Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells

Abstract

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

Metabolic precision labeling enables selective probing of O-linkedN-acetylgalactosamine glycosylation

“…Our initial attempts of using a per-acetylated precursor failed, as we did not observe 5 in cell lysates by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). This was in line with previous findings on the low promiscuity of both endogeneous biosynthetic enzymes GALK2 and AGX1 towards chemically modified substrate analogs (16, 25, 33). Mutants of AGX1 with enlarged active sites have been used by us and Yu et al to successfully transform analogs of GlcNAc-1-phosphate or GalNAc-1-phosphate into the corresponding UDP-sugars and bypass the GALK2 phosphorylation step ( see Fig.…”

“…S1B). GALE-mediated epimerization of linear, but not branched UDP-GalNAc analogs was corroborated by performing the reactions in the presence of cytosolic extracts of K-562 cells with or without functional GALE (control-sgRNA or GALE-KO, respectively) (25). An extract containing GALE epimerized compounds 1 - 3 , but not 4 - 10 , whereas an extract from GALE-KO cells was devoid of epimerization in all cases (Fig.…”

“…Mutants of AGX1 with enlarged active sites have been used by us and Yu et al to successfully transform analogs of GlcNAc-1-phosphate or GalNAc-1-phosphate into the corresponding UDP-sugars and bypass the GALK2 phosphorylation step ( see Fig. 1A) (25, 34). We thus synthesized a caged, membrane-permissive version of GalNAzMe-1-phosphate 11 (Fig.…”

“…We thus synthesized a caged, membrane-permissive version of GalNAzMe-1-phosphate 11 (Fig. 2A) (25) and equipped cells with the capacity to biosynthesize UDP-GalNAzMe 5 ( see Fig. 1A).…”

“…Further complexity is added by the interplay of 20 GalNAc transferase (GalNAc-T1…T20) isoenzymes that mediate the first O-GalNAc biosynthesis step (22, 23). In a “bump-and-hole” (BH) approach, we have recently engineered GalNAc-Ts to carry a double mutation to preferentially accept UDP-GalNAc analogs with bulky chemical, editable tags (24, 25). Although this technique produced bioorthogonal reporters with great specificity for particular GalNAc-T isoenzymes, epimerization of GalNAc analogs by GALE was still a challenge and resulted in background N-glycan labeling (25).…”