Molecular Biochemistry II

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Molecular Biochemistry II
Lectures 41-44
Glycoconjugates
Dr G BantingA100 Medical Schoolg.banting_at_bristol
.ac.uk
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The following articles should also be of
use/interest Trends in Cell Biology Trends in
Biochemical Science Volume 7, pages 193-200 ,
May 1997 Volume 23, pages 321-324,
Sept.1998 Calnexin, calreticulin and the folding
of Evolving views of protein glycosylation glyco
proteins Drickamer, K. and Taylor,
M.E. Helenius, A., Trombetta, E.S., Hebert, D.N.
and Simons, J.F. Why Mammalian Cell
Surface Proteins are Glycoproteins Gahmberg and
Tolvanen TIBS 21 308-311 1996 Sorting sweet
sorting Ponnambalam and Banting Current Biology 6
1076-1078 1996 The role of N-glycans in the
secretory pathway Fiedler and Simons Cell 81
309-312 1995
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D-Glucose
D-Fructose
(aldose)
(ketose)
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L-Glucose
L-Mannose
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Alcohol
Aldehyde
Hemiacetal
Alcohol
Ketone
Hemiketal
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?-D Glucopyranose (Hawthorn Projection)
D-Glucose
Pyran
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D-Fructose
Furan
?-D-Fructoranose (Hawthorn Projection)
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Equitorial Axial
Chair Form of ?-D-Glucopyranose
Boat Form of ?-D-Glucopyranose
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Methyl-?-D -Glucoside
?-D-Glucose
Methyl-?-D -Glucoside
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Glucose
Glucose
H Bonds
Cellulose
n
Chain
Cellulose
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?-Amylose
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N Glycosidic
bond
Adenosine
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Common Disaccharides
Sucrose glucose ?(1?2) fructose
Lactose galactose ?(1?4) glucose
Maltose glucose ?(1?4) glucose
Cellobiose glucose ?(1?4) glucose
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N-Acetylmannosamine
?
N-Acetylneuraminic Acid

Pyruvic Acid
N-Acetylneuraminic Acid
(An example of a Sialic acid)
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Methylation Analysis ? Monsacchanide Linkages

• Methyl ethers not at the anomeric C atom are
  resistant to acid hydrolysis but glycosidic
  bonds are not.
• If an oligosaccharide is exhaustively methylated
  and then hydrolysed, the free OH groups on the
  resulting methylated monnosaccharides mark the
  former positions of the glycosidic bonds.
• Methylated monnosaccharides are identified by
  gas-liquid chromotography and mass spectroscopy.

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UTP
Inorganic pyrophosphatase
G1P
UDP-Glucose
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Overall Reaction for the Formation of UDPG
O
?G (kJ.mol )
-1
- O
G1P UTP ? UDPG PPi
- 33.5
H O PPi ? 2Pi
2
G1P UTP ? UDPG 2Pi
- 33.5
Overall
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Donor Sugar
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UDP-Galactose
UDP-Glucose
UDP-Galactose -4-Epimerase
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N-Linked
Asparagine
(N)
NB
GlcNac
X not Proline
O-Linked
R H or CH?
Linked to OH by either Serine/Threonine
GalNac
(S)
(T)
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The branching of
glycogen.Branches are formed by




transferring a seven- residue terminal segment
from



an ?(1?4)-linked
glucan chain to the



C(6)-OH
group of a glucose residue




on the same or another chain.
(1?4) - terminal chains of glycogen
Transferred segment 7 residues
long from chain at least 11 residues long
Branching Enzyme
amylo(1,4?1,6) - transglycosylase
Branch points at least four residues apart
? 1,6 link
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Sugar Nucleotides and Their Corresponding
Monosaccharides In Glycosyl Transferase Reactions
UDP
GDP
CMP
Gal Nac
Fucose
Sialic Acid
Mannose
Glc Nac
Galactose
Glucose
Nucleotide Sugars Are Of Vital Importance In
Oligosaccharide Synthesis
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Distribution Of O-GlcNac Modified Proteins
Nucleus
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Cytosol
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Microsomes
6
Golgi
3
Plasma Membrane
2
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Synthesis Of N Linked Glycoproteins
1) Synthesis of lipid-linked oligosaccharide
precurser.
2) Transfer of precurser to NH? group of Asn on
nascent


polypeptide.
3) Trimming of precurser.
4) Addition of further residues to core
oligosaccharide.
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Isoprene unit
Saturated ?-isoprene




unit
14 - 24
n
Dolichol
The carbohydrate precursers of N-linked
glycosides are sythesised as dolichol phosphate
glycosides.
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Pathway of Dolichol-PP-Oligosaccharide Synthesis
Endoplasmic Reticulum
Lumen
GlcNAc
Mannose
Glucose
dolichol
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Inhibitors
Rough Endoplasmic Reticulum
Nojirimycin
deoxy nojirimycin
Castanospermin
Bromoconduritol
glucosidase I
glucosidase II
ER mannosidase
Processing the same for most glycoproteins up to
this stage.
Cis Golgi
golgi mannosidase
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L Fucose
medial Golgi
GlcNac transferase II
GlcNac transferase 1
Golgi mannosidase II
Fucose transferase
Swainsonine acts here.
trans Golgi
Sialyl transferase
Gal transferase
Sialic acid
Galactose
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Human Immunoglobulin M (IgM)
Human Transferrin
N- acetylneuramic acid (Neu)
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Glycosyl transferases
add other
monnosaccharides.
GalNAc Transferase
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Paulson TIBS 14 p272 1989
(a) N-Linked
I
II
III
(b) O-Linked
IV
VI
VI
VII
VIII
Examples of N-linked (a) and (b) sugar chains of
glycoproteins. Structures were selected to
contrast the two classes of sugar chains, which
show difference in the core sequences, and
similarities in the peripheral sequences.
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N-glycosylation and glucose trimming in the
endplasmic reticulum (ER).
Glucose
Mannose
N-acetylglucosamine
Glucosyl-transferase
Glucosidase I
Glucosidase II
Glucosidase II
The core oligosaccharide (Glc3Man9GlcNAc2) is
synthesized in the ER membrane and transferred
to the consensus sequence N-glycosylation sites
(Asn-X-Ser/Thr) on nascent polypeptide chains.
Immediately after transfer, the glucose
residues are removed by the sequential action of
glucosidases I and II. Glucosidase I removes the
terminal ?1-2-linked glucose, whereas glucosidase
II removes the two remaining ?1-3-linked
residues. Fully glucose-trimmed high- mannose
glycans can be re-glucosylated by
UDP-glucoseglycoprotein glucosyltransferase if
present on incompletely folded or assembled
proteins.
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Incompletely
folded
Folded
Transferase
Glucosidase II
Glucosidase II
Glucosidases I and II
CNX/CRT
CHO-protein
interaction
Model for calnexin and calreticulin binding to
their substrate glycoproteins. Glucose residues
(G) in the core oligosaccharides are trimmed by
glucosidases I and II. When trimmed to the
monoglucosylated form, the oligosaccharides
mediate the binding of the glycoprotein to
calnexin (CNX) and calreticulin (CRT). These
function as lectins. Release of the substrate
glycoprotein depends on hydrolysis of the
remaining glucose residues by glucosidase II. If
the glycoproteins are not completely folded, the
high-mannose glycans are selectively
re-glycosylated by the glucosyltransferase,
allowing the oligosaccharide to rebind to the
chaperones. Once glycoproteins reach their mature
conformation, they are no longer recognised by
the glucosyltransferase.
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Incompletely
folded
Folded
Glucosidases I and II

_
Transferase
CNX
CNX
ATP?
CHO-protein
Protein-protein
interaction
interaction
(weak)
(strong)
Dual-mode model. According to this model, the
monoglucosylated oligosaccharides are merely
needed to bring glycoproteins into contact with
calnexin (CHO-protein interaction). After the
initial carbohydrate-mediated association,
stronger protein-protein interactions occur
between the chaperone and peptide elements
exposed on the surface of the incompletely folded
substrate protein.These stabilise the complex
until the peptides are hidden in the folded
protein. Thus, release of the sustrate depends on
conformational changes in the substrate
glycoprotein that eliminate the protein-protein
interaction. It may also involve ATP-mediated
conformational changes in calnexin.
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Comparison of the Chemical Structures of (a)
tunimycin and (b)
dolichol-P UDP-N-acetylglucosamine.
(a) Tunimycin
(b)
Dolichol phosphate
UDP-N-Acetylglucosamine
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Bacitracin
Chemical structure of bacitracin. Note that this
dodecapeptide has four amino acid residues and
two unusual intachain linkages. Orn represents
the nonstandard amino acid residue ornithine.
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Glucuronic Acid
Sulphated N-acetylglucosamine
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The Glycosaminoglycans

Sulfates per Disaccha-ride Unit
Other Sugar Compo-nents
Linked to Protein
Tissue Distribution
Repeating Disaccharide (A-B)n
Glycosamino-glycan
Molecular Weight
Group
Monosaccharide
Monosaccharide
A
B
Various
connective
tissues,
skin,
N-acetyl- D-glucosamine
vitreous
4,000 to 8 x 106
D-glucuronic acid
0
Hyaluronic acid
1
0
body,
cartilage,
synovial fluid
cartilage,
N-acetyl-
D-galctosamine

D-galactose
5,000 - 50,000
D-glucuronic acid
0.2 - 2.3
Chondroitin sulfate
cornea.
2
D-xylose
bone, skin
arteries
Skin,blood
15,000- 10,000
D-glucuronic acid

D-galactose
Dermatan sulfate
N-acetyl-
D-galctosamine
1.0-2.0
vessels,
or
D-xylose
heart, heart
L-iduronic acid
valves
lung,arteries,
5,000-25,000
D-glucuronic acid
N-acetyl- D-glucosamine

D-galactose
Heparin sulfate
0.2-2.0
3
or
cell surfaces,
D-xylose
L-iduronic acid
basal laminae
6,000-25,000
D-glucuronic acid
N-acetyl- D-glucosamine
2.0-3.0

D-galactose
lung,liver,
Heparin
or
skin,
D-xylose
L-iduronic acid
mast cells
D-galactos-amine
cartilage,
4,000-19,000

Keratin sulfate
N-acetyl- D-glucosamine
D-galactose
0.9-1.8
cornea,
4
D-mannose
invertebral
L-fucose
disc
sialic acid
L-iduronic acid is produced by the epimerization
of D-glucuronic acid at the position where the
carbonly group is located. Thus dermatan sulfate
is a modified form of chondroitin sulfate, and
the two types of repeating disaccharide sequences
usually occur as alternating segments in the
same glycosaminoglycan
chain.
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???
???
Link Trisaccharide
Glycosaminoglycan
GAG
Core Protein
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Proteoglycans
Glycoproteins
Carbohydrate Composition




up to 97
Carbohydrate Composistion 1- 60
Short, branched, N and O-linked carbohydrate
chains lt 20 residues
1
100s of GAG chains
GAG chains of 80 sugars
Mixture of sugars
No sialic acid
Often terminal sialic acid
3
Up to 3 x 10 Kd
Up to 2 - 300 Kd
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Proteoglycan Aggregate From Cartilage
Hyaluronic Acid
Mix of keratin sulphate and chondroitin sulphate
Link Protein
Core Protein
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N-Acetylglucosamine (NAG)
N-Acetylmuramic acid (NAM)
L-Ala
Isoglutamate
L-Lys
D-Ala
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A segment of a teichoic acid with a glycerol
phoshphate backbone that bears alternating
residues of D-Ala and NAG
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?
Globular Domain(s)
?
?
Glycocalyx (Up to 100A)
lots of
poly-N-acetlyllacosamine on
erthrocytes
O-linked sugar region ? extended conformation of
protein backbone
Plasma Membrane
?
Cytoplasmic Tail
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Structures of the A, B, and H Antigenic
Determinants in Erythrocytes
Antigen
Type
Gal?(1 ? 4)GlcNAc
H
?
1,2
L-Fuc ?
A
GalNAc ?(1 ? 3) Gal?(1 ? 4)GlcNAc
?
1,2
L-Fuc ?
B
Gal?(1 ? 3) Gal?(1 ? 4)GlcNAc
?
1,2
L-Fuc ?