Glycoproteins
Glycoproteins
Mammalian glycoproteins contain
three major types of oligosaccharides
(glycans):
 N-linked,
 O-linked, and
 glycosylphosphatidyl- inositol (GPI)
lipid anchors.
Glycoproteins
N-Linked glycans are linked to the protein backbone via an
amide bond to asparagine residues in an Asn-X-Ser/Thr
motif, where X can be any amino acid, except Pro.
O-Linked glycans are linked to the hydroxyl group of
serine or threonine.
GPI-anchored proteins are attached at their carboxyterminus through a phosphodiester linkage of
phosphoethanol- amine to a trimannosyl glucosamine core
structure. The reducing end of the latter moiety is bound to
the hydrophobic region of the membrane via a
phosphatidyl- inositol group.
Chemical diversity of glycans
Symbolic Representation of
Oligosaccharides
Symbol and Text Nomenclature for
Representation of Glycan Structure
Nomenclature Committee Consortium for
Functional Glycomics
The Nomenclature committee evaluated symbol
nomenclatures in wide use, consulted with a variety
of interested parties, and selected a version
adapted by the Editors of “Essentials of
Glycobiology” from that originally put forward by
Stuart Kornfeld, and further modified for the
upcoming second edition. The selected version fits
Consortium needs based on the following criteria:
Consortium for Functional Glycomics
The nomenclature must be convenient for the annotation of
mass spectra. To this end, it was decided that:
 Each sugar type (eg sugars of the same mass: hexose,
hexosamine and N-acetylhexosamine), should have the same
shape.
Isomers of each sugar type (eg
mannose/galactose/glucose) should be differentiated by
color or shading.
 Where possible, the same color or shading should be
used for derivatives of hexose (eg the corresponding Nacetylhexosamine and hexosamine).
 Using the same shape but different orientation to represent
different sugars should be avoided so structures can be
represented either horizontally or vertically.
Consortium for Functional Glycomics
The color version of the nomenclature should
appear indistinguishable from the black and
white version when copied or printed in black
and white.
Because 10% of the population is color blind,
the use of both red and green for the same
shaped symbols should be avoided.
If desired, linkage information can be
represented in text next to a line connecting
the symbols (e.g. α4, β4).
Consortium for Functional Glycomics
Text nomenclature
The committee recommends a “modified IUPAC
condensed” text nomenclature which includes the anomeric
carbon but not the parentheses, and which can be written in
either a linear or 2D version. The Committee felt that:
Including the anomeric carbon is important, and is likely
to become increasingly more so in the future as more
complicated structures are discovered.
The presence of parentheses (which then necessitates the
use of brackets to indicate branching structures) is
unnecessarily cumbersome, particularly when representing
the structure in 2D form.
Consortium for Functional Glycomics
Consortium for Functional Glycomics
Examples of symbol nomenclature used to illustrate Nand O-linked glycans written in the 2D version of the
text nomenclature.
Note that symbol structures will be used to annotate
data where linkages have not been defined (e.g MALDI
profiling), and if linkages between monosaccharides
are known, they can be added above or to the side of
the line connecting the symbols if desired (e.g. α6 or
β4).
Consortium for Functional Glycomics
Objections to USA Scheme
The linkage information is only conveyed by the use of
numeric notation .
 This makes the symbols clumsy and when the size is
reduced the numeric notation becomes impossible
to read.
 using the angle and dotting of the lines to represent
linkage information this can be displayed clearly
The symbols and shadings/colours are arbitrary.
A scheme where derivatives of the basic monosaccharide
are filled or shaded is clearer
A scheme where all basic monosaccharides have different
shapes is clearer in print and reduced size
Comparison of Symbols
Simplified Text and Symbolic
Representation
of Glycosaminoglycans (GAGs)
GalNAc4Sb4GlcAb3GalNAc4Sb4IdoAa3GalNAc4Sb4IdoA2Sa3GalNAc4Sb4GlcAb3
Chondroitin/Dermatan Sulfate
EuroCarbDB
GlycanBuilder
Users can select from five graphical display schemes for
glycan structures. As an example structure a complex Nglycan is shown in
The IUPAC Text mode,
the CFG symbolic format with linkage labels,
the CFGL format with linkage positions shown
geometrically,
the Oxford black & white (UOXF)
and color (UFOXCOL) schemes, where linkage positions
are shown by geometry and anomeric configurations are
denoted by dashed (α) or solid (β) lines.
GlycanBuilder
Note that this structure is indefinite since the linkage
positions of the terminal GalNAc residues are not defined.
Glucose
Mannose
Glucuronic acid
Iduronic acid
Galactose
Fucose
N-Acetylglucosamine
N-Acetylneuraminic
acid (Sialic Acid)
Xylose
N-linked Glycoproteins
All eukaryotic cells express N-linked
glycoproteins. Protein glycosylation of N-linked
glycans is actually a co-translational event,
occurring during protein synthesis. N-linked
gly- cosylation requires the consensus
sequence Asn-X-Ser/Thr. Glycosylation occurs
most often when this consensus sequence
occurs in a loop in the peptide.
Basic N-linked Structure
N-linked Glycoproteins
In the Golgi, high mannose N-glycans
can be converted to a variety of
complex and hydrid forms which are
unique to vertebrates.
N-linked Glycoproteins
The diverse assortment of N-linked glycans
are based on the common core
pentasaccharide, Man3GlcNAc2.
Further processing in the Golgi results in
three main classes of N-linked glycan subtypes;
High-mannose,
Hybrid,
and Complex..
High-Mannose Structure
Hybrid Structure
Complex Structure (tetraantennary)
N-linked Glycoproteins
The oligosaccharide precursor is transferred en bloc from dolichol to Asn
residues in the sequence Asn -X-Ser/Thr by oligosaccharyltransferase.
N-linked Glycoproteins
N-linked Glycoproteins
Complex glycans contain the common
triman- nosyl core. Additional
monosaccharides may occur in repeating
lactosamine units. Additional modifications
may include a bisecting GlcNAc at the
mannosyl core and/or a fucosyl residue on
the innermost GlcNAc. Complex glycans
exist in bi-, tri- and tetraantennary forms
Different N-linked glycans structures
α(2,3)
β(1,4)
β(1,2)
α(1,6)
α(1,6)
β(1,4)
β(1,4)
Human
proteins
β(1,4)
β(1,4)
Plant
proteins
α(1,3)
α(2,6)
β(1,4)
β(1,2)
α(1,4)
β(1,2)
α(1,6)
β(1,3)
α(1,3)
α(1,3)
β(1,2)
α(1,4)
β(1,2)
β(1,3)
‘Lewis a’ epitope
GlcNAc
Man
Gal
NeuAC
Xyl
Fuc
Examples of oligosaccharides found in
N-linked glycoproteins
Example oligosaccharides found in
N-linked glycoproteins
Different N-glycosylation in Golgi-complexes
Dolichol
Ribosome
mRNA
Oligosaccharyl
Transferase
a-Glucosidase I
Calnexin
Folding
: Folded peptide chain
a-Glucosidase II
: GlcNAc
Endoplasmic
Reticulum Man GlcNAc
Glc Man GlcNAc
ER a-Mannosidase
3
9
2
8
: Glucose
Transporter
a-1,2-Mannosidase I
a-1,2-Mannosidase I
GnT-I
a-Mannosidase II
⇒ Fuc-Transferase
a-Mannosidase II
GnT-II
UDP-Gal
Transporter
GalT
CMP-NeuAc
synthetase
SAT
a-1,6-Mannosyl
Transferase (OCH1)
N-acetyl-glucosaminyltransferase I (GnT-I)
UDP-GlcNAc
Transporter
F
F
GnT-II
Xyl-Transferase
a-1,3-Mannosyl
Transferase
(MNN1)
Mannose-6Phosphate
Synthesis
(MNN4,6 and others)
a-1,2-Mannosyl
Transferase
(MNN2,5?)
F X N-acetyl-Glucosaminidase
F X
Complex Type
: Phosphoric acid
Yeast
Golgi
n
Complex Type
: Sialic acid
2
Plant Golgi
Mammalian
Golgi
UDP-GlcNAc
: Galactose
: Mannose
Mannan Type
Mammalian N-glycans
O-linked Glycoproteins
 Function in many cases is to adopt an
extended conformation
 These extended conformations resemble
"bristle brushes"
 Bristle brush structure extends functional
domains up from membrane surface
O-linked Glycoproteins
O-Linked glycans are usually attached to the peptide
chain through serine or threonine residues. OLinked glycosylation is a true post-translational
event and does not require a consensus sequence
and no oligosaccharide precursor is required for
protein transfer.
The most common type of O-linked glycans contain
an initial GalNAc residue (or Tn epitope), these are
commonly referred to as mucin-type glycans. Other
O-linked glycans include glucosamine, xylose,
galactose, fucose, or manose as the initial sugar
bound to the Ser/Thr residues.
O-linked Glycoproteins
Glycosylation generally occurs in high-density
clusters and may contribute as much as 50-80%
to the overall mass. O-Linked glycans tend to be
very heterogeneous, hence they are generally
classified by their core structure.
Di- and Trisialated O-Linked Core
O-Linked Core 2 Hexasaccharide
Core 1
Core 2
Core 3
Core 4
Core 5
Core 6
Core 7
Core 8
 Extracellular aggregate of
protein and
glycosaminoglycans
 Core protein
 Oligosaccharide glycosidic
bond to O of Ser or Thr
Proteoglycans
Blood Group Substances
O-Linked glycans are involved in hematopoiesis,
inflammation response mechanisms, and the
formation of ABO blood antigens.
Blood Group Substances
Membranes of animal plasma cells have large
numbers of relatively small carbohydrates bound
to them
 these membrane-bound carbohydrates act as
antigenic determinants
 among the first antigenic determinants
discovered were the blood group substances
 in the ABO system, individuals are classified
according to four blood types: A, B, AB, and O
 at the cellular level, the biochemical basis for
this classification is a group of relatively small
membrane-bound carbohydrates
Type A Blood
Type B Blood
Type O Blood
These glycoproteins are found in
The blood of Arctic and Antarctic
fish enabling these to live at subzero water temperatures
GPIAnchor
Glycosylphosphatidylinisotol (GPI) anchored
proteins are membrane bound proteins found
throughout the animal kingdom. GPI anchored
proteins are linked at their carboxy- terminus
through a phosphodiester linkage of phosphoethanolamine to a trimannosyl-non-acetylated
glucosamine (Man3-GlcN) core. The reducing end
of GlcN is linked to phosphatidylinositol (PI). PIis
then anchored through another phosphodiester
linkage to the cell membrane through its
hydrophobic region.
GPIAnchor
Classification of human diseases
known to be related to glycans
 Infectious disease
Bacterial and viral infection
Parasite infection
 Genetic disorders
Glycan synthesis/degradation related
Glycosylation related
 Acquired diseases
 Cancer
Altered glycosylation in cancer
 Increased β1-6GlcNAc branching of N-glycans
 Changes in the amount, linkage, and acetylation
of sialic acids
 Truncation of O-glycans, leading to expression
of Tn and sialyl Tn antigens
 Expression of the nonhuman sialic acid Nglycolylneuraminic acid, likely incorporated
from dietary sources
 Expression of sialylated Lewis structures and
selectin ligands;
Altered glycosylation in cancer
Altered expression and enhanced shedding
of glycosphingolipids
Increased expression of galectins and
poly-N-acetyllactosamines
Altered expression of ABH(O) blood-grouprelated structures
Alterations in sulfation of glycosaminoglycans
Increased expression of hyaluronan
Loss of expression of GPI lipid anchors
TASK
For these 36 PDB entries
3gwj
2dw2
1igt
2vuo
1p8j
2c4l
1h3y
1t89
3hn3
2c4a
1gai
1t83
2b8h
1zag
1gah
1i1a
1mco
1yy9
1e4k
1h3x
1h4p
1xc6
1bhg
1h3v
3og2
1tg7
3ogr
1glm
2rgs
1mwe
3h32
1cvi
2j6e
1igy
3gly
1ckl
Glycan sizes from 12 to 8 sugar residues
12
11
10
9
3gwj
1p8j
3hn3
3og2
2c4l
1igy
1e4k
8 3ogr
8 1h4p
(x6)
(x2)
3hn3
2c4a
(x2)
1bhg
3h32
1h3x
2b8h
2rgs
2b8h
1igt
(x2)
3gly
(x2)
1mco
(x2)
(x3)
(x2)
1h4p
2rgs 2j6e (x2) 2dw2 (x2)
1zag 1yy9 1xc6 1tg7 1mwe
1h3y (x2) 1gai 1gah 1e4k (x2)
2vuo 1t89 (x2) 1t83 (x2) 1i1a (x2)
1h3v 1glm 1cvi 1ckl (x2)
TASK
Run getsite with options –o –g 8
e.g. o/p for 3gwj
view the *.xyz files rasmol 3gwj_ASN_A_196.xyz
and check if files match one of the known N-glycan
cores
3gwj_ASN_A_196.xyz
3gwj_ASN_B_196.xyz
3gwj_ASN_C_196.xyz
3gwj_ASN_D_196.xyz
3gwj_ASN_E_196.xyz
3gwj_ASN_F_196.xyz
3gwj_site.cif
contacts.3gwj
rem800.3gwj
site.3gwj