Glycobiology Probes

What is Glycobiology?

Glycobiology is the study of the structure, function, and biology of saccharides. Saccharides are essential components of all living organisms; monosaccharides can be linked by glycosidic bonds to form unbranched, branched or tree-like structures known as polysaccharides or glycans. The monosaccharides that constitute a glycan can vary widely in type and number, this variety provides chemical and structural diversity, and a high level of complexity of glycans in cells. Glycans can be covalently linked to other biomolecules via glycosylation to form glycoconjugates. For example, in eukaryotic cells, glycans are attached to amino acid side chains of proteins during a co-translational or post-translational modification, without the rigid guidance of genetic codes on nucleic acid templates, to form glycoproteins. Many secreted extracellular proteins, such as IgG antibodies produced by mammalian cells, are glycosylated and glycosylation of antibodies is a key regulator of humoral immune activity. In bacterial cells, the cell walls are made of covalently linked polysaccharide and polypeptide chains, known as peptidoglycans, which form a framework that completely encases the single cell organism. Studies of peptidoglycan and bacterial cell wall glycoconjugates have led to the development of a range of antibiotics.

Glycobiology Probes and the Chemical Reporter Strategy

Glycobiology probes are molecular tools for studying glycobiology and are used for studying glycan biosynthesis and glycosylation. Common genetic tags, such as fluorescent proteins, cannot be easily translated to non-protein biomolecules such as glycans, lipids and nucleic acids. Bioorthogonal chemistry offers an alternative strategy to study these biomolecules. Bioorthogonal chemistry refers to a type of rapid, selective, and high-yielding chemical reaction that can proceed in biological environments without side reactions interfering with native biochemical processes. It enables probing of biological systems through selective covalent bond formations. To apply bioorthogonal chemistry to cellular systems, a type of glycobiology probe (known as a chemical reporter) containing non-natural functional groups, must first be incorporated into the target biomolecule. The reporter can then be detected with complementary reagents via bioorthogonal ligation. This two-step process is known as the chemical reporter strategy.

Metabolic Glycoengineering with Glycobiology Probes

Metabolic Glycoengineering (MGE) is a non-genetic approach in which unnatural derivatives of monosaccharide building blocks (also known as chemical reporters) are incorporated into glycans via cellular metabolic pathways. These glycobiology probes may contain functional groups such as reactive handles (e.g. for bioorthogonal chemistry or photo-crosslinking). This technique allows the study of important chemical functions and interactions on the cell surface.

MGE offers the following advantages over genetic approaches:

• Versatile: MGE can equip cell surfaces with various functional groups, such as thiol-, azide-, alkyne- or ketone-modified glycans. It is applicable to all cell types with conserved glycobiology-related enzymes.
• Biocompatible: the incorporated monosaccharide derivatives are stable under physiological conditions and innocuous to the modified cell.
• Many are reversible after washout and incubation with a controlled precursor.

MGE can be used in the following applications:

• Modulation of cell biological behaviors, such as altering cell-cell, cell-matrix and cell-molecule interactions.
• Metabolic glycan labeling for monitoring aberrant protein glycosylation in various cancers.
• In vivo cell imaging and tracking, through conjugation of click-reactive dyes or via direct labeling with metabolic probes containing a fluorophore.
• Cell-based therapies, such as introducing specific cell surface antigens for immunotherapy.

MGE Glycobiology Probes for Cell Membrane Glycoconjugates

Glycosylation is the process by which a saccharide is covalently attached to a target macromolecule. Glycosylation of cell surface biomolecules increases their complexity and function and is important in cell-cell interactions and immune responses. Almost all of the membrane-associated proteins of eukaryotic cells are glycosylated co-translationally or post-translationally in the endoplasmic reticulum (ER) and Golgi apparatus. Many lipids are glycosylated and assembled in the Golgi apparatus and embedded in the membrane of vesicles which are then transported to the cell surface. Small noncoding RNAs can also be glycosylated via canonical N-glycan biosynthetic machinery, and the majority of glycoRNAs are found on the cell surface in multiple cell types and mammalian species. These processes of glycosylation are carried out through highly conserved metabolic pathways by enzymes such as glycoside hydrolases and glycotransferases. Some enzymes in these metabolic pathways recognize the core structure of monosaccharide precursors and have some freedom for the functional groups on their substrate. For example, both the natural precursor N-acetylmannosamine (ManNAc) and its azide derivative N-azidoacetylmannosamine (ManNAz) (Cat. No. 7479) can be converted by the Roseman-Warren pathway into their corresponding sialic acid derivatives, which are then incorporated into protein, lipid, and RNA by sialyltransferases to form natural and azide functionalized glycoconjugates, respectively (Figure 1).

Figure 1: Overview of metabolic glycoengineering for incorporating glycobiology probes (chemical reporters) onto the mammalian cell surface, depicted with an example of ManNAc derivative. An azide functional group can be introduced onto the cell surface via addition of Ac₄ManNAz (Cat. No. 7479)to the metabolic pathways for glycosylation of cell surface proteins, lipids or RNA.

Table 1. Glycobiology probes for the study of glycobiology in mammalian cells

MGE Glycobiology Probes for the Bacterial Envelope

The bacterial cell envelope is an essential structure that allows a bacterium to protect itself from and directly interact with its surrounding environment. It is covered with a dense array of glycan structures that are associated with colonization and pathogenesis. Bacterial cells produce glycans containing rare saccharides that are refractory to traditional glycan analysis such as mass spectrometry. This makes the study of bacterial carbohydrate metabolism and glycan biosynthesis a challenge. Glycobiology probes and metabolic labeling approaches have been applied to solve this problem. When added to living bacteria, reporter probes can be metabolically incorporated into the envelope and then used for a variety of applications including live-cell imaging, functional modulation and diagnostic or therapeutic targeting (Figure 2). For example, peptidoglycans (PG) form an interwoven glycan-peptide meshwork around bacteria cells; this meshwork has critical physiological functions, and its biosynthesis has been targeted by broad-spectrum antibiotics, such as β-lactams and Vancomycin (Cat. No. 5506). PG is composed of alternating N-acetyl muramic acid (NAM) and N-acetyl glucosamine (NAG) monosaccharide units with short peptide chains linked to the muramic acid residue. A range of macromolecular structures can be produced from the combination of these molecular building blocks, with NAM and NAG consistently present in all bacteria. Click N-Acetylmuramic acid-azide (Cat. No. 7506) is a derivative of N-acetylmuramic acid (NAM). By simply adding this glycobiology probe into the culture media, it can be installed into the backbone of Gram-positive and Gram-negative bacterial PG via metabolic cell wall recycling and biosynthetic machineries. The azide groups on click N-Acetylmuramic acid-azide enable fluorescence modification via click chemistry onto the NAM backbone of bacterial PG.

Figure 2: Overview of the two-step bioorthogonal chemical reporter strategy as applied to the study of bacterial envelope. In the first step, non-natural functional groups X (see Table 2) on the glycobiology probes are incorporated into bacterial glycans via metabolic glycoengineering. In the second step, bioorthogonal ligation takes place between the incorporated chemical reporter X and the functional group Y (see Table 2) from the complementary reagent.

Table 2. Glycobiology probes and complementary reagents

References

Spiro et al (2002) Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 12 43R. PMID: 12042244.

Li et al (2012) Sialic acid metabolism and sialyltransferases: natural functions and applications. Appl.Microbiol.Biotechnol. 94 887. PMID: 22526796.

Wratil et al (2016) Metabolic glycoengineering with N-Acyl side chain modified mannosamines. Angew.Chem.Int.Ed.Engl. 55 9482. PMID: 27435524.

Liang et al (2017) Metabolic labelling of the carbohydrate core in bacterial peptidoglycan and its applications. Nat.Commun. 8 15015. PMID: 28425464.

Flynn et al (2021) Small RNAs are modified with N-glycans and displayed on the surface of living cells. Cell 184 3109. PMID: 34004145.

Banahene et al (2022) Chemical reporters for bacterial glycans: development and applications. Chem.Rev. 122 3336. PMID: 34905344.

Varki et al (2022) Essentials of glycobiology. Cold Spring Harbor Laboratory Press. PMID: 35536922.

Our sister brand R&D Systems also offers Glycobiology Assay Kits and Reagents.