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.
Bio-orthogonal chemistries
have revolutionized many fields. For
example, metabolic chemical reporters (MCRs) of glycosylation are
analogues of monosaccharides that contain a bio-orthogonal functionality,
such as azides or alkynes. MCRs are metabolically incorporated into
glycoproteins by living systems, and bio-orthogonal reactions can
be subsequently employed to install visualization and enrichment tags.
Unfortunately, most MCRs are not selective for one class of glycosylation
(e.g., N-linked vs O-linked), complicating the types of information
that can be gleaned. We and others have successfully created MCRs
that are selective for intracellular O-GlcNAc modification by altering
the structure of the MCR and thus biasing it to certain metabolic
pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do
the same for the core GalNAc residue of mucin O-linked glycosylation.
The most widely applied MCR for mucin O-linked glycosylation, GalNAz,
can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz.
This results in a mixture of cell-surface and O-GlcNAc labeling. We
reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would
lock the stereochemistry of this position in place, causing the MCR
to be more selective. After synthesis, we found that 4FGalNAz labels
a variety of proteins in mammalian cells and does not perturb endogenous
glycosylation pathways unlike 4FGalNAc. However, through subsequent
proteomic and biochemical characterization, we found that 4FGalNAz
does not widely label cell-surface glycoproteins but instead is primarily
a substrate for OGT. Although these results are somewhat unexpected,
they once again highlight the large substrate flexibility of OGT,
with interesting and important implications for intracellular protein
modification by a potential range of abiotic and native monosaccharides.
Oligosaccharides are essential for interactions of cells with their environments. These complex carbohydrates are often found covalently attached to proteins embedded in eukaryotic cell membranes. Protein glycosylation is heterogeneous; this heterogeneity stems from the biosynthesis of these polymers. As proteins destined for secretion or cell‐surface presentation traffic through the endoplasmic reticulum and the Golgi apparatus, they are modified with sugars in a stepwise fashion by enzymes called glycosyltransferases. The differential expression of these enzymes leads to a multiplicity of specific oligosaccharides both among and within cells because not all cells contain all enzymes and because not all substrate proteins will encounter every enzyme. Although myriad oligosaccharides are found attached to proteins, most of these diverse structures can be grouped into several classes of glycans. In this article, we will discuss some of the most common forms of Golgi protein glycosylation: mucin‐type
O
‐linked glycosylation,
N
‐linked glycosylation, and the formation of glycosaminoglycans. In addition, we will briefly consider some less common, but essential, forms of glycosylation.
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