Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

Abstract

O-Linked -N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide Nacetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bumphole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly-synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme-substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme-substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and-T2 that prefer non-glycosylated peptide substrates and GalNAcT-10 that prefers a pre-glycosylated peptide substrate. A detailed investigation of enzyme

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

“…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).…”

An Enzymatic N‐Acylation Step Enables the Biocatalytic Synthesis of Unnatural Sialosides

“…Subsequent epimerizations (to generate the galactose and mannose derivatives 4 and 5 , respectively) and further elaboration to sialic acids ( 6 ) and glycoconjugates can also be achieved enzymatically. However, there remains a gap in the enzyme toolkit in that the acylation of glucosamine ( 2 ) needs to be performed chemically, requiring the activation of carboxylic acids or stoichiometric coupling agents and protecting strategies …”

Chemical precision glyco-mutagenesis by glycosyltransferase engineering in living cells

“…We next sought to confirm that DM-GalNAcTs localize to the Golgi compartment and glycosylate alkyne chain led to substantial labeling, consistent with 4 being a substrate for WT-GalNAcTs (8,26). These results were confirmed using soluble GalNAcTs and a membrane fraction of nontransfected cells (Fig.…”

“…1B) re-programmed GalNAcTs to accept alkynecontaining uridine diphosphate (UDP)-GalNAc analogs such as compounds 1-4 instead of the native substrate UDP-GalNAc ( Fig. 1B) (8). We endowed living cells with the capacity to biosynthesize a UDP-GalNAc analog that was complementary to engineered DM-GalNAcTs.…”