Transcript glyco revision 2004

Functional Glycobiology
Course Outline
• Structure of Glycoconjugates
• sugar residues, linkage, sequence, conformation
• types of glycoconjugate, protein and lipid
• Biosynthesis
• N- and O-linked Glycosylation, GP Is, PGs, GSLs
• molecular biology of enzymes
• Determination of Structure
• sugar sequencing, HPLC, MS, NMR
• endo- and exo-glycosidases
• Function of Glycoconjugates
• glycoproteins and glycolipids in biology
• metabolic diseases
Functional Glycobiology
Revision Topics
Function of Glycoconjugates
• Structure of Glycoconjugates
• Determination of Structure
• Biosynthesis
Carbohydrate/protein interactions
• Intracellular functions - protein folding pathways
• Extracellular functions - leukocyte surveillance
Lessons from Gene Manipulation or Mutations
N-Linked Glycosylation
• GPT KO in mouse
• GlcNAcT KO in mouse
• CDG
GSLs
• GlcCer, NeuAc & GalNAc transferase KO in mouse
• lysosomal storage disorders
PGs
• Inhibition of CS synthesis using RNAi
• Hyaluronan synthase disruption
GPIs
• Paroxysmal nocturnal haemoglobinurea
Mutations in Early GPI-anchor Biosynthesis
• Mutations in the enzyme leads to a loss of all GPI-anchored proteins
• Paroxysmal nocturnal haemoglobinurea
• Intravascular haemolysis, cytopenia and thrombosis
• Loss of GPI-anchored complement inactivating proteins in blood
UDP-GlcNAc
UDP
GPI-anchored proteins
P
GlcNAc
P
Takeda & Kinoshita (1995), TIBS 20:367
Early Embryogenesis
in C.elegans
Inhibition of chondroitin
sulphate (CS) synthesis by
RNAi
• inhibition of cell division
• inhibition of cytokinesis
Sugahara et al (2003)
COSB 13:612
Targeted Disruption of GSL-Specific Glycosyltransferases in Mice
Gene
Major GSL
Phenotype
CGT
GM3S
GalNAcT
none
LacCer
GM3+GD3
Lethal at E7.5. Ectodermal apoptosis
Viable. Enhanced insulin sensitivity
Viable. Impaired nerve conductance.
Wallerian degeneration and age related
motor function defects
GD3S
GM1+GD1a Viable. Impaired nerve regeneration
GalNAcT
and GD3S
GM3
Viable. Peripheral nerve degeneration.
Spontaneous adult lethal phenotype and
sensitivity to audiogenic seizures
Effects of GPT Deletion on Pre-implantation Embryos
Tunicamycin
P-Dol
GPT
PP-Dol
GlcNAc-PP-Dol
Glc3Man9(GlcNAc) 2 -PP-Dol
(GlcNAc) 2 -PP-Dol
Man9(GlcNAc) 2 -PP-Dol
oligosaccharide
transfer
Glc3Man9(GlcNAc) 2
Asn
GlcNAc-Transferase I KO in Mouse is Embryonically Lethal
n
Mannosidase
n
mannosidase
n
n
GlcNAcTI
n
mature glycoproteins
n
n
Protein N-Glycosylation - why is it so critical?
• Disruption of early events in lipid cycle (complete absence of N-glycans)
ablates early differentiation processes.
• Disruption of later events (lack of complex N-glycans) is embryonically lethal
Points to early events in the ER as having important role
Oligosaccharide sequences are recognised by ER resident
chaperones calnexin and calreticulin
Quality Control in the Protein Folding Pathway
• ER co-translational addition of N-linked oligosaccharides
• Trimming with a-glucosidases
• Recognition of monoglucosylated glycans by chaperones
calnexin (membrane bound)
careticulin (soluble)
• Disulphide bond formation and folding
• Glucosyltransferase ‘senses’ folded state
• Re-glucosylation and chaperone binding
• Elimination from the ER or maturation via Golgi
ERp57
(disulphide isomerase)
protein folding
on/off calnexin
Golgi
cx
G II
a-glucosidase II
P
ERGIC-53
EDEM
P
ER
glucosyltransferase
other
chaperones
Glc
Glc
Glc
Man
P
mannosidase
P
EDEM
a-glucosidase
I & II
Man
P
Sec61
proteasome
Calnexin - a protein with multifunctional motifs
The Glc1Man9GlcNAc2 Oligosaccharide Ligand for Calnexin
a-Glucosidase II
binding
Calnexin
binding
Glycoprotein Biosynthesis
n
ER Gl c' a se I
n



DNJ
an alogu es
n
n
ER Gl c' a se II
n


ER Glc' ase I I
n
DNJ
an alogues

n
n
ER Mann' as e
n
n
DNJ
an alogues
Golgi
Mann' a se I
Golgi e ndom ann' a se
Golg i
Ma nn' a se I
n
n
n
n
N- Ac e t y lgluc osam i nyl
t ra nsf e ra se I
Golgi
Ma nn' a se II
m a t ur e
gly copr ot ei ns
n
n
n
sw ain sonine
n
n
n
MHC Class I Requires Folding Chaperones
MHC Class I Molecule
peptide
NB-DNJ
NN-DNJ
protein folding
on/off calnexin
cx
G II
a-glucosidase II
P
P
ER
P
other
chaperones
glucosyltransferase
Glc
Glc
Glc
Man
P
a-glucosidase
I & II
NB-DNJ
NN-DNJ
Man
P
Golgi
Therapeutic Opportunities
Inhibitors of Processing Glucosidases as Antivirals
• N-alkylated imino sugars are micromolar inhibitors
• Virus encoded glycoproteins synthesised by host
• Prevention of calnexin-mediated pathway creates
misfolded proteins
• Aberrant virus envelope assembly leads to non-infectious
particles
• No possibility of ‘escape’ mutations
• In vitro efficacy for HIV and Hepatitis B
OH
HO
OH
N
CH2OH
CH3
Lipids are presented to T cells by CD1 Molecules
Zeng et al.,(1997) Science 277, 339
CD1 Molecules bind Lipid Ligands
CD1b
CD1b,d
CD1c
CD1d
Green = Apolar; Red = Polar
CD1 Pathway of Lipid Presentation
bacteria
CD8
CD4
DN
?
IL-4, IFNg
Perforin
FAS
Granzyme B
MR
GOLGI
ER
LE (pH 4.0)
nascent CD1
The ligand binding groove of Human CD1b
A’channel
Phe144
VAL12
T’tunnel
F’channel
VAL12
C’channel
A’channel
Gadola et al., (2002)
Nat Immunol 3, 721
Extracellular Protein - Carbohydrate Interactions
Transmigration of lymphocytes from lymph to blood
HEV cells in lymph nodes
Allows antigen-specific B and T cells to survey all
possible sites of pathogen entry
Leukocyte response to venous endothelial damage
mechanical or chemical injury
pathogen induced inflammation
GROUPS WITHIN THE C-TYPE LECTIN FAMILY
Carbohydrate Ligands for Selectins
sialyl Lewis x
NeuAca2,3Galß1,4GlcNAcß1-R
Granulocytes
Monocytes
Lymphocytes
P-selectin
E-selectin
Lymph node HE Vs
Peyer’s patch HEVs
NK cells
L-selectin
P-selectin
E-selectin
Fuca1,3
6-sulphated
sialyl Lewis x
SO3-
NeuAca2,3Galß1,4GlcNAcß1-R
Fuca1,3
E-Selectin binding to Sialyl Lewis X Oligosaccharide
Cell Attachment and Rolling is Carbohydrate mediated
• Requires expression of correct oligosaccharide
• Terminal carbohydrate sequence found on protein and lipid
• Weak monomeric affinity but fast Kon- and Koff-rates
• Multimeric interactions increase affinity
• Bonds have a low fractional spring slippage, which means
that as the bond is subjected to strain it has a low tendency to break.
Flexible Ligands, Flexible Friends
Endothelial cell expression of selectins is spatially and temporally restricted
Therapeutic Opportunities
Carbohydrate based drugs to control inflammation
sLeX for P-selectin-dependent acute lung injury (ARDS)
carbohydrate based dendrimers (increase valency)
peptidemimetics
Glycosylation and Disease
N-linked Glycosylation Biosynthesis
Congenital Disorders of Glycosylation - 8 diseases
rare (<500 cases) deficiencies in enzymes or proteins in ER/Golgi pathway
multi-organ involvement, psychomotor retardation
few therapies
Glycolipid biosynthesis
GM3 synthase deficiency (Simpson et al., 2004, Nat Gen 36, 1225)
Cancer and Metastasis
Changes in N-linked glycosylation and GSL expression
secondary effects?
glycosylation inhibitors reduce metastasis in some forms of disease
Lysosomal Storage Disorders
deficiencies in catabolic enzymes
storage of GSL and glycoprotein
relatively rare (1:18000 live births ww)
disease can be severe (infantile death)
few therapies
Further Reading/Sources
Control of protein folding
Schrag et al., (2003) TIBS 28, 49
Oda et al., (2003) Science 299, 1394
Molinari et al., (2003) Science 299, 1397
Selectins
http://hsc.virginia.edu/medicine/basic-sci/biomed/ley/index.html
http://lewis.sfsu.edu/glyco/Lsel.html
CDG
Grunewald et al., (2002) Pediatric Res 52, 618
Lysosomal Storage Diseases
Butters et al., (2000) Chem Rev 100, 4683
Web-based resources
Schwartz, Nancy B (December 1998 ) Proteoglycan. In:
Nature Encyclopedia of Life Sciences. London: Nature Publishing Group. http://www.els.net/
Sen–itiroh Hakomori and Ineo Ishizuka (September 2001 ) Glycolipids: Animal. In:
Nature Encyclopedia of Life Sciences. London: Nature Publishing Group. http://www.els.net/
Verbert, André ; Cacan, René and (July 2000 ) Cell Surface Glycoconjugates. In:
Nature Encyclopedia of Life Sciences. London: Nature Publishing Group. http://www.els.net/
Butters, Terry D (March 2001 ) Glycoproteins. In:
Nature Encyclopedia of Life Sciences. London: Nature Publishing Group. http://www.els.net/