Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells

• Published in: STAR protocols 2023-03, Vol.4 (1), p.101974, Article 101974
• Main Authors: Calle, Beatriz, Gonzalez-Rodriguez, Edgar, Mahoney, Keira E., Cioce, Anna, Bineva-Todd, Ganka, Tastan, Omur Y., Roustan, Chloe, Flynn, Helen, Malaker, Stacy A., Schumann, Benjamin
• Format: Article
• Language: English
• Subjects: Cell-based Assays; Glycosylation; Humans; Mass Spectrometry; Molecular/Chemical Probes; N-Acetylgalactosaminyltransferases - chemistry; N-Acetylgalactosaminyltransferases - genetics; N-Acetylgalactosaminyltransferases - metabolism; Peptides - chemistry; Polysaccharides - chemistry; Protein Biochemistry; Proteins - metabolism; Proteomics; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101974

Despite the known disease relevance of glycans, the biological function and substrate specificities of individual glycosyltransferases are often ill-defined. Here, we describe a protocol to develop chemical, bioorthogonal reporters for the activity of the GalNAc-T family of glycosyltransferases using a tactic termed bump-and-hole engineering. This allows identification of the protein substrates and glycosylation sites of single GalNAc-Ts. Despite requiring transfection of cells with the engineered transferases and enzymes for biosynthesis of bioorthogonal substrates, the tactic complements methods in molecular biology. For complete details on the use and execution of this protocol, please refer to Schumann et al. (2020)1, Cioce et al. (2021)2, and Cioce et al. (2022)3 [Display omitted] •Generation of bump-and-hole reporter systems for GalNAc-T glycosyltransferases•Artificial biosynthetic pathway to biosynthesize bioorthogonal substrate analogues•Protein substrates of individual GalNAc-Ts bioorthogonally tagged in living cells•Substrate identification and glycan site localization achieved by MS-glycoproteomics Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Despite the known disease relevance of glycans, the biological function and substrate specificities of individual glycosyltransferases are often ill-defined. Here, we describe a protocol to develop chemical, bioorthogonal reporters for the activity of the GalNAc-T family of glycosyltransferases using a tactic termed bump-and-hole engineering. This allows identification of the protein substrates and glycosylation sites of single GalNAc-Ts. Despite requiring transfection of cells with the engineered transferases and enzymes for biosynthesis of bioorthogonal substrates, the tactic complements methods in molecular biology.