Generating orthogonal glycosyltransferase and nucleotide sugar pairs as next-generation glycobiology tools

Abstract

Protein glycosylation fundamentally impacts biological processes. Nontemplated biosynthesis introduces unparalleled complexity into glycans that needs tools to understand their roles in physiology. The era of quantitative biology is a great opportunity to unravel these roles, especially by mass spectrometry glycoproteomics. However, with high sensitivity come stringent requirements on tool specificity. Bioorthogonal metabolic labeling reagents have been fundamental to studying the cell surface glycoproteome but typically enter a range of different glycans and are thus of limited specificity. Here, we discuss the generation of metabolic 'precision tools' to study particular subtypes of the glycome. A chemical biology tactic termed bump-and-hole engineering generates mutant glycosyltransferases that specifically accommodate bioorthogonal monosaccharides as an enabling technique of glycobiology. We review the groundbreaking discoveries that have led to applying the tactic in the living cell and the implications in the context of current developments in mass spectrometry glycoproteomics.

Keywords: Bioorthogonal; Click chemistry; Glycoprotein; Glycosylation; Glycosyltransferase; Mucin; Protein engineering.

Figure 1

Using glycosyltransferase bump-and-hole engineering to understand cell surface glycosylation. (a) Diversity of cell surface glycans. (b) Bump-and-hole engineering enlarges the active site of a GT to accommodate a chemically modified nucleotide-sugar containing a bioorthogonal tag. (c) A blueprint of key steps to establish a cellular GT bump-and-hole system [11]. (d) Co-crystal structure of WT-B4GALT1 (PDB 1OQM) with UDP-GalNAc. B4GALT1 was subsequently engineered to accommodate bioorthogonal UDP-GalNAc analogs [25]. Adapted from Molecular Cell, Vol 78/5, B. Schumann et al., Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells, 824–834.e15, Copyright (2020), with permission from Elsevier. GTs, glycosyltransferases.

Figure 2

Reprogramming metabolism to deliver UDP-sugar analogs. (a) Schematic representation of the GalNAc salvage pathway applied to chemically modified GalNAc analogs. Suitable membrane-permeable precursors can be used to circumvent the GALK2 step if needed, but AGX1 engineering is necessary to deliver bumped UDP-GalNAc analogs. GALE-mediated epimerization to UDP-GlcNAc analogs can be suppressed by using branched acylamide side chains [12]. (b) Co-crystal structure of WT-AGX1 with UDP-GalNAc (PDB 1JV3) used to rationalize the F383G/A mutation that biosynthesizes bumped UDP-sugar analogs. (c) Structures of UDP-sugar analogs used in conjunction with GT engineering. GTs, glycosyltransferases.

Figure 3

Structural basis for GalNAc-T bump-and-hole engineering. (a) Gatekeeper residues identified in the crystal structures of GalNAc-T1 (PDB 1XHB), T2 (PDB 4D0T), T4 (PDB 5NQA), T7 (PDB 6IWR), and T10 (PDB 2D7I). (b) Sequence alignment of gatekeeper residues in all 20 GalNAc-Ts. (c) Co-crystal structure of BH-GalNAc-T2 (PDB 6NQT) with UDP-GalNAc6yne 3. Gatekeeper residues are mutated to Ala to accommodate the aliphatic alkyne. (d) Superposition of WT-GalNAc-T2 (PDB 2FFU) and BH-GalNAc-T2 (PDB 6E7I) with EA2 substrate peptide (overlay of both structures), Mn²⁺ and UDP. (e) Metabolic labeling of cells transfected with AGX1 (WT or mut) and either GalNAc-T1 or T2 (WT or BH-mutant) constructs. Dox-inducible GalNAc-T expression was used in conjunction with feeding a caged precursor of UDP-GalNac6yne 3. DMSO and the tagged sialic acid precursor Ac₄ManNAl served as negative and positive controls, respectively. Panel E reprinted from Molecular Cell, Vol 78/5, B. Schumann et al., Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells, 824–834.e15, Copyright (2020), with permission from Elsevier.

Figure 4

Glycoproteomic workflows enable analysis of the glycoproteome complexity. (a) Typical workflow before MS analysis. The complex sample contains unmodified peptides, differentially modified peptides, and glycopeptides. To enrich for the latter, samples are subjected to lectin columns, SPE extraction, or chemical enrichment procedures. The elution is then separated by RP-HPLC and electrospray ionized into the mass spectrometer. (b) Schematic of the instrumentation parameters often used in glycoproteomic analysis. The RP-HPLC elution trace is shown in the top panel and consists of a series of full mass spectra (MS1s). Typically, ions are selected in an abundance-dependent manner and subjected to HCD fragmentation (2nd panel). If a glycopeptide is present, a HexNAc fingerprint will be visible (3rd panel), which can then be used to trigger electron-based fragmentation (bottom panel). (c) Overview of glycopeptide fragmentation methods and the information they can provide. ETD (purple dashes) only fragments the peptide backbone, giving complementary c/z type ions with the glycan attached to the peptide. HCD (green dashes) fragments the peptide backbone as well as the glycosidic linkages, allowing for peptide and glycan sequencing, but often loses site-specificity. EThcD (red lines) combines the two techniques and allows for the most information to be gleaned from a single spectrum. Adapted from a study by Reiding et al. [92]. (d) Bump-and-hole chemical glycoproteomics provides a gain-of-function enrichment strategy (top). An HCD spectrum is shown under this, where 2 new fingerprint ions are present (491, 330) that can be used to trigger ETD (bottom). The ETD spectrum allows for site-localization of the modification and also demonstrates a new fingerprint ion (194) that can be used in search algorithms for more confident scoring. SPE, solid-phase extraction; RP, reverse-phase; HCD, higher-energy collisional dissociation; ETD, Electron transfer dissociation; HPLC, high performance liquid chromatography.