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Histochem Cell Biol (2017) 147:119–147
DOI 10.1007/s00418-016-1526-4
REVIEW
Eukaryotic protein glycosylation: a primer for histochemists
and cell biologists
Anthony Corfield
1
Accepted: 25 November 2016 / Published online: 23 December 2016
© The Author(s) 2016. This article is published with open access at Springerlink.com
awareness that their structure is an ideal platform to store
information [Winterburn and Phelps
1972; Gabius 2009,
2015; please see also the introduction to this theme issue
(Gabius and Roth 2017)], a survey of their characteristics
is timely. In connection with the overview on glycolipids
(Kopitz 2017, this issue), an introduction to protein glyco-
sylation is provided here. Present in archae- and eubacte-
ria and in Eukaryotes (Reuter and Gabius 1999; Patsos and
Corfield 2009; Wilson et al. 2009; Zuber and Roth 2009;
Corfield 2015; Corfield and Berry 2015; Tan et al. 2015),
protein glycosylation is shared by organisms of all three
urkingdoms, associated with diseases when aberrant (Hen-
net 2009; Hennet and Cabalzar 2015). Starting with struc-
tural aspects, functional implications are then exemplarily
discussed.
Glycosylation of proteins: general aspects
Most of the proteins are subject to glycosylation by a wide
variety of enzymatic mechanisms. The length of the conju-
gated glycan ranges from a single sugar moiety to branched
structures and the long glycosaminoglycan chains (Fig. 1;
for information on proteoglycans, please see Buddecke
2009).
This wide spectrum of structural modes of glycosyla-
tion requires access to detailed information available on the
presence of glycans. Representative techniques are listed as
follows:
• Detection of glycans as carbohydrates in glycoproteins
using chemical assays. This can be applied for screen-
ing in standard fractionation techniques such as high-
performance liquid chromatography, size fractionation
chromatography, ion-exchange chromatography, elec-
Abstract Proteins undergo co- and posttranslational modi-
fications, and their glycosylation is the most frequent and
structurally variegated type. Histochemically, the detection
of glycan presence has first been performed by stains. The
availability of carbohydrate-specific tools (lectins, mono-
clonal antibodies) has revolutionized glycophenotyping,
allowing monitoring of distinct structures. The different
types of protein glycosylation in Eukaryotes are described.
Following this educational survey, examples where known
biological function is related to the glycan structures car-
ried by proteins are given. In particular, mucins and their
glycosylation patterns are considered as instructive proof-
of-principle case. The tissue and cellular location of gly-
coprotein biosynthesis and metabolism is reviewed, with
attention to new findings in goblet cells. Finally, protein
glycosylation in disease is documented, with selected
examples, where aberrant glycan expression impacts on
normal function to let disease pathology become manifest.
The histological applications adopted in these studies are
emphasized throughout the text.
Keywords Eukaryocyte · Glycans · Glycoprotein ·
Glycosylation · Histochemistry · Mucin
Introduction
Histochemists and cell biologists are familiar with the
ubiquitous presence of glycans. In view of the increasing
* Anthony Corfield
corfielda@gmail.com
1
Mucin Research Group, School of Clinical Sciences, Bristol
Royal Infirmary, University of Bristol, Bristol BS2 8HW, UK
120 Histochem Cell Biol (2017) 147:119–147
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trophoretic methods and density gradient centrifuga-
tion (Brockhausen et al. 1988; Nakagawa 2009; Marino
et al. 2010).
• Detection of glycans as carbohydrates in tissue sections
using chemical assays to provide morphological data
regarding the localization of the carbohydrate/glycopro-
tein (Filipe and Branfoot 1983; Buk and Filipe 1986;
Warren
1993; Filipe and Ramachandra 1995; Corfield
and Warren 1996) (for an example on the identification
of O-acetylated sialic acids in human colon using the
mild-PAS method, please see Fig. 2).
• Detection of glycan by probes with specificity to gly-
cans, i.e. monoclonal antibodies (such as the CD-based
reagents specific for the T/Tn antigens; for an over-
view, please see Gabius et al. 2015) or lectins (for an
introduction to lectins and their application in cyto- and
histochemistry, please see Kaltner et al. 2017; Man-
ning et al. 2017, this issue). Working with cytological
Fig. 1 Classes of vertebrate glycan structures. Membrane and
secreted proteins have N-glycan, GlcNAc to asparagine as oligoman-
nose, complex or hybrid forms, or O-glycans linked through GalNAc
to serine/threonine with eight core structures and extension. Glycosa-
minoglycans have a core linkage tetrasaccharide to protein, with sub-
sequent disaccharide repeats and characteristic sulphation patterns.
They may be secreted, transmembrane or GPI-anchored. Hyaluronan
is not linked to a protein. O-Mannosyl residues may be extended.
O-Glucose and O-fucose are found in EGF domains of some proteins.
C-Mannose is attached to protein tryptophan side chains. Single β-O-
GlcNAc is found on many cytosolic and nuclear proteins. The col-
lagen disaccharide is linked to hydroxylysine and through galactose.
Glycogen is linked through glucose unit to a tyrosine in glycogenin.
Glycosphingolipids contain glycans linked to a ceramide carrier;
from Moremen et al. (
2012), with permission
121Histochem Cell Biol (2017) 147:119–147
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specimen or tissue sections, glycophenotyping is read-
ily feasible with labelled lectins by various microscopi-
cal techniques (Roth 1993, 1996, 2011; Habermann
et al. 2011). Using chemically prepared compounds as
inhibitors (Murphy et al. 2013; Roy et al. 2016), struc-
tural and topological aspects of the specificity of lectin
binding can be analysed (André et al. 2016; Roy et al.
2017, this issue). In addition to their application, lec-
tins have found a broad range of applications for gly-
coprotein analysis (for compilation, please see Table 1
in Solís et al. 2015). These versatile assays also shape
the notion that such interplay will have physiological
relevance (for information on tissue lectins, please see
Gabius et al. 2016; Kaltner et al. 2017; Manning et al.
2017; Mayer et al. 2017; Roth and Zuber 2017, this
issue).
Glycosylation: biological roles
Glycosylation is a flexible co- and posttranslational mod-
ification that has been adopted by Eukaryotes to create a
dynamic strategy applicable in modern biology. As many
options are possible, an overview of the biological rele-
vance of glycan chains in glycoproteins is shown in Fig. 3.
Backed by exemplary references, special aspects are
highlighted:
• Impact on the physicochemical properties of the gly-
coprotein molecule. The secreted mucins are an exam-
ple, where viscoelasticity and gel formation establish
a protective barrier on mucosal surfaces (Newton et al.
2000; Pearson et al. 2000; Atuma et al. 2001; Allen and
Flemström 2005; Gustafsson et al. 2012; Johansson and
Hansson 2012; Verdugo 2012; Berry et al. 2013; Birch-
enough et al. 2015).
• Docking sites for tissue lectins, hereby serving a broad
range of functions including adhesion, growth regu-
lation or routing (for further information, please see
Gabius et al. 2011, 2016 and in this issue, Kaltner et al.
2017; Manning et al. 2017; Mayer et al. 2017; Roth and
Zuber 2017). The quality control and the specific deliv-
ery of glycoproteins in tissues and cells are illustrative
examples. Specific functions of individual glycopro-
teins are related to their location and selective expres-
sion. The glycans serve as postal code for routing and
delivery, for example for asialoglycoproteins, lysoso-
mal enzymes carrying mannose-6-phosphate or glyco-
proteins in galectin-dependent apical/axonal transport
(Kornfeld et al. 1982; Stechly et al. 2009; Velasco et al.
2013; Higuero et al. 2017; Manning et al. 2017, this
issue).
In order to illustrate the importance and scope of protein
glycosylation it is necessary to enumerate the range glycan
structures that have been identified and which are carried
by glycoproteins. Table 1 gives an overview of the broad
scope of glycan structures found in Eukaryotes. The main
Fig. 2 mPAS detection of sialic acids in human colon. Mucus stored
in goblet cell thecae. Staining of the colonic mucosa with the mild
periodic acid-Schiff reaction stains non-O-acetylated sialic acids and
demonstrates the location of the mucus prior to secretion; from Cor-
field (
2011), with permission
Fig. 3 Biological roles of glycans. A general classification of the
biological roles of glycans is presented, emphasizing the roles of
organism proteins in the recognition of glycans; from Varki and Lowe
(
2009), with permission
122 Histochem Cell Biol (2017) 147:119–147
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types of glycosylation are N-linked and O-linked glycans,
with a considerably smaller group of C-linked glycans.
N-Linked glycans are attached through an N-glycosidic
bond between asparagine and β-N-acetyl-D-glucosamine
(GlcNAc). The asparagine residues are associated with the
Table 1 Main types of glycan structures
Glycan Group Glycan Structure
Proteoglycans
Hyaluronan
Glycoproteins
N-Glycans
Mannose 6-phosphateglycans
Glycoproteins
O-Glycans
Table 1 continued
Glycoproteins
O-GlcNAcylation
Glycoproteins
Glycophosphatidylinositol
(GPI) anchor
Glycoproteins
C-Mannose
Glycosphingolipids
Major groups of eukaryotic glycans. Examples of the general types of gly-
can, largely drawn from animal examples, are shown. Key: yellow circles,
D-galactose; yellow squares, N-acetyl-D-galactosamine; blue circles, D-glu-
cose; blue squares, N-acetyl-
D-glucosamine; blue/white squares, D-glucosa-
mine; green circles,
D-mannose; red triangles, L-fucose; purple diamonds,
N-acetyl-
D-neuraminic acid; light blue diamonds, N-glycolyl-D-neuraminic
acid; blue/white diamonds
D-glucuronic acid; orange/white diamonds,
L-iduronic acid; orange stars, D-xylose; white diamonds, myo-inositol.
All glycosidic linkages are shown as α or β, with the corresponding posi-
tion; for example, β4, β1,4 linkage. 2S 2-O-sulphate, 3S 3-O-sulphate, 4S
4-O-sulphate, 6S 6-O-sulphate, 2P 2-O-phosphate, 6P 6-O-phosphate, Asn
asparagine, CH
2
CH
2
NH
2
ethanol amine, FA fatty acid, predominantly pal-
mitate, Hyd hydroxylysine, Hyp hydroxyproline, NS N-sulphate, Tryp tryp-
tophan, R various glycan substitutions occur at the initial mannose in GPI
anchors; from Corfield and Berry (
2015), with permission
123Histochem Cell Biol (2017) 147:119–147
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recognition sequence Asn-X-Ser/Thr. This sequence and the
associated synthetic pathway are conserved in evolution for
all of the metazoan (Aebi 2013; Breitling and Aebi 2013).
The N-glycans contain a common, branched core compris-
ing Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAcβ1-
Asn-X-Ser/Thr and this is extended to yield three differ-
ent types, oligomannose, complex and hybrid (Zuber and
Roth 1990). Common features occur in the extension of
the N-glycan core, generation of two antennae from the
Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAcβ1Asn-
X-Ser/Thr core. Second, the core is extended to yield oli-
gomannose forms containing only mannose, formation of
complex types having antennae terminated with a sialylated
N-acetyllactosamine trisaccharide, plus a fucose on the
internal GlcNAc linked to the asparagine and finally hybrid
types containing both oligomannose linked to Manα1,6
and complex units attached to the Manα1,3 residues (Aebi
2013; Breitling and Aebi 2013).
The process of N-glycosylation, starting co-translation-
ally, is common across the Eukaryotes in accordance with
their comprehensive range of biological functions. The
enzymes responsible for the stepwise generation of the
precursor glycan utilize a dolichol pyrophosphate lipid car-
rier and follow a series of trimming and processing steps
that are conserved across the Eukaryotes. A series of three
cytoplasmic glycosyltransferases, initially a GlcNAc trans-
ferase followed by mannosyltransferases, result in the for-
mation of the Man5GlcNAc2 pentasaccharide. Subsequent
extension occurs in the lumen of the endoplasmatic reticu-
lum and the dolichol-oligosaccharide is translocated by a
flippase. In the ER lumen a series of manipulations occur
to generate the range of N-glycans required for the tis-
sue (Zuber and Roth 2009; Aebi 2013; Breitling and Aebi
2013).
Oligosaccharyltransferase (OST) is the principal
enzyme in the N-glycan pathway. It catalyses the transfer
of the glycan from the dolichol phosphate-oligosaccharide
to an asparagine in Asn-X-Ser/Thr motifs on acceptor poly-
peptides. OST is a hetero-oligomeric complex comprising
8 subunits in most Eukaryotes. The transfer reaction cata-
lysed by OST is exclusive, showing strict substrate speci-
ficity applicable to wide range of protein acceptors (Zuber
and Roth 2009; Aebi 2013; Breitling and Aebi 2013).
N-Glycosylation is closely linked with important gly-
coprotein regulatory events. Protein folding is mediated
by the chaperones calnexin and calreticulin and ensures
that glycoproteins that exit the ER are correctly folded
(Roth 2002). Trimming of the terminal triglucosyl unit by
α-glucosidases I and II is followed my monitoring of the
glycoprotein. In the case that folding is incomplete a sin-
gle α-glucose residue is transferred to the α1,2mannose
unit on the α1,3mannosyl antenna. Recycling ensues and
the glycoprotein is reassessed in the same manner. Those
glycoproteins that do not fold properly are eliminated by
ER-associated degradation (Roth 2002; Aebi 2013; Brei-
tling and Aebi 2013; Roth and Zuber 2017).
The second most common type of glycosylation, the
O-glycosidic linkage coupling serine or threonine to α-N-
acetyl-D-galactosamine (GalNAc), also known as mucin-
type glycosylation, as it is the major glycosylation found
in this large group of heavily glycosylated proteins (Cor-
field 2015). Other non-mucin-type O-glycans have been
detected, and these are described later. The O-glycans
present in mucins are located in variable number tandem
repeat domains, which vary in size and sequence between
the different mucins (Hattrup and Gendler 2008; Thorn-
ton et al. 2008; Bafna et al. 2010; Kreda et al. 2012; Cor-
field 2015). O-Glycans do not have a peptide recognition
sequon, as established for N-glycans, but are characterized
by eight different core structures, as shown in Table 2. The
most frequently observed are cores 1, 2, 3 and 4.
The initial transfer of a GalNAc to serine and threonine
residues in proteins is catalysed by a family of GalNAc
transferases (Patsos and Corfield 2009; Tabak 2010; Ger-
ken et al. 2011; Bennett et al. 2012; Gerken et al. 2013;
Revoredo et al. 2016), the site of action localized immuno-
histochemically by electron microscopy (Roth et al. 1994).
The core structures are extended through N-acetyllactosa-
mine backbone repeat unit of type 1 (Galβ1,3GlcNAc-)
or type 2 (Galβ1,4GlcNAc-) or the blood group antigens
I (Galβ1,3GlcNAcβ1,3(GlcNAcβ1,6)Galβ1,4-) and I
(Galβ1,4GlcNAcβ1,3Galβ1,4-R). Peripheral glycosyla-
tion of these structures is extensive and includes ABO and
Lewis blood groups together with sialylated, fucosylated
and sulphated glycans. The pathways responsible for the
biosynthesis of these glycans are well studied (Schachter
and Brockhausen 1992; Brockhausen and Schachter
1997; Patsos and Corfield 2009; Corfield 2015; Corfield
and Berry 2015). Unique for mucin glycosylation is the
α-GlcNAc terminus of core 2 O-glycans in the gastrointes-
tinal tract, which is readily detectable with the plant lectin
GSA-II (Nakayama et al. 1999; André et al. 2016).
A large group of cytosolic and nuclear proteins, which
carry multiple additions of a single β-O-GlcNAc unit
linked to serine and threonine hydroxyl residues, has been
reported. The same serine and threonine residues are also
sites for phosphorylation, prompting consideration of a
mutual relationship between these two modifications (But-
kinaree et al. 2010; Ma and Hart 2014). The cycling of β-O-
GlcNAc and phosphate has functional roles and is mediated
by an O-GlcNAc transferase (Zimmerman et al. 2000) and
an N-acetyl-D-glucosaminidase (Zhu-Mauldin et al. 2012).
O-GlcNacylation is common throughout the metazoans.
Further O-glycan families have been identified. O-Man-
nose α-linked to serine and threonine residues is commonly
found in the metazoans, largely in skeletal muscle and