, T.W.; Gable, A.L.; Schmolka, N.; Manzo, M.; et al. ChromID identifies the protein interactome at chromatin marks. Nat. Biotechnol. 2020, 38, 728–736. [Google Scholar] [CrossRef] [PubMed]
- Padrón, A.; Iwasaki, S.; Ingolia, N.T. Proximity RNA Labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules. Mol. Cell 2019, 75, 875–887.e875. [Google Scholar] [CrossRef] [PubMed]
- Qin, W.; Cho, K.F.; Cavanagh, P.E.; Ting, A.Y. Deciphering molecular interactions by proximity labeling. Nat. Methods 2021, 18, 133–143. [Google Scholar] [CrossRef]
- Rees, J.S.; Li, X.-W.; Perrett, S.; Lilley, K.S.; Jackson, A.P. Protein Neighbors and Proximity Proteomics. Mol. Cell. Proteom. 2015, 14, 2848–2856. [Google Scholar] [CrossRef] [PubMed]
- Ke, M.; Yuan, X.; He, A.; Yu, P.; Chen, W.; Shi, Y.; Hunter, T.; Zou, P.; Tian, R. Spatiotemporal profiling of cytosolic signaling complexes in living cells by selective proximity proteomics. Nat. Commun. 2021, 12, 71. [Google Scholar] [CrossRef] [PubMed]
- Shuster, S.A.; Li, J.; Chon, U.; Sinantha-Hu, M.C.; Luginbuhl, D.J.; Udeshi, N.D.; Carey, D.K.; Takeo, Y.H.; Xie, Q.; Xu, C.; et al. In situ cell-type-specific cell-surface proteomic profiling in mice. Neuron 2022, 110, 3882–3896.e3889. [Google Scholar] [CrossRef] [PubMed]
- Kwak, C.; Shin, S.; Park, J.-S.; Jung, M.; Nhung, T.T.M.; Kang, M.-G.; Lee, C.; Kwon, T.-H.; Park, S.K.; Mun, J.Y.; et al. Contact-ID, a tool for profiling organelle contact sites, reveals regulatory proteins of mitochondrial-associated membrane formation. Proc. Natl. Acad. Sci. USA 2020, 117, 12109–12120. [Google Scholar] [CrossRef] [PubMed]
- Cho, K.F.; Branon, T.C.; Rajeev, S.; Svinkina, T.; Udeshi, N.D.; Thoudam, T.; Kwak, C.; Rhee, H.-W.; Lee, I.-K.; Carr, S.A.; et al. Split-TurboID enables contact-dependent proximity labeling in cells. Proc. Natl. Acad. Sci. USA 2020, 117, 12143–12154. [Google Scholar] [CrossRef] [PubMed]
- Qin, W.; Cheah, J.S.; Xu, C.; Messing, J.; Freibaum, B.D.; Boeynaems, S.; Taylor, J.P.; Udeshi, N.D.; Carr, S.A.; Ting, A.Y. Dynamic mapping of proteome trafficking within and between living cells by TransitID. Cell 2023, 186, 3307–3324.e30. [Google Scholar] [CrossRef]
- Yao, X.; Wang, X.; Hu, X.; Liu, Z.; Liu, J.; Zhou, H.; Shen, X.; Wei, Y.; Huang, Z.; Ying, W.; et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res. 2017, 27, 801–814. [Google Scholar] [CrossRef] [PubMed]
- Wang, S.; Ran, W.; Sun, L.; Fan, Q.; Zhao, Y.; Wang, B.; Yang, J.; He, Y.; Wu, Y.; Wang, Y.; et al. Sequential glycosylations at the multibasic cleavage site of SARS-CoV-2 spike protein regulate viral activity. Nat. Commun. 2024, 15, 4162. [Google Scholar] [CrossRef] [PubMed]
- Branon, T.C.; Bosch, J.A.; Sanchez, A.D.; Udeshi, N.D.; Svinkina, T.; Carr, S.A.; Feldman, J.L.; Perrimon, N.; Ting, A.Y. Efficient proximity labeling in living cells and organisms with TurboID. Nat. Biotechnol. 2018, 36, 880–887. [Google Scholar] [CrossRef] [PubMed]
- Roux, K.J.; Kim, D.I.; Raida, M.; Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 2012, 196, 801–810. [Google Scholar] [CrossRef] [PubMed]
- Li, P.; Chen, Z.; You, S.; Xu, Y.; Hao, Z.; Liu, D.; Shen, J.; Zhu, B.; Dan, W.; Sun, S. Application of StrucGP in medical immunology: Site-specific N-glycoproteomic analysis of macrophages. Front. Med. 2022, 17, 304–316. [Google Scholar] [CrossRef] [PubMed]
- Tan, Z.; Cao, L.; Wu, Y.; Wang, B.; Song, Z.; Yang, J.; Cheng, L.; Yang, X.; Zhou, X.; Dai, Z.; et al. Bisecting GlcNAc modification diminishes the pro-metastatic functions of small extracellular vesicles from breast cancer cells. J. Extracell. Vesicles 2020, 10, e12005. [Google Scholar] [CrossRef] [PubMed]
- Fang, Z.; Qin, H.; Mao, J.; Wang, Z.; Zhang, N.; Wang, Y.; Liu, L.; Nie, Y.; Dong, M.; Ye, M. Glyco-Decipher enables glycan database-independent peptide matching and in-depth characterization of site-specific N-glycosylation. Nat. Commun. 2022, 13, 1900. [Google Scholar] [CrossRef]
- Tan, Z.; Ning, L.; Cao, L.; Zhou, Y.; Li, J.; Yang, Y.; Lin, S.; Ren, X.; Xue, X.; Kang, H.; et al. Bisecting GlcNAc modification reverses the chemoresistance via attenuating the function of P-gp. Theranostics 2024, 14, 5184–5199. [Google Scholar] [CrossRef] [PubMed]
- Li, C.-W.; Lim, S.-O.; Xia, W.; Lee, H.-H.; Chan, L.-C.; Kuo, C.-W.; Khoo, K.-H.; Chang, S.-S.; Cha, J.-H.; Kim, T.; et al. Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity. Nat. Commun. 2016, 7, 12632. [Google Scholar] [CrossRef] [PubMed]
- Console, L.; Scalise, M.; Salerno, S.; Scanga, R.; Giudice, D.; De Bartolo, L.; Tonazzi, A.; Indiveri, C. N-glycosylation is crucial for trafficking and stability of SLC3A2 (CD98). Sci. Rep. 2022, 12, 14570. [Google Scholar] [CrossRef] [PubMed]
- Zhu, Q.; Wang, H.; Chai, S.; Xu, L.; Lin, B.; Yi, W.; Wu, L. O-GlcNAcylation promotes tumor immune evasion by inhibiting PD-L1 lysosomal degradation. Proc. Natl. Acad. Sci. USA 2023, 120, e2216796120. [Google Scholar] [CrossRef]
- Schiffmann, R. Fabry disease. Pharmacol. Ther. 2009, 122, 65–77. [Google Scholar] [CrossRef] [PubMed]
- Joeh, E.; O’Leary, T.; Li, W.; Hawkins, R.; Hung, J.R.; Parker, C.G.; Huang, M.L. Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling. Proc. Natl. Acad. Sci. USA 2020, 117, 27329–27338. [Google Scholar] [CrossRef] [PubMed]
- Chang, L.; Chen, Y.-J.; Fan, C.-Y.; Tang, C.-J.; Chen, Y.-H.; Low, P.-Y.; Ventura, A.; Lin, C.-C.; Chen, Y.-J.; Angata, T. Identification of Siglec Ligands Using a Proximity Labeling Method. J. Proteome Res. 2017, 16, 3929–3941. [Google Scholar] [CrossRef]
- Stephen, H.M.; Praissman, J.L.; Wells, L. Generation of an Interactome for the Tetratricopeptide Repeat Domain of O-GlcNAc Transferase Indicates a Role for the Enzyme in Intellectual Disability. J. Proteome Res. 2020, 20, 1229–1242. [Google Scholar] [CrossRef]
- Liu, Y.; Nelson, Z.M.; Reda, A.; Fehl, C. Spatiotemporal Proximity Labeling Tools to Track GlcNAc Sugar-Modified Functional Protein Hubs during Cellular Signaling. ACS Chem. Biol. 2022, 17, 2153–2164. [Google Scholar] [CrossRef] [PubMed]
- Zhang, T.; Fassl, A.; Vaites, L.P.; Fu, S.; Sicinski, P.; Paulo, J.A.; Gygi, S.P. Interrogating Kinase–Substrate Relationships with Proximity Labeling and Phosphorylation Enrichment. J. Proteome Res. 2022, 21, 494–506. [Google Scholar] [CrossRef] [PubMed]
- Huang, H.-T.; Lumpkin, R.J.; Tsai, R.W.; Su, S.; Zhao, X.; Xiong, Y.; Chen, J.; Mageed, N.; Donovan, K.A.; Fischer, E.S.; et al. Ubiquitin-specific proximity labeling for the identification of E3 ligase substrates. Nat. Chem. Biol. 2024, 20, 1227–1236. [Google Scholar] [CrossRef] [PubMed]
- Mukhopadhyay, U.; Levantovsky, S.; Carusone, T.M.; Gharbi, S.; Stein, F.; Behrends, C.; Bhogaraju, S. A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates. Sci. Adv. 2024, 10, eadp3000. [Google Scholar] [CrossRef]
- May, D.G.; Scott, K.L.; Campos, A.R.; Roux, K.J. Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation. Cells 2020, 9, 1070. [Google Scholar] [CrossRef]
- Xiong, Z.; Lo, H.P.; McMahon, K.-A.; Martel, N.; Jones, A.; Hill, M.M.; Parton, R.G.; Hall, T.E. In vivo proteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish. eLife 2021, 10, e64631. [Google Scholar] [CrossRef] [PubMed]
- Wang, X.; Cao, L.; Jiang, H.; Zhou, L.; Hu, Z.; Xu, G. Proximity Proteomics and Biochemical Analysis Reveal a Noncanonical Function for UFM1-Specific Protease 1 in the p62 Body Formation. J. Proteome Res. 2023, 22, 2352–2363. [Google Scholar] [CrossRef] [PubMed]
- Bennett, E.P.; Mandel, U.; Clausen, H.; Gerken, T.A.; Fritz, T.A.; Tabak, L.A. Control of mucin-type O-glycosylation: A classification of the polypeptide GalNAc-transferase gene family. Glycobiology 2011, 22, 736–756. [Google Scholar] [CrossRef] [PubMed]
- Hashimoto, Y.; Kawade, H.; Bao, W.; Morii, S.; Nakano, M.; Nagae, M.; Murakami, R.; Tokoro, Y.; Nakashima, M.; Cai, Z.; et al. The K346T mutant of GnT-III bearing weak in vitro and potent intracellular activity. Biochim. Biophys. Acta (BBA)-Gen. Subj. 2024, 1868, 130663. [Google Scholar] [CrossRef] [PubMed]
- Li, J.; Xu, J.; Li, L.; Ianni, A.; Kumari, P.; Liu, S.; Sun, P.; Braun, T.; Tan, X.; Xiang, R.; et al. MGAT3-mediated glycosylation of tetraspanin CD82 at asparagine 157 suppresses ovarian cancer metastasis by inhibiting the integrin signaling pathway. Theranostics 2020, 10, 6467–6482. [Google Scholar] [CrossRef] [PubMed]
- Kizuka, Y.; Kitazume, S.; Fujinawa, R.; Saito, T.; Iwata, N.; Saido, T.C.; Nakano, M.; Yamaguchi, Y.; Hashimoto, Y.; Staufenbiel, M.; et al. An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer’s disease. EMBO Mol. Med. 2015, 7, 175–189. [Google Scholar] [CrossRef]
- Rudman, N.; Kifer, D.; Kaur, S.; Simunović, V.; Cvetko, A.; Pociot, F.; Morahan, G.; Gornik, O. Children at onset of type 1 diabetes show altered N-glycosylation of plasma proteins and IgG. Diabetologia 2022, 65, 1315–1327. [Google Scholar] [CrossRef] [PubMed]
- Cheng, L.; Cao, L.; Wu, Y.; Xie, W.; Li, J.; Guan, F.; Tan, Z. Bisecting N-Acetylglucosamine on EGFR Inhibits Malignant Phenotype of Breast Cancer via Down-Regulation of EGFR/Erk Signaling. Front. Oncol. 2020, 10, 929. [Google Scholar] [CrossRef] [PubMed]
- Lu, J.; Isaji, T.; Im, S.; Fukuda, T.; Kameyama, A.; Gu, J. Expression of N-Acetylglucosaminyltransferase III Suppresses α2,3-Sialylation, and Its Distinctive Functions in Cell Migration Are Attributed to α2,6-Sialylation Levels. J. Biol. Chem. 2016, 291, 5708–5720. [Google Scholar] [CrossRef] [PubMed]
- Nakano, M.; Mishra, S.K.; Tokoro, Y.; Sato, K.; Nakajima, K.; Yamaguchi, Y.; Taniguchi, N.; Kizuka, Y. Bisecting GlcNAc Is a General Suppressor of Terminal Modification of N-glycan. Mol. Cell. Proteom. 2019, 18, 2044–2057. [Google Scholar] [CrossRef]
- Kizuka, Y.; Taniguchi, N. Enzymes for N-Glycan Branching and Their Genetic and Nongenetic Regulation in Cancer. Biomolecules 2016, 6, 25. [Google Scholar] [CrossRef]
- Zhang, Y.; Li, J.; Yin, X. High-expression of Galactosidase alpha is correlated with poor prognosis and immune infiltration in low-grade glioma. J. Cancer 2023, 14, 646–656. [Google Scholar] [CrossRef]
- Bird, S.; Hadjimichael, E.; Mehta, A.; Ramaswami, U.; Hughes, D. Fabry disease and incidence of cancer. Orphanet J. Rare Dis. 2017, 12, 150. [Google Scholar] [CrossRef]
Name | Sequence (5′–3′) |
---|---|
shGLA-1 | CTGCAATCACTGGCGAAATTT |
shGLA-2 | TGCTCCTTTATTCATGTCTAA |
Name | Sequence (5′–3′) |
---|---|
GAPDH-Forward | GGAGCGAGATCCCTCCAAAAT |
GAPDH-Reverse | GGCTGTTGTCATACTTCTCATGG |
GLA-Forward | TTGGATACTACGACATTGATGCC |
GLA-Reverse | TTCTGCCAGTCCTATTCAGGG |
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Wang, B.; He, X.; Zhou, Y.; Tan, Z.; Li, X.; Guan, F.; Lei, L. Proximity Labeling-Based Identification of MGAT3 Substrates and Revelation of the Tumor-Suppressive Role of Bisecting GlcNAc in Breast Cancer via GLA Degradation. Cells 2025, 14, 103. https://doi.org/10.3390/cells14020103
Wang B, He X, Zhou Y, Tan Z, Li X, Guan F, Lei L. Proximity Labeling-Based Identification of MGAT3 Substrates and Revelation of the Tumor-Suppressive Role of Bisecting GlcNAc in Breast Cancer via GLA Degradation. Cells. 2025; 14(2):103. https://doi.org/10.3390/cells14020103
Chicago/Turabian StyleWang, Bowen, Xin He, Yue Zhou, Zengqi Tan, Xiang Li, Feng Guan, and Lei Lei. 2025. "Proximity Labeling-Based Identification of MGAT3 Substrates and Revelation of the Tumor-Suppressive Role of Bisecting GlcNAc in Breast Cancer via GLA Degradation" Cells 14, no. 2: 103. https://doi.org/10.3390/cells14020103
APA StyleWang, B., He, X., Zhou, Y., Tan, Z., Li, X., Guan, F., & Lei, L. (2025). Proximity Labeling-Based Identification of MGAT3 Substrates and Revelation of the Tumor-Suppressive Role of Bisecting GlcNAc in Breast Cancer via GLA Degradation. Cells, 14(2), 103. https://doi.org/10.3390/cells14020103