Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

Ganka Bineva-Todd,Lauren J. S. Wagner,Jun Won Choi ,Holly Douglas ,Carolyn R. Bertozzi,Aristotelis Antonopoulos,Deg Briggs ,Zhen Li,Benjamin Schumann,William M. Browne,Leonhard Moeckl ,Ambrosius P. Snijders,Michael C. Bassik,Stacy A. Malaker,Ömür Y. Tastan ,Gaelen T. Hess,Chloë Roustan ,Simon Wisnovsky ,Stuart M. Haslam,W. E. Moerner,Svend Kjær ,Helen Flynn,Marjoke F. Debets,Vivian Li ,Anna Cioce,Nikolaos Angelis,Anthony J. Agbay

doi:10.1073/pnas.2007297117

Abstract

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

Highlights

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis
We envisioned that a chemically modified UDPGalNAc analog would be O-GalNAc specific if it was 1) not epimerized to the uridine diphosphate (UDP)-GlcNAc analog by GALE and 2) used by either wild-type (WT) or BH-engineered GalNAc-Ts to be incorporated into cell surface O-GalNAc glycans (Fig. 1A)
Investigation of the cocrystal structure of human GalNAc-T2 and UDP-GalNAc suggested that the GalNAc acetamide group is embedded in a pocket that allows for some three-dimensional freedom (Fig. 1A)

Summary

Introduction

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. We use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)–linked N-acetylgalactosamine (GalNAc) glycosylation. A particular drawback of most current chemically modified monosaccharides is their low specificity: UDP-GalNAz enters mucin-type (Ser/Thr–N-acetylgalactosamine [O-GalNAc]) glycans but is converted to the corresponding GlcNAc derivative UDPGlcNAz by the cellular UDP-GlcNAc/N-acetylgalactosamine–4epimerase (GALE) (Fig. 1A) [15]. Forays have been made into developing reagents that are specific for the structurally simple nucleocytoplasmic O-GlcNAc modification [18,19,20] No such reagents are available to probe the complex cell surface O-GalNAc glycosylation that has fundamental relevance in many aspects of cancer [21, 22]

Methods

Results

Conclusion