Global view of human protein glycosylation pathways and functions

Abstract

Glycosylation is the most abundant and diverse form of post-translational modification of proteins that is common to all eukaryotic cells. Enzymatic glycosylation of proteins involves a complex metabolic network and different types of glycosylation pathways that orchestrate enormous amplification of the proteome in producing diversity of proteoforms and its biological functions. The tremendous structural diversity of glycans attached to proteins poses analytical challenges that limit exploration of specific functions of glycosylation. Major advances in quantitative transcriptomics, proteomics and nuclease-based gene editing are now opening new global ways to explore protein glycosylation through analysing and targeting enzymes involved in glycosylation processes. In silico models predicting cellular glycosylation capacities and glycosylation outcomes are emerging, and refined maps of the glycosylation pathways facilitate genetic approaches to address functions of the vast glycoproteome. These approaches apply commonly available cell biology tools, and we predict that use of (single-cell) transcriptomics, genetic screens, genetic engineering of cellular glycosylation capacities and custom design of glycoprotein therapeutics are advancements that will ignite wider integration of glycosylation in general cell biology.

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Fig. 1: Main classes of glycoconjugates of the human cellular glycome.
Fig. 2: Subcellular organization of protein glycosylation.
Fig. 3: Human glycosylation pathways and enzymes.
Fig. 4: Protein glycosylation serves general roles and specific roles for protein functions.
Fig. 5: Common dysregulated glycosyltransferase genes in cancer.

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Acknowledgements

The authors are grateful to R. Schnaar and B. Henrissat for discussions and critical comments on the manuscript. They thank H. Wandall, A. Halim, C. Büll, Y. Zhang and L. Hansen for help with the manuscript, and all members of the Copenhagen Center for Glycomics for discussions. Supported by the Lundbeck Foundation, the Novo Nordisk Foundation and the Danish National Research Foundation (DNRF107).

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The authors contributed equally to all aspects of the article.

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Correspondence to Hiren J. Joshi or Henrik Clausen.

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Competing interests

The University of Copenhagen has filed patent applications related to engineering cellular glycosylation capacities and cell-based glycan arrays. H.C. and Y.N. are named inventors on these applications. GlycoDisplay Aps has license rights to these patent applications, and H.C. and Y.N. hold shares in GlycoDisplay Aps. The remaining authors declare no competing interests.

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Nature Reviews Molecular Cell Biology thanks S. Flitsch, C. Reis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Carbohydrate-Active enZYmes database: http://www.cazy.org

Copenhagen Center for Glycomics, University of Copenhagen: https://glycomics.ku.dk

Essentials of Glycobiology, 3rd edition: https://www.ncbi.nlm.nih.gov/books/NBK310274/

ExPADSy glycomics: https://www.expasy.org/glycomics

GlycoDomainViewer: https://glycodomain.glycomics.ku.dk

GlyGen: https://www.glygen.org

GlyTouCan: https://glytoucan.org

Handbook of Glycosyltransferases and Related Genes. Editors: Taniguchi, N., Honke, K., Fukuda, M., Narimatsu, H., Yamaguchi, Y., Angata, T. (Eds.) Springer Nature, 2014. Society for Glycobiology: http://glycobiology.org

International Glycoconjugate Organization: https://intl-glyco.org

NetOGlyc 4.0: http://www.cbs.dtu.dk/services/NetOGlyc/

Supplementary information

Glossary

O-GlcNAcylation

The enzymatic process directed by the N-acetyl-d-glucosamine (GlcNAc) glycosyltransferase (OGT) that transfers GlcNAc to proteins (Ser and Thr residues) occurring in the cytosol and nucleus of cells.

Isoenzymes

Enzymes that catalyse the same reactions but differ in amino acid sequence and often have partially distinct (non-redundant) functions.

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins

A class of proteins that are attached to the membrane lipid bilayer via a carboxy-terminal glycolipid anchor consisting of phosphoethanolamine, an oligosaccharide core and phosphatidylinositol.

Proteoglycans

Proteins carrying one or more glycosaminoglycan chains attached covalently.

Glycocalyx

The cell coat comprising glycans and glycoconjugates surrounding animal cells found as an electron-dense layer by electron microscopy. It protects the cell from physical stress and mediates a plethora of macromolecular and cell–cell interactions.

Xenoantigens

Antigens found in multiple species that elicit antibodies in a species without the antigen after transplantation of tissues and organs. A major xenoantigen in porcine to human transplantation is the Galα1–3Galβ1–R glycan epitope (αGal).

Lectins

Proteins that bind to glycans. Major animal lectin families include Galectin, C-type, P-type and I-type lectins. Lectins, mostly from plants, with well-characterized binding specificities are frequently used as tools in glycobiology. Lectins are often multivalent with binding affinities in the low micromolar range and binding avidities approaching the nanomolar range for larger glycans with multiple epitopes.

Dolichol

(Dol). A polyisoprenol lipid that serves as an acceptor for the lipid-linked oligosaccharides in N-glycan biosynthesis.

Type II transmembrane glycoproteins

Single-pass transmembrane glycoproteins with the amino terminus oriented towards the cytosol and the carboxyl terminus facing the lumen of the secretory pathway or cell exterior.

COPI-coated vesicles

Coat protein complex I-coated vesicles that mediate intra-Golgi and Golgi-to-endoplasmic reticulum retrograde transport.

Multipass transmembrane proteins

Proteins spanning the membrane more than once.

KDEL signals

A carboxy-terminal Lys-Asp-Glu-Leu (KDEL) retention sequence found on endoplasmic reticulum (ER)-resident proteins. The KDEL receptors recognizing this signal facilitate the retrograde movement of ER-based proteins from the Golgi and back to the ER by coat protein complex I (COPI) vesicles.

Sialylation

Modification by the addition of sialic acids, which are a large family of glycans derived from the neuraminic acid (Neu) monosaccharide with a nine-carbon backbone. In humans, N-acetylneuraminic acid (Neu5Ac) is the most common sialic acid, often found in the non-reducing terminal of glycoconjugates.

O-Glycan core structures

The initiating O-GalNAc glycan can be extended to form four different common core structures. Core1, Galβ1–3GalNAcα1–O-Ser/Thr; Core2, GlcNAcβ1–6(Galβ1–3)GalNAcα1–O-Ser/Thr; Core3, GlcNAcβ1–3GalNAcα1–O-Ser/Thr; and Core4, GlcNAcβ1–6(GlcNAcβ1–3)GalNAcα1–O-Ser/Thr. The core structures can be further elongated or branched.

Oligosaccharyltransferase (OST) complex

A membrane protein complex in the endoplasmic reticulum that transfers an oligosaccharide from a dolichol pyrophosphate-activated donor to N-linked acceptor sequences on secreted proteins.

Translocon

A protein complex that mediates translocation of newly synthesized polypeptides from the cytosol across the endoplasmic reticulum membrane.

Oxidoreductase

An enzyme that catalyses thiol–disulphide exchange reactions. In vivo oxidoreductases are important in the oxidative protein folding that takes place in the endoplasmic reticulum. Well-known examples are PDI and ERp57.

Lectin domains

Carbohydrate-binding protein domains.

Sulfation

An enzymatic process that transfers a sulfo group to another molecule, for example a glycan, by modifying a hydroxyl group on a monosaccharide by addition of a sulfo group. Sulfotransferases catalyse the reaction using 3′-phospho-5′-adenylyl sulfate (PAPS) as a donor.

EGF-like repeats

Common motifs of 30–40 amino acids found in the extracellular domain of transmembrane proteins or in proteins known to be secreted. The epidermal growth factor (EGF)-like repeats include six conserved cysteines forming three disulfide bonds.

Thrombospondin type 1 repeats

(TSRs) Common protein motifs of 50–60 amino acids (6 conserved cysteines forming 3 disulfide bonds) found on transmembrane proteins and proteins in the extracellular matrix.

Mucin

A large viscous heavily O-glycosylated protein. Mucins are the most abundant macromolecule in biofluids and mucus, covering most epithelial surfaces in the body.

α-Dystroglycan

Dystroglycan is encoded by the DAG1 gene and comprises two non-covalently-bound subunits (α and β). The extracellular α-subunit with the O-Man matriglycan provides binding to laminin, and the transmembrane β-subunit provides binding to dystrophin and the cytoskeleton.

IPT domains

(also known as TIG domains). Immunoglobulin–plexin–transcription (IPT) protein domains found on cell-surface transmembrane receptors and intracellular transcription factors with an immunoglobulin-like fold.

Glycosaminoglycan chains

(GAGs). Extended linear polysaccharides comprising repeating disaccharides.

Stoichiometry

The fraction of a glycosylation site in a glycoprotein that is occupied by a glycan.

ABH

Carbohydrate antigens of the ABO blood group system.

Lipoproteins

Large complexes of lipids and proteins that transport lipids in the blood.

Lipopolysaccharide

A bacterial glycolipid endotoxin and a major constituent of the outer membrane of Gram-negative bacteria.

Proprotein convertases

A family of seven secretory mammalian serine proteinases that post-translationally activate proproteins in the secretory pathway by limited proteolysis after multiple basic residues with a general recognition motif, (R/K)Xn(R/K). A prototypical proprotein convertase is furin.

Epithelial–mesenchymal transition

The physiological process in which epithelial cells transition to a mesenchymal state by loss of polarity and other characteristics, and acquire migratory and invasive properties. This occurs during embryonic development and during cancer progression.

Immune checkpoint

An inhibitory pathway in the immune system that is crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses.

Sialyl-Lewisx antigen

A glycan determinant/epitope and selectin ligand (NeuAcα2–3Galβ1–4(Fucα1–3)GlcNAcβ1-R) found on glycoproteins and glycolipids.

Sialyl-Lewisa antigen

A glycan determinant/epitope and selectin ligand (NeuAcα2–3Galβ1–3(Fucα1–4)GlcNAcβ1-R) found on glycoproteins and glycolipids.

Warburg effect

The capacity for tumours to metabolize glucose anaerobically when oxygen is available, increase glucose uptake and produce lactate. The enhanced flux of metabolites through the hexosamine biosynthetic pathway may lead to increased O-GlcNAcylation.

Paucimannose

An N-glycan structure consisting of two N-acetyl-d-glucosamines (GlcNAcs) and only one to three mannose residues (core Fuc optional).

Haploid genetic screen

A genetic screen performed in haploid cells to ease the search for recessive functions in mammalian cells.

Tau protein

Tubulin binding protein involved in neurodegenerative diseases called tauopathies where Tau aggregates into intracellular neurofibrillary tangles.

Native mass spectrometry

An analytical technique used to study non-denatured proteoforms and protein complexes in the gas phase.

Glucocerebrosidase

(GBA). A lysosomal enzyme that hydrolyses glucosylceramide (GlcCer) cerobroside into glucose and free ceramide.

Gaucher disease

A rare genetic lysosomal storage disease caused by mutations in the GBA gene encoding glucocerebrosidase (GBA). Damaging mutations in GBA cause progressive accumulation of the glycolipid glucosylceramide (GlcCer) substrate for the enzyme with damaging effects on tissues and organs. Enzyme replacement therapy is currently the treatment of choice for this disease.

α-Galactosidase

(GLA). A lysosomal enzyme that hydrolyses α-galactose from glycolipids and glycoproteins.

Fabry disease

A rare, genetic X-linked lysosomal storage disease caused by damaging mutations in the GLA gene encoding α-galactosidase A, which catalyses the hydrolysis of glycolipid globotriacylceramide (Gb3).

αGal epitope

A Galα1–3Galβ1–4GlcNAc-R glycan epitope (also known as the Galili antigen) found on glycoproteins and glycolipids of non-primate mammals and new world monkeys, but absent in humans, apes and old world monkeys because of an inactivated GGTA1 gene.

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Schjoldager, K.T., Narimatsu, Y., Joshi, H.J. et al. Global view of human protein glycosylation pathways and functions. Nat Rev Mol Cell Biol 21, 729–749 (2020). https://doi.org/10.1038/s41580-020-00294-x

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