Protein Glycosylation, an
Overview
Elwira Lisowska, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy,
Wroclaw, Poland
Glycosylation is the most common posttranslational modification of proteins. It is a
complex process involving many functional proteins and resulting in a great diversity of
structures. Biological role of glycosylation and molecular and genetic basis of
glycosylation disorders have recently been extensively explored. The goal of this short
article is to signalize the variety of problems of this vast field of research.
Types of Glycosylation
Analysis of the SWISS-PROT database indicated that
more than half of all proteins are glycosylated. There are
various types of carbohydrate–protein linkage (Table 1),
involving most known monosaccharides and functional
groups of amino acid side chains. The protein-linked
monosaccharides are usually extended (exceptions are
GlcNAcb-Ser/Thr and Mana-Trp) by attachment of other
monosaccharides that gives multiple oligosaccharide
strtuctures.
The most common protein-linked oligosaccharides are
N-glycosidic chains (linked to Asn via GlcNAcb) which
exist in two major forms: (1) oligomannosidic (or ‘highMan’)
N-glycans with branched or linear oligomannosidic
chains attached to both a-mannose residues of the core
structure shown in Table 1 and (2) complex chains containing
2–4 linear or branched antennae composed of one or
more LacNAc (Galb1-4GlcNAcb) units and linked to
a-mannose residues of the core structure. These antennae
(and also b-mannose and Asn-linked GlcNAc) are ‘decorated’
with sialic acid, fucose and other monosaccharides,
and also may contain phosphate, sulfate and O-acetyl residues
that gives in effect a variety of structures. There are
also various hybrid N-glycans combining features of both
forms.
Mucin type O-glycans are attached to Ser/Thr by the
GalNAca residue, and the monosaccharides (and their
linkages) attached to this GalNAc define various core
structures (Table 1). Further elongation of these structures
yields a large number of different O-glycans. The most
common O-glycans are represented either by the core1
structure substituted with one or two sialic acid residues
linked to Gal or/and GalNAc, or by more complex core2
O-glycans containing LacNAc-type chains. Generally, the
structures (or arrays of structures) of protein-linked glycans
are determined by the type of carbohydrate–protein
linkage. However, the LacNAc-type chains present in
N- and O-glycans can carry the same terminal nonreducing
units, e.g. blood group ABH/Lewis (Le) antigens. See also:
Blood Groups: Molecular Genetic Basis
In addition to other types of carbohydrate–protein linkage
listed in Table1, carbohydrates can be linked to proteins
via phosphoester linkage. A distinct form of protein-linked
oligosaccharides is a glycosylphosphatidylinositol (GPI,
linked to the C-terminal group of the protein) which anchors
some proteins in the cell membrane lipid bilayer.
The diverse glycan structures can be found in GlycosuiteDB
(see http://www.glycosuite.com). This database
contains 3238 unique glycan structures (Release 8.0,
August 2005), and if known, the proteins to which the glycans
are attached are described.
Multiple Proteins Involved in
Glycosylation Process
Oligosaccharide chains are indirect products of genes, because
many direct gene products (proteins) are involved in
their biosynthesis. Protein glycosylation proceeds by the
stepwise addition of monosaccharides, first to the protein
and then to the growing oligosaccharide chain. Exceptions
are GPI anchor, attached to protein in preassembled form,
and N-glycans (with GlcNAcb-Asn bond), where a preassembled
dolichol-linked triglucosylated nanomannosidic
chain is transferred to protein and after partial enzymatic
trimming is further extended by addition of monosaccharides.
Glycosyltransferases are strictly specific in respect to
the donor, the acceptor residue and the type of linkage.
Their catalytic efficiency is also more or less dependent on
the location of the acceptor residue (type of carrier, underlying
sugar chain). For each glycosidic bond, one or
several glycosyltransferases exist, and many of them have
been cloned. Other multiple enzymes are involved in
Introductory article
Article Contents
. Types of Glycosylation
. Multiple Proteins Involved in Glycosylation Process
. Biological Role of Glycosylation
. Protein Glycosylation and Disease
Online posting date: 30th
April 2008
ELS subject area: Cell Biology
How to cite:
Lisowska, Elwira (April 2008) Protein Glycosylation, an Overview. In:
Encyclopedia of Life Sciences (ELS). John Wiley & Sons, Ltd: Chichester.
DOI: 10.1002/9780470015902.a0006211.pub2
ENCYCLOPEDIA OF LIFE SCIENCES & 2008, John Wiley & Sons, Ltd. www.els.net 1
Table 1 Protein-linked saccharides: major types of carbohydrate–protein linkage
Glycans Recognition motif Occurence
N-glycans, core structure – Common in cellular and
secreted proteins, wide
phylogenetic distribution
Mana1,3
Manb1,4GlcNAcab1,4GlcNAcb-Asn -N-X-S/TMana1,6
–
Glcb-N-Asn Laminin, archaebacteria
Mucin type O-glycans, core structures
Core1: Galab1,3GalNAca-O-Ser/Thr Mutiple chains (clusters) in
mucin-type glycoproteins,
glycophorins, leukosialins,
single or few chains in some
other proteins
Core2: Galb1,3(GlcNAcb1,6)GalNAca-O-Ser/Thr
Core3: GlcNAcb1,3GalNAca-O-Ser/Thr
Core4: GlcNAcb1,3(GlcNAcb1,6)GalNAca-
O-Ser/Thr
Core5: GalNAca1,3GalNAca-O-Ser/Thr
Core6: GlcNAcb1,6GalNAca-O-Ser/Thr
Core7: GalNAca1,6GalNAca-O-Ser/Thr
Core8: Gala1,3GalNAca-O-Ser/Thr
O-mannosylation
OligoMan-Mana-O-Ser/Thr Yeast mannoprotein
O-fucosylation Epidermal growth factor
(EGF)-like domains:
coagulation factors, Notch,
thrombospondin, properdin,
F-spondin
NeuAca2,6Galb1,4GlcNAcb1,3Fuca-O-Ser/Thr EGF module
Glcb1,3Fuca-O-Ser/Thr -C-X-X-G-G-S/T-CXyla1,3Xyla1,3Glcb-O-Ser
-C-X-S-X-P-C- EGF domains
O-GlcNAc glycosylation
GlcNAcb-O-Ser/Thr Nuclear and cytosolic proteins
(dynamic glycosylation)
Gala1,2Galb-O-HyLys Collagen repeats: -G-X-HyK-G- Collagens, C1q complement,
core-specific lectin
Xylb-O-Ser S-G/A Proteoglycans
(Ara,Gal)-Araa or b-O-HyPro Plant proteins
Galab-O-HyPro
(Glc)n-Glca-O-Tyr Glycogenin
C-mannosylation
Mana1-C-Trp -W-X-X-W, found in over 300
proteins
Ribonuclease 2, IL-12b,
properdin, thrombospondin-1
F-spondin, hypertrehalosemic
hormone, five complement
components
Notes: Abbreviations of sugar names:Ara – arabinose, Fuc – fucose,Gal – galactose,GalNAc – N-acetylgalactosamine,Glc – glucose, GlcNAc –
N-acetylglucosamine, Man – mannose, NeuAc – N-acetylneuraminic acid and Xyl – xylose.
Abbreviations of amino acid names (three and one-letter code): Ala (A) – alanine, Asn (N) – asparagine, Cys (C) – cysteine, Gly (G) – glycine,
HyLys (HyK) – hydroxylysine, Hypro (HyP) – hydroxyproline, Lys (K) – lysine, Pro (P) – proline, Ser (S) – serine, Thr (T) – threonine, Trp (W) –
tryptophan and Tyr (O) – tyrosine.
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biosynthesis of ‘activated’ donors of sugars and other glycan-modifying
residues (acetyl,sulfate). Moreover, various
hydrolases participate in glycosylation process and catabolism
of glycoproteins.
Formation of each glycosidic bond in glycoproteins
or lipid-linked precursors requires a nucleotide- or
lipid-linked sugar donor, acceptor (containing a monosaccharide
or amino acid residue to be glycosylated) and
respective transferase, all present in a proper cell compartment.
Most glycosylation reactions take place in the
lumen of the endoplasmic reticulum (ER) and Golgi
compartments where transferases are located in the membranes
in a defined order with their functional domains
directed into the lumen. Nucleotide–sugar donors are
synthesizedin cytosoland are transportedintothe lumen of
the Golgi by specific transporters that act as antiporters
removing ‘used’ mononucleotides from the Golgi. It is an
example indicating that the glycosylation process is
dependent not only on enzymes, but also on many other
proteins responsible for intracellular trafficking. See also:
Protein Degradation and Turnover
The great variability of protein-linked glycan structures
is dictated by tissue-specific regulation of genes encoding
enzymes involved in glycosylation process, availability of
the reaction components in the proper cell compartment,
competition between glycosyltransferases for acceptor
during glycan elongation, and finally by the structure of
the glycosylated polypeptide and microenvironment of the
growing glycan chain. The differences in glycan structures
exist not only between different glycoproteins, but also between
molecules of an individual glycoprotein produced by
the same cells and between different glycosylation sites of
one molecule, that results in various protein glycoforms.
See also: Protein: Cotranslational and Posttranslational
Modification in Organelles
Biological Role of Glycosylation
A rapidly growing evidence has indicated that proteinlinked
glycans not only protect proteins against proteolytic
degradation and denaturation, but also interact directly
with various carbohydrate-specific proteins and these interactions
initiate or modulate many important biological
events. Moreover, intramolecular interactions between
glycan and peptide backbone affect the conformation and
flexibility of both glycan and peptide that can modulate
protein–protein interactions.
The glycosylation plays a role in ‘quality control’ of
synthesized proteins. The triglucosylated oligomannosidic
N-glycans, linked to nascent proteins in the ER, undergo
trimming of two glucose residues and monoglucosylated
glycans bind the lectin-like chaperonins, calnexin and calreticulin,
which promote the protein folding. The correctly
folded and assembled proteins proceed further to the Golgi
compartments (also with the help of mannose-specific lectins,
ERGIC-53 and VIP36) and misfolded proteins are
reglucosylated and returned to the calnexin–calreticulin
cycle or trimmed by the ER-mannosidase I and eliminated
by ER-associated degradation (ERAD). Another intracellular
process involving glycans is targeting the lysosomal
hydrolases to lysosomes with participation of mannose-
6-phosphate receptors which recognize Man-6P residues
specifically present in oligomannosidic N-glycans of hydrolases.
See also: Protein Folding and Chaperones
Glycans linked to the proteins exported to the plasma
membrane participate in the overall architecture of the cell
surface. Glycans of cell membrane-bound or soluble glycoproteins
react with various carbohydrate-specific proteins
that also exist in a membrane-bound or secreted form. The
number of discovered and characterized mammalian lectins
and information on their roles is rapidly increasing.
Apart from mentioned intracellular lectins, there are E-,
L- and P-selectins specific for sialyl-Lex
/Lea
and related
structures, Siglec family (12 members identified in humans)
of sialic acid binding lectins, galectins reactive with bgalactose-terminated
glycans, endocytic macrophage and
hepatocyte receptors and many others. Interactions of glycans
with lectins play various roles in recirculation of cells,
cell–cell and cell–matrix adhesion, cell growth and viability,
cellular signalling, etc.
An interesting example of a role of glycosylation is
glycodelin (Gd). It is the glycoprotein containing two occupied
N-glycosylation sites. Gd produced by several reproductive
tissues has the same polypeptide backbone but
represents tissue-specific glycoforms and different biological
activities. For example, GdA (from amniotic fluid),
which contains sialylated complex N-glycans, inhibits
sperm–oocyte interaction and shows immunosuppressive
activity, while GdS (from seminal plasma) carries oligomannosidic
chains and nonsialylated/highly fucosylated
complexN-glycansand maintains an uncapacitatedstate in
the spermatozoa.
A role of protein O-fucosylation (Table1) has been shown
in studies on the Notch signalling pathway. Highly conserved
Notch cellular receptors have a central role in signal
transducing pathways that influence many cell-fate decisions
in metazoans and play a role in a variety of developmental
processes in higher animals and humans. Fringe
proteins have been known as modulators of activation of
Notch receptors by Notch ligands. It was reported in 2000
that Fringe proteins have b1,3-N-acetylglucosaminyltransferase
activity, initiating elongation of fucose residues
O-linked to epidermal growth factor (EGF)-like repeats of
Notch. This puzzling finding initiated further studies which
showed various aspects of an important role of O-fucosylation
in regulating Notch activity, and suggested a quality
control function of protein O-fucosyltransferase-1 in ER.
Particular roles has O-glycosylation of Ser/Thr residues
of nuclear and cytoplasmic proteins by b-GlcNAc.
The O-GlcNAc glycosylation has dynamic character regulated
by two enzymes: uridine diphosphate (UDP)GlcNAc:polypeptide
b-N-acetylglucosaminyltransferase
and b-N-acetylglucosaminidase. It is an ubiquitous and
essential protein modification. The diversity of proteins
modified by O-GlcNAc and dynamic interplay between
Protein Glycosylation, an Overview
ENCYCLOPEDIA OF LIFE SCIENCES & 2008, John Wiley & Sons, Ltd. www.els.net 3
O-glycosylation and O-phosphorylation imply importance
of this type glycosylation in signal transduction. Moreover,
it has a role in protein expression, degradation and trafficking
and in the aetiology of diabetes and neurodegeneration.
Protein glycosylation has a great impact on the immune
system. Almost all key molecules involved in the adaptive
and innate immunity (receptors, immunoglobulins, cytokines,
lectins, etc.) are glycosylated and some specific
glycoforms are involved in recognition events. There are
significant changes of glycosylation and transient appearing
of certain structures on the cell surface, dependently on
the cell differentiation or functional state. For example, the
disialylated core1 O-glycans of resting T cells are shifted
during T-cell activation into more complex branched core2
O-glycans (carriers of selectin ligands) that modulates
intercellular interactions. Glycosylated protein antigens
can induce humoral and cellular immune responses against
oligosaccharidic, glycopeptidic and glycosylation-oriented
peptidic epitopes. The existence of many natural anticarbohydrate
antibodies in human sera and immune responses
against ‘foreign’ oligosaccharide structures (of animal or
plant origin) have great clinical importance in blood transfusion,
transplantation and allergic diseases. Interactions
of various viruses, bacteria and parasites with host cell
glycans, and interactions of human and animal lectins with
glycans of pathogens play a role in infection and host defence
processes.
Protein Glycosylation and Disease
Protein glycosylation, which is dependent on so many factors,
is altered in multiple diseases. Characteristic alterations
are observed in rheumatoid arthritis (decreased
galactosylation of immunoglobulin G, IgG), cystic fibrosis
(undersialylation and overfucosylation of plasma
membrane glycoconjugates), Wiskott–Aldrich syndrome
(WAS) and acquired immunodeficiency syndrome (AIDS)
(aberrant expression of core2 O-glycans on T lymphocytes)
and in many other disorders. Altered glycosylation (loss of
expression or excessive expression of certain structures) is a
universal feature of cancer cells and affects glycoproteins
and glycosphingolipids. Most typical alterations include
increased branching of complex N-glycans, expression of
truncated O-glycans (Thomsen–Friederich, Tn and sialylTn
antigens), overexpression of the sialylated Lewis
stuctures (selectin ligands), differentially altered expression
of the blood group ABH-related structures, alterations
in sialylation. These disease-related changes in
protein glycosylation are acquired and are likely to be the
effect, and not the primary reason, of the disease. However,
some of these altered structures have functional significance,
and/or serve as diagnostic or prognostic markers of
the disease, or as therapeutic targets.
Nevertheless, there is a group of diseases evidently
caused by altered glycosylation. The rare congenital disorders
of glycosylation (CDGs) occur due to mutations in
genes encoding some key components of the glycosylation
‘machinery’ that most frequently results in severe clinical
symptoms (malformation, psychomotor retardation, dysfunction
of some organs and others). These diseases demonstrate
the importance of protein glycosylation for the
development and functions of the organism.
There are two major groups of CDGs. Group I includes
various defects in the assembly of lipid-linked
N-glycan precursor and its transfer to proteins in the ER
that results in decreased number of protein-linked Nglycans.
The 12 defects identified so far concern the
following proteins: phosphomannomutase 2 (type Ia, most
frequent, over 300 patients diagnosed worldwide), phosphomannose
isomerase (Ib), dolichy(Dol)l-P-Glc:Man9
GlcNAc2-PP-Dol a1,3-glucosyltransferase (Ic), Dol-PMan:Man5GlcNAc2-PP-Dol
a1,3-mannosyltransferase
(Id), Dol-P-Man synthase 1 (Ie), protein faciltating
utilization of Dol-P-Man (If ), Dol-P-Man:Man7GlcNAc2-PP-Dol
a1,6-mannosyltransferase (Ig), Dol-PGlc:Glc1Man9GlcNAc2-PP-Dol
a1,3-glucosyltransferase
(Ih), GDP-Man:Man1GlcNAc2-PP-Dol a1,3-mannosyltransferase
(Ii), UDP-GlcNAc:Dol-P GlcNAc-1P transferase
(Ij), GDP-Man:GlcNAc2-PP-Dol b1,4-mannosyltransferase
(Ik), Dol-P-Man:Man6 or 8GlcNAc2-PP-Dol a1,2-mannosyltransferase
(IL).
In CDG group II defects affect the processing of proteinlinked
N-glycans and also biosynthesis of O-glycans. It
may concern a deficient function of enzymes: b1,
2-GlcNAc-transferase II which initiates elongation of
a1,6-Man-linked antenna (IIa), a1,2-glucosidase I removing
the first glucose residue from triglucosylated N-glycan
precursor (IIb) and b1,4-galactosyltransferase (IId). Two
types of CDG-II are connected with mutations in genes
encoding nucleotide–sugar transporters: GDP-fucose
transporter (IIc, known as leukocyte adhesion deficiency,
LAD-II) and CMP-sialic acid transporter (IIf). The LADII
patients, due to lack of fucosylation, do not express the
blood group ABH epitopes (‘Bombay’ phenotype) and selectin
ligands. Recently, new types of CDG-II were identified
related to defects of genes encoding components of
the conserved oligomeric Golgi complex (COG). The COG
complex is an eight-subunit (Cog1-8) peripheral Golgi
protein required for normal intracellular trafficking and
activity of multiple proteins involved in glycosylation machinery.
Three types of defects were identified so far, concerning
Cog7 (CDG-IIe), Cog1 (CDG-IIg) and Cog8
(CDG-IIh). These data show that a defect in one component
destabilizes the complex. See also: Cell Adhesion
Molecules and Human Disorders
In the congenital dyserythropoietic anaemia type II
(CDA-II, known also as HEMPAS, with heterogeneous
genetic background) defective N- and O-glycosylation on
erythroid lineage cells is observed. However, the lack of
linkage of genetically determined alterations with the activity
of proteins involved in glycan synthesis suggests that
hypoglycosylation is not the primary defect but a consequence
of the dyserythropoiesis. Galactosaemia refers to a
group of inherited diseases caused by defects in genes encoding
enzymes of galactose metabolism and manifested
Protein Glycosylation, an Overview
ENCYCLOPEDIA OF LIFE SCIENCES & 2008, John Wiley & Sons, Ltd. www.els.net4
by hypogalactosylation of glycoproteins and glycosphingolipids
and accumulation of toxic galactose metabolites.
Some congenital muscular dystrophies are associated with
genetically determined defects in glycosylation of a-
dystroglycan.
Normal functions of the organism also depend on the
proper catabolism of glycoproteins. There are several ‘lysosomal
storage diseases’ resulting from deficiency of one of
lysosomal glycosidases and accumulation of respective undegraded
glycans. Deficient GlcNAc-phosphotransferase
involved in phosphorylation of mannose residue on lysosomal
hydrolases is the reason of the I-cell disease. Hydrolases
lacking Man-6P residue are directed to plasma
instead to lysosomes and undegraded cellular components
accumulate in lysosomes of fibroblasts and macrophages.
See also: Tay–Sachs Disease
The examples (listed here and elsewhere) of glycosylation-related
disorders show that protein glycosylation and
catabolism of glycoproteins have profound patophysiological
significance, not fully understood yet. Better understanding
of these processes can help in finding new
therapeutic approaches for treatment of these diseases.
See also: Glycoproteins; Glycosylation and Disease;
Lysosomal Transport Disorders
Further Reading
Davies GJ, Gloster TM and Henrissat B (2005) Recent structural
insights into the expanding world of carbohydrate-active enzymes.
Current Opinion in Structural Biology 15: 637–645.
Fraser-Reid B, Tatsuta K and Thiem J (eds) (2001) Glycosciences.
Heidelberg: Springer.
Freeze HH and Aebi M (2005) Altered glycan structures: the molecular
basis of congenital disorders of glycosylation. Current
Opinon in Structural Biology 15: 490–498.
Garcia-Vallejo JJ, Gringhuis SI, van Dijk W and van Die I (2006)
Gene expression analysis of glycosylation-related genes by realtime
polymerase chain reaction. Methods in Molecular Biology
347: 187–209.
Hart GW, Housley MP and Slawson C (2007) Cycling of bN-acetylglucosamine
on nucleocytoplasmic proteins. Nature
446: 1017–1022.
Helenius A and Aebi M (2004) Roles of N-linked glycans in the
endoplasmic reticulum. Annual Review of Biochemistry 73:
1019–1049.
Lisowska E (2002) The role of glycosylation in protein antigenic
properties. Cellular and Molecular Life Sciences 59: 445–455.
Rampal R, Luther KB and Haltiwanger RS (2007) Notch signaling
in normal and disease states: possible therapies related to
glycosylation. Current Molecular Medicine 7: 427–445.
Seppa¨ la¨ M, Koistinen H, Koistinen R, Chiu PC and Yeung WS
(2007) Glycosylation-related actions of glycodelin: gamete, cumulus
cell, immune cell and clinical associations. Human
Reproduction Update 13: 275–287.
Spiro R (2002) Protein glycosylation: nature, distribution,
enzymatic formation and disease implications of glycopeptide
bonds. Glycobiology 12: 43R–56R.
Web Links
GlycosuiteDB http://www.glycosuite.com
Entrez PubMed http://nslij-genetics.org./search_pubmed.html
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