REVIEWS

Global view of human protein

glycosylation pathways and functions
Katrine T. Schjoldager   , Yoshiki Narimatsu   , Hiren J. Joshi    ✉ and Henrik Clausen    ✉
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.

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.
Center for Glycomics,
Department of Cellular and
Molecular Medicine, Faculty of
Health Sciences, University
of Copenhagen, Copenhagen,
Denmark.
✉e-mail: joshi@sund.ku.dk;
hclau@sund.ku.dk
https://doi.org/10.1038/
s41580-020-00294-x
Glycosylation of proteins is arguably the most diverse
post-translational modification. Proteins are glycosylated
by enzymes or through non-enzymatic glycation where
glucose (aldehyde form) reacts with lysine and arginine
residues in proteins, and undergo further changes that
eventually lead to advanced glycation end products
that serve important functions in ageing and disease,
especially diabetes1. The enzymatic protein glycosylation
processes (herein referred to as glycosylation) mostly
involve sequential concerted steps in the endoplasmic
reticulum (ER) and the Golgi system resulting in glyco-
sylation of most (>85%) secretory proteins2,3. In addition,
the majority of nuclear and cytoplasmic proteins undergo
dynamic O-GlcNAcylation4, perhaps making glycosylation
the most abundant post-translational modification (even
more abundant than phosphorylation)5.
Protein glycosylation is a complex, multistep pro-
cess that employs around 200 glycosyltransferase
enzymes that determine which proteins are to become
glycoproteins, the positions of glycans on those pro-
teins and the glycan structures assembled (Table 1).
Furthermore, the initial attachment of glycans may be
differentially regulated in cells by expression of distinct
glycosyltransferase isoenzymes, and thus some proteins
may or may not become glycoproteins depending on
their cell of origin and in response to functional needs.
Glycosylation greatly amplifies the proteome by pro-
ducing diverse proteoforms with different properties,
thereby instructing myriad functions6–8. The ensem-
ble of glycans found on glycoproteins, including
glycosylphosphatidylinositol (GPI)-anchored proteins and
proteoglycans, alongside glycosphingolipids and free
oligosaccharides and polysaccharides constitutes the
glycome of a cell, and the ensemble of glycoconjugates
at the cell surface constitutes the glycocalyx (Fig. 1).
The glycome is diverse, involving different types
of glycoconjugates and oligosaccharides with varying
compositions, sequences and linkages of sugar moieties.
Nevertheless, despite this diversity, the glycoconjugates
do share certain features, such as common structural
scaffolds and terminal modifications9. The diversity in
types of glycans and structures has expanded during
evolution of eukaryotes likely in response to needs for
increased molecular cues and regulation10,11. Protein
glycosylation pathways are nearly identical across mam-
malian cells, although several glycan features were elim-
inated late in evolution by gene inactivation resulting in
xenoantigens12–14.
Studies of deficiencies in glycosylation enzymes
in animal models and human diseases have advanced
understanding of biological functions of protein glyco-
sylation and demonstrated that most glycosyltransferases
serve essential roles in mammalian physiology15–18.
Glycosylation of proteins is thus integral to their func-
tion and should be considered in functional studies of
the proteome. However, our understanding of specific

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Reviews

Table 1 | initiation steps for human protein glycosylation pathways

Type Symbol linkage initiation glycosylation sites in target proteins
enzymes
Sequence motifs Specific
domain
N-Glycosylation LLO/GlcNAcβ–Asn OST complex N-X-S/T, X≠P None
β (STT3A/B)
N-X-C; N-G: N-X-V; X≠P
Asn

O-Glycosylation GalNAcα–Ser/Thr GALNT1–20 Weak isoform-specific None

α (Tyr) motifs51
Ser/Thr/Tyr GALNT11 C6-X-X-X-T-C1 LA
Fucα–Ser/Thr POFUT1 C -X-X-X-X-(S/T)-C
2 3
EGF
α POFUT2 C-X-X-(S/T)-C-X-X-G TSR
Ser/Thr

GlcNAcβ–Ser/Thr EOGT C5XX(G/P/S)(Y/F/W)(T/S) EGF

β GXXC6
Ser/Thr

Glycosylphosphatidylinositol Manα–Ser/Thr POMT1, POMT2 ND None

(GPI)-anchored α TMTC1–4 ND EC
glycoproteins Ser/Thr
A class of proteins that are
Unknown ND IPT
attached to the membrane Glcβ–Ser POGLUT1 C1-X-S-X-(A/P)-C2 EGF
lipid bilayer via a carboxy- β POGLUT2–3 C3-X-N-T-X-G-S-(F/Y)-X-C4
terminal glycolipid anchor Ser
consisting of phosphoethanola-
mine, an oligosaccharide core Xylβ–Ser XYLT1, XYLT2 a-a-a-a-G-S-G-a-(a/G)-a None
and phosphatidylinositol. β (a = D/E)
Ser
Proteoglycans
Proteins carrying one or more Galβ–Hyl COLGALT1–2 Collagen repeats Collagen
glycosaminoglycan chains β
attached covalently.
Hyl

Glycocalyx GlcNAcβ–Ser/Thr OGT None None

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.
β
Ser/Thr
Glcα–Tyr GYG Y194 in glycogenina NA
α
Tyr
C-Mannosylation Manα–Trp DPY19L1–4 W-X-X-W TSR
α
Trp
Glypiation NA Protein–C(O) Transamidase Carboxy-terminal None
EthNP–Man hydrophic segment
(GPI anchor)
The minimal protein–glycan linkages for the distinct human protein glycosylation pathways are shown with the monosaccharide
linkage (including the anomeric α/β configuration of linkage) to amino acid residues in proteins (Figs 1 and 3), which include
11 types of O-glycosylation when the 2 O-Fuc, 3 O-Man and 2 O-Glc pathways are considered distinct. For references and details,
please refer to the main text. COLGALT, collagen O-Gal transferase; DPY19L, dpy-19 like C-Man transferase; EC, extracellular
cadherin immunoglobulin-like; EGF, epidermal growth factor-like repeat; EOGT, epidermal growth factor-domain specific
O-GlcNAc transferase; EthNP, ethanolamine phosphate; Fuc, l-fucose; Gal, d-galactose; GalNAc, N-acetyl-d-galactosamine;
GALNT, polypeptide GalNAc transferase; Glc, d-glucose; GlcNAc, N-acetyl-d-glucosamine; GPI, glycosylphosphatidylinositol;
Hyl, hydroxylysine; IPT, immunoglobulin-like, plexin and transcription factor; LA, low-density lipoprotein receptor (LDLR) class A
repeats; LLO, lipid-linked oligosaccharide; Man, d-mannose; NA not applicable; ND, not determined; OGT, O-GlcNAc transferase;
OST, oligosaccharyltransferase; POFUT, protein O-fucosyltransferase; POGLUT, protein O-Glc transferase; POMT, protein O-Man
transferase; TMTC, transmembrane O-Man transferase targeting cadherin (also transmembrane and tetra-trico-peptide repeat
(TPR) repeat-containing protein); TSR, thrombospondin type 1 repeat; unknown, transmembrane O-Man transferase to be
reported; X, any of the 20 amino acids; Xyl, d-xylose; XYLT, protein O-Xyl transferase. aThe unique glycosylation of glycogenin
(GYG) involves autoglucosylation of Tyr residue 194 (Glcα–Tyr) followed by formation of the glycogenin polymer, and is not
further discussed in the text.
structure–function relationships and roles of specific glycan-specific antibodies and glycan-binding proteins
glycans on specific proteins is incomplete. (GBPs, such as lectins). It is therefore often perceived as
The glycome is produced and regulated by the gly- a daunting task to uncover and dissect specific biolog-
cosylation machinery in a single cell, yet analysis of ical functions of glycans and the underlying molecular
glycans at the single-cell level is not possible with current mechanisms. Advances in next-generation sequencing
glycomics methods, which are limited to probing with and proteomics are beginning to provide single-cell

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ol
ol
n n

2S 4S
n 6S

2S
LDLR 6S
3S
class A NOTCH NS n
repeats
3S Plasma
P membrane

GPI anchor Glyco- N-Linked O-GalNAc EGF TSR domain α-Dystro- Cadherin Plexin/ Proteo- Collagen
sphingolipid repeats glycan EC IPT domain glycan Hyl–Gal
(for example, NOTCH) domain O-Xyl

O-GlcNAc O-Fuc/O-Glc/ O-Man

O-GlcNAc/ C-Man

Nucleus and cytoplasm Domain specific

Ethanolamine phosphate Phosphate d-Glucose (Glc) l-Fucose (Fuc) N-Acetylneuraminic acid (NeuAc)

d-Glucuronic acid (GlcA) P Phosphoinositol d-Galactose (Gal) N-Acetyl-d-glucosamine (GlcNAc) D-Xylose (Xyl)

ol d-Ribitol (Rbo) d-Glucosamine (GlcN) d-Mannose (Man) N-Acetyl-d-galactosamine (GalNAc) Sulfation (S)

Fig. 1 | Main classes of glycoconjugates of the human cellular glycome.
Depiction of the key components of the cellular glycome, highlighting types
of glycosylation that are specific to distinct protein classes or protein
domains. The glycans depicted are only illustrative examples of the glycan
structures that can be synthesized by the different types of glycosylation
pathways. N-glycans and most GalNAc-type O-glycans are widely found
on most proteins trafficking the cellular secretory pathway, whereas the
occurrence of domain-specific glycans is limited to specific protein
domains. The enzymatic processes that orchestrate glycosylation of the
different types of glycoconjugates are partly distinct and partly overlapping.
The initial attachment of the first monosaccharide (or oligosaccharide for
N-glycosylation) to proteins represents the key initiation step that
determines which proteins and positions become glycosylated. These
initiation steps are distinct for the different types of glycosylation pathways
and, to large extent, direct the structures of glycans that are generated.
Some overlap in the later processing steps that involve elongation,
branching and capping of oligosaccharides is found among N-glycosylation
and several types of O-glycosylation as well as in glycosphingolipid
biosynthesis (Fig. 3). The background colour scheme is organized according
to the colour of the first monosaccharide attached to the core protein,
except for glycosylphosphatidylinositol (GPI)-anchored proteins and
glycolipids (shown in grey). The colouring scheme is useful for distinguishing
the protein glycosylation pathways involved in the synthesis of different
types of glycans215 (see also Table 1). Glycan symbols are drawn according to
the Symbol Nomenclature for Glycans (SNFG) format246. Sugar repeat units
are indicated by square brackets with ‘n’ to indicate a number of possible
repeats. EC, extracellular cadherin; EGF, epidermal growth factor; Hyl,
hydroxylysine; IPT, immunoglobulin-like, plexin, transcription factor; LDLR,
low-density lipoprotein receptor; NS, non-specific; TSR domain,
thrombospondin type 1 repeat domain.

transcriptomes and proteomes, which has opened
the way for global analysis of the network of enzymes
that orchestrate protein glycosylation and the assess-
ment of the glycosylation capacities of any given cell.
Accompanying these are the nuclease-based gene edit-
ing technologies that — through precise manipulation
of glycosylation enzymes — provide virtually unlimited
opportunities for engineering, exploration and custom
design of cellular glycosylation capacities. We can now
probe glycosylation systematically through a genetic
entry point, and we foresee that with additional efforts
we will be able to connect information on cellular gly-
cosylation capacities with the actual outcome of the
glycome and roles of glycosylation in cells.
Here, we provide an overview of the current know­
ledge of the human protein glycosylation pathways
and the genes encoding the orchestrating enzymes.
We also discuss the emerging genetic and systems bio­
logy approaches to the study of protein glycosylation and
dissection of biological functions. We are fully aware that
the inherent space limitations necessitate some level of
generalization with omittance of numerous details (and
referencing available literature). Our aim here is to pro-
vide a global view of how the human glycome is estab-
lished and highlight our current understanding of its
roles in physiology.
Basis of protein glycosylation
Glycans are not primary gene products and, in contrast to
proteins, their synthesis occurs without a template. The
human genome contains around 700 genes encoding
enzymes, transporters and chaperones required for the

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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.
cellular glycosylation machinery, glycan modifications
and their degradation (corresponding to 3–4% of the
genome)19–22. Over 200 of these genes encode glycosyl-
transferases, and 173 of these work sequentially to estab-
lish the complex patterns of sugars found on glycoproteins
and lipids20 (Table 1). Tremendous efforts by a large num-
ber of laboratories allowed isolation, cloning, expression
and characterization of these enzymes, thereby estab-
lishing insight into their substrate and kinetic pro­perties
and roles in glycosylation pathways (Supplementary
Table 1) (for comprehensive reviews, see refs16,23–26).
With complete genome sequences available, the reper-
toire of glycosyltransferase genes is well characterized,
and most known glycosidic linkages established between
sugar moieties are accounted for20. Nevertheless, new
glycosyltransferase genes and even distinct glycosylation
pathways are still being discovered27–30.
Protein glycosylation takes place in the secretory path-
way (ER and Golgi), nucleus, cytoplasm and mito­chondria
of all eukaryotic cells (Fig. 2). Ten mono­saccha­rides —
d-glucose (Glc), d-galactose (Gal), N-acetyl-d-glucosamine
(GlcNAc), N-acetyl-d-galactosamine (GalNAc), l-fucose
(Fuc), d-glucuronic acid (GlcA), d-mannose (Man),
N-acetylneuraminic acid (Neu5Ac), d-xylose (Xyl) and
d-ribose (Rib) — derived from activated donor sugar
nucleotides or dolichol (Dol)-linked donors are used to
build the human glycome. Glycans are attached to pro-
teins in four different ways — N-linked to asparagine
(Asn), O-linked to the hydroxyl groups of serine (Ser),
threonine (Thr) or tyrosine (Tyr), C-linked to trypto-
phan (Trp) and glypiation — and there are several dif-
ferent O-linked sugars including GalNAc, Fuc, GlcNAc,
Man, Glc, Xyl and Gal (Table 1). Different types of pro-
tein glycosylation are broadly defined by the sugar–
protein linkage, the initial monosaccharide linked to
proteins and, for some types of O-glycosylation, by the
enzymes directing the first step in protein glycosylation
(Fig. 3). Many types of protein glycosylation (Table 1) start
in the ER, two start in the early Golgi (GalNAc-type and
Xyl-type) and O-GlcNAcylation takes place in the cyto-
plasm and nucleus (GalNAc-type O-glycosylation has
also been reported in the nucleus31, but this result may
be an experimental artefact resulting from glycosylation
occurring during cellular fractionation). Most glyco-
syltransferases are type II transmembrane glycoproteins
with ER and Golgi lumen-oriented catalytic domains
that make use of activated sugar nucleotides (UDP-Glc/
GlcNAc/Gal/GalNAc/Xyl/GlcA, GDP-Man/Fuc,
CMP-NeuAc and CDP-ribitol) as donor substrates,
and have a short carboxy-terminal segment required
for retrograde transport from the Golgi to the ER via
COPI-coated vesicles32,33. Type II glycosyltransferases are
prone to proteolytic cleavage in stem regions, unteth-
ering their catalytic domains from the ER or Golgi
mem­branes34–36 and accounting for the presence of glyco­
syltransferases in body fluids37. Some ER-resident
glycosyltransferases are multipass transmembrane proteins
and utilize Dol-linked donor substrates (Dol-P-Man,
Dol-P-Glc or the lipid-linked oligosaccharide precursor),
whereas some glycosyltransferases are soluble ER-resident
enzymes (protein O-fucosyltransferase 1/2 (POFUT1/2),
protein O-Glc transferase 1–3 (POGLUT1–3), collagen
O-Gal transferase 1/2 (COLGALT1/2) and epidermal
growth factor-domain specific O-GlcNAc transferase
(EOGT) — note that throughout the article we refer to
glycosyltransferases by their gene names and when refer-
ring specifically to genes we use italics) retained in the
ER by C-terminal KDEL signals and use activated sugar
nucleotides for glycosylation38,39.
Structural elaboration of glycans by sequential
addition of monosaccharides to extend, branch and
cap growing oligosaccharides occurs largely in the
Golgi — one exception has been reported in the pro-
tein O-Man transferase (POMT)-directed synthesis of
O-Man glycans, with the first elongation step mediated
by POMGNT2 occurring in the ER40 (Fig. 3). Following
anterograde transport of glycoproteins to the surface,
further glycosylation (in particular, sialylation) during
recycling of membrane glycoproteins can occur41–43,
and more extensive modifications including change of
O-glycan core structures have been suggested44–47.
Glycosylation is orchestrated mainly by the kinetic
properties of glycosyltransferases and their compart-
mentalization in Golgi stacks, with a distribution related
to sequential biosynthetic steps19,20,22,48 (Fig. 2). Formation
of multimeric (homomeric and heteromeric) enzyme
complexes may contribute to the orchestration of these
glycosylation steps49. Insight into the structures and
catalytic mechanisms of glycosyltransferases reveals
common structural scaffolds with distinct acceptor
substrate specificities partly conferred by variable loop
regions extending from the core catalytic unit50,51. It is
further proposed that evolutionary diversification of
the functions of glycosyltransferases involves mutations
in the common core sugar nucleotide binding region
and varying loop regions, which drive the divergence
in donor sugars and acceptor substrate recognition,
respectively50–52. Glycosyltransferases utilizing activated
donor nucleotide sugars have high specificity for the
nucleotide (although they may have some flexibility for
the donor monosaccharide) and, in general, form only
one type of glycosidic linkage structure50. The final glyco­
sylation outcome is influenced by many other factors,
including the availability of substrates and sugar nucleo­
tides, competing glycosylation reactions, co-factors
(such as Mn2+), intracellular transport, pH and actions
of protein chaperones and glycosidases, as well as by
general factors, for example stress, that may affect the
normal cellular state.
Human protein glycosylation pathways
The known glycome is generated through 16 distinct
glycosylation pathways — distinguished on the basis of
the sugar–protein linkage, the initial monosaccharide
linked to proteins and the unique initiating enzymes —
which are directed by at least 173 distinct glycosyltrans-
ferases (Fig. 3; see also Supplementary Fig. 1). These gly-
cosylation pathways include, apart from 2 types of lipid
glycosylation, 14 distinct types of protein glycosylation,
including N-glycosylation, 11 types of O-glycosylation,
C-mannosylation and generation of GPI-anchored pro-
teins (Table 1). Protein glycosylation involves a series of
sequential steps to build characteristic oligosaccharide
structures, including an initiation step determining
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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.
Nature Reviews | Molecular Cell Biology
proteins to be glycosylated, immediate core extension
steps with options for different core structures, elon-
gation/branching steps that expand (and repeat) com-
mon structural motifs and capping steps that terminate
oligosaccharide chains (Fig. 3).
Initiation of protein glycosylation. The initiation step for
each type of protein glycosylation is distinct and regu­
lated by one or more unique glycosyltransferase, or, for
N-glycosylation, an oligosaccharyltransferase (OST) complex
or, for GPI-anchored proteins, a GPI–transamidase
complex that transfers the preassembled GPI anchor to
the C-terminus of select proteins in the ER53,54 (Table 1).
A total of 47 of the 173 glycosyltransferases direct initiation
steps of protein glycosylation. Initiation of N-glycosylation
and likely POMT-directed O-mannosylation occur
co-translationally.
N-glycosylation is initiated in the ER by the oligo-
saccharyltransferase (OST) complex assembled with
STT3A or STT3B catalytic subunits for co-translational
and post-translational glycosylation, respectively, and
these subunits appear to provide some regulation of
N-glycosites55,56. The OST–STT3A complex is asso-
ciated with the ER peptide translocon54, whereas the
OST–STT3B complex includes MAGT1 or TUSC3
oxidoreductase subunits57. The OST–STT3B complex
appears to be the main source of released oligosac-
charides derived from deglycosylation of misfolded
N-glycoproteins destined for proteasomal degradation
and found widely in the cytosol56,58,59.
GalNAc-type O-glycosylation of Ser/Thr and pos-
sibly Tyr is initiated in the Golgi by up to 20 poly­
peptide GalNAc transferase (GALNT) isoenzymes
with distinct and partly overlapping specificities
(of note, only 15 of those have so far been confirmed
to be active enzymes25,60), which leads to the genera-
tion of the simple GalNAcα1–O-Ser/Thr monosac-
charide structure also known as the cancer-associated
Tn antigen. GALNTs generally lack clear acceptor
sequence motifs in target proteins, but they do exhibit
differences in substrate specificities and orchestrate
regulation of sites and patterns of O-glycans in pro-
teins in cooperative ways25,51. Some GALNTs initiate
GalNAc transfer directly to peptides, whereas others
only transfer to prior GalNAc-glycosylated peptides
(designated follow-up glycosylation). Follow-up glyco-
sylation can occur within a short range (1–3 residues)
or a long range (6–17 residues) from the initial glyco-
sylation site, and the latter is mediated by C-terminal
GalNAc-binding lectin domains , a unique prop-
erty among metazoan glycosyltransferases 51. The
GalNAc-type O-glycoproteome is extensive, with
more than 3,000 human O-glycoproteins and over
15,000 identified O-glycosites61. Analysis of differen-
tial O-glycoproteomes of pairs of isogenic cells with
disabled GALNT genes confirms that the expressed
repertoire of GALNTs in a given cell determines
its O-glycoproteome, with individual isoenzymes
making distinct non-redundant contributions 62–64.
Some GALNTs, for example GALNT1 and GALNT2,
have major contributions to the O-glycoproteome,
whereas others serve specific proteins and functional
Reviews
roles (for example, GALNT11 serves specifically the
low-density lipoprotein receptor (LDLR)-related recep-
tor family (LRPs) and activates their ligand binding
properties)65. GalNAc-type O-glycosylation cross-talks
with other post-translational modifications (examples
include FAM20C Ser phosphorylation66,67, VLK Tyr
phosphorylation68 and TPST1/2 Tyr sulfation69).
Fuc, Glc and GlcNAc types of O-glycosylation are initi-
ated in the ER. The most prominent targets for these types
of O-glycosylation are the NOTCH receptor epidermal
growth factor (EGF)-like repeats (Fig. 1). NOTCH receptor
O-glycosylation represents one of the most complex types
of glycan-mediated regulation of receptor functions (see
also Functions of glycosylation below)39,70–72. Initiation
of Fuc-type O-glycosylation is directed by two POFUTs,
wherein POFUT1 serves EGF repeats and POFUT2
serves related thrombospondin type 1 repeats. Glc-type
O-glycosylation of EGF repeats in NOTCH is differen-
tially regulated by the three POGLUTs: POGLUT1 has
wide specificity for many NOTCH EGF repeats, whereas
POGLUT2 and POGLUT3 have specificities for a single
functionally important repeat (NOTCH1 EGF11 and
NOTCH3 EGF10) and glycosylate at a different position73
(Table 1). GlcNAc-type O-glycosylation of EGF repeats
is regulated by EOGT74. All of these initiation enzymes
require folded repeat domains for activity and acceptor
sites have defined sequence motifs.
Man-type O-glycosylation initiates in the ER and
involves at least three distinct types of initiation enzymes.
A yeast-related O-mannosylation type is directed by the
POMT1/2 heteromeric complex75. Interestingly, whereas
the yeast Man O-glycoproteome is diverse and similar
to the GalNAc-type O-glycoproteome76, the human
POMT1/2 complex appears to selectively target the
mucin-like region in α-dystroglycan and a very limited
number of other proteins27. Two additional types of
animal O-mannosylation were recently discovered.
These are driven by four transmembrane O-Man trans-
ferase targeting cadherins (TMTCs), dedicated primar-
ily to modifying the cadherin superfamily, and an as yet
unreported enzyme that selectively targets IPT domains
found in plexins and in receptor tyrosine kinases c-MET
and RON27 (Table 1).
Mannosyl moieties can also be attached to Trp resi-
dues (in the consensus WxxW motif; shown for throm-
bospondin type 1 repeats and type I cytokine receptors),
known as C-mannosylation. This modification is driven
by four dpy-19 like C-Man transferase (DPY19L) gly-
cosyltransferases and occurs in the ER, presumably
co-translationally77.
Xyl-type O-glycosylation is characteristic of pro-
teoglycans and initiated in the Golgi by protein O-Xyl
transferase 1 (XYLT1) and XYLT2 that both have a rela­
tively defined sequence motif (Table 1). Xyl-type glyco-
sylation at select Ser residues primes the biosynthesis of
glycosaminoglycan chains (GAGs). XYLT1 and XYLT2 are
differentially expressed and have overlapping substrate
specificities78. The diversity of proteoglycans is limited,
but a recent sensitive glycoproteomics strategy almost
doubled the number of known proteoglycans79.
Hydroxylysine (HYL)-Gal O-glycosylation is
limited to collagens (important components of the
Reviews

Cell-surface Galectins
glycoprotein

Plasma membrane NEUs

Single-pass
Golgi resident
Endocytosis 7
Soluble enzyme
with KDEL

5 Secretion
Degradation
Multipass
transmembrane protein
Secretion
Retrograde
SPPL3/BACE1 transport ACP2/
ACP5
Adaptor proteins
COPI
CMP Lysosome
Sugar nucleotide SIATs
transporter CMP
CMP

UDP Sugar UMP TGN

GDP Sugar UDP Capping
GMP
4
CMP Sugar GMP
Sugar nucleotide
donors
GDP ol
Elongation and branching
CDP Lysosomal
enzyme
Dol-P-Man/Glc Clathrin
M6PR 6
Core extension or IGF2R
N-Acetylneuraminic GNPTAB/G
acid (NeuAc) Golgi
3
d-Xylose (Xyl) UMP
Phosphate
UMP
UDP Initiation
ol d-Ribitol (Rbo)

d-Glucuronic acid UDP

(GlcA) XYLT1/2 GALNTs
UDP UDP
d-Glucose (Glc) 2
d-Galactose (Gal) COPI
d-Mannose (Man)
GMP
l-Fucose (Fuc)
GDP
1 KDELR UMP
N-Acetyl-d-
glucosamine
(GlcNAc)
N-Acetyl-d- UDP COLGALT1/2
galactosamine EOGT POFUT1/2
POGLUTs
(GalNAc) UMP
POMTs,
TMTCs, COPII
ER
UDP UMP UDP UDP DPY19Ls UDP
GDP
Ribosome
UDP
OST
OGT UDP
OGT UDP
Nucleus Cytoplasm

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◀ Fig. 2 | Subcellular organization of protein glycosylation. The initiation steps of
most types of protein glycosylation occur in the endoplasmic reticulum (ER) and involve
transfer of a first monosaccharide to an amino acid (to Ser, Thr, Tyr, Trp), or in the case of
N-glycosylation to a preformed oligosaccharide (to Asn). Two types of O-glycosylation
(GalNAc-type and Xyl-type) are initiated in the Golgi. O-GlcNAcylation directed by
O-GlcNAc transferase (OGT) occurs in the cytosol and nucleus. The glycosyltransferases
directing these steps are indicated by their gene name (or for N-glycosylation, the oligo-
saccharyltransferase (OST) enzyme complex). Further glycosylation steps (core extension,
elongation and capping) occur throughout the Golgi and trans-Golgi network (TGN).
ER-resident glycosyltransferases directing initiation of protein glycosylation are
transmembrane proteins (type III) or are equipped with a Lys-Asp-Glu-Leu (KDEL) retrieval
signal recognized by the KDEL receptor (KDELR) associated with COPI vesicles (1).
Glycosyltransferases in the Golgi are type II transmembrane proteins with luminal catalytic
domains and short cytoplasmic carboxy-terminal sequences (2). Glycosyltransferases
use dolichol (Dol)-linked donor substrates (Dol-P-Man, Dol-P-Glc or the lipid-linked
oligosaccharide precursor) or activated sugar nucleotides transported from the cytosol
into the ER and/or Golgi lumen by members of the SLC35 family of solute carriers (3).
Although there are over 30 human members of the SLC35 family247, only 9 of these have
been demonstrated to serve in sugar nucleotide transport and their specificities for
donors are not fully clarified. The cisternal maturation model dictates that Golgi-resident
glycosyltransferases are maintained and distributed across stacks by retrograde coat
protein complex I (COPI) vesicular transport directed by motifs in short cytoplasmic tail
sequences (4). Stem/transmembrane regions of glycosyltransferases can undergo
proteolytic cleavage by, for example, signal peptide peptidase-like 3 (SPPL-3) or
β-secretase 1 (BACE-1) proteases in the secretory pathway releasing catalytic domains
to the extracellular milieu (5). N-Glycoproteins acquiring mannose-6-phosphate (M6P)
in the early Golgi by action of the GlcNAc-1-phosphotransferase complex (complex of
GNPTA, GNPTB and GNPTG, which catalyses formation of GlcNAc1-P-Man linkages)
followed by the uncovering enzyme GlcNAc-1-phosphate hydrolase (NAGPA, which
removes the GlcNAc residue leaving M6P) are recognized by the cation-dependent
(M6PR) and cation-independent (insulin-like growth factor 2 receptor (IGF2R)) M6P
receptors, and transported in clathrin-coated vesicles to the lysosome where mannose is
dephosphorylated by lysosomal acid phosphatases (ACP2 and ACP5), and the receptors
are recycled back to the Golgi (6). From the cell surface, glycosylated proteins can
undergo endocytosis followed by recycling, degradation in lysosomes or retrograde
transport to the TGN/Golgi (7). Neuraminidases (NEU1–4) may remove sialic acids
previously attached during the capping step of glycosylation (sialylation), and such
desialylated glycoproteins may be recognized by different glycan-binding proteins,
such as galectins, and upon internalization undergo resialylation by sialyltransferases
(SIATs) again in the TGN. CDP, cytosine diphosphate; CMP, cytosine monophosphate;
COLGALT, collagen O-Gal transferase; DPY19L, dpy-19 like C-Man transferase; EOGT,
epidermal growth factor-domain specific O-GlcNAc transferase; GALNT, polypeptide
GalNAc-transferase; GDP, guanosine diphosphate; GMP, guanosine monophosphate;
GNPTAB/G, GlcNAc-1-phosphotransferase; POFUT, protein O-fucosyltransferase;
POGLUT, protein O-Glc transferase; POMT, protein O-Man transferase; TMTC,
transmembrane O-Man transferase targeting cadherins (also transmembrane and
tetra-trico-peptide repeat (TPR) repeat-containing protein); UDP, uridine diphosphate;
UMP, uridine monophosphate; XYLT, protein O-Xyl transferase.
extracellular matrix). HYL residues are generated by the
α-Dystroglycan
Dystroglycan is encoded by the activity of pro-collagen lysine hydroxylases (PLOD1–3),
DAG1 gene and comprises two and these are subsequently glycosylated by COLGALT1/2
non-covalently-bound subunits isoenzymes. These events take place in the ER before
(α and β). The extracellular formation of the mature collagen triple helix, which is
α-subunit with the O-Man
matri­glycan provides binding to
secreted80. In addition to catalysing lysine hydroxyla-
laminin, and the transmembrane tion, PLOD3 also harbours galactosyltransferase and
β-subunit provides binding to glucosyltransferase activity, at least in vitro, suggesting
dystrophin and the cytoskeleton. that this enzyme may also be involved in generating the
collagen-attached sugar chain81.
IPT domains
(also known as TIG domains). Finally, O-GlcNAcylation is a highly abundant
Immunoglobulin–plexin– glycosylation affecting most cytosolic and nuclear
transcription (IPT) protein proteins that serves as a nutrient sensor and a master
domains found on cell- switch in regulating signalling, transcription and cellu-
surface transmembrane
receptors and intracellular
lar metabolism4,82. O-GlcNAcylation in the cytosol and
transcription factors with nucleus is directed by a single soluble O-GlcNAc trans-
an immunoglobulin-like fold. ferase (OGT) glycosyltransferase that in complex with
Nature Reviews | Molecular Cell Biology
Reviews
an O-GlcNAc hydrolase (OGA; also known as MGEA5)
serves to dynamically regulate on/off O-GlcNAc modi-
fications on proteins in concert with phosphorylations4.
OGT contains amino-terminal transmembrane and
tetra-trico-peptide repeats (TPRs) that mediate protein–
protein interactions and orchestrate access to substrates83.
The residues modified by O-GlcNAcylation are not fur-
ther glycosylated but pose particular analytic challenges,
being labile and of low stoichiometry.
Further processing of protein glycosylation. The process-
ing of protein glycosylation involves sequential steps
adding further monosaccharides to growing oligosac-
charide chains by glycosyltransferases, resulting in core
extension, elongation and branching, and final capping
of glycans (Fig. 3). The core extension refers to glycosyla-
tion steps and glycosyltransferases unique to particular
types of protein glycosylation. For N-glycosylation, the
initially attached preformed N-glycan oligosaccharide
is trimmed by α-mannosidases in the ER, and sequen-
tial addition of β-GlcNAc residues by MGATs gen-
erates complex-type bi-antennary, tri-antennary and
tetra-antennary N-glycan core structures. GalNAc-type
O-glycosylation involves four distinct O-glycan core
structures (Core1–Core4). POMT1/2-driven Man-type
O-glycosylation involves three distinct core struc-
tures (Cores M1–M3). Interestingly, TMTC-initiated
O-mannosylation does not appear to be processed
and appears as the Man monosaccharide. Xyl-type
O-glycosylation involves a common tetrasaccharide
that is extended by either chondroitin sulfate or heparan
sulfate polysaccharides.
The elongation and branching biosynthetic steps may
be shared among different types of protein glycosylation,
and the involved glycosyltransferases therefore function
in multiple glycosylation pathways (Fig. 3). Elongation
primarily involves N-acetyllactosamine (LacNAc
type 2 chain Galβ1–4GlcNAc), often in the form of
repeating disaccharides (polyLacNAc) and branches
(Galβ1–4GlcNAcβ1–3(Galβ1–4GlcNAcβ1–6)Galβ1–
4GlcNAc). The isomeric disaccharide type 1 LacNAc
(Galβ1–3GlcNAc) or the N,N′-Diacetyllactosamine
(LacDiNAc, GalNAcβ1–4GlcNAc) disaccharide are
also found as terminal disaccharides on a common
scaffold of LacNAc glycans. Most of these elongation
and branching reactions are shared with glycolipids.
The capping step mainly involves terminal decoration
of the oligosaccharide chains with Fuc and sialic acid
N-acetylneuraminic acid (Neu5Ac) and is directed
by the large sialyltransferase and fucosyltransferase
families9,84,85 (Fig. 3).
Processing steps may be specific for certain types
of protein glycosylation (pathway specific) or shared
among several types (pathway non-specific). Most
pathway-specific enzymes do not have close paralogues,
which infers that expression of these enzymes allows
predictions to be made about the cellular glycosylation
capacity and the glycan structures being produced (Fig. 3)
(Supplementary Box 1). This concerns glycosyltrans-
ferases involved in initiation and most core extension
steps of the different types of protein glycosylation, with
a few notable exceptions: initiation of GalNAc-type
Reviews

Specific Specific Non-specific

Elongation
and
Initiation Core extension branching Capping Sulfation

PGAP4 3S
P
PIGA R ST3GAL1–6 GAL3ST1
B3GALT1/2/5
PIGM 3S
PIGZ
PIGV PIGB R
R
Type 1 ST6GAL1/2
Lipid UGT8
glycosylation LacNAc R
B3GNT5 R 4S

B3GALNT1 R CHST8/9
ST6GALNAC1–6 GAL3ST3
UGCG A4GALT B4GALNT3/4 3S
B4GALT5/6 B3GALT4 LacDiNAc R R
R
B4GALNT1 6S
R
MGAT1 ST8SIA1–6 GAL3ST2 CHST1/3*
B4GALT1–4
(MGAT4D) MGAT4A–C 3S
Type 2
R GAL3ST4
LacNAc R
N-Glycosylation STT3A/B (OST) N MGAT3 B4GALNT2 A4GNT R
R 6S 6S
MGAT2
CHST5/
QC FUT8 CHST7/
B3GNT2–4 CHST4/
UGGT1 UGGT2 MGAT5 B3GNT7–9 R
R R CHST2/
C1GALT1 B3GAT1/2 CHST6
(C1GALT1C1) Core1 PolyLacNAc
ABO
Core2
Secretory GALNT1–20 S/T/Y Tn antigen GCNT1/3/4 R Lex Lea R
O-glycosylation B3GNT6 Core3 6S 6S
R R
Core4 GCNT2/7 R 3S CHST10*
FUT1/2 FUT3/5
MFNG/ FUT4–7/
POFUT1 S/T LFNG/ FUT9–11 R
RFNG

B3GLCT ER
POFUT2 S/T
DPM1 Dolichol

EOGT S/T
GDP
POMGNT1 Core M1 ALG6
ALG8
S/T ALG2 ALG11
POMT1/2 MGAT5B Core M2 RXYLT1 LARGE1/2
ALG13/14
S/T ol ol ALG3 ALG10
POMK ALG10B
B3GALNT2 FKTN/ B4GAT1 Core M3 DPAGT1 ALG9
FKRP Matriglycan ALG1
POMGNT2
TMTC1–4 S/T
? (IPT domains) ALG12

Secretory N-Linked LLO precursor biosynthesis

DPY19L1–4 W
C-mannosylation
2S DS
POGLUT1 XXYLT1
GXYLT1/2 CHPF/
Secretory CHPF2/ CHST15 DSE/DSEL
O-glycosylation POGLUT2/3 S CSGALNACT1/2 CHSY1/ 2S UST CS
CHSY3
B3GAT3 6S 4S CHST11–14
B3GALT6 3S CHST3* Sulfation
2S
XYLT1/2 S CHST10* HS 3S
B4GALT7 NDST1–4 GLCE R
NS 3S 2S 6S

COLGALT1/2 Hyl EXT1/2 NS 3S

6S
EXTL1/2
PLOD3 EXTL3 HS6ST1–3 HS3ST3A1/B1 HS2ST1 R
Nucleocytoplasmic OGT S/T
HS3ST1/2/4/5/6 6S 4S
O-glycosylation
2S 3S
Ethanolamine phosphate Phosphate d-Glucose (Glc) N-Acetyl-d-glucosamine (GlcNAc) R

Ceramide P Phosphoinositol d-Galactose (Gal) N-Acetyl-d-galactosamine (GalNAc) Linkage

2 3
d-Glucuronic acid (GlcA) l-Iduronic acid (IdoA) d-Mannose (Man) N-Acetylneuraminic acid (NeuAc)
R 4
ol d-Ribitol (Rbo) d-Glucosamine (GlcN) l-Fucose (Fuc) d-Xylose (Xyl)
8 6

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◀ Fig. 3 | Human glycosylation pathways and enzymes. A global view of glycosylation
pathways, the major structural elements of the glycans and the assigned (predicted)
biosynthetic roles for glycosyltransferases as well as carbohydrate sulfotransferases
(indicated are genes encoding these enzymes; please note that the enzymes that initiate
the novel O-mannosylation specific for immunoglobulin-like, plexin, transcription factor
(IPT) domains are currently not reported (question mark)). The 16 known glycosylation
pathways are organized into major biosynthetic steps specific for pathways (initiation
and core extension) and those that are non-specific (elongation and branching, and
capping). For pathways involving isoenzymes, all isoforms are listed (with a dash or solidus
character). The background colours for the different protein glycosylation pathways
mimic the colours of the initiating monosaccharide (lipid glycosylation pathways are
shown in grey), and these are maintained for the pathway-specific glycosylation steps.
Dashed lines indicate alternative pathways for chondroitin sulfate (CS) and dermatan
sulfate (DS) or heparan sulfate (HS) biosynthesis on the common tetrasaccharide linker.
The display is useful to peruse individual genes as well as the glycosylation processes
as an integrated system. Please note that the major structural variations characteristic
of the glycosylation pathways are illustrated, but the diagram is not intended to show all
structural permutations and variations found in cells (linkages only indicated for select
structures). Supplementary Table 1 provides detailed information of the glycosyltrans-
ferase genes mapped. For information on transcriptional regulation of glycosylation
enzymes shown and their association with congenital diseases and genome-wide
association study traits, see Supplementary Figs 1 and 2 (note that these present the
biosynthetic pathways in a vertical orientation as compared with horizontal orientation
shown here and used before215). *Transferases that appear twice in the figure due to dual
pathway-specificity. COLGALT, collagen O-Gal transferase; DPY19L, dpy-19 like C-Man
transferase; EOGT, epidermal growth factor-domain specific O-GlcNAc transferase;
ER, endoplasmic reticulum; GALNT, polypeptide GalNAc-transferase; GDP, guanidine
diphosphate; Hyl, hydroxylysine; LacNAc, N-acetyllactosamine (Galβ1–4GlcNAc);
LAcDiNAc, N,N′-Diacetyllactosamine (GalNAcβ1–4GlcNAc); LLO, lipid-linked
oligosaccharide; NS, non-specific; OGT, O-GlcNAc transferase; OST, oligosaccharyl­
transferase; POFUT, protein O-fucosyltransferase; POGLUT, protein O-Glc transferase;
POMT, protein O-Man transferase; QC, quality control; R, variable underlying core
glycan; S, sulfation; TMTC, transmembrane O-Man transferase targeting cadherins
(also transmembrane and tetra-trico-peptide repeat (TPR) repeat-containing protein);
XYLT, protein O-Xyl transferase.
O-glycosylation is regulated by 20 GALNT isoenzymes;
tri-antennary branching of N-glycans is regulated by
MGAT4A and MGAT4B (note that role of MGAT4C is
unclear86); branching of GalNAc-type O-glycans to form
Core2 or Core4 O-glycans is mediated by three isoen-
zymes (GCNT1, GCNT3 or GCNT4); extension of the
POFUT1-mediated O-Fuc glycan is regulated by three
isoenzymes (MFNG, LFNG or RFNG); and the impor-
tant steps determining chondroitin sulfate or heparan
sulfate GAG biosynthesis on the tetrasaccharide linker
are governed by CSGALNACT1 or CSGALNACT2 and
by EXTL2 or EXTL3, respectively.
Glycosyltransferases considered pathway non-
specific include 17 enzymes that are involved in elon-
gation steps, including B3GNTs, B4GALTs, B3GALTs
and B4GALNTs, and 35 glycosyltransferases involved
in capping steps, including FUTs, ST3GALs, ST6GALs,
ST6GALNACs and ST8SIAs as well as B3GATs, A4GNT
and ABO (Fig. 3). Arguably, these glycosylation pathway
non-specific enzymes direct the greatest diversity in the
glycome, although they also produce common struc-
tural scaffolds that may reduce this diversity in terms
Glycosaminoglycan chains
(GAGs). Extended linear of distinct functional binding epitopes of the glycome9.
polysaccharides comprising Moreover, most of these glycosyltransferases belong to
repeating disaccharides. isoenzyme families with overlapping properties and
poorly understood non-redundant functions, which
Stoichiometry
The fraction of a glycosylation
at least partly hampers the ability to predict the glycan
site in a glycoprotein that is structures produced by analysis of enzyme expression
occupied by a glycan. (Supplementary Box 1).
Nature Reviews | Molecular Cell Biology
Reviews
Side-chain modifications of glycans. Sulfation is the most
abundant and diverse glycan modification. Whereas 35
Golgi sulfotransferases are involved in glycan sulfation,
only two sulfotransferases (TPST1/2) direct tyrosine pro-
tein sulfation87. The majority of these sulfotransferases
serve in decorating GAGs, and different sulfation pat-
terns produced on these large polysaccharides serve as
distinct binding motifs for proteins and regulate wide
essential biological roles (Fig. 3). Whereas the biosynthe-
sis and sulfation of GAGs is well understood, insight into
the specific structures of biologically active GAG motifs
and the sulfotransferase isoenzymes directing these is
still limited88,89. A different type of GAG, keratan sulfate,
is found on N-glycoproteins and O-glycoproteins and is
built on polyLacNAc repeats. Keratan sulfate synthesis
is initiated by 6-O-sulfation of GlcNAc carried out by
CHST6/2 and subsequent galactosylation by B4GALT4
and elongation by B3GNT7 with further 6-O-sulfation of
Gal involving CHST1/3 (ref.90). Some 14 sulfotransferases
are predicted to direct sulfation of N-glycoproteins
and O-glycoproteins, but the specific roles of these
isoenzymes are only partly understood87 (Fig. 3).
Phosphorylation of glycans regulates glycosylation
and serves as a gatekeeper for progression to elonga-
tion steps. For example, the extracellular kinase POMK
phosphorylates the O-Man residue in α-dystroglycan
to induce synthesis of the elaborate matriglycan — an
extracellular matrix-binding motif on α-dystroglycan91,92
(Fig. 3). FAM20B phosphorylates the Xyl residue in the
forming tetrasaccharide linker, and this phosphorylation
affects the third synthesis step directed by B3GALT6
regulating GAG synthesis93,94.
Acetylation of sialic acids is an abundant modifica-
tion that regulates the interaction of sialoglycoproteins
with cellular receptors such as the sialic acid-binding
immunoglobulin-like lectins (Siglecs)95. Acetylation
also conveys resistance to sialic acid removal by most
sialidases7. Sialic acid acetylation occurs through 9-O
or 7-O-acetylation of the activated Neu5Ac donor sugar
nucleotide by CASD1, and incorporation of acetylated
Neu5Ac into glycans by sialyltransferases in the Golgi96.
Sialate 9-O-acetylesterases serve as NeuAc deacetylases97.
Of interest, certain viral receptors bind 9-O-acetyl
Neu5Ac (ref.98). Thus, acetylation of sialic acids in glycans
may function in regulating interactions with endogenous
receptors, while being exploited by pathogens.
Context-specificity of glycosylation
The glycosylation of proteins, in general, reflects the
glycosyltransferase repertoire and the glycosylation
capacities of the producing cell. However, the individ-
ual protein may not be efficiently glycosylated, leading
to heterogeneities, and certain glycosylation features are
targeted to specific proteins and, hence, not universally
found. Moreover, the secretory pathway and glycosyl-
ation machinery, in general, are influenced by numer-
ous cellular and environmental factors that affect the
glycosylation efficiency.
Heterogeneity in protein glycosylation. Different effi-
ciencies in the initiation of glycosylation at specific sites
in proteins (stoichiometry of glycan attachment) and
Reviews

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.
variable processing of glycans at sites in proteins (struc-
tures of the glycan) will result in macroheterogeneity
and microheterogeneity of glycosylation, respectively.
Glycosylation may be influenced by the overall pro-
tein structure and conformational constraints around
glycosites. For example, the presence of high-Man
N-glycans at select sites in mature glycoproteins can
correlate with steric hindrance of the action of manno-
sidases at these sites in the ER. Preferences for acquir-
ing bi-antennary or multi-antennary glycans regardless
of the available glycosylation capacity are found; for
example, generation of the conserved IgG1 biantennary
N-glycan (Asn297) in the Fc region shows that glycan–
peptide backbone interactions and steric hindrance
for processing enzymes result in inefficient branching,
galactosylation and sialylation99–102. Assessing hetero­
geneity in glycosylation of specific proteins is inherently
challenging in glycoproteomics and requires elabo-
rate site-specific analysis with isolated glycoproteins,
but progress towards quantitative glycosite-specific
glycoproteomics workflows is being made103.
Protein-specific glycosylation features. Some gly-
cosylation features are only observed in a subset of
cellular proteins. The most notable example here is
tagging of lysosomal hydrolases (some ~60 different
N-glycoproteins) with mannose-6-phosphate (Man-6-P)
by the GlcNAc-1-phosphotransferase (GNPTAB–
GNPTG) and the uncovering enzyme (UCE)23 (Fig. 2),
which serves as a ligand for the Man-6-P receptors in the
trans-Golgi network to direct transport of these enzymes
to the endo-lysosomal system104. Mechanistically, the
GlcNAc-1-phosphotransferase appears to recognize
conformational motifs in the diverse lysosomal enzyme
proteins to select high-Man N-glycans105. Polysialylation
(α2–8NeuAc) is another protein-specific modification
found on select N-glycans of the neural cell adhesion
molecule and few other proteins that depends on inter-
actions with protein motifs106,107. Polysialylation may also
be found on O-glycans of Neuropilin-2 (ref.108).
Glycosylation features derived from the extracellular
milieu. The glycome of a cell may to some degree also
depend on the glycosylation capacities of other cells
through transfer of glycoconjugates and extracellular
glycosylation reactions. For example, uptake of ABH
blood group glycolipids from plasma lipoproteins to
red blood cells109 and uptake of GPI-anchored glyco­
protein CD52 in sperm during maturation110 both serve to
introduce new glycan structures. Glycosylation enzymes
can also be secreted. Secreted ST6GAL1 can contri­
bute to extracellular sialylation of IgGs and remodel-
ling of cell-surface glycans111–114. Glycosyltransferases
and glycoproteins can also be transferred between
cells via extracellular vesicles and non-membranous
nanoparticles (exomeres), having functional conse-
quences in recipient cells115. Finally, pathogenic bacte-
ria encode and can inject virulent glycosyltransferases
that O-glycosylate and N-glycosylate host proteins and
interfere with cellular functions, as well as glycoside
hydrolases that reprogramme cell-surface glycans116–118.
Functions of glycosylation
Glycosylation has roles in folding, quality control, sta-
bility, transport and function of proteins7, and many
of these functions serve the proteome globally. For
example, in the ER, the initial steps of N-glycosylation
guide protein folding and their quality control 119.

Fig. 4 | Protein glycosylation serves general roles and specific roles for
protein functions. a | Specific roles of glycosylation in regulating protein
functions. Intracellularly, in the nucleus and cytosol, dynamic
O-GlcNAcylation regulates numerous cellular processes, including
transcription and signalling, and serves in nutrient sensing. In the secretory
pathway, endoplasmic reticulum (ER)-initiated types of glycosylation may
serve in folding, stability and trafficking of proteins. Several types of
glycosylation serve specific roles for select proteins driven by differentially
regulated glycosylation at specific glycosites, for example, co-regulating
processing (for activation or inactivation) by proprotein convertases
(GalNAc-type). Within the glycocalyx at the cell surface (zoom-in box),
specific glycosites serve to (from left to right): regulate the stability and/or
activity of glycoproteins, by affecting their susceptibility to regulated
proteolytic processing (for example, G protein-coupled receptor (GPCR)
amino-terminal cleavage to regulate their signalling or entire ectodomain
shedding of membrane proteins) (GalNAc-type); by providing contact
points for cell adhesion (Poly-Sia and Man-type); by modulating the ligand
specificity and/or affinity of receptors like low-density lipoprotein receptors
(LDLRs) and NOTCH (GalNAc-type, Fuc-type and Glc-type); by modulation
effector function of immunoglobulins (N-glycans); or by regulating receptor
dimerization and signalling (GalNAc-type). Glycans also serve in a myriad of
interactions with endogenous glycan-binding proteins (GBPs) (intrinsic, left)
and microbial GBPs (extrinsic, right). Intrinsic glycan interactions serve in
cell–cell adhesion and communication, and are mediated by, for example,
sialic acid-binding immunoglobulin-like lectins (Siglecs) binding to sialic
acid capped glycans for immunomodulation; selectins binding to, for
example, sialyl-Lewisa antigen for cell trafficking; and galectins binding to
different β-Gal glycans for numerous functions, for example, cross-linking
cell-surface glycoproteins by Galectin-3 pentamers in a dynamic lattice that
regulates endocytosis and compartmentalization248. The large Siglec family ◀
promotes cell–cell interactions and regulates important functions in the
immune system by engaging numerous sialylated glycan epitopes. Selectins
(P-selectin, L-selectin and E-selectin) specifically require both sialic acid and
fucose, and in some cases also sulfate, for binding. Extrinsic glycan
interactions involve binding by bacterial lectins or adhesins mediating
bacteria–host interactions, including initial attachment and potential
invasion and colonization; for example, Helicobacter pylori encodes
numerous adhesins that recognize different histo-blood group-related
glycans in the human gastrointestinal tract mucosa249–251. Bacteria also
produce glycoconjugates like lipopolysaccharides and glycan structures
that mimic host glycans, allowing the bacteria to avoid recognition by host
immune cells (mimicry). b | Overview of the common scaffolds and capping
motifs, and the combinations of these that help generate diversity of
glycans, which serve as the major ligands for GBPs (see Fig. 3 for enzymes
involved). Core glycan structures (bottom left) are elongated with one of
three types of chains (type 1 N-acetyllactosamine (LacNAc, Galβ1–3GlcNAc),
type 2 LacNAc (Galβ1–4GlcNAc) or N,N′-Diacetyllactosamine (LacDiNAc,
GalNAcβ1–4GlcNAc); labelled R1–R3, respectively). The core glycan
structures can be extended by chains from R1–R3 (except for O-Man cores,
which can only be extended by R2). Type 2 chains may be repeated to form
poly-LacNAc chains (indicated by square brackets, ‘n’ indicating the number
of repeat units). The terminal core structures may be variously fucosylated,
sulfated and capped by sialic acids, as may residues along the poly-LacNAc
chain, and the combinatorial action of relatively few transferases results in
complex glycan structures with large diversity. To better indicate which
residues have been added in a synthesis step, the residues added
by previous steps are presented in grayscale. Pol II, polymerase II; X, any
amino acid.

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 Reviews

a Further, O-glycosylation via O-Fuc and O-Glc of folded

EGF-like and thrombospondin type 1 repeat domains
Cell Ligand Effector
binding function
stabilizes these after folding, enabling secretion71,120,121.
adhesion
Interestingly, other types of O-glycosylation do not
appear to be required for protein transport and secre-
Antibody tion. On the cell surface, glycoconjugates constitute the
Processing and Receptor glycocalyx that provides a barrier and a protective layer
Ectodomain dimerization
activity regulation
cleavage shielding the plasma membrane from physical stress
Signalling and shaping the cell surface. The glycocalyx serves to
mediate numerous intrinsic and extrinsic interactions.
Signalling
GPCR Signalling Excellent recent reviews cover these topics7–9, and here
we focus on non-global roles of glycosylation, high-
Processing by lighting examples where specific glycosyltransferases
pro-protein regulate specific functions at the level of glycosites or
convertases glycostructures (Fig. 4a).

R-X-X-R-T/S Functions determined at the glycosite level. The NOTCH

receptor illustrates how multiple types of O-glycosylation —
Cell–cell Intracellular Bacteria–host O-Fuc (directed by POFUT1), O-GlcNAc (directed by
interaction trafficking interaction EOGT)122 and O-Glc (directed by the POGLUT1–3
Golgi isoenzymes) — converge to modulate complex protein
Siglecs
Adhesins functions by decorating distinct glycosites in the many
Selectins
ER NOTCH EGF repeats39 (Table 1). With 20 isoenzymes,
Glycoprotein
Galectins folding GalNAc-type O-glycosylation by GALNTs provides the
Nucleus greatest opportunity for differential regulation of specific
DNA Pol II Pathogen mimicry sites in proteins, and GALNT isoenzymes serve highly
specific co-regulatory roles in fundamental processes,
Nutrient sensing including in the inhibition of protein processing by
Endocytosis and signalling
proprotein convertases35,123, in the inhibition (or, in some
cases, activation) of proteolytic cleavage by ADAM pro-
b teases with altered shedding of ectodomains of surface
Sialylation Fucosylation Sulfation proteins124, in the activation of β1-adrenergic receptor
n
by modulating its N-terminal cleavage125, in the modu-
n n lation of peptide hormone function and promoting their
6S stability126,127, and in promoting ligand binding by LDLR
6S
n and LRPs65 (Fig. 4a). These functions are subject to tight
OR n regulation by expression levels of specific GALNTs128,
which may be controlled by feedback loops as shown
n
R2 for GALNT3: GALNT3 uniquely co-regulates propro-
R1 R2
Sialylation tein convertase-mediated processing of FGF23 that
Sialylation Fucosylation
R1 R2 R3 S/T/Y Fucosylation serves to regulate phosphate homeostasis129, and in turn
Sulfation
the expression of GALNT3 is responsive to blood phos-
Elongation 3S phate levels130. Man-type O-glycosylation by TMTC1–4
n n
also provides for differential regulation of O-glycans on
3S
n cadherins and protocadherins that may regulate their
functions in cell adhesion131. Initiation of GAG synthesis
R1 R2 R3
R1 R2 R3 by XYLT1 and XYLT2 also likely directs different subsets
Type 1 Type 2 LacDiNAc R1 R2
LacNAc LacNAc of proteoglycans and corresponding functions78.
Fucosylation The dynamic and widespread O-GlcNAcylation in
Core extension Sulfation the nucleus and cytosol regulated by the OGT and OGA
N-Glycan pair of enzymes has a major role in nutrient sensing
S/T/Y through the hexosamine biosynthetic pathway and modu­
Core2 4S 3S
lating the cellular response to stress4. O-GlcNAcylation
at specific glycosites regulates functions of proteins,
S/T/Y S/T/Y R3 S/T/Y including their enzymatic activity, stability and locali-
N-X-S/T S/T Core1
Lipids zation, and the dynamic on/off nature of O-GlcNAc on
O-Man GalNAc-
(only R2) type Fucosylation glycosites enables O-GlcNAcylation to regulate tran-
Sulfation
scription and signalling events in crosstalk with protein
d-Glucose d-Mannose N-Acetyl-d-glucosamine N-Acetylneuraminic phosphorylation4. O-GlcNAcylation regulates transcrip-
(Glc) (Man) (GlcNAc) acid (NeuAc)
tion by modifying drivers of gene expression like Pol II,
d-Galactose l-Fucose N-Acetyl-d-galactosamine Sulfation (S)
(Gal) (Fuc) (GalNAc)
where C-terminal addition and removal of GlcNAc by
OGT and OGA during the transcription cycle regulates

Nature Reviews | Molecular Cell Biology

Reviews
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.
transcription activation and repression. Site-specific
O-GlcNAcylation also regulates important processes
such as neuronal depolarization132 and intermediate fil-
ament morphology and cell migration133. The OGT sub-
strate selectivity is partly regulated through interactions
between the TPR domains, adaptor proteins and target
protein substrates83.
Functions regulated at the glycan structure level. There is
abundant evidence that the structure of glycans on pro-
teins regulates diverse protein interactions and biological
functions. For N-glycan, for example, tetra-antennary
branching by MGAT5 favours synthesis of polyLacNAc
chains that promote multivalent interactions with lectin
receptors, for example galectins134–136. Functionally,
MGAT5-mediated branching on α5β1 integrin serves to
enhance cell motility and growth137. By contrast, MGAT3
introduces a bisecting GlcNAc residue to the early stages
of complex N-glycans that is not elongated and leads to
inhibition of further N-glycan branching, and there-
fore is a major determining factor for the N-glycome
architecture138. In line with this, MGAT3-directed gly-
cosylation of α5β1 integrin reduces its ability to bind to
its ligands in the extracellular matrix and interferes with
cell migration139. As another example, FUT8-directed
fucosylation of the N-glycan core modulates several
specific protein interactions, including EGF–EGFR
binding140, interactions between the T cell receptor and
the major histocompatibility complex II during T cell
activation141, and IgG1 interactions with the FcγRIIIa
receptor99,142 (Fig. 4a).
Protein function can also be modulated by specific
GalNAc-type O-glycan core extensions. GCNT1-
directed Core2 O-glycosylation promotes Galectin-
1-induced T cell death of immature T cell precursors and
activated T cells143. GCNT3-directed Core2 O-glycans
are also required for surface expression of intestinal
cell differentiation markers144. The β3GlcNAc exten-
sion of O-Fuc on NOTCH regulates the interaction of
the receptor with its ligands, Delta and Jagged, and the three
isoenzymes, MFNG, LFNG or RFNG, directing the
O-Fuc elongation appear to target distinct EGF repeats
and determine differential binding between these two
ligands71. Another example is assembly of the matri­
glycan on α-dystroglycan. In the ER, POMGNT2 primes
matriglycan synthesis by elongating select O-Man gly-
cans on α-dystroglycan145, and in the Golgi, LARGE1/2
produce the functional glycosaminoglycan-like
matriglycan polymer28.
The common structural capping motifs on glycans
found on several types of glycoproteins (Fig. 4b) comprise
the major interactome for GBPs, including the endog-
enous lectin receptors galectins, Siglecs and selectins,
and diverse microbial lectins and adhesins7,9,146 (Fig. 4).
Common for most mammalian lectins are shallow
binding sites and low affinity interactions (micromolar
to millimolar) for single glycan epitopes, allowing for
dynamic and reversible glycan–receptor associations.
GBPs may also recognize more complex structures (clus-
tered saccharides or discontinuous glycans) involving a
higher-order presentation of glycans that can be within
a context of specific glycoconjugates and even specific
proteins resulting in increased affinity towards their
target glycans147. Glycan binding motifs are generally
directed by multiple isoenzymes that are glycosylation
pathway non-specific and highly regulated. However,
these isoenzymes may have non-redundant activities
and selectively regulate glycosylation: in megakaryo-
cytes, the B4GALT1-driven LacNAc epitope regulates
β1 integrin activity required for the formation of mature
platelets148; terminal sialylation of LacNAc or LacDiNAc
O-glycans by ST6GAL1 was found to be important to
regulate B cell activation via lectin Siglec-2 (CD22)95,149;
and ST3GAL1-directed capping of Core1 O-glycans
controls CD8+ T lymphocyte homeostasis by inhibiting
apoptosis and regulating memory cell formation150,151.
Changes of glycosylation in disease
Glycosylation processes in cells are highly sensitive to
the physiological state, and glycans are prevalent report-
ers of disease152,153. An overwhelming number of studies
using lectins, antibodies and direct structural analyses of
glycan features have documented diverse changes in gly-
cosylation in human diseases and especially cancers153.
However, our insight into how these changes in gly-
cosylation occur, what the functional consequences,
if any, are and the nature of specific mole­cular mech-
anisms is still limited. Congenital disorders of glyco-
sylation (CDGs) and genome-wide association studies
(GWAS) are beginning to enable us to deconvolve highly
specific roles of glycosylation in common diseases (for
a global overview of the glycosylation steps and path-
ways served by glycosyltransferase genes known to
cause CDGs and/or implicated by GWAS traits, see
Supplementary Fig. 2).
Lessons from congenital disorders of glycosylation.
The importance of glycosylation is clearly under-
scored by decades of studies of rare monogenic CDGs,
where more than 60 of the 100 CDGs identified to
date are caused by complete or partial loss of function
of a glycosyltransferase17,18 (Supplementary Table 1).
CDGs may be caused by mutations in glycosyltrans-
ferase genes in all types of glycosylation pathways, but
currently most glycosyltransferase-related CDGs are
caused by deficiency in unique enzymes functioning in
pathway-specific glycosylation steps where loss of func-
tion results in global glycome changes (Supplementary
Fig. 2a). These CDGs are generally characterized by
multisystemic disorders, with a wide spectrum and
severity of clinical manifestations predicted to arise
from a myriad of impaired direct and indirect biologi­
cal functions of glycans, and these are often difficult to
trace and dissect17,18.
CDGs were often identified by profiling N-glycans
on an abundant serum glycoprotein (usually transfer-
rin), but today CDGs are also discovered by genome
sequencing that is uncovering CDGs with more subtle
phenotypes154. This has resulted in the discovery of novel
CDGs caused by deficiencies in isoenzymes with subtle
non-global glycosylation functions20,155, and these are
pointing us to highly specific regulatory roles of protein
glycosylation. The most illustrative examples so far are
CDGs caused by loss of function within the GALNT
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 Reviews

isoenzymes, which have uncovered how site-specific
O-glycosylation fine-tunes highly specific protein func-
tions and regulates major physiological processes such as
blood phosphate homeostasis (GALNT3) and likely also
kidney function (GALNT11)129,156,157 (Fig. 5).
Lessons from genome-wide association studies. Survey­
ing glycosyltransferase genes implicated by GWAS for
diverse traits or predispositions interestingly revealed
that GWAS candidate genes represent almost a mirror of
the glycosyltransferase genes so far identified as causing

Specific Healthy Cancer

Core elongation and branching
Initiation MGAT3↓ • Adhesion
• Migration ECM
Non-specific
Epithelial/
SLeX endothelial EMT
MGAT5↑ cell
α2-6-SA α2-6-SA
ST6GAL1↑ Metastasis
FUT8↑↓ ↑FUT6
• Introduction of bisecting GlcNAcs
• Changes in branching
• Changes in core Fuc SLea T cell
FUT3↑ NK cell Siglec 7/9
Macrophage
T Immune
GCNT1↓ TGFβ
Tn • Switch from 2-3 to 2-6 recognition
C1GALT1C1↓ sialic acid capping Activation Activation Siglec 15
COSMC↓ • Increased SLex and
SLea epitopes Immunosuppression
B3GALT5↓
↑GALNT3/T6 ↓B3GNT6 GCNT3↓
STn
ST6GALNAC1↑ Signalling

• Reduced Core2 branching Signalling

• Reduced Core1 elongation
• Reduced Core3/4 structures
• Accumulation of Tn and STn

Placental CS 2S

6S 4S
Apoptosis
NS 3S

6S Cell death Cell death

Changes in GAGs signalling signalling

d-Galactose (Gal) N-Acetyl-d-glucosamine (GlcNAc) d-Xylose (Xyl) T cell receptor, Death

Selectin Siglec receptor
NK cell activated
d-Mannose (Man) N-Acetyl-d-galactosamine (GalNAc)
receptor
l-Fucose (Fuc) N-Acetylneuraminic acid (NeuAc)
d-Glucuronic acid (GlcA) E-Cadherin Integrin CSF3R EGFR

Fig. 5 | common dysregulated glycosyltransferase genes in cancer.
Three major glycosylation pathways (N-glycosylation, GalNAc-type
O-glycosylation and O-Xyl glycosaminoglycans) that undergo characteristic
changes in cancer (left) and examples of the biological effects these
may exert in cancer (right). Glycosylation pathways are simplified
accord­ing to the scheme in Fig. 3, and the key glycosyltransferase genes
undergo­ing altered expression in cancer are indicated (arrows indicate
up/downregulation of expression). Examples of specific effects of altered
glycosylation in cancer are illustrated. Adhesion and migration: increased
branching of N-glycans (MGAT5↑, MGAT3↓) and core fucosylation (FUT8) on
adhesion receptors (for example, E-cadherin, integrins) modulates both cell–
cell adhesion252,253 and, alongside increased α2-6-sialic acid (α2-6-SA)
capping (ST6GAL1), cell–extracellular matrix (ECM) adhesion through
integrin receptors253. Mechanisms behind the changes in adhesiveness can
be related to the abundance of receptors, conformational changes or
presentation of binding epitopes to adhesive partners. These glycosylation
changes are one of the drivers of epithelial–mesenchymal transition (EMT).
Metastasis: alongside increased propensity for EMT, the metastatic potential
of cells is increased by the upregulation of expression of sialyl-Lewisx (SLex)
(FUT6 and ST6GAL1) and sialyl-Lewisa (SLea) (FUT3) glycan epitopes, which
are recognized by selectins on epithelial and endothelial cells promoting
tumour cell extravasation from vasculature and metastatic site homing196–198.
Immune recognition: altered sialylation patterns on tumour cells allow for
evasion from immune surveillance. This is mediated by the sialic acid-binding
immunoglobulin-like lectin (Siglec) family of receptors, both inhibitory (for
example, Siglec 7/9) and activating (for example, Siglec 15), which recognize
different sialic acid ligands and modulate the signalling responses to tumour
ligands resulting in immunosuppression254. Changes in sialylation may be
attributed to cancer-associated changes in glycosyltransferase genes in the
GalNAc-type O-glycosylation pathway, most notably the ST6GALNAC1
gene that induces expression of the STn epitope185–187. Signalling and
apoptosis: glycosylation can modulate the proximity of cell-surface
receptors to each other, affecting the ability to directly interact or cluster,
thereby affecting their signalling capability. For example, loss of a single
GalNAc-type O-glycosylation site (unknown polypeptide GalNAc
transferase (GALNT) isoform) on one of the cytokine receptors of
granulocytes, colony-stimulating factor 3 receptor (CSF3R), is suggested to
result in increased ligand independent dimerization and aberrant
signalling255. Similarly, increased N-linked branching and core fucosylation
(FUT8) on epidermal growth factor receptor (EGFR) increase receptor
dimerization and signalling promoting growth and progression of cancer181.
By contrast, clustering of death receptors is reduced in cancers due to the
truncated GalNAc-type O-glycans and modified N-glycosylation, which
leads to tumour cells escaping apoptotic fates256. CS, chondroitin sulfate;
GAG, glycosaminoglycan; NK cell, natural killer cell; NS, non-specific;
S, sulfation; TGFβ, transforming growth factor-β.

Nature Reviews | Molecular Cell Biology

Reviews
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.
CDGs, and they concentrate around those found to
exhibit high transcriptional regulation158 (Supplementary
Fig. 2a). This suggests the somewhat controversial con-
clusion that dysregulation of glycosyltransferase iso­
enzymes, rather than loss of function of these enzymes,
is involved in more common disease conditions, such as
coronary disease, osteoporosis, chronic kidney disease
and schizophrenia identifiable by GWAS158. This predic-
tion was first supported by studies of GALNT2 impli-
cated in regulation of high-density lipoprotein (HDL)
and triglycerides important for cardiovascular health159.
Individuals with disrupted GALNT2 show lower HDL
levels155, which has been recapitulated in several animal
models with loss of function of Galnt2 (ref.160). The GWAS
signal for GALNT2 is located close to a liver-specific
regulatory element that induces differential allele-
specific transcription161,162, supporting the idea that
liver-specific dysregulation of GALNT2 is the cause
of the altered HDL metabolism. GALNT2 serves
non-redundant O-glycosylation on ANGPTL3 and phos-
pholipid transfer protein (PLTP), two proteins involved
in regulating HDL. GALNT3 is a GWAS candidate for
bone mineral density163, which is consistent with CDG
loss of function causing hyperphosphataemia and familial
tumoral calcinosis with ectopic bone formations129,156,164.
GALNT11, a GWAS candidate for chronic kidney decline,
likely relates to its role in directing O-glycosylation of
the ligand binding regions of LDLR and LRPs, includ-
ing LRP2 (also known as megalin) that serves as the
major endocytic receptor in the proximal tubules of
the kidney 65. GALNT11 O-glycosylation enhances
ligand affinity of LDLR65, and a mouse Galnt11–/– model
revealed an essential role of O-glycosylation in LRP2
regulation and kidney function157.
Dysregulation of glycosyltransferases in cancer. Recent
reviews detail the prevalent glycome changes that equip
cancer cells with distinct glycan features required dur-
ing different stages of tumour growth and dissemination,
including immune evasion and metastasis153,165–167 (Fig. 5).
Given such common aberrations of glycosylation in can-
cer, it may be surprising that somatic mutations in genes
controlling cellular glycosylation are extremely rare, and
very few validated mutations in glycosyltransferases have
been reported in cancer. Mutations in COSMC encoding
a private chaperone required for C1GALT1 that directs
GalNAc-type O-glycan Core1 elongation were reported
in a few cervical cancers168,169, but studies in colorec-
tal and pancreatic cancers found hypermethylation of
COSMC rather than somatic mutations to be the major
reason for the O-glycan truncation that is a hallmark
of epithelial cancers (see also below)169,170. Relatively
rare heterozygous missense mutations in GALNT12
were reported in colorectal cancers, and these were also
shown to affect catalytic properties in recent structural
analysis171. An analysis of data from The Cancer Genome
Atlas (TCGA) reveals that cancer genes are more often
mutated (for example, KRAS is mutated in 8% of can-
cers) than typical glycosyltransferases, which have pro-
tein coding mutations in only around 1% of cancers,
underlining the rarity of somatic mutations deleterious
to the glycosylation machinery.
In contrast to protein-coding somatic mutations,
there is more evidence to support aberrant expression
of glycosyltransferases in cancer153,172–175; for example,
as seen with a hotspot of non-coding mutations around
the ST6GAL1 gene in B cell non-Hodgkin lymphomas176.
Still, only a few studies have provided compelling insights
into the biosynthetic, structural and functional conse-
quences of misexpression of specific glycosyltransferases
in cancer, and we limit our discussion to these (of note,
we excluded discussion of studies using RNA silencing to
validate involvement of specific glycosyltransferase genes
as these generally require further validation due to ineffi-
ciencies in reduction of enzyme activities and off-target
effects). The mechanisms behind altered expression
may include epigenetic regulation169,177 or the action
of miRNAs178,179. Only a handful of key glycosyltrans-
ferases currently emerge as consistent players in cancer
(Fig. 5). In the N-glycosylation pathway, tetra-antennary
branching by MGAT5 (ref.135), bisecting GlcNAc by
MGAT3 (ref.180) and core fucosylation by FUT8 (ref.181)
have important roles in modulating growth factor recep-
tor activation and signalling, cell–cell and cell–matrix
adhesion receptors182 and immune checkpoint inhibitors183.
A characteristic feature of most cancers is incom-
plete biosynthesis of GalNAc-type O-glycans and
accumulation of immature, short, truncated O-glycan
structures (including Tn and STn antigens) that are
normally not found on cells. Tn accumulation appears
to occur primarily through silencing of COSMC169,170,
and this can provide the substrate for ST6GALNAC1 to
produce STn184, which cannot be further glycosylated.
Overexpression of the ST6GALNAC1 enzyme in cells
alone may also override O-glycan extension and induce
STn expression185. These truncated O-glycans are recog-
nized by GBPs including macrophage galactose binding
lectin (MGL) and Siglecs, and serve important roles
in modulating tumour immunity185–187. Expression of
GALNTs is also commonly altered in cancer188–191; for
example, de novo expression of GALNT6 may be directly
involved in inducing characteristic features of colon can-
cer cells including increased proliferation, compromised
adhesion and loss of normal differentiation192.
Yet another glycosylation alteration often associated
with cancer is the change from Core2 to Core1 O-glycans
that can impact interactions with galectins and Siglecs.
This change may be driven by altered expression lev-
els of GCNT1 and ST3GAL1 and competition for the
common unsialylated Core1 substrate193. Increased
expression of ST6GAL1 in cancer may lead to a switch
from α2-3 to α2-6-sialic acid capping of N-glycans and
some O-glycans, which can contribute to cancer cell
survival as α2-6-sialylation of tumour necrosis factor
receptor 1 (TNFR1) inhibits TNF-induced internaliza-
tion of TNFR1 and prevents death receptor-mediated
apoptosis194,195. Furthermore, upregulation of FUTs
directs selectin-mediated vascular extravasation of
cancer cells and cancer cell metastasis196. One exam-
ple here is capping of LacNAc-terminated oligosac-
charides by sialyl-Lewisx antigen directed by FUT3 or
FUT6, which has key roles in mediating bone meta­
stasis through E-selectin binding in the vascular niche197.
Furthermore, FUT3 together with B3GALT5 generate
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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.
Nature Reviews | Molecular Cell Biology
the isomeric sialyl-Lewisa antigen, which has been shown
to induce hyperactivation of EGFR signalling and lead to
pancreatitis, thereby promoting pancreatic cancer198.
O-GlcNAcylation and OGT are commonly dysregu-
lated and have profound regulatory roles in cancer4,199,200.
Enhanced O-GlcNAcylation is caused by the altered
cellular metabolism of cancer cells shifting to anaerobic
glycolysis (the Warburg effect). This results in increased
glucose flux through the hexosamine biosynthetic path-
way with elevated levels of the UDP–GlcNAc donor
sugar that enhances the activity of the OGT enzyme and
increases cellular O-GlcNAcylation.
Also, GAGs and the proteoglycans they are attached
to show wide roles in cancer as they modulate signal-
ling from growth factors and chemokines through
their receptors and affect adhesion between cells and
to the extracellular matrix88,89,201. It may be surprising
that the many glycosyltransferase and sulfotransferase
genes involved in their biosynthesis have not yet stood
out as having direct functions in cancer. Of note, one
GAG epitope widely expressed on malignant cells — a
4-O-sulfated form of chondroitin sulfate that normally
is restricted to the placenta and appears to be associated
with cell proliferation and invasion — does not so far
appear to be regulated by specific changes in expression
of the genes of the predicted biosynthetic pathway202,203.
Overall, cancer cells show prominent alterations to
their glycome, with functional consequences for cancer
pathogenesis. However, it appears that most of these
changes do not have a genetic basis, and more complex
mechanisms seem to be at play in modulating glycosyl-
ation in cancer. Perhaps the main cause of aberrancies
of glycosylation in cancer cells relates to general cellular
perturbations associated with malignancy, including
disorganization of the secretory pathway, Golgi frag-
mentation, ER stress, hypoxia and general metabolic
perturbations204–206. Myriad associated effects, such as
ectopic localization of glycosyltransferases207,208, altered
pH homeostasis in the Golgi209 and metabolic availabil-
ity of donor sugars, impact the glycome in cancer cells.
General disorganization of the secretory pathway is pre-
dicted to have a predominant role in defining the can-
cer glycome. For example, the presence of paucimannose
N-glycans on cell-surface glycoproteins in cancer is sug-
gested to be a result of inappropriate exposure to lyso-
somal enzymes by as yet unknown ways210. As another
example, relocation of GALNTs from the Golgi to the ER
was proposed to induce expression of Tn211, and forced
expression of GALNT1 in the ER in a mouse liver cancer
model strongly enhanced tumour growth through hyper-
glycosylation and activation of matrix metalloprotease
MMP14 (ref.212). Finally, a recent CRISPR–Cas screen
revealed that the zinc transporter SLC39A9 is essential
for efficient O-glycosylation as well as N-glycosylation,
and deficiency in this transporter can lead expression of
the truncated cancer-associated O-glycan Tn213.
Glycome engineering and its prospects
Despite a rapidly increasing wealth of experimental
support for diverse functions of protein glycosylation,
current insight into specific structure–function rela-
tionships and the regulation of these is still fragmented7.
Reviews
This void is likely a consequence of the great structural
complexity and functional (and mechanistic) diversity
of glycans — with few universal and simple rules gov-
erning the process of glycosylation and the need for
experts demanding analytic technologies to address
them — which overwhelm much of the scientific com-
munity and deter from considering glycosylation in their
studies. As enhanced tools and knowledge in the field
expand, the opacity of glycoscience is giving way and a
plethora of opportunities emerge to explore functions of
protein glycosylation and to improve therapeutic protein
functionalities.
Genetic engineering of the glycome. Genetic approaches
to the study of the glycome and its many functions are
appealing because they can be performed at single-cell
resolution (in contrast to current structural approaches
that are not yet at the single-cell level) and they employ
commonly available tools in cell biology today. Tools for
single-cell transcriptomics are available and proteom-
ics are following, and we foresee that such data can be
used to develop in silico approaches to transform these
data into cellular glycosylation capacities (repertoire
of expressed glycosyltransferases) and enable prediction of
the architecture of the glycome of a cell (Supplementary
Box 1). Such in silico glycomics approaches would be
amenable to multi-omics workflows by allowing inte-
gration of the glycome in proteomics and genomics.
Enabling analysis of glycome information by computa-
tional transformation of genetic and proteomic informa-
tion will undoubtedly increase the number and diversity
of studies that address glycosylation and greatly advance
our insight into regulation and functions of glycosyl-
ation. We envision the development of an atlas of gly-
comes for human cells in the near future, much like the
emerging protein and gene expression atlas. Resources
sprouting from genetic approaches to survey the gly-
come have started to emerge, including collections of
validated, efficient gene targeting constructs for human
glycosyltransferase genes (GlycoCRISPR214), libraries
of isogenic cells with combinatorial engineered glyco­
sylation capacities that display the glycome on cells
in a tractable manner for interrogation of functions
(GAGOme203 and GlycoDisplay215) and libraries of
mammalian host cells for recombinant production
of proteins with different glycans for direct testing of
glycan functions216 (Box 1). Such tools offer simple and
direct ways to explore and exploit glycosylation.
Genetic dissection of glycosylation pathways and
the biological functions of their products already have
wide support from studies in cell systems217 and model
organisms15,16, and with the rapid progress in develop-
ing efficient precise gene editing tools, more systematic
and global dissection of glycosylation pathways is now
possible218. A first example illustrating the power of a
whole-genome dissection approach to analysing glyco-
sylation uncovered most of the genes required for syn-
thesis of the matriglycan on α-dystroglycan (Fig. 3) with
the use of a haploid genetic screen in human cells219. More
generally applicable are genome-wide loss-of-function
screens using CRISPR–Cas9 knockout, which have
identified not only glycosyltransferase genes but also
Reviews
Box 1 | Next-generation cell-based glycan arrays
Glycan arrays are essential tools for exploring the structural features required for
binding to glycans, and they have been instrumental in decoding the informational
content of complex glycans257. Glycan arrays require continued synthesis (or isolation)
of glycans with limitations on size and complexity that can be synthesized, and they
generally present glycans in high densities without the natural context of their protein
or lipid carriers258. to circumvent some of these limitations, it is possible to use a genetic
‘tree-pruning’ approach — glycotopiary — to engineer and extensively reprogramme
the glycosylation capacities of a human cell line to construct large libraries of isogenic
cell lines that each differ in expression of one or more glycosyltransferases and, hence,
display the glycome with loss or gain of distinct glycosylation features215. the creation
of smaller sub-libraries of such engineered cells rationally designed to display specific
features of the glycome — for example, elaborated glycans only on select types of
glycoconjugates (glycoconjugate sub-library), N-glycoproteins with different branching
(N-glycan sub-library) or glycans with different types of sialic acid capping (sialome
sub-library) — enables step-by-step dissection of the importance of these features for
biological interactions226. Cell-based glycan arrays can be interrogated by traditional
cell-binding assays such as flow cytometry, but also with any other types of assay
applicable to cells. the cell-based arrays can be used to express proteins of interest
on the cell surface or, when possible, as secreted glycoproteins with different glycan
structures for studying the effects of specific glycan structures. For example, the
cell-based array can be used readily to produce various distinct glycoforms of a
glycoprotein therapeutic that can be tested in preclinical studies for identifying the
optimal design of glycans. importantly, using cell-based glycan arrays for probing
biological interactions provides knowledge of the genes required (and not required)
for display of the specific glycan binding epitope and this result informs on the
biosynthetic basis, which ultimately enables prediction of the structural features of
the glycans necessary for the binding interaction studied. the extended information
provided by cell-based glycan arrays enables scientists to readily use genomics and
proteomics approaches to further study and dissect identified functions in other
models. Moreover, this information also provides design matrices for engineering other
mammalian host cells for recombinant production of glycoprotein therapeutics with
desirable and reproducible glycan patterns216,239.
led to the discovery of unexpected genes and functions
required for glycosylation. Examples of the discover-
ies made with such screens include the demonstration
of N-glycan core fucosylation (mediated by FUT8) as
a positive regulator of cell-surface expression of the
immune checkpoint receptor on T cells183, the require-
ment of sialic acid biosynthesis (with a role of sialic acid
transporter SLC35A1) for avian H5N1 influenza virus
replication220 and the role of tetra-antennary N-glycans
(instigated by MGAT5) for CD8+ T cell-mediated glio-
blastoma tumour-cell killing221. The genetic approach is
particularly useful in dissecting the structural features
that guide interactions with GAGs and confer their func-
tions due to difficulties in sequencing the GAG poly-
saccharide chains and structural determination of such
binding motifs203,222. One example here is the demon-
stration of the role of specific GAG chains on heparan
sulfate in the cellular uptake of Tau protein223.
Despite the various fruitful insights, a limitation
of genetic screens is identifying glycosylation features
covered by isoenzyme redundancies. For example, a
screen for avian H5N1 influenza virus replication medi-
ators only identified the sialic acid transporter as being
Tau protein involved in the process, but not any of the sialyltrans-
Tubulin binding protein ferases and other genes expected to be involved in the
involved in neurodegenerative sialylation pathway224. In this case, focused screens with
diseases called tauopathies
where Tau aggregates into
combinatorial targeting of multiple isoenzymes covering
intracellular neurofibrillary redundant steps are needed to identify the candidate iso-
tangles. enzyme genes involved. Targeted screens with individual
and combinatorial gene knockout to systematically cover
known individual biosynthetic steps are being employed
and can take advantage of validated efficient tools. Select
screens may also be used with organotypic tissue models
for dissection of functions of different types of glyco-
sylation and distinct isoenzymes, as recently shown in
human skin organoids64,225.
More rational engineering of glycosylation capacities
in cells offers opportunities to interrogate established
genetic and biosynthetic rules with the added potential
of surprises for discovery of novel glycosylation path-
ways, such as the TMTC-directed O-mannosylation
discovered by the observation that knockout of the
POMT1/2 genes eliminated only select O-mannosylation
capacities27. Libraries of engineered isogenic cells that
each lack one or more glycosyltransferases can be used
to display parts of the human glycome in the natu-
ral environment of the cell surface and the context in
which glycans are naturally found on different types
of glycoconjugates203,215 (Box 1). Such cell-based glycan
libraries may be probed by any assay commonly used
with cells, and may provide a sustainable self-renewable
resource to survey glycan interactions of GBPs, substi-
tuting analyses with traditional glycan arrays compris-
ing individual immobilized glycans. Whereas studies
with immobilized glycans provide information on the
structural requirement for interactions with glycans,
studies with the genetic engineered cell libraries pro-
vide information on the biosynthetic and genetic basis
of the glycosylation required for the interacting glycans.
The structural features of interacting glycans are pre-
dicted using maps of glycosylation pathways226 (Fig. 3).
This extended information greatly facilitates further
studies into functions using genetic strategies commonly
available to non-experts.
Overall, despite the complexity of the glycome, our
knowledge is expanding, mainly owing to the application
of ever-more sophisticated analytical techniques. We
predict an explosion in genetic dissection applications
including not only disruption of genes but also gene
activation. Popularization of such tools and continued
progress in this space now hold promise for the devel-
opment of an atlas of cellular glycosylation and glycomes
of all human cells in the near future, which could then
be explored — with readily available genetic dissection
tools — for gaining functional insight into the glycome.
Glycoengineering of therapeutics. Glycans are often
regarded as ‘necessary evils’ in biologics production
pipelines. The freedom to perform extensive engineer-
ing of glycosylation capacities in mammalian host cells
provides wide opportunities for the improvement and
optimization of current biologics production pipelines
and for the development of custom-designed glycopro-
tein therapeutics216. Engineering glycans on therapeu-
tic proteins has been pursued for decades by chemical,
chemo-enzymatic and genetic approaches in differ-
ent cell types (Escherichia coli, yeast, plant, insect and
mammalian227–230), but engineering cells already tooled
for human glycosylation provide a simpler and more
versatile starting point and opportunities for develop-
ing more complex and physiologically relevant designs
www.nature.com/nrm

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.
Nature Reviews | Molecular Cell Biology
as well as novel designs offering improved pharmacoki-
netic and pharmacodynamic properties. Availability of
comprehensive cell libraries of isogenic cells with dif-
ferent glycosylation capacities that enable production
of glycoproteins with controlled diversity in glycan
structures attached should greatly facilitate advances
in this respect, and the increased understanding of the
outcomes of genetic engineering are providing relia-
ble design matrices for custom-designed glycoprotein
therapeutics216 (Box 1).
The primary objectives for glycoengineering are
desires to produce safe, consistent and efficient biolog-
ics. Species differences in glycosylation and immunity
to non-human glycans are posing challenges for use of
non-human cell lines for production of biologics. The
Chinese hamster ovary (CHO) cell line is currently
the lead production host for human protein biologics
because of its simple human-like glycosylation capaci-
ties, but heterogeneity of the glycans found on recom-
binant CHO-expressed glycoproteins still challenges
regulatory demands for consistency and imposes exten-
sive analytic efforts to produce pure glycoprotein species
with the desired properties231. However, with engineered
homogeneous glycosylation to reduce proteoform com-
plexity, the downstream analysis can be largely simpli-
fied and perhaps reduced to native mass spectrometry232,
which improves quality control of biologics.
The first glycoengineered biologic agent approved for
clinical application was the lysosomal glucocerebrosidase
(GBA) used for treatment of the lysosomal storage disor-
der, Gaucher disease. GBA was engineered to remove M6P
from its N-glycans that mediates uptake by Man-6-P
receptor233, and instead expose Man residues to achieve
efficient mannose receptor-mediated uptake by macro­
phages, which are the main cell type affected by GBA
storage defect, and efficient targeting to lysosomes233.
Major progress in glycoengineering has been seen in
the area of immunotherapy for cancer142. Engineering
therapeutic IgG antibodies with low core fucose on the
Fc N-glycan234,235 to increase antibody-dependent cell
cytotoxicity is a major advancement and such antibod-
ies are now used in cancer therapies142. Unnatural short
trisaccharide N-glycans have been engineered by intro-
duction of an endo-β-N-acetylglucosaminidase (endoT)
in the Golgi to serve a similar purpose236. A novel ele-
gant strategy using installation of inducible expression
of glycosyltransferase genes (FUT8 and B4GALT1)
has also shown the ability to fine-tune control of IgG
N-glycosylation 237. Finally, extensive engineering
of N-glycosylation capacities demonstrates that multiple
homogeneous glycoforms can be produced238.
Relying only on endogenous glycosylation capac-
ities of mammalian cells severely limits the discovery
potential for novel, improved glycan designs for thera-
peutics. However, with engineering, there are now only
a few limitations in achievable designs and options for
testing these203,216. Stably engineered CHO and HEK293
cells offer production hosts for a wide range of glyco-
forms for experimental screening and discovery of
new improved designs. Inspired by the above-noted
success for Gaucher disease233, we engineered variants
of α-galactosidase used for the treatment of another
Reviews
lysosomal storage disorder, Fabry disease, and found,
surprisingly, that glycodesigns without exposed man-
nose, but capped with α2-3-linked sialic acid, showed
improved circulation and biodistribution with efficient
reduction of accumulated glycolipid species in a mouse
model239. Glycoengineering may also be used to improve
immunogenicity of recombinant produced vaccines by
introducing glycan structures known to target immune
regulatory lectin receptors240,241, and for example, to
better mimic high-mannose glycans often found on
viral envelope glycoproteins and recognized by broadly
neutralizing monoclonal antibodies242.
In addition to approaches in cells, glycoengineering
of entire animals to remove immunogenic glycans for
organ sourcing for human transplants has a long his-
tory, with approaches to remove the strongly immuno­
genic αGal epitope widely expressed on non-primate
mammalian cells and, recently, the generation of a
triple-knockout pig with elimination of three immuno-
genic glycoepitopes, αGal, N-glycolylneuraminic acid
(Neu5Gc) and terminal GalNAc243. Although increased
immunotolerance achieved by these approaches may not
solve all issues with xenotransplantation, it still holds
great promise for more specific uses of animal tissues
in human medicine, such as for heart valve curation
and as acellular dermal matrices for reconstructive sur-
gery. Currently, porcine dermal matrices treated with an
effective α-galactosidase to remove αGal epitopes244 are
in wide clinical use (Strattice™)245.
Conclusions and perspective
Most proteins are glycosylated through 1 or more of
14 distinct glycosylation pathways that in humans involve
at least 173 glycosyltransferases as well as many other
enzymes that modify glycans. Glycosylation produces
a vastly larger number of glycan structures through
combinatorial use of enzymes and of repeated com-
mon scaffolds. As such, glycosylation of proteins clearly
provides the greatest and most diverse expansion of the
proteome. This complexity, compounded by analytical
limitations (sensitivity, site-specific glycan analysis of
glycoproteins and sequencing of GAGs), has for many
years been challenging to capture and dissect func-
tionally, and studies of the glycome have lagged behind
studies of other omes. However, protein glycosylation
pathways are now sufficiently outlined to start applying
systems biology approaches to explore regulation of the
glycome through quantitative transcriptomics and/or
proteomics data, opening new avenues in understanding
structure–function relationships of protein glycosylation.
Genetic approaches to studying assembly and func-
tions of the glycome have a long history. The new gene
editing tools are now allowing more comprehensive
genetic dissection and genome-wide screens of func-
tions of glycans, generation of cell-based glycan dis-
play platforms for interrogation of glycan interactions
and functions, and custom design of glycoproteins —
advances that will ignite wider integration of glyco-
sylation in cell biology and systems biology. From a
translational standpoint, these advances provide oppor-
tunities to discover and test improved drug and vac-
cine designs95,240. To fully benefit from these resources,
Reviews

it is important to develop approaches to efficiently and
reproducibly translate cellular glycosylation capacities
(transcriptome/proteome information) into the archi-
tecture of the glycome generated (Supplementary Box 1).
We are now in an era where it is simpler — and per-
haps more informative — to use genetic approaches to
study protein glycosylation and functions, rather than
traditional analysis of the structures of glycans on pro-
teins, but considerable experimentation and validation
of these approaches are still needed. We posit that the
biosynthetic and genetic rules for human glycosylation
pathways may provide sufficient foundation for devel-
oping global predictions of cellular glycosylation capac-
ities and the resulting glycome at the single-cell level
based on transcriptomics and proteomics data — an
era that could be designated in silico glycomics. We
envision the development of a comprehensive atlas of
cellular glycomes across the entire human body and
integration of protein glycosylation features in gene
and protein databases.
Published online xx xx xxxx

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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).
Author contributions
The authors contributed equally to all aspects of the article.
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.
Peer review information
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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Supplementary information
Supplementary information is available for this paper at
https://doi.org/10.1038/s41580-020-00294-x.
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/
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