Contract for a DESIGN STUDY Implemented as a
SPECIFIC SUPPORT ACTION
EuroCarbDB
http://www.eurocarbdb.org/
Design Studies related to the development of
distributed, Web-based European Carbohydrate Data
Bases
Life Sciences, Genomics and Biotechnology for Health
RIDS Contract number 011952
Design Study DS1: Definition of standards, rules and formats for the biological and
analytical data to be collected. Definition of good practice, recommendations of
procedures for quality control.
Task Title: Recommendations for standards, digital formats and quality measures
Deliverable DS1-SUB1: Report: “Survey structural complexity of carbohydrates and their
profiles of occurrence in various tissues, species and cells”.
Dissemination: PU
Partners : 4, SUB
Due date of deliverable:
31.03.2006
Actual submission date:
31.03.200
Start date of project: 01.03.2005
Duration: 48 months
Organisation name of lead contractor for this deliverable:
EBI-EMBL, European Molecular Biology Laboratory, European Bioinformatics Institute,
Hinxton (Cambridge),United Kingdom
Author: Dr. Tony Merry
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Survey of the structural complexity of carbohydrates and their
profiles in disease and in various tissues, species and cells.
Tony Merry, Glycosciences Consultancy, Charlbury, OXON OX7 3HB UK
Abstract
Glycoconjugates are undoubtedly complex and it is difficult to present a summary of data
in a condensed form without omitting some structures but this report contains a review
of the available data whilst trying to provide a comprehensive summary.
The major classes of glycoconjugate are listed along with the non carbohydrate moiety .
It should be noted that since the biosynthetic machinery is not generally specific for the
glycoconjugate particularly in the terminal parts of the molecule, it is frequently found
that these areas have a sequence which is common to different glycoconjugates.
In contrast to this some glycoconjugates have structures which may be unique even
down to a single species level. This is particularly true for bacteria where there are
conjugates of lipid or protein that have a carbohydrate moiety which may be unique to
that particular bacterium. The glycophosphatidylinositol (GPI) types of glycoconjugate
also may have very individual structures particularly those found in parasites.
All these considerations need to be taken into account in compiling databases of
carbohydrate related data particularly if they are to be related to other databases. This
is in contrast to databases of other types of macromolecule such as the nucleic acid or
proteins. Here the macromolecule will be characteristic of the species or organism but
will always have the same general structural characteristics. That is to say they have the
same basic monomer residues and have the same type of linkage.
There is another feature of glycoconguates which separates them from the other
macromolecules, in that their sequence is not template derived. Nucleic acids and
proteins are both derived from a universal genetic code.
The sequence of
monosaccharides in any glycan is a product of many factors. The biosynthetic
machinery comprises the enzymes involved in monosaccharide addition to the polymer,
the glycosyltransferases, the activated monosaccharide donors and a whole host of
environmental factors as well as the cellular localisation and compartmentalisation of the
enzymes.
The main aim of this survey is to consider the degree of structural complexity that needs
to be considered in designing the databases EUROCarbDB.
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Contents
Abstract ...............................................................................................................................................................2
1.
Introduction................................................................................................................................................4
2.
Nomenclature.............................................................................................................................................5
3.
Diversity of complex carbohydrates in nature ............................................................................................7
Which residues occur in which species ?.....................................................................................7
3.1.
Human .........................................................................................................................................7
3.2.
Mammalian...................................................................................................................................7
3.3.
Plants...........................................................................................................................................7
3.4.
Bacteria .......................................................................................................................................7
3.5.
Viruses.........................................................................................................................................7
3.6.
Sialic Acids ..................................................................................................................................7
3.7.
Linkages present in glycoconjugates...........................................................................................8
4.
Distribution of Glycans ...............................................................................................................................9
4.1.
By Species ...................................................................................................................................9
4.2.
By Tissue ...................................................................................................................................10
5.
Glycosyltransferases................................................................................................................................11
6.
Disease related changes in glycosylation .................................................................................................11
6.1.
Genetic Disorders (Congenital Disorders of Glycosylation – CDG) ............................................12
6.2.
Genetic Disorders (Abnormal catabolism of glycoconjugates - Lysosomal Storage Diseases) ..14
6.3.
Inflammatory Diseases...............................................................................................................15
6.4.
Infectious Diseases....................................................................................................................16
6.4.1.
Bacterial ....................................................................................................................................17
6.4.2.
Viral ...........................................................................................................................................17
6.4.3.
HIV .............................................................................................................................................18
6.4.4.
Parasitic.....................................................................................................................................18
6.5.
Cancer and Metastasis ..............................................................................................................19
6.6.
Other Diseases ..........................................................................................................................19
References.........................................................................................................................................................21
A Bacterial Lipopolysaccharide (Lipid A) ...........................................................................................................25
B Bacterial Lipopolysaccahride O-antigen .........................................................................................................26
d Bacterial Peptidoglycan ..................................................................................................................................27
e Eukaryote GPI anchor .....................................................................................................................................27
f Plant cell wall cellulose ....................................................................................................................................28
g Plant lipochito-oligosaccharides NOD factors .................................................................................................29
h Plant glycoprotein N Glycan ............................................................................................................................29
i Plant glycoprotein O Glycan .............................................................................................................................30
j Sulphated fucans..............................................................................................................................................31
Amphibian muins................................................................................................................................................32
l Reptilian venom and toxins .................................................................................... Error! Bookmark not defined.
m Insect polysaccharide chitin...........................................................................................................................32
n Human hyaluronic acid ....................................................................................................................................33
o Human glycolipids ...........................................................................................................................................35
p Human glycoprotein N Glycans ............................................................................ Error! Bookmark not defined.
q Human glycoprotein O Glycan .........................................................................................................................37
r Human GPI anchors .........................................................................................................................................38
s Human mucins .................................................................................................................................................39
t Human GAG Heparan Sulphate ........................................................................................................................40
u Human GAG Keratan Sulphate ........................................................................................................................41
Appendix B Some Examples of Disease Related Changes in Glycosylation and Disease ...................................42
1.
Respiratory Tract Mucin Genes and Mucin Glycoproteins in Health and Disease ......................42
Tumour-cell fusion as a source of myeloid traits in cancer ............................................................................42
2.
Advanced Glycation end products and disease .........................................................................43
3.
Glycans In Cancer And Inflammation .........................................................................................43
4.
Role of O-GlcNAc play a in neurodegenerative diseases ...........................................................44
5.
Prion protein glycosylation ........................................................................................................45
6.
Muscular dystrophies caused by abnormal glycosylation. .........................................................45
7.
Cancer related changes in O-glycosylation................................................................................45
8.
Glycosylation and HIV infection - Mannose binding lectin (MBL) and HIV...................................46
9.
Glycosylation and Influenza virus infection and transmission. Influenza virus entry and infection
require host cell N-linked glycoprotein..........................................................................................................46
10.
Human influenza virus recognition of sialyloligosaccharides.....................................................46
11.
Human-specific regulation of alpha 2-6-linked sialic acids.........................................................47
12.
Receptor determinants of human and animal influenza virus isolates: differences in receptor
specificity of the H3 hemagglutinin based on species of origin......................................................................47
Appendix C Current On-line resources ..............................................................................................................50
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Introduction
Chemically, complex carbohydrates are the most diverse of all biological
macromolecules, a fact often overlooked by many scientists. Not only are there a very
large number, running into thousands, of possible monomers but they may also be linked
together in a variety of way and also in branched chains. This is in contrast to nucleic
acids and proteins where they are a relatively small number of monomers and they are
always in the same linkage in linear chains. In addition complex carbohydrates are most
often found in combination with other molecules where a generic term of glycans is now
commonly used for carbohydrates attached to proteins or lipids although the term
oligosaccharide is also used.
A consequence of this is that the reference and cataloguing of complex carbohydrates is
an immense task and construction of databases not a trivial matter. This complexity is
undoubtedly a factor in the generally poor understanding of glycans and the lack of
suitable databases contributes in a large way to this.
Relating structure to function in glycans is even more problematic as it is very difficult to
obtain glycoconjugates in a single form as there diverse configurations lead to structures
with very similar physical properties. Added to this is the fact that they are not template
driven so that the exact type of glycocongugates produced in any organism depend on
many factors in addition to genetic makeup.
In fact there are several factors which limit this potentially vast range of structures.
Firstly, the biosynthetic enzymes, glycosyltransferases generally have a very high
degree of specificity and frequently will make only one type of linage of a
monosaccharide to a glycan chain. Secondly the biosynthetic processes of many
complex carbohydrates are such that some common ‘core’ region adjacent to the
attachment point of the glycan is present. Finally the availability of the activated
monosaccharide building blocks is limited to generally around 10 monosaccharides.
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2.
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Nomenclature
The classification of glycconjugates tends to be much more confused than other
macromolecules for historical reasons [1]. The glycoconjugates were named according
to their occurrence before much structural data was available and generally this
nomenclature has been retained for consistency.
It should also be noted that non-enzymatic glycosylation (glycation) plays an important
role in many illnesses such as diabetes. Indeed the advanced glycation end products
(AGE) are very important in the development and symptoms of many human diseases.
This is probably beyond the scope of the EuroCarbDB project as different techniques
tend to be employed but it should be noted that this is a very important aspect of
glycosylation.
Glycans
The nomenclature and way in which glycans are represented are the subject of a
separate report .
Glycoconjugates – the features of the major glycoconjugates are as
follows and are summarised in Table 1
Glycoproteins
•
N-Glycans – 1 type of linkage GlcNAc to Asn
•
O-Glycans – Several core structures GalNAc to Ser or Thr
•
Species-specific and unusual types of protein glycosylation
e.g. O-GlcNAc, O-xyl, Pro-OH-Glu
•
Glycospingolipids – glycan chain attached to lipid core
•
GPI-anchors – N-terminal linkage of glycan through PI
Glycolipids
Proteoglycans and Glycosaminoglycans
•
Monomer units – usually 2 types but may be changed e.g.
IdA
•
Modifications – commonly O- and N- sulphation
Polysaccharides
•
Homo polysaccharides – single monomer
•
Polysaccharides with more than 1 residue – may be
disaccharide repeat
•
Cell wall polysaccharides
•
Bacterial polysaccharides
•
Lipopolysaccharides
Common structural motifs
•
Core Structures – common N glycan, different O-glycan
•
Antigens – some monosaccharides antigenic e.g. α-gal
•
Epitopes – often found in terminal parts of chains e.g. SLex
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Table 1
The Major Types of Glycoconjugate and Complex Carbohydrates
GLYCOCONJUGATE
CONSTITUENTS
GLYCAN TYPE OCCURRENCE
EXAMPLES
POLYSACCHARIDE
Repeating
monosaccharide
units – may have core
peptide
Branched or
linear chain 1
(usually 1 or 2
types)
GLYCOLIPID
O-linked glycan In most organisms Globoside
Lipid component
chain through
–most frequently
derivative of glycerol glucose
GLYCOSPHINGOLIPID
As above
O-linked glycan Predominantly in
chain through nervous tissue
glucose
GLYCOPROTEIN
Peptide, N- (Asn) or
O-linked glycans
N- , O- O-Fuc,
O man, O Xyl
MUCIN
In many secretions Muc-1
Peptide with multiple O-linked
O-inked glycans
through serine but also found on
cell surfaces
or threonine
COLLAGEN
Always linked to
hydroxyproline
Glucose,
galactose
PROTEOGLYCAN
May have several
glycosaminoglycan
chains always Olinked to peptide
GAG
Frequently
charged
In most organisms Starch,
cellulose inulin
Sphingomyelin
In all cells and
secretions
Mainly in
connective tissue
but also in
basement
membrane
Very commonly in
intracellular matrix
but also on cell
surfaces
Type 1 collagen
Type IV collagen
Heparan
Sulphate
Different glycoconjugates are located in different cellular or extracellular compartments
[1] as shown below
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All the factors of the different types of glycoconjugate of need to be taken into
consideration when designing a database of the functional activity of glycans. The work
performed during the current year has been to examine the range and diversity of
glycans found in living organisms and the range of the glycosyltransferase enzymes (see
below) responsible for their biosynthesis on a cell by cell basis as far as possible.
3.
Diversity of complex carbohydrates in nature
-
Which residues occur in which species ?
The following list is of residues found in glycoconjugates. There are several other
monosaccharide residues or derivatives of them which play important roles in
metabolism such as fructose or in other biological conjugates such as ribose in RNA or 2deoxyribose in DNA but they are not listed here.
3.1.
Human
Glc, Gal, Man, Fuc, GalNAc, GlcNAc, GlcA, IdA, Neu5Ac (NOT Neu5Gc) , Xyl
3.2.
Mammalian
Glc, Gal, Man, Fuc, GalNAc, GlcNAc, GlcA, IdA, Neu5Ac, Neu5Gc, Xyl
3.3.
Plants
Lyx Alt , Man , Ara, Ara-ol,Fru,Rha, Rha3,4Me2, GalN, GalNAc,Rib, Glc, Rib5P, GlcN, Rul,
GlcN3N, Sor, Glc-ol ,GlcNAc,Tal,GlcA , Xyl, Xul, Xyl2Cme
3.4.
Bacteria
Abe, IdoA , All, Lyx Alt , Man , Api , Mur,Ara, Ara-ol, ,Fru, Neu5Gc, Kdo, Fuc-ol,Rha,
Rha3,4Me2, GalN, Psi ,GalNAc,Qui,b-D-Galp4S,Rib Glc Rib5P,GlcN,Rul,GlcN3N,Sor, Glcol ,Tag GlcNAc,Tal,GlcA , Xyl, GlcpA6Et , Xul,Gul, Xyl2Cme,Ido
3.5.
Viruses
Depends on the host cell biosynthetic capabilities
3.6.
Sialic Acids
The sialic acids are especially important in considering the biological functions of
glycans especially in humans. It has been pointed out by Varki [2] that the sialic acids
are the only major change in the constituents of glycoconjugates from apes to man [3, 4]
and they probably represent the most recent evolutionary change in glycosylation in our
evolution. They are the most complex of the monosaccharides found in mammals and
exist in a variety of linkages and with modification such as O-acetylation [5-7].
Chemically they are based on a nine carbon backbone as shown below ;
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5-N-acetyl-neuraminic acid (Neu5NAc)
Linkages are to the anomeric carbon (C2 in this case) and are found to positions 2,6, and
8 of the molecule.
3.7.
Linkages present in glycoconjugates
Linkages are from the anomeric carbon on one monosaccharide (generally C1 although
C2 in sialic acids) to a carbon on another monosaccharide. Linkage is found to C2, C3,
C4, C6 and C8 in sialic acids. The anomericity of the linkage may be either alpha or beta
however some monosaccharides are only linked in one way
Numbering of carbon atoms in a hexasaccharide
Example of α-linkage (α-gal 1,4 glc)
Example of β- linkage (β -gal 1,4 glc)
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Distribution of Glycans
4.1.
By Species
Table 2 shows the major classes of organisms, and of these the viroids and viruses are
the only ones which have no glycosylation machinery. Viruses are however commonly
glycosylated as they use the glycosylation biosynthetic pathways of their host to produce
glycoconjugates such as the cell surface glycoproteins.
In the eukaryotes there are generally some common biosynthetic pathways, quite often
to build a core which is then extended in ways that may be species specific [8-10]. There
are considerable differences in glycosylation between plants and animals [11] but a
range of complex carbohydrates and glycoconjugates is found in both. The most diverse
and unusual glycosylation is seen in bacteria and in general there is no common pathway
of biosynthesis for bacterial complex carbohydrates
The major glycoconjugates in different species are shown in Table 2 along with some
representative structures ;
In view of the complexity and different types of glycosylation found in bacteria a
diagrammatic representation of the bacterial cell wall is given below ;
Diagrammatic Structure of gram positive bacterial cell wall
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TABLE 2 Occurrence of Glycoconjugates in Different Species
POLYSACCHARIDES
GLYCOLIPIDS
GLYCOPROTEINS
PROTEOGLYCANS
Membrane derived
oligosaccharides (a)
Capsular
polysaccharide (c, K
antigens)
Lipopolysaccharide
(Lipid A) and endotoxin
(b) O-antigen. (c)
(GPI) Glycosyl
Phosphatidyl Inositol
very rare
GPI anchors in all
species (e)
lipochitooligosaccharide
s (g, Nod factors,)
Peptidoglycan (d) N- and Oglycans in some species
None as such
N- linked glycans (h) O-linked
None as such
Glycosphinolipids,
gangliosides
N- and O-linked glycans,
Glycogen Hyaluronic
acid
Gangliosides
N- and O-linked glycans,
Insects
Chitin (m)
Several types present
Birds
Glycogen, hyaluronic
acid
Several
anchors
Fish
Glycogen
Ganglioside,
glycospingolpids
Some examples with
diverse functions e.g.
hyaluronic acid (n)
Several types often in Glycoproteins with N- (p) and Heparan sulphate (t)
cell walls and tissue O-linked (q) glycans and GPI keratan sulphate (u)
anchors (r) Mucins (s)
specific (o)
and many other forms
BACTERIA
EUCARYOTA
Many examples e.g.
cellulose (f)
Plants
Amphibia
Suphated fucans (j)
Reptiles
Mammals
Man
VIRUSES
glycans (i)
mucins (k)
venoms and toxins (l)
types,
N- linked glycans
GPI N and O-linked glycans,
oligomannose, IgY
Sevaer types present
N- and O-linked glycans
Most cell surface
proteins
glycosylated – may
have N- or O-linked
glycans
4.2.
Several types present
including heparan
sulphate, sydecan
Proteoglycans
By Tissue
The distribution of glycan in different tissues has not been studied systematically but it
has been apparent for some time that certain carbohydrate epitopes only occur on one
cell type. It was also found that when glycoproteins were produced from cells grown in
culture that the same gene product when expressed in different cells lines gave rise to a
number of quite differently glycosylated proteins.
Although most cells in humans produce glycoconjugates with the same core structures
either the N-and O-linked glycans, glycolipids or proteoglycans the extent and nature of
the glycosylation can vary considerably. These differences are partly due to the levels of
glycosyltransferase expression (see below) but glycosylation is also influenced by
growth factors [12], hormones [13, 14] or environmental factors [15, 16]. This makes
prediction of the nature of cell-specific glycosylation difficult [17].
Studies in related cell types which perform different functions have shown that there is
structural diversity and specific distribution of O-glycans in normal human mucins along
the intestinal tract. [18]
It has also been shown that different glycoforms of the human GPI-anchored antigen
CD52 associate differently with lipid microdomains in leukocytes and sperm membranes.
[19]
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There is evidence for regulatory function of different types of glycosylation such as a
proposed regulatory mechanism for beta1 integrins. [20]
Cell type specific glycosylation is often seen in neural tissue and a comparison of Nglycosylation patterns of HSA/CD24 from different cell lines and brain homogenates
showed that some types of glycoosylatiuon were related to certain cell types and that
this could be correlated with different function of cells in nervous tissue [21]
The mechanisms by which this diversity are not well understood and cannot be
accounted for solely by the presence of certain transferase genes in different cell or
even in the level of their expression. It seems that special distribution of the transferases
may be very important and the roles of enzyme localization and complex formation in
glycan assembly within the Golgi apparatus have been studied. [22]
Studies in the area of development have shown how glycosylation changes are very
much related to development and some very good examples of developmental genes that
are either transferases in their own right [23] or are involved in transferase level
regulation [24] now exist.
Another area where changes in glycosylation have been carefully studied is in the
immune system where considerable changes in glycosylation have been found upon T
cell activation [25].
5.
Glycosyltransferases
The enzymes which are involved in the biosynthesis of glycans, those making the lipid
donor precursors, those involved in nucleotide sugar biosynthesis and those which
modify glycans such as sulphotransferases and epimerases are all closely regulated.
The glycosyltransferases are highly specific, both in terms of the monosaccharide added
to the glycan but generally in the anomeric configuration of the linkage created. They
may also be specific for some features of the glycan chain to which the monosaccharide
is to be attached and in the case of the GalNAc transferase family responsible for the
intitiation of O-glycan chain for the peptide sequence as well.
Most types of glycosyltransferase have now been characterised and expressed. They are
identified by the monosaccharide which they transfer and a number which indicate the
specificity of the linkage. It is now apparent that there may be several different genes
coding for transferases with the same activity but they are often expressed in a tissue
specific manner. A total of almost 150 different glycosyltransferases have now been
identified along with a number of sulphotransferases and N-glycan transferases.
Databases exist where all the known transferases are recorded notably in CAZy
(www.cazy.org )
Several examples of all of these genes are available in the gene microarray from the
Glycomics consortium in the US and studies now underway on the tissue distribution of
all these genes which will provide an invaluable resource in future.
6.
Disease related changes in glycosylation
There are a growing number of diseases where some change in glycosylation of
glycoconjugates has been observed. The changes are sometimes difficult to interpret in
terms of the factors which have changed the glycosylation pattern. There is even more
difficulty in relating the change in glycosylation to the symptoms of the disease and to
know how it may effect disease progression.
The importantance of glycosylation in disease can be highlighted in two examples which
are currently in the public eye and which both involve viral infection. Firstly the outbreak
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of avian flu and the possibility of transmission of the virus to the human population is very
much concerned with glycobiology. Recent studies of the specificity of the virus for
glycoconjugates in strains from the 1918 out break have shown crucial differences in the
specificity when the strain crossed from avian to human subjects. However this
important observation is largely overlooked and there are less than 20 references to
work in this area ! The second example is of HIV infection where it is frequently forgotten
that the surface of the HIV virus is largely covered by glycans. It is the interaction of
these with cells of the human immune system that are responsible for the transmission of
this devastating disease but again the number of research groups who are actively
looking at this aspect is disappointingly small despite several excellent papers showing
the importance of glycosylation. There is obviously a big gap in knowledge in this area.
Reviewing the literature at present will reveal a wide variety of changes in glycosylation
reported in various diseases but after examination it may be found that many of these are
unconfirmed, may rest on dubious or unproven approaches, and are often frankly
contradictory. A thorough review of the literature has been performed for this report and
some of the better understood and substantiated examples of diseased related changes
in glycosylation will be discussed. Frequently, with a few notable exceptions, these
reports are from those groups who are not very familiar with glycoscience and details of
the changes may not be clear. This is an area where there is a need for much better
bioinformatics to support this area
Much better knowledge and understanding of these changes will be required to realise
the full potential of glycotherapeutics and this highlights the role of bioinformatics and
the need for appropriate databases and links between them. This will be a goal for the
EuroCarbDB design study. However a summary of some existing data is now presented.
6.1.
Genetic Disorders (Congenital Disorders of Glycosylation – CDG)
These are the best characterised examples of disease-related changes in glycosylation.
They are inherited conditions where specific enzymes involved in the biosynthesis or
degradation of complex carbohydrates are either missing or defective. These conditions
affect the biosynthesis of N- and O-linked glycans glycolipids and glycosaminoglycans.
The changes in the structures found can generally be related to the defective enzymes
although sometimes studies of such disorders has led to the recognition of alternative
biosynthetic pathways that can compensate to some degree for the deficiency. The term
CDG is generally more specifically applied to the defects in N- and O-glycan biosynthetic
pathways of which some 18 different types have been described at the present time
Another genetic disorder which has recently received a lot of attention is that related to
muscular dystrophies. One particular aspect of this which deserves mention is that of
the changes in glycosylation of an important muscle glycoprotein alpha-dystroglycan
which has been the subject of intense investigations in recent years. This glycoprotein is
important in the development of muscle tissue and the interaction of muscle fibres with
the extracellular matrix [26]. It has been shown that there is a particular type of Oglycosylation in which glycans like NeuAcα 2,3Galβ1,4GlcNAcβ 1,2Man are O-linked to
ser or thr [27]
It has been shown that there are changes in activity of several transferases during the
progression of some neuromuscular diseases such as Walker-Warberg disease and
muscle-eyebrain disease, although the precise nature of the changes in glycan
structures and their significance in the progression of these serious developmental
diseases remains to be fully elucidated.
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Table 3
Causes and symptoms of CDG.
CDG
Gene
Enzyme
Typical symptoms
Mental retardation (MR), hypotonia,
esotropia, lipodystrophy, cerebellar
hypoplasia, seizures
Hepatic fibrosis, protein-losing enteropathy
(PLE), coagulopathy, hypoglycemia
CDG-Ia
PMM2
Phosphomannomutase II
CDG-Ib
MPI
CDG-Ic
ALG6
CDG-Id
ALG3
CDG-Ie
DPM1
CDG-If
MPDU1
CDG-Ig
ALG12
CDG-Ih
ALG8
CDG-Ii
ALG2
CDG-Ij
DPAGT1
CDG-Ik
ALG1
CDG-IL
ALG9
Phosphomannose isomerase
Dol-P-Glc: Man9GlcNAc2-PP-Dol
glucosyltransferase
MR, hypotonia, epilepsy
Dol-P-Man: Man5GlcNAc2-PP-Dol
mannosyltransferase
Severe MR, optic nerve atrophy
Dol-P-Man synthase I GDP-Man:
Severe MR, epilepsy, hypotonia, mildly
Dol-P-mannosyltransferase
dysmorphic, coagulopathy
Mannose-P-dolichol utilization
defect 1/Lec35
Short stature, icthyosis, MR, retinopathy
Dol-P-Man: Man7GlcNAc2-PP-Dol Hypotonia, MR, facial dysmorphism,
microcephaly, frequent infections
mannosyltransferase
Dol-P-Glc: Glc1Man9GlcNAc2-PPHepatomegaly, coagulopathy, PLE, renal
failure
Dol glucosyltransferase
GDP-Man: Man1GlcNAc2-PP-Dol
Normal at birth, hepatomegaly,
coagulopathy, MR, hypomyelination,
mannosyltransferase
UDP-GlcNAc: dolichol phosphate N acetylglucosamine-1 phosphate
Severe MR, hypotonia, seizures,
transferase
microcephaly
Severe MR, hypotonia, acquired
GDP-Man: GlcNAc2-PP-Dol
microcephaly, intractable seizures, fever,
coagulopathy, nephrotic syndrome
mannosyltransferase
Dol-P-Man: Man6 and 8 GlcNAc2-PP- Severe microcephaly, hepatomegaly,
hypotonia, seizures
Dol mannosyltransferase
CDG-IIa
MGAT2
GlcNAcT-II
CDG-IIb
GLS1
Glucosidase I
CDG-IIc
SLC35C1/FUCT1
GDP-fucose transporter
CDG-IId
B4GALT1
β1,4-galactosyltransferase
CDG-IIe
COG7
COG complex, subunit 7
CDG-IIf
SLC35A1
CMP-sialic acid transporter
MR, facial dysmorphism, seizures
Dysmorphism, hypotonia, seizures,
hepatomegaly, hepatic fibrosis (death at 2.5
months), normal Tf
Recurrent infections, neutrophilia, MR,
microcephaly, hypotonia, normal Tf
Hypotonia and myopathy, spontaneous
hemorrhage
Fatal in infancy, dysmorphism, hypotonia,
intractable seizures, hepatomegaly,
progressive jaundice, recurrent infections,
cardiac failure
Thrombocytopenia, abnormal platelet
glycoproteins, but no neurologic symptoms
and normal Tf
What is more difficult is to correlate the changes in glycan structure to the observed
symptoms. These conditions are frequently serious and affect development so are
observed in childhood . The most common aspect of CDGs involving biosynthetic
enzymes for glycan chains is in impaired neural or neuromuscular development although
other changes are also observed such is in the immune system.
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6.2. Genetic Disorders (Abnormal catabolism of glycoconjugates Lysosomal Storage Diseases)
The defects in glycolipids storage associated with impaired ability to break down
glycolipids have been well described such as in Gaucher, Fabry and other conditions.
There are other conditions affecting polysaccharides (inulin) and glycosaminoglycans
(such as hyaluronic acid). These have been collectively called the lysosomal storage
disorders and several have been linked to specific diseases as shown below (from [28,
29]
Table I. Known lysosomal disorders.
Mucopolysac
charidoses
(MPS)
MPS I
Glycoproteinoses
Sphingolipidoses
Aspartylglucosa
minuria
Fabry's disease
Other
lipidoses
NiemannPick disease
type C
Lysosomal
transport
defects
Cystinosis
MPS II
Fucosidosis
Farber's
disease
Wolman's
disease
Sialic
storage
disease
MPS IIIA
α-Mannosidosis
Gaucher's
disease
Neuronal
ceroid
lipofuscinosi
s
Other
disorders due
to defects in
lysosomal
proteins
MPS IIIB
α -Mannosidosis
GM1
gangliosidosis
Glycogen
storage
disease
Danon
disease
Hyaluronidase
deficiency
MPS IIIC
Mucolipidosis
(sialidosis)
Tay-Sachs
disease
Glycogen
storage
disease type
II (Pompe's
disease)
MPS IIID
Schindler
disease
Sandhoff's
disease
Multiple
enzyme
deficiency
MPS IVA
Krabbe's
disease
Multiple
sulphatase
deficiency
MPS IV B
Metachromatic
leucodystrophy
Galactosiali
dosis
MPS VI
Niemann-Pick
disease, types A
and B
Mucolipidos
is II/III
MPS VII
acid
Mucolipidos
is IV
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There are diverse symptoms of all these diseases ranging from mild to very severe and
life threatening and they affect many different systems in the body
6.3.
Inflammatory Diseases
Studies of glycosylation in inflammatory diseases provided one of the first examples of a
disease related change in a glycosylation profile. It was noted that not only the amount
but also the glycosylation of several acute phase reactant proteins present in serum
changed following injury [30-35]. The best characterised of these changes is in the
highly glycosylated acute phase protein alpha-1 acid glycoprotein. Alpha 1-acid
glycoprotein (AGP) is a serum acute phase glycoprotein which possesses five N-linked
complex type heteroglycan side chains which may be present as bi-, tri- and
tetraantennary structures. Depending upon the content of biantennary structure on AGP,
up to four glycoforms of AGP are present in serum. These glycoforms can be easily
estimated in body fluids by means of crossed affinity-immunoelectrophoresis (CAIE) with
the lectin, Concanavalin A (Con A). Con A selectively binds biantennary structures; the
more biantennary structures on AGP, the stronger the binding. In acute inflammation, a
relative increase of AGP glycoforms with biantennary units is observed-a type I
glycosylation change. In some chronic inflammatory states there is an relative decrease
of AGP glycoforms with biantennary heteroglycans-a type II glycosylation change.
Moreover, in certain other states such as pregnancy, oestrogen administration or liver
damage, type II glycosylation changes are also seen. A detailed analysis of the clinical
applications of the assessment of AGP glycoforms in sera of patients with rheumatic
diseases, AIDS and various types of cancers has been performed [34]. The data showed
that AGP glycoforms may be very useful in the detection of intercurrent infections in the
course of rheumatoid arthritis, systemic lupus erythematosus, or myeloblastic
leukaemia, and in the detection of secondary infections in human immunodeficiency virus
infected individuals.
The occurrence of differences in acute-phase response, with respect to concentration
and glycosylation of alpha 1-acid glycoprotein (AGP) was studied in the sera of patients,
surviving or not from septic shock [32]. Crossed affino-immunoelectrophoresis was used
with concanavalin A and Aleuria aurantia lectin for the detection of the degree of
branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection
of sialyl Lewisx (SLeX) groups on AGP. Septic shock apparently induced an acute-phase
response as indicated by the increased serum levels and changed glycosylation of AGP.
In the survivor group a transient increase in diantennary glycan content was
accompanied by a gradually increasing fucosylation and SLeX expression, comparable to
those observed in the early phase of an acute-inflammatory response. In the non-survivor
group a modest increase in diantennary glycan content was accompanied by a strong
elevation of the fucosylation of AGP and the expression of SLeX groups on AGP, typical
for the late phase of an acute-phase response. These results suggest that changes in
glycosylation of AGP can have a prognostic value for the outcome of septic shock.
In another experimental study with injection to stimulate the acute phase (AP) response
in rats, the N-acetylneuraminic acid content of plasma proteins increases and that of
fucose was found to decrease by about 60%.[32]. The NeuAc/Gal ratio increased from
the normal 0.75 to 1.0 on day 2 of the AP.. This indicated that NeuAc caps the normally
Gal-terminated chains. Study of alpha1-Acid glycoprotein (a positive AP protein), alpha1macroglobulin (a non-AP protein), and alpha1-inhibitor3 (a negative AP protein) also
showed similar alterations in NeuAc/Gal ratio and decreases in Fuc. alpha2Macroglobulin, which arises only during the AP, does not contain significant amounts of
Fuc. Sambucus nigra agglutinin (alpha2,6-linked NeuAc-specific) binds a majority of
plasma proteins, and binding was increased during the AP response. Maackia amurensis
lectin (alpha2,3-linked NeuAc-specific) binds only three proteins in normal plasma and
three additional proteins in AP plasma. The Fuc-specific Aleuria aurantia agglutinin and
Lens culinaris agglutinin each detect five proteins in normal plasma. Their binding
decreased during the AP response. The authors concluded that these results show that:
(1) sialylation and defucosylation of preexisting plasma proteins occur rapidly in the AP
response; (2) sialylation caps the preexisting Gal-terminating oligosaccharides; and (3)
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the oligosaccharides of even the non-AP and negative AP proteins are modified. These
changes are distinct from the elevation in the levels of protein-bound monosaccharides
and the altered concanavalin A-binding profile the oligosaccharides of AP proteins
acquire in diseases Thus the glycosylation of alpha-1 acid glycoprotein is changed in
inflammatory conditions [34, 36, 37] where decreased branching and increased α,3
fucosylation is found [38]
When the Immunoglobulin IgG was investigated in rheumatoid arthritis a consistent
change in that reduced galactosylation was present on the bi-antennary glycans of IgG
and this was observed by many investigators [39-41]. This has been found to revert to
normal following remission from the disease [42]. It was found that the percentage of
IgG-associated agalactosyl N-linked oligosaccharides (G0) falls during normal human
pregnancy and rises to values higher than before conception following delivery (n = 10,
39-55 days after delivery). Serial bleeds from a normal pregnant woman showed a fall in
the percentage G0 during gestation and a rapid rise post-partum. A similar study on a
pregnant arthritic woman with a pathologically elevated percentage G0 also showed a
fall in percentage G0 during pregnancy and a rapid rise post-partum. The changes in IgG
glycosylation in the pregnant arthritic woman occurred simultaneously with the
pregnancy-induced remission and post-partum recurrence of disease. A further seven
pregnant women with rheumatoid arthritis were studied and analysis of their G0 values
pre- and post-partum confirmed the result. In a further series of experiments using an
animal model of rheumatoid arthritis, DBA/1 mice with collagen-induced arthritis were
found to have elevated G0 levels compared with control mice. The percentage G0 was
found to fall simultaneously with pregnancy-induced remission to the same value as nonarthritic pregnant mice. Post-partum recurrence of arthritis in these mice was also
accompanied by a simultaneous and rapid rise in percentage G0. Pseudopregnancy did
not result in a change in the percentage G0, confirming the effect of true pregnancy.
Since the proportion of agalactosyl IgG is abnormally high in the serum of patients with
rheumatoid arthritis these changes in IgG glycoform levels, or the factors which control
them, may be related to the mechanisms underlying remission of arthritis in humans
during pregnancy.
6.4.
Infectious Diseases
There are many reports of changes in host glycosylation following various types of
infection which go back many years and report a wide variety of changes. This are is
particularly confusing in the literature as a large number of different techniques are
employed and sometimes the conclusions drawn from the studies is not justified by the
observations. This is important in considering the database requirement as the
relationship between host and infectious agent glycosylation do present good potential
routes for immunisation, diagnostic or therapeutic approaches but frequently lack of
knowledge of glycoscience means these are not properly exploited.
One example which is particularly relevant at present is that of the avian flu virus H5N1
strain and the possibility of its spread to human hosts with possible emergence of a
major flu pandemic as in 1918. Studies have been made which show the major influenza
virus serotype components haemagluttinin (H) and neuraminidase (N) have been shown
to have undergone subtle changes in glycan specificity in the transmission from birds to
humans. There is some excellent research which is available on this aspect but there
seems to have been little exploitation of these basic findings. The neuraminidase
inhibitor drugs as famous for their antiviral activities but detailed knowledge of the
specificities of the H and N should allow the design of much more specific and relevant
inhibitors of viral activity and entry into cells.
Further details of glycosylation and its
relevance to influence virus infection in humans and in its transmission from birds may b
may be found in Appendix B page 47
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Bacterial
There are many references to the role of the complicated and variable glycosylation
found on bacterial cells walls (see above) in the interaction in infection. It has been
shown that the carbohydrate and pathogen-specificity of DC-SIGN identifies this lectin to
be central in pathogen-DC interactions.[43]
Indigenous microbes and their soluble factors differentially modulate intestinal
glycosylation steps in vivo. A "lectin assay" has been used to survey in vivo glycosylation
changes. [44-46]
Mutations in the srf-3 locus in C. elegans confer resistance to infection by
Microbacterium nematophilum and Yersinia pseudotuberculosis. The srf-3 gene has been
cloned and shown to encode a multitransmembrane hydrophobic protein homologous to
mammalian Golgi-localized nucleotide-sugar transporters [34••]. The srf-3 gene is
exclusively expressed in secretory cells, consistent with its proposed function in
cuticle/surface modification, and encodes a UDP-Gal/UDP-GlcNAc transporter. It is
suggested that bacterial resistance in srf-3 mutant worms is due to absence of a glycan
required for binding of bacteria to the nematode cuticle. This study is an example of
using an invertebrate organism as a model for studying vertebrate innate immunity and
infection by an organism that forms a biofilm (defined as a community of bacteria
enclosed in a self-produced exopolysaccharide matrix that adheres to a biotic or abiotic
surface). Biofilm formation by pathogens is of great clinical importance because bacteria
embedded in biofilms have been shown to be more resistant to antibiotics, to
components of the host immune system and to removal by mechanical forces [35].
A report of a new form of complete IFNR2 deficiency, characterized by surfaceexpressed nonfunctional receptors has recently appeared [47]. The T168N IFNR2
mutation results in a protein carrying an N-linked carbohydrate moiety attached at
Asn168. This polysaccharide is both necessary and sufficient to account for the
pathological effect of the T168N mutation. Despite this glycosylation, the T168N IFNR2
molecules have the same intracellular localization as wild-type molecules. Similar
complete deficiencies due to nonfunctional receptors expressed at the cell surface have
been reported in other individuals with inherited defects of the IL12/23-IFN axis involving
the other two known receptor defects: complete deficiencies of IFNR1 and IL-12R1
Surface-expressed nonfunctional IFNR1 and IL-12R1 molecules have been associated
with missense mutations and in-frame small and large deletions; the mutant molecules
fail to bind their natural ligands, IFN and IL-12/2, respectively9, 11. By contrast, the
mechanism by which the T168N-associated neoglycosylation of IFNR2 affects IFN
signaling is still unknown.
The T168N mutation in IFNR2 is the first reported germline mutation for which a causal
relationship has been unequivocally established between the gain of glycosylation and
the loss of function. Six other previously described missense mutations associated with a
primary immunodeficiency are also characterized by gains of glycosylation. Such
mutations are not confined to primary immunodeficiencies, as indicated by the 16
pathogenic mutations in 11 genes previously shown to involve gains of glycosylation.
Moreover, among 577 genes bearing missense mutations and encoding proteins that
migrate through the secretory pathway, up to 77 (13.3%) may be subject to potential
gains of glycosylation (corresponding to 1.4% of pathogenic missense mutations found in
the 577 genes; Gain-of-glycosylation mutations may therefore affect many thousands of
individuals worldwide.
6.4.2.
Viral
Since viruses do not have any glycosylation machinery the glycosylation seen reflects
that of the host they have infected. Glycosylation is important in several ways. Firstly
most of the viral coat proteins are glycoprotein and many of the recognition elements
involve the coat glycoproteins. They are also important for viris assembly and transport
from infected cells. They may also bring about some change in host glycosylation as a
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result of infection. Changes in fucosylation of glycoproteins following HBV infection in
the development of primary hepatocellular carcinoma have been reported. [48]
An example of the importance of glycosylation in recognition of virus is that of the
involvement of the collectins. Collectins are secreted collagen-like lectins that bind,
agglutinate, and neutralize influenza A virus (IAV) in vitro [49]. Surfactant proteins A and
D (SP-A and SP-D) are collectins expressed in the airway and alveolar epithelium and
could have a role in the regulation of IAV infection in vivo. Previous studies had shown
that binding of SP-D to IAV is dependent on the glycosylation of specific sites on the HA1
domain of hemagglutinin on the surface of IAV, while the binding of SP-A to the HA1
domain is dependent on the glycosylation of the carbohydrate recognition domain of SPA. In this report, using SP-A and SP-D gene-targeted mice on a common C57BL6
background, it was shown that viral replication and the host response as measured by
weight loss, neutrophil influx into the lung, and local cytokine release are regulated by
SP-D but not SP-A when the IAV is glycosylated at a specific site (N165) on the HA1
domain. SP-D does not protect against IAV infection with a strain lacking glycosylation at
N165. With the exception of a small difference on day 2 after infection with X-79, we did
not find any significant difference in viral load in SP-A(-/-) mice with either IAV strain,
although small differences in the cytokine responses to IAV were detected in SP-A(-/-)
mice. Mice deficient in both SP-A and SP-D responded to IAV similarly to mice deficient in
SP-D alone. Since most strains of IAV currently circulating are glycosylated at N165, SPD may play a role in protection from IAV infection.
6.4.3.
HIV
Changes in T cell surface glycosylation in HIV-1 infection with increased susceptibility to
galectin-1-induced cell death. [50] The mechanisms by which HIV virus evades the
immune system are unclear but he surface glycoprotein GP 120 is extensively
glycosylated with oligomannose structures which may make it resemble a ‘self’ antigen
and thus be undetected by the immune system.
The envelope protein (gp120/gp41) of HIV-1 is highly glycosylated with about half of the
molecular mass of gp120 consisting of N-linked carbohydrates. While glycosylation of
HIV gp120/gp41 provides a formidable barrier for development of strong antibody
responses to the virus, it also provides a potential site of attack by the innate immune
system through the C-type lectin mannose binding lectin (MBL) (also called mannan
binding lectin or mannan binding protein). A number of studies have clearly shown that
MBL binds to HIV. Binding of MBL to HIV is dependent on the high-mannose glycans on
gp120 while host cell glycans incorporated into virions do not contribute substantially to
this interaction. It is notable that MBL, due to its specificity for the types of glycans that
are abundant on gp120, has been shown to interact with all tested HIV strains. While
direct neutralization of HIV produced in T cell lines by MBL has been reported,
neutralization is relatively low for HIV primary isolates. However, drugs that alter
processing of carbohydrates enhance neutralization of HIV primary isolates by MBL.
Complement activation on gp120 and opsonization of HIV due to MBL binding have also
been observed but these immune mechanisms have not been studied in detail. MBL has
also been shown to block the interaction between HIV and DC-SIGN. Clinical studies
show that levels of MBL, an acute-phase protein, increase during HIV disease. The
effects of MBL on HIV disease progression and transmission are equivocal with some
studies showing positive effects and other showing no effect or negative effects.
Because of apparently universal reactivity with HIV strains, MBL clearly represents an
important mechanism for recognition of HIV by the immune system. However, further
studies are needed to define the in vivo contribution of MBL to clearance and destruction
of HIV, the reasons for low neutralization by MBL and ways that MBL anti-viral effects can
be augmented. Further details of glycosylation in relation to HIV may be found in
Appendix B page 46
6.4.4.
Parasitic
Glycosylation is also important in recognition of parasites. A review of glycan-lectin
interactions in schistosomiasis showed that serum levels of soluble adhesionmolecules
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including E-selectin are correlated with the differentpathological manifestations of
schistosomiasis. It may be expected that the glycoconjugates expressed by
schistosomes interact with host lectins, most notably of the selectin family which have
LeX-related oligosaccharides as their ligands .
Selectins have also been shown to interact strongly with LDNF [139]. Indeed it has been
demonstrated that host-soluble Lselectin enters tissue-trapped eggs and binds to
glycosylated antigens on the surface of miracidia [140], but that E- and Pselectin do not.
This implies that miracidia express L-selectin ligands and potentially directly influence Lselectin-mediated
processes.
In
parallel
with
the
selectin-mediated
endotheliumleukocyte interaction [141], it has been shown that adhesion of eggs to
endothelial cells under flow conditions is E-selectin mediated, and that process could be
blocked by an anti-LeX antibody [142]. Even more remarkable in this parallel between
host and parasite adhesion molecules is the finding that S. mansoni itself expresses
selectin-like molecules with affinity for LeX and sialyl-LeX [143]. These schistosome
lectins and their human glycan counterparts, as well as the opposite combination of
human selectins and LeX-containing schistosome glycoconjugates are all required as coreceptors in the antibody-dependent cell-mediated cytotoxicity of macrophages and
eosinophils to schistosomula [143,144]. Interestingly, other helminths also express Ctype lectins homologous to human lectins involved in immunological events [145].
Parasitic infections have also been reported as changing host glycosylation . This seems
to take place in the site of infection following exposure to the parasite. Alterations of
mouse intestinal mucins were found after infection caused by the parasite
Nippostrongylus brasiliensis. [51]. Experimental studies of infection with Trichinella
spiralis where there was an enteric mucin-related response resulting in changes in Oglycosylation in conventional and SPF pigs.[52, 53]
The changes that have been found in glycosylation following exposure to infectious
diseases may result from a number of factors. Firstly they may be a result of glycosidase
activity of the infectious agent. Secondly they may result fro the immunological response
to the infection such as the activation of T cells. Thirdly they may result from some other
factor which has then increased susceptibility to infection.
Modification of glycosylation of the host lymphocytes following T cell activation are well
documented and involves changes in the degree of sialylation and the size of the glycan
chains. An example of such a change is the changes recognised by CD22 (Siglec-2) [54]
a well-known regulator of B cell signalling Other changes in glycosylation undoubtedly
take place upon activation of the immune systems and are subject of intense
investigations.
6.5.
Cancer and Metastasis
There are many reports of changes in glycosylation in cancer patients, however in many
cases no rigorous investigations of the actual nature of the change or how it has brought
about have been made.
The evidence suggests that glycosylation can play a key role in controlling tissue growth
and development so it is not surprising that glycosylation changes are found in many
cancers. Of particular importance may be the attachment of cells in metastasis.
Changes in gastric epithelium in cancer have been reported [55] There are also some
consistent and well documented examples such as the change of the O-linked mucin
glycan in breast cancer [56]. Further details of glycosylation changes in cancer may be
found in Appendix B pages 43 and 45
6.6.
Other Diseases
A number of other diseases have now been associated with specific changes in
glycosylation. One well characterised example is IgA nephropathy where there are
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specific changes in the glycosylation of O-glycans on IgA1 [57-59] where there is a defect
in galactosylation caused by a mutant in a protein folding chaperone Cosmc [58] which
may lead to complement activation and the subsequent nephropathy. Another condition
which has recently been linked to a glycosylation change is in muscular dystrophy [6065], where there is a change in the O-glycosylation of alpha-dystroglycan.
Changes is glycosylation have also been reported in diabetes although this is primary
non-enzymatic in nature [66] and as mentioned above such modifications are termed
advanced glycation end products (AGE). A specific receptor has been shown to be
present (RAGE) which recognises the glycosylation and is responsible for the symptoms
of the diseases. More details are given on page 43
It is certain that many more diseases involve glycosylation but it is often difficult to obtain
conclusive evidence for this in light of the relative scarcity of reliable data on
glycocongugate analysis that has, until recently, not been available.
To quote from ‘Essential of Glycobiology’ by Varki et al (2005) [1]
‘”Available data indicate that considerable diversity of glycan structure and
expression exists in nature. However, partly because of the inherent
difficulties in studying glycan structure, relatively little is known about the
details of this diversity (there are very few published reviews on this
subject).”
“Approaches taken to understand the biological roles of glycans include the
prevention of initial glycosylation, alteration of oligosaccharide processing,
enzymatic or chemical deglycosylation of completed chains, genetic
elimination of glycosylation sites, and the study of naturally occurring
variants and genetic mutants in glycosylation. In reviewing many such
studies, the consequences of altering glycosylation range from being
essentially undetectable to the complete loss of particular functions, or
even loss of the entire glycoprotein itself. Even within a particular class of
proteins (e.g., cell surface receptors), the effects of altering glycosylation
are highly variable and unpredictable. Moreover, the same glycosylation
change can have markedly different effects when studied in vivo or in vitro.
The answer obtained may depend on the structure of the glycan, the
biological context, and the specific biological question being asked. Overall,
it is difficult to predict a priori the functions that a given oligosaccharide on
a given glycoconjugate might mediate or its relative importance to the
organism.”
In conclusion this report shows that there are a wide range of gyconjugate structures
that should be represented in the EurocarbDB databases. However the proposed
scheme for design of the databases should be able to encompass all of these and provide
a resource that is readily searchable, validated by experimental data and is in a format
that is exchangeable with other databases.
The need for reliable data on the biological function and disease relationship of
glycosylation is certainly apparent and should provide an invaluable resource for
understanding the complex relationships. At present much of the data available is
confused and it is difficult to get a clear idea of how glycosylation relates to many
diseases. This is a clear role for the bioinformatics project which should help greatly in
the more general understanding of glycoscience.
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Appendix A Examples of Structures from Table 1
A Bacterial Lipopolysaccharide (Lipid A)
b-D-Galp-(1-3)-b-D-GalNAc(1-4)-b-D-Galp- (1-3) -b-D-Galp (1-3) -a- D-Hep- (1-3) -a- DHep-(1-5)-Kdo-(2-2)+
|
a-D-Neup5Nac-(2-3)+
|
a-D-Galp-(1-2)+
|
b-D-Glc-(1-2)+
|
b-D-Glc-(1-
From Campylobacter Jejuni NCTC 11168
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B Bacterial Lipopolysaccahride O-antigen
-4-b-D-Glcp-(1-3)-a-D-GalNAc-(1-2)-a-D-Rha4NAc-(1-3)-a-L-Fuc(1-
E.Coli O157 H7 Salmonella O30 Serogroup N
C Bacterial Capsular Polysaccharide (K antigen)
a-D-Neup5Nac-(2-8)-a-D-Neup5Nac-(2-8)-a-D-Neup5Nac-(2-8)-a-D-Neup5Nac-(2-8)-
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d Bacterial Peptidoglycan
Major Staph A peptidoglycans
GlcNAc)n
glycan (GlcNAc-[ -1,4]-MurNAc)n or (MurNAc-[ -1,4]-
b-D-GlcNAc-(1-4)-b-D-MurNAc-(1,4)-b-D-GlcNAc-(1-4)-b-D-MurNAc-(1,4)reduced muropeptide N-acetylglucosamine (GlcNAc or "G") ß-1,4–linked to N-acetylmuramic acid
(MurNAc or "M"), substituted with a tripeptide group
e Eukaryote GPI anchor
a-D-Manp-(1-2)-a-D-Manp-(1-6)-a-D-Manp-(1-4)-b-D-GlcN-(1-6)-inositol
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f Plant cell wall cellulose
[b-D-Glcp-(1-4)-b-D-Glcp-(1-4)-]
n
Celluose (n>100)
[a-D-Glcp-(1-4)-a-D-Glcp-(1-4)-]
n
Amylose (n>100)
(a) Superposition of cellohexaose and cellulose II 3D models derived using 13C
chemical shifts constraints (blue) and a cellulose II polymorph structural model.
(b) Lowest energy conformer for cellohexaose obtained through SA when
chemical shift constraints are omitted [67]
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g Plant lipochito-oligosaccharides NOD factors
Nod factors
Most Nod factors consist of a backbone of three, four, or five ß-1,4-linked Nacetylglucosaminyl residues, N-acylated at the nonreducing-terminal residue by either a
"common" fatty acid, such as vaccenic (C18:1) and stearic (C18:0) acid, or by a
(poly)unsaturated fatty acid, such as C20:1 (Mesorhizobium loti NZP2213) or C18:4 (R.
leguminosarum bv. viciae A1) (Table I). Often, N-methyl, O-acetyl, and O-carbamoyl
groups are found at the nonreducing-terminal residue and L-fucosyl, 2-O-Me-fucosyl, 4-OAc-fucosyl, acetyl, and sulfate ester at the reducing-terminal residue
b-D-GlcNAc-(1-4)-b-D-GlcNAc-(1-4)-b-D-GlcNAc-(1-4)-b-D-GlcNAc-(1-4)|
N-acylation with fatty acid
h Plant glycoprotein N Glycan
a-D-Fuc-(1-4)-b-D-GlcNAc (1-2)-a-D-Manp-(1-6)+
|
|
b-D-Galp-(1-3)+
|
|
a-L-Fucp-(1-3)+
|
a-D-Fuc-(1-4)-b-D-GlcNAc (1-2)-a-D-Manp-(1-4)--b-D-Manp-(1-4)-b-D-GlcpNAc-(1-4)-b-D-GlcpNAc-(1-4)-Asn
|
|
b-D-Galp-(1-3)+
b-D-Xyl-(1-2)+
29
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i Plant glycoprotein O Glycan
Structures
of
veracylglucan
A
(1),
B
(2),
C
(3)
and
malic
acid
(4).
6-
O-(1-L-maloyl)-alpha-,beta-D-Glcp (veracylglucan A), alpha-D-Glcp-(1-->4)-6-O-(1-Lmaloyl)-alpha,-beta,-D-Glcp (veracylglucan B) and alpha-D-Glcp-(1-->4)-tetra-[6-O-(1-Lmaloyl)-alpha-D-Glcp-(1-->4)]-6-O-(1-L-maloyl)-alpha,-beta-D-Glcp (veracylglucan C).
b-Ara-(1-3)-b-Ara-(1-2)-b-Ara-(1-6)-b-D-Gal+
|
b-D-Gal-O-HYP
|
b-D-Galp-(1-3)-b-D-Galp-(1-3)+ b-D-Galp-(1-3)+
|
b-D-Galp-(1-6)+
b-Ara-(1-3)-b-Ara-(1-3)-b-Ara-(1-2)-b-Ara-b-1-O-HYP
b-D-Galp-(1-3)-a-D-Gal-1-O-Ser
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j Sulphated fucans
(1-3)-a-L-Fucp+
|
a-L-Fucp-(1-3)-a-L-Fucp-(1-3)-a-L-Fucp-(1-3)-a-L-Fucp-(1-3)-a-L-Fucp-(1-3)|
|
|
(1-4)-a-L-Fucp+
3OS+
3OS+
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Amphibian muins
m Insect polysaccharide chitin
(1-6)b-D-GlcNAc+
|
(1-3)-a-L-Fucp-(1-6)-b-L-GlcNAcp-(1-3)b-D-Galp-(1-3)-a-D-GalpNAc-(1-3)-Ser
|
(
1-3)-b-D-GalpNAc-(1-3)-b-D-Galp-(1-4)+
[(1-4)-b-D-GlcpNAc-(1-4)-b-D-GlcNp-(1-4)-b-D-GlcpNAc-(1-4)-b-D-GlcNp-]
N
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n Human hyaluronic acid
[(1-3)-b-D-GlcpNAc-(1-4)-b-D-GlcAp-(1-3)-b-D-GlcpNAc-(1-4)-b-D-GlcAp-]
n
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34
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o Human glycolipids
NeuAc 3Galß4GlcßCerGM3, 3s ganglioside
Galß3GalNAcß4(NeuAc 3)Galß4GlcßCerGM1
NeuAc 3Galß3GalNAcß4(NeuAc 3)Galß4GlcßCerGD1a
Galß3GalNAcß4(NeuAc 8NeuAc 3)Galß4GlcßCerGD1b
NeuAc 3Galß3GalNAcß4(NeuAc 8NeuAc 3)Galß4GlcßCerGT1b
NeuAc 3Galß4GlcNAcß3Galß4GlcßCerS-3-PG, NeuAc-3-paragloboside, sialyl-3-paragloboside,5s ganglioside
NeuAc 6Galß4GlcNAcß3Galß4GlcßCerS-6-PG, NeuAc-6-paragloboside, sialyl-6-paragloboside,5s ganglioside
NeuAc 3Galß4GlcNAcß3Galß4GlcNAcß3Galß4GlcßCer7-sugar NeuAc-3-neolacto ganglioside, 7s ganglioside
NeuAc 6Galß4GlcNAcß3Galß4GlcNAcß3Galß4GlcßCer7-sugar NeuAc-6-neolacto ganglioside, 7s ganglioside
NeuAc 3Galß4(Fuc 3)GlcNAcß–Sialyl-Lewis x epitope
NeuAc 3Galß4GlcNAcß3Galß4(Fuc 3)GlcNAcß–"VIM-2" epitope (epitope reacting with CDw65/clone
VIM-2 monoclonal antibody
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p Human glycoprotein N Glycans
May be oligomannose, complex or hybrid
Oligomannose
a-D-Manp-(1-2)+
|
a-D-Manp-(1-2)-a-D-Manp-(1-6)+
|
a-D-Manp-(1-6)+
|
a-D-Manp-(1-6)+
|
b-D-Manp-(1-4)-b-D-GlcpNAc-(1-4)-b-D-GlcpNAc-(1-4)-Asn
|
a-D-Manp-(1-3)+
|
a-D-Manp-(1-2)+
Hybrid
a-D-Manp-(1-2)+
|
a-D-Manp-(1-2)-a-D-Manp-(1-6)+
|
a-D-Manp-(1-6)+
|
a-D-Manp-(1-6)+
|
b-D-Manp-(1-4)-b-D-GlcpNAc-(1-4)-b-D-GlcpNAc-(1-4)-Asn
|
a-D-Manp-(1-3)+
|
b-D-Galp-(1-4)-b-D-GlcNAcp-(1-2)+
complex
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q Human glycoprotein O Glycan
Core Structures
Type 1 core
a-D-Neup5Nac-(2-)6+
|
b-D-Galp-(1-3)-a-D-GalpNAc-Ser
|
a-D-Neup5Nac-(2-3)+
a-D-Neup5Nac-(2-6)+
|
b-D-GlcNAcp-(1-6)+
|
b-D-Galp-(1-3)-a-D-GalpNAc-Ser
|
a-D-Neup5Nac-(2-3)+
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r Human GPI anchors
38
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s Human mucins
Proposed acidic oligosaccharide structures identified in human intestinal mucins by nano-ESI MS/MS [18]
I, ileum; C, cecum; T, transverse colon; S, sigmoid colon; R, rectum; a, structures recovered from donor 2; b, structures recovered
fro donor 1. The upper branch of the oligosaccharides is indicated in bold.
I
C T S R
Oligosaccharides with one NeuAc residue
NeuAc
Gal
6GalNAc-ol
513 + + + + + + + + + +
3(NeuAc
6)GalNAc-ol
3GalNAc-ol
675 + + + + + + - - + +
(NeuAc
3)Gal
GalNAc
3(NeuAc
6)GalNAc-ol
716 + + + + + + + + + +
GlcNAc
3(NeuAc
6)GalNAc-ol
716 + + + + + + + + + +
(Fuc
2)Gal
3(NeuAc
3GlcNAc
Gal
675 - - - - - - + + - -
6)GalNAc-ol
3(NeuAc
821 + - + - - - - - - -
6)GalNAc-ol
878 + + + + + + + + + +
(NeuAc
3)Gal
3(GlcNAc
(NeuAc
3)Gal
4GlcNAc
3GalNAc-ol
878 - - - - - - - + - -
GlcNAc
3Gal
3(NeuAc
6)GalNAc-ol
878 + - + - + + + + + +
6)GalNAc-ol
878 - - - - - - - + - -
Gal
3(Fuc
4)GlcNAc
3(NeuAc
6)GalNAc-ol
Gal
4(Fuc
3)GlcNAc
3(NeuAc
6)GalNAc-ol
1024 + + + + + + + + + +
3)GlcNAc
3GalNAc-ol
1024 - - - - - - - + - -
(NeuAc
3)Gal
4(Fuc
HexNAc
3Gal
3(NeuAc
6)GalNAc-ol
1081 - + - + - - - - - -
GalNAc
4(NeuAc
3)Gal
4GlcNAc
3GalNAc-ol
1081 - - - - + + + + + +
GalNAc
4(NeuAc
3)Gal
3GlcNAc
3GalNAc-ol
1081 - - - - + + + + + +
(Fuc
2)Gal
HexNAc
3(Fuc
3(NeuAc
4)GlcNAc
2)Gal
3GlcNAc
4GlcNAc
3(Fuc
3Gal
4)GlcNAc
3(Fuc
3)Gal
HexNAc
NeuAc
3(Fuc
3Gal
HexNAc
(NeuAc
4GlcNAc
1024 + + + + + + + + + +
3Gal
2)Gal
6)GalNAc-ol
3(NeuAc
6)GalNAc-ol
3Gal
1227 + + + + + - - - - 1227 + - + - - - - - - -
3GalNAc-ol
4)GlcNAc
3)GlcNAc
1170 + + + + + - - - - -
3(NeuAc
GlcNAc
3(Fuc
4(Fuc
6)GalNAc-ol
3(NeuAc
3[Gal
1243 - - - - - - + + - 6)GalNAc-ol
4(Fuc
3)GlcNAc
1373 + + + + - - - - - 6]GalNAc-ol
1697 - - - - - - + + + +
Oligosaccharides wih one sulphate residue
(SO3-)3Gal
Gal
4GlcNAc
3GalNAc-ol
667 - - - + - - + + + +
4(SO3-)6GlcNAc
3GalNAc-ol
667 + - - + - - + + + +
4(SO3-)6GlcNAc
2)Gal
(Fuc
(SO3-)3Gal
3GalNAc-ol
813 + - - + - - - - - +
4(Fuc
3)GlcNAc
3GalNAc-ol
813 - - + + + + + + + +
Gal
3[(SO3-)3Gal
4GlcNAc
6]GalNAc-ol
829 - - - - + - + + + +
Gal
3[Gal
4(SO3-)6GlcNAc
6]GalNAc-ol
(SO3-)3Gal
4GlcNAc
3[(SO3-)3Gal
Gal
3Gal
4(Fuc
829 - - - - + - - - + +
3GalNAc-ol
3)GlcNAc
829 - - + + + - - - - -
6]GalNAc-ol
(SO3-)3Gal
4(Fuc
3)GlcNAc
3Gal
3GalNAc-ol
(SO3-)3Gal
4(Fuc
3)GlcNAc
3Gal
3[Gal
975 - - - + + + - + + +
975 - - - - + - + + + +
4(Fuc
3)GlcNAc
6]GalNAc-ol
1486 - - - - - - - + - -
Oligosaccharides with two acidic residues
(SO3-)3Gal
(NeuAc
4GlcNAc
3)Gal
(SO3-)3Gal
3
(SO )3Gal
3(NeuAc
3(NeuAc
3[(SO3-)3Gal
4(Fuc
4(Fuc
4GlcNAc
6]GalNAc-ol
1120 - - - - - - + - + +
3[(SO3-)3Gal
4GlcNAc
3)Gal
3(NeuAc
3)GlcNAc
4(Fuc
4GlcNAc
1055 - - - - - - - + - 1104 + + + + + + + + + +
3)Gal
(NeuAc
6]GalNAc-ol
6)GalNAc-ol
3)Gal
(SO3-)3Gal
3)GlcNAc
3(NeuAc
(NeuAc
4(Fuc
958 - - - - + + + + + +
966 + + + + + + + + - -
3)GlcNAc
(NeuAc
(SO3-)3Gal
6)GalNAc-ol
6)GalNAc-ol
3Gal
3)GlcNAc
3Gal
6)GalNAc-ol
1169 + + - + + + + + + +
3(NeuAc
1266 - - - - + + - - - -
3(NeuAc
4GlcNAc
6)GalNAc-ol
6)GalNAc-ol
3(NeuAc
1315 - - + + + + + + + +
6)GalNAc-ol
GalNAc
4(NeuAc
3)Gal
4GlcNAc
3(NeuAc
6)GalNAc-ol
GalNAc
4(NeuAc
3)Gal
3GlcNAc
3(NeuAc
6)GalNAc-ol
(SO3-)3Gal
4GlcNAc
(SO3-)3Gal
4(Fuc
3Gal
3)GlcNAc
3[(SO3-)3Gal
3Gal
4(Fuc
4GlcNAc
1372 - - - + + + + + + +
1372 - - - + + + + + + +
3)GlcNAc
3(NeuAc
1323 - - - + - - - + - -
6]GalNAc-ol
6)GalNAc-ol
1420 - - - - - - - + - 1469 - - + + - - + + + -
3 Gal, 2 HexNac, NeuAc, SO3-, GalNAc-ol
1485 - - - - - - - - + -
(SO3-)3Gal
4(Fuc
3)GlcNAc
3Gal
3[(SO3-)3Gal
(SO3-)3Gal
4(Fuc
3)GlcNAc
3Gal
4(Fuc
2 Gal, 2 HexNAc, Fuc, 2 NeuAc, GalNAc-ol
4(Fuc
3)GlcNAc
3)GlcNAc
3(NeuAc
6]GalNAc-ol 1566 - - - + - - + + + +
6)GalNAc-ol
1615 - - + + + + + + + +
1680 - - + - - - - - - -
2Gal, 2 HexNAc, 2 Fuc, 2 NeuAc, GalNAc-ol
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t Human GAG Heparan Sulphate
FGFR4 binding heparan sulfate octasaccharide sequences.
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u Human GAG Keratan Sulphate
Biosynthesis of Keratan Sulphate
Di saccharide repeat unit
[b-D-Galp-(1-4)-b-D-GlcpNAc-(1-3)-]
n
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Appendix B
Some Examples of Disease Related Changes in Glycosylation and Disease
1. Respiratory Tract Mucin Genes and Mucin Glycoproteins in Health and Disease
[68]
Chronic Obstructive Pulmonary Disorder (COPD) is defined clinically as a complex of
diseases characterized by airflow obstruction due to chronic bronchitis or emphysema. It
is the fourth leading cause of patient deaths in adults in the United States. Approximately
14 million people in the United States have COPD; the majority of these patients have
chronic bronchitis
Airway mucins from chronic bronchitis patients overall are similar in size and structure to
mucus from healthy individuals, but appear to be less acidic. However, glycosylation
patterns vary during acute exacerbations, as chronic bronchitis mucins are highly
sialylated with increased sialylated and sulfated Lex structures. Despite biochemical
characterization of the mucus gel, information about specific mucin glycoproteins in
chronic bronchitis airway secretions is limited to a few studies. MUC5AC and MUC5B
mucins are highly expressed constituents both in normal and chronic bronchitis mucus.
The ratio of MUC5B:MUC5AC is increased, and MUC5B differs in charge in chronic
bronchitis sputum compared with normal airway mucus .
Several studies support altered glycosylation of CF mucins, although this remains to be
conclusively demonstrated. Initially, studies focused on mucoprotein fractions from the
duodenum, where increased levels of fucose and sulfate and decreased levels of sialic
acid in CF samples relative to non-CF samples are observed . Increased sulfation in CF
glycoconjugates has also been reported. The structures of several dozen neutral,
sialylated and sulfated O-glycans from airway mucins of patients with CF or bronchitis
have been determined. A consistent finding has been increased expression of sialyl-Lex
epitopes in mucin-rich fractions isolated from patients with CF. However, a similar finding
has also been observed in mucins isolated from patients with chronic bronchitis, leading
to the suggestion that increased sialyl-Lex expression in airway mucins correlates with
severe infection or inflammation
Tumour-cell fusion as a source of myeloid traits in cancer
.[69]
One of the strongest associations of a specific type of glycosylation with disease is the
finding that frequently there is an increased expression of complex type glycans with
β1,6-branched oligosaccharides from the trimannosyl core associated with increased
activity of the GlcNAc transferase GNT-V. Although an association between GNT-V, β1,6branched oligosaccharides, and tumour progression has been known for two decades,
the nearly universal expression of β1,6-branched oligosaccharides in human solid
tumours had not been recognised. Generation of these structures on N-glycans is
initiated by ß1,6-N-acetylglucosaminyltransferase V and used by both myeloid cells and
cancer cells in systemic migration.
In summary, supported by two decades of research on GnT-V, aberrant glycosylation,
and tumour progression, these studies firmly establish a role for ß1,6-branched
oligosaccharides in breast carcinoma metastasis and their prognostic value as
indicators of outcome, notably in primary tumours with no nodal involvement. Thus, ß1,6branched oligosaccharides, the enzymes regulating their synthesis and degradation, and
their associated glycoprotein conjugates present new targets for diagnosis and therapy
of this difficult and highly prevalent cancer.
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In addition it has been reported that β 1-6 branching of Asn-linked oligosaccharides is
directly associated with metastasis [70]
Neoplastic transformation has been associated with a variety of structural changes in
cell surface carbohydrates, most notably increased sialylation and beta 1-6-linked
branching of complex-type asparagine (Asn)-linked oligosaccharides (that is, -GlcNAc
beta 1-6Man alpha 1-6Man beta 1-). However, little is known about the relevant
glycoproteins or how these transformation-related changes in oligosaccharide
biosynthesis may affect the malignant phenotype. It has been reported that a cell surface
glycoprotein, gp 130, is a major target of increased beta 1-6-linked branching [70] and
that the expression of these oligosaccharide structures is directly related to the
metastatic potential of the cells. Glycosylation mutants of a metastatic tumor cell line
were selected that are deficient in both beta 1-6 GlcNAc transferase V activity and
metastatic potential in situ. Moreover, induction of increased beta 1-6 branching in
clones of a nonmetastatic murine mammary carcinoma correlated strongly with
acquisition of metastatic potential. The results presented indicated that increased beta
1-6-linked branching of complex-type oligosaccharides on gp 130 may be an important
feature of tumor progression related to increased metastatic potential.
2.
Advanced Glycation end products and disease
The AGE field sprung from the work of Louis Camille Maillard. The Maillard reaction
begins with the reaction of the carbonyl (aldehyde or ketone) of a reducing sugar to form
a reversible Schiff base with the amino group of a biomolecule The initial product is
called a Schiff base; Schiff bases may undergo intramolecular rearrangements to form
Amadori products. These Amadori products may undergo further rearrangements, such
as dehydration and condensation, to form irreversible end products, called AGEs linked
this pathway to diabetes in human subjects by the observation that a naturally occurring
minor human hemoglobin, HbA1c, is a posttranslational adduct of glucose with the Nterminal valine amino group of the β chain of hemoglobin. Since then, a plethora of AGEs
have been described in human tissues and fluids
The initial Schiff base adducts formed from glucose and lysine and N-terminal amino-acid
residues rearrange to form fructosamine. Fructosamine degradation and the direct
reaction of -oxoaldehydes with protein form many AGEs. Oxidative reactions may be
increased by oxidative stress arising from mitochondrial dysfunction and activation of
NADPH oxidase. Some AGEs are cross-linked, for example, the bis(lysyl)imidazolium
salts may denature proteins and confer resistance to proteolysis. When AGEs are formed
at critical sites in enzymes or proteins, they may be associated with enzyme inactivation.
Cross-linked AGEs, GOLD [glyoxal-derived lysine dimer, 1,3-di(N -lysino imidazolium
salt], MOLD [methylglyoxal-derived lysine dimer, 1,3-di(N -lysino)-4-(methyl-imidazolium
salt], DOLD [3-deoxyglucosone-derived lysine dimer, 1,3-di(N -lysino)-4 (2,3,4trihydroxybutyl)imidazolium salt], and pentosidine may alter protein structure and
function
3.
Glycans In Cancer And Inflammation
Changes in glycosylation are often a hallmark of disease states. For example, cancer
cells frequently display glycans at different levels or with fundamentally different
structures than those observed on normal cells. This phenomenon was first described in
the early 1970s, but the molecular details underlying such transformations were poorly
understood. In the past decade advances in genomics, proteomics and mass
spectrometry have enabled the association of specific glycan structures with disease
states. In some cases, the functional significance of disease-associated changes in
glycosylation has been revealed. This review highlights changes in glycosylation
associated with cancer and chronic inflammation and new therapeutic and diagnostic
strategies that are based on the underlying glycobiology.
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Glycans, which decorate all eukaryotic cell surfaces, undergo changes in structure with
the onset of diseases such as cancer and inflammation. This article highlights some
examples of disease-associated glycans and the possibility of exploiting these glycans
for therapeutic or diagnostic strategies.
Cancer-associated changes in glycosylation include both the under- and overexpression
of naturally-occurring glycans as well as neoexpression of glycans normally restricted to
embryonic tissues. These structures most often arise from changes in the expression
levels of glycosylating enzymes (glycosyltransferases and glycosidases) in cancerous
versus healthy cells.
To dissect the roles of glycans in metastasis and tumour formation, cellular glycans have
been structurally perturbed in a number of ways. The general conclusion of these studies
is that certain glycans seem to play a role in cancer progression.
Given the functional link between aberrant glycosylation and malignancy, therapeutics
that block the formation of cancer-associated glycans might have an effect on tumour
progression. The immune system can be recruited to target cancer cells on the basis of
their altered glycosylation.
Several glycan-based vaccines are presently undergoing clinical evaluation with some
encouraging preliminary results.
Existing diagnostic methods used to monitor tumour-specific glycosylation require
surgical biopsy followed by histological analysis with lectins or monoclonal antibodies.
An interesting future direction in the field is to target aberrant glycosylation with probes
for non-invasive imaging.
Specific carbohydrate epitopes, such as 6-sulpho sialyl Lewis x, initiate leukocyte
homing to sites of chronic inflammation by enabling leukocyte-endothelial cell adhesion
via the leukocyte receptor L-selectin and are specifically expressed at disease sites.
Drugs that block the selectins or the biosynthesis of their glycan ligands are under
investigation in the pharmaceutical industry. In addition, there is an opportunity for the
development of noninvasive diagnostics that might identify sites of chronic inflammation
prior to the presentation of disease symptoms.
4. Role of O-GlcNAc play a in neurodegenerative diseases [71]
There are several lines of evidence that the modification of proteins by cytosolic- and
nuclear-specific O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is closely
related to neuropathologies, particularly Alzheimer's disease. Several neuronal proteins
have been identified as being modified with O-GlcNAc; these proteins could form part of
the inclusion bodies found, for example, in the most frequently observed neurologic
disorder (i.e., Alzheimer's disease; Tau protein and beta-amyloid peptide are the well
known aggregated proteins). O-GlcNAc proteins are also implicated in synaptosomal
transport (e.g., synapsins and clathrin-assembly proteins). Inclusion bodies are partly
characterized by a deficiency in the ubiquitin-proteasome system, avoiding the
degradation of aggregated proteins. From this perspective, it appears interesting that
substrate proteins could be protected against proteasomal degradation by being
covalently modified with single N-acetylglucosamine on serine or threonine, and that the
proteasome itself is modified and regulated by O-GlcNAc (in this case the turnover of
neuronal proteins correlates with extracellular glucose). Interestingly, glucose uptake
and metabolism are impaired in neuronal disorders, and this phenomenon is linked to
increased phosphorylation. In view of the existence of the dynamic interplay between OGlcNAc and phosphorylation, it is tempting to draw a parallel between the use of
glucose, O-GlcNAc glycosylation and phosphorylation. Lastly, the two enzymes
responsible for O-GlcNAc dynamism (i.e., O-GlcNAc transferase and glucosaminidase)
are both enriched in the brain and genes that encode the two enzymes are located in two
regions that are found to be frequently mutated in neurologic disorders. The data
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presented in this review strongly suggest that O-GlcNAc could play an active role in
neurodegenerative diseases.
5. Prion protein glycosylation. [72]
The finding of a unique glycoform ratio of PrPSc that provided a clue to the origin of vCJD
and its links with BSE. How the degree of PrP glycosylation can influence the phenotype
of TSE strains has yet to be determined. One possibility is that TSE strains target regions
of the brain where the neurones express a compatible PrPC glycosylation pattern. This is
supported by electrophoretic analysis, which has identified differences in PrPC
glycosylation in different regions of the brains of Syrian hamstersand mice (and by the
demonstration that the glycosylation pattern of PrPSc changes when a TSE strain
replicates in different regions of the brain or in the lymphoreticular system It was
recently proposed that TSE infection might even be able to perturb host cell
glycosylation patterns and thus encode a strain-specific glycosylation pattern, following
the observation that the glycosylation state of select cell glycoproteins are altered in
infected (ScN2a) vs. uninfected (N2a) cells
6.
Muscular dystrophies caused by abnormal glycosylation.[64]
An increasing number of genes encoding for putative or demonstrated
glycosyltransferases are being associated with muscular dystrophies of variable
severity, ranging from severe congenital onset and associated structural eye and brain
changes, to relatively mild forms with onset into adulthood. Five of these genes (POMT1;
POMGnT1; FXRP; Fukutin; LARGE) encode for proteins involved in the glycosylation of
alpha-dystroglycan and, indeed, abnormal glycosylation of this molecule is a common
finding in all the respective conditions (Walker Warburg syndrome; Muscle-Eye-Brain
disease; congenital muscular dystrophy type 1C and Limb girdle muscular dystrophy
type 21; Fukuyama muscular dystrophy; congenital muscular dystrophy type 1D). A 6th
gene, GNE, responsible for the hereditary form of inclusion body myositis, encodes for a
glycosyltransferase the substrate(s) of which is, however, still unclear. This article
provides an overview of the clinical, biochemical and genetic features of this group of
disorders.
7.
Cancer related changes in O-glycosylation
There is a strong association with tuncated forms of the Type 1 core O-glycans structure
Tn (GalNAc-Ser) or the sialyated form sTn. The sTn antigen is recognised by the mouse
monoclonal antibody CC49, which is being extensively investigated for the development
of radioimmunotherapeutic protocols.
Alternatively, the Tn antigen can be elongated to form one of the eight distinct cores and
terminated with sulphate, sialic acid, fucose, galactose, N-acetylglucosamine, and Nacetylgalactosamine. Depending on the physiologic conditions, the peripheral sugar may
vary and gives rise to tumor-associated antigens, such as Lea (Lewisa), Leb, Lex, Ley,
sLea, sLec, and sLex. The CA 19-9 described previously recognises the sLea and
DUPAN-2 recognises the sLec.
Aberrant expression of MUC3 and MUC4 membrane-associated mucins and sialyl lex
antigen in pancreatic intraepithelial neoplasia has been reported [73, 74]
MUC3 showed a progressive increase in expression in PanINs of increasing dysplasia
and was also highly expressed in ductal adenocarcinoma. In contrast, neoexpression of
MUC4 and sialyl Lex antigen was observed, mainly in PanIN-3 and ductal
adenocarcinoma. In addition, a decrease in the expression of MUC3 and MUC4 was
correlated with the degree of de-differentiation of the tumor. Aberrant expression of
membrane mucins MUC3 and MUC4 and of a mucin-associated carbohydrate tumor
antigen Sialyl Lex in PanINs and adenocarcinoma further supports the progression
model for pancreatic adenocarcinoma.
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8. Glycosylation and HIV infection - Mannose binding lectin (MBL) and HIV
[75]
The envelope protein (gp120/gp41) of HIV-1 is highly glycosylated with about half of the
molecular mass of gp120 consisting of N-linked carbohydrates. While glycosylation of
HIV gp120/gp41 provides a formidable barrier for development of strong antibody
responses to the virus, it also provides a potential site of attack by the innate immune
system through the C-type lectin mannose binding lectin (MBL) (also called mannan
binding lectin or mannan binding protein). A number of studies have clearly shown that
MBL binds to HIV. Binding of MBL to HIV is dependent on the high-mannose glycans on
gp120 while host cell glycans incorporated into virions do not contribute substantially to
this interaction. It is notable that MBL, due to its specificity for the types of glycans that
are abundant on gp120, has been shown to interact with all tested HIV strains. While
direct neutralization of HIV produced in T cell lines by MBL has been reported,
neutralization is relatively low for HIV primary isolates. However, drugs that alter
processing of carbohydrates enhance neutralization of HIV primary isolates by MBL
9. Glycosylation and Influenza virus infection and transmission. Influenza
virus entry and infection require host cell N-linked glycoprotein. [76]
HA is the surface glycoprotein through which virus particles bind to cell surface
receptors containing sialic acid . The HA is synthesized as a polyprotein precursor (HA0)
that is posttranslationally cleaved into two subunits. This cleavage step is necessary for
virus infectivity. Major factors affecting tissue tropism, systemic spread, and
pathogenicity of avian influenza viruses are the amino acids at the cleavage site of HA0
and the distribution of proteases in the host (reviewed in reference 32). The acquisition
of virulence in the field correlates with changes in glycosylation patterns of HA and with
addition of polybasic amino acids at the HA cleavage site. The presence of polybasic
amino acids at the cleavage site of HA is characteristic of HP influenza A viruses of the
H5 and H7 subtypes. This polybasic amino acid region is the target of not only trypsin-like
proteases but also intracellular proteases, such as furin, which enable systemic spread
of the virus and thereby increase its virulence.
Several studies indicate that the NA plays some role in pathogenicity . The NA protein
facilitates the mobility of virions by removing sialic acid residues from the viral HA during
entry and release from cells. Virus particles with low NA activity cannot be efficiently
released from infected cells. A balance in HA and NA activities is crucial: there must be
enough HA activity to facilitate virus binding and enough NA activity to allow release of
virus progeny. Studies showing a link between pathogenicity of influenza viruses and NA
have been done mostly on the A/WSN/33 (H1N1) virus.
10. Human influenza virus recognition of sialyloligosaccharides. [77].
Sialic acids are essential components of cell-surface receptors utilized by influenza
viruses. To evaluate the recognition of asialic sugar parts of the receptor, three
representative strains of human influenza A and B viruses were tested for their binding of
a panel of sialyloligosaccharides. The highest affinity binding carbohydrate determinants
recognized by the viruses in a context of different core structures were Neu5Acα2-3Gal
for the type B virus, Neu5Acα2-6Gal for the H3 subtype virus, and Neu5Acα2-6Ga/β14GlcNAc for the H1 subtype virus. Penultimate to these determinants parts of
sialyloligosaccharides studied either contributed less significantly to the binding affinity,
or interfered with the binding
The specificity of the virus receptors also change on transmission of the virus from birds
to humans. Differences have been shown between influenza virus receptors on target
cells of duck and chicken and receptor specificity of the 1997 H5N1 chicken and human
influenza viruses from Hong Kong. [78].
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Chicken cells contained Neu5Ac alpha(2-6)Gal-terminated receptors recognized by
Sambucus nigra lectin and by human viruses. This finding explains how some recent
H9N2 viruses replicate in chickens despite their human virus-like receptor specificity.
Duck virus bound to gangliosides with short sugar chains that were abundant in duck
intestine. Human and chicken viruses did not bind to these gangliosides and bound more
strongly than duck virus to gangliosides with long sugar chains that were found in
chicken intestinal and monkey lung tissues. Chicken and duck viruses also differed by
their ability to recognize the structure of the third sugar moiety in Sia2-3Gal-terminated
receptors. Chicken viruses preferentially bound to Neu5Ac alpha(2-3)Gal beta(14)GlcNAc-containing synthetic sialylglycopolymer, whereas duck viruses displayed a
higher affinity for Neu5Ac alpha(2-3)Gal beta(1-3)GalNAc-containing polymer. The data
indicate that sialyloligosaccharide receptors in different avian species are not identical
and provide a potential explanation for the differences between the hemagglutinin and
neuraminidase proteins of duck and chicken viruses.
11.
Human-specific regulation of alpha 2-6-linked sialic acids.
[79]
Many microbial pathogens and toxins recognize animal cells via cell surface sialic acids
(Sias) that are alpha 2-3- or alpha 2-8-linked to the underlying glycan chain. Human
influenza A/B viruses are unusual in preferring alpha 2-6-linked Sias, undergoing a switch
from alpha 2-3 linkage preference during adaptation from animals to humans. This
correlates with the expression of alpha 2-6-linked Sias on ciliated human airway
epithelial target cells and of alpha 2-3-linked Sias on secreted soluble airway mucins,
which are unable to inhibit virus binding. Given several known differences in Sia biology
between humans and apes, we asked whether this pattern of airway epithelial Sia
linkages is also human-specific. Indeed, we show that since the last common ancestor
with apes, humans underwent a concerted bidirectional switch in alpha 2-6-linked Sia
expression between airway epithelial cell surfaces and secreted mucins. This can
explain why the chimpanzee appears relatively resistant to experimental infection with
human Influenza viruses. Other tissues showed additional examples of human-specific
increases or decreases in alpha 2-6-linked Sia expression and only one example of a
change specific to certain great apes. Furthermore, while human and great ape
leukocytes both express alpha 2-6-linked Sias, only human erythrocytes have markedly
up-regulated expression. These cell type-specific changes in alpha 2-6-Sia expression
during human evolution represent another example of a human-specific change in Sia
biology. Because the data set involves multiple great apes, we can also conclude that Sia
linkage expression patterns can be conserved during millions of years of evolution within
some vertebrate taxa while undergoing sudden major changes in other closely related
ones.
Receptor determinants of human and animal influenza virus isolates: differences
12.
in receptor specificity of the H3 hemagglutinin based on species of origin.[80]
The binding of influenza virus to erythrocytes and host cells is mediated by the
interaction of the viral hemagglutinin (H) with cell surface receptors containing sialic
acid (SA). The specificity of this interaction for 19 human and animal influenza isolates
was examined using human erythrocytes enzymatically modified to contain cell surface
sialyloligosaccharides with the sequence SA alpha 2,6Gal beta 1,4GlcNAc; SA alpha
2,3Gal beta 1,4(3)GlcNAc; SA alpha 2,3Gal beta 1,3GalNAc; or SA alpha 2,6GalNAc.
Although none of the viruses agglutinated cells containing the SA alpha 2,6GalNAc
linkage, differential agglutination of cells containing the other three sequences revealed
at least three distinct receptor binding types. Several virus isolates exhibited marked
receptor specificity, binding only to cells containing the SA alpha 2,6Gal or the SA alpha
2,3Gal linkage, while others bound equally well to cells containing either linkage.
Moreover, some viruses could distinguish between two oligosaccharide receptor
determinants containing the terminal SA alpha 2,3Gal linkage when present in the SA
alpha 2,3Gal beta 1,4(3)GlcNAc sequence or the SA alpha 2,3Gal beta 1,3GalNAc
sequence binding cells containing only the former. The observed receptor specificities
were not significantly influenced by the viral neuraminidases as shown by the use of the
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potent neuraminidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. Receptor
specificity appeared, to some extent, to be dependent on the species from which the
virus was isolated. In particular, human isolates of the H3 serotype all agglutinated cells
containing the SA alpha 2,6Gal linkage, but not cells bearing the SA alpha 2,3Gal beta
1,3GalNAc sequence. In contrast, antigenically similar (H3) isolates from avian and
equine species preferentially bound erythrocytes containing the SA alpha 2,3Gal linkage.
This is of particular interest in view of the identification of the avian virus H3
hemagglutinin as the progenitor of the H3 hemagglutinin present on the current human
Hong Kong viruses.
Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza
viruses revealed different receptor specificities. [60] Avian viruses preferentially bind to
alpha2-3-linked sialic acids on receptors of intestinal epithelial cells, whereas human
viruses are specific for the alpha2-6 linkage on epithelial cells of the lungs and upper
respiratory tract. To define the receptor preferences of a number of human and avian H1
and H3 viruses, including the 1918 H1N1 pandemic strains, their hemagglutinins were
analyzed using a recently described glycan array. The array, which contains 200
carbohydrates and glycoproteins, not only revealed clear differentiation of receptor
preferences for alpha2-3 and/or alpha2-6 sialic acid linkage, but could also detect fine
differences in HA specificity, such as preferences for fucosylation, sulfation and
sialylation at positions 2 (Gal) and 3 (GlcNAc, GalNAc) of the terminal trisaccharide. For
the two 1918 HA variants, the South Carolina (SC) HA (with Asp190, Asp225) bound
exclusively alpha2-6 receptors, while the New York (NY) variant, which differed only by
one residue (Gly225), had mixed alpha2-6/alpha2-3 specificity, especially for sulfated
oligosaccharides. Only one mutation of the NY variant (Asp190Glu) was sufficient to
revert the HA receptor preference to that of classical avian strains. Thus, the species
barrier, as defined by the receptor specificity preferences of 1918 human viruses
compared to likely avian virus progenitors, can be circumvented by changes at only two
positions in the HA receptor binding site. The glycan array thus provides highly detailed
profiles of influenza receptor specificity that can be used to map the evolution of new
human pathogenic strains, such as the H5N1 avian influenza.
With this powerful glycan microarray technology, it is now possible to map the fine
specificity of emerging influenza viruses and to revisit and complete the analyses on
earlier human, pig and bird isolates. Changes in receptor specificity can now be quickly
monitored and correlated with mutations in the receptor binding site to aid in prediction
of new pandemics or epidemics. In-depth cellular studies will also now be needed to
ascertain the range, levels and distribution of different carbohydrates on lung epithelial
tissue and human airways, especially since HA specificities for sialylated sugars can be
assessed not only for α2-3 or α2-6 linkages, but also can now include preferences for
GalNAc versus GlcNAc at position 3 of sialylated sugars as well as for additional
substituents, such as sulfate, fucose and extra sialic acid moieties.
The structure and receptor specificity of the Hemagglutinin (HA) from an H5N1 Influenza
Virus. [60] has now been studied at 2.9 angstrom resolution, from a highly pathogenic
Vietnamese H5N1 influenza virus. This showed that it is more related to the 1918 and
other human H1 HAs than to a 1997 duck H5 HA. Glycan microarray analysis of this
Viet04 HA reveals an avian alpha2-3 sialic acid receptor binding preference. Introduction
of mutations that can convert H1 serotype HAs to human alpha2-6 receptor specificity
only enhanced or reduced affinity for avian-type receptors. However, mutations that can
convert avian H2 and H3 HAs to human receptor specificity, when inserted onto the
Viet04 H5 HA framework, permitted binding to a natural human alpha2-6 glycan that
suggests a path for this H5N1 virus to gain a foothold in the human population.
Proposed glycan binding site of Hemagglutinin (HA) from an H5N1 Influenza Virus . [60]
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Histidine–rich patches in the 1918 H1 and H5 structures. (A) Tube representation of the
Viet04 monomer with a box surrounding the vestigial esterase domain. This region is
enlarged [box (B)] to show the histidine and lysine–rich patch. Equivalent regions for
avian H5 (A/Duck/Singapore/3/1997; PDB: 1jsm) and the 1918 HA (A/South Carolina/1918;
PDB: 1rd8) are shown in boxes (C) and (D), respectively. Of all the current H5 strains that
have
been
isolated
from
infected
humans,
A/Vietnam/1203/2004,
and
A/Vietnam/1204/2004 are the only two to have a lysine mutation introduced at position 46,
thus making this region even more basic compared to the other H5 sequences. (E )
Electrostatic surface representation of the Viet04 lysine/histidine–rich region with the
internal ionizable HisA295 residue that is accessible through a cavity as indicated by the
green ellipse. Taken from Stevens et al [81]
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Appendix C
Current On-line resources
1. ESSENTIALS OF GLYCOBIOLOGY
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowTOC&rid=glyco.TOC&dep
th=2
2. CAZy - Carbohydrate-Active enzymes
http://www.cazy.org/
3. Japanese glycoforum web-pages
www.glycoforum.gr.jp/
4. Consortium for functional glycomics – USA
www.functionalglycomics.org/static/consortium/
5. Locus-Specific Mutation Databases (for congenital diseases)
http://archive.uwcm.ac.uk/uwcm/mg/docs/oth_mut.html
6. KEGG: Kyoto Encyclopedia of Genes and Genomes
www.genome.jp/kegg/
7. Bacterial Carbohydrate Structure DataBase
www.glyco.ac.ru/bcsdb/start.shtml
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