Review of Projections Synthesis and Structure of Glycolipidsglycobiology/lecture011607.pdf · GPI...
Transcript of Review of Projections Synthesis and Structure of Glycolipidsglycobiology/lecture011607.pdf · GPI...
Synthesis and Structure of Glycolipids
1/16/07
Review of Projections
HO
CHO
H
HHO
OHH
OHH
CH2OH
HO
O
H
H
HO
OH
OH
HH
H
OH
HO
OH
H
OH
OH
HH
OH
CH2OH
H
1
2
3
4
5
6
1
23
4
5
6
12
3
4
5
6
Fischer Hayworth Stereochemical
1. What monosaccharide is this?
2. Which chiral center defines the stereochemistry?
3. How are axial and equitorial positions related to up/down in Haworth projections?
Fischer Haworth Stereochemical
1. Where is the aglycone?
2. Where is the “reducing” end?
3. How many "-glycosidic linkages?
4. How many !-glycosidic linkages?
5. How many 3-substituted residues?
6. How many 6-substituted residues?
1. How many non-reducing termini?
2. How many glycosidic bonds?
3. Other than the NANA (N-acetylneuraminic acid, a type of sialic acid) residues,how many are !-glycosides?
About 20 years ago, bovine brain GT1b retailed for $300/mg (Matreya Lipids). A graduatestudent purified 75 mg during a single 3-4 month lab rotation (street value = $22,500).
GlycoEconomics lesson #1.
From 2007 Matreya Lipids Catalog:
Bovine Brain GQ1b retailed for $500/10 µg at that time. Same purification yielded
approximately 10 mg pure GQ1b (street value = $500,000).
Graduate student stipend was approximately $12,000/year (or $4,000/rotation). Studentvalue multiplier = 130-fold. Tell your PI how valuable you can be!!!
!Large O-linked
Glycosaminoglycans and poly-
lactosamine structures
!Glycoprotein N-linked and O-
linked oligosaccharides
!Glycolipid oligosaccharides
The "Primary" Sialic Acids
H-C-OH
H-C-OH
H-C-OH
C OH-C-C-N
H
H
C C
C C
H
OH
H
OH
1
2
4 3
6
5
C-O
9
8
7
O
H O H
H
H
-C C
H-C-OH
H-C-OH
H-C-OH
C OHO
H
H
H
C C
H
OH
H
OH
1
2
4 3
6
5
C-O
9
8
7
O
-
!-D-KDN!-D-Neu5Ac
Thought to be the metabolic precursors of all other sialic acids
N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-
D-glycero-D-galacto-nonulosonic acid)
“Neu5Ac” “NANA”, “NeuAc”
KDN (2-keto-3-deoxy-D-glycero-
D-galacto-nonulosonic acid)
After Varki, A
Phylogenetic
relationships
of enzymes
involved
in the
metabolism
of sialic
acids
HOMOLOGYHOMOLOGY
Neu5Ac/bacteria
GlcNAc-6-phosphate
ManNAc
Neu5Ac
CMP-Neu5Ac
ManNAc-6 -phosphate
Pi
PEP
Pi
CTP
PPi
PhosphatasePhosphatase
GlcNAcGlcNAc-6-phosphate-6-phosphate2-2-epimeraseepimerase
Neu5AcNeu5Acsynthetasesynthetase
CMP-Neu5AcCMP-Neu5Acsynthetasesynthetase
Neu5Ac/Vertebrates
UDP-GlcNAc
ManNAc
Neu5Ac-9-phosphate
Neu5Ac
CMP-Neu5Ac
ATP
ADP
UDP
PEP
Pi
Pi
CTP
PPi
ManNAc-6 -phosphate
ManNAcManNAc kinase kinase
UDP-UDP-GlcNAcGlcNAc2-2-epimeraseepimerase
Neu5Ac-9-Neu5Ac-9-phosphatephosphatesynthetasesynthetase
CMP-Neu5AcCMP-Neu5Acsynthetasesynthetase
Neu5Ac-9-phosphateNeu5Ac-9-phosphatephosphatasephosphatase
Angata and VarkiChemical Reviews
102,439-469, 2002.
After Varki, A
THE SIALIC ACIDS
R2
R2
R5
R5N-acetyl
Hydrogen or:!-linkage to: -Gal (-3 -4 -6) or -GalNAc (-6) or -GlcNAc(-4 -6) or -Sialic Acid (-8 -9) or"-linkage to CMP orAbsent in: - 2,3dehydro or - 2,7anhydro (double-bond when R2 absent)
R5
R5
R5
N-glycolyl
Hydroxyl
Amino
*
-at physio-logical pH,ionized or lactonized
R111
22
33
44
55
66
77
88
99
*
CARBON OXYGEN HYDROGEN
R1
= hydrogen or:
R8
R9
R7
R4
R8R9 R7 R4
Acetyl
R8R9 R7 R4
Phosphate R9
Methyl
R8
Sulfate R8
LactylR9
After Varki, A
Two Major Kinds of Sialic Acids
in Mammalian Cells
N-ACETYLNEURAMINIC ACID
(Neu5Ac)88
77
1122
33
55
66
99
LINKAGE TOUNDERLYING
SUGAR CHAIN
44
N-GLYCOLYLNEURAMINIC ACID
(Neu5Gc)
8877
11
44
22
33
55
66
99
LINKAGE TOUNDERLYING
SUGAR CHAIN
After Varki, A
!Large O-linked
Glycosaminoglycans and poly-
lactosamine structures
!Glycoprotein N-linked and O-
linked oligosaccharides
!Glycolipid oligosaccharides
Glycan synthesis
in a cellular context
Overview
From ER
through
Trans-Golgi and
points
inbetween
On and into the ER
Dolichol-P-X
Glycosyl phosphatidylinositol (GPI)
Glycosphingolipids (GSL)
Schenk, B. et al. Glycobiology 2001 11:61R-70R; doi:10.1093/glycob/11.5.61R
Topological model for the enzymatic reactions leading to Dol-P biosynthesis denovo on the cytoplasmic face of the ER
Biosynthesis of N-Glycans:
Production of GlcNAc-P-P-Dolichol
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAcMan
GalSia
Fuc
Glc
Dolichol
TunicamycinBlocks - notvery specific!
Schenk, B. et al. Glycobiology 2001 11:61R-70R; doi:10.1093/glycob/11.5.61R
Topological model for lipid intermediate synthesis, translocation and the role forDol-P-P/Dol-P phosphatases in the recycling of Dol-P-P/Dol-P in the ER
ER glycolipid synthesis
Dolichol-P-X
Glycosyl phosphatidylinositol (GPI)
Basic Glycosylphosphatidylinositol (GPI) Anchor
Phospholipid
After Hart, G
Examples of GPI-Anchored Proteins
Cell surface hydrolases
alkaline phosphatase
acetylcholinesterase
5! nucleotidase
Adhesion molecules
neural cell adhesion molecule
heparan sulfate proteoglycan
Others
decay accelerating factor
scrapie prion protein
folate receptor
Protozoal antigens
trypanosome VSG
leishmanial protease
plasmodium antigens
Mammalian antigens
Thy-1
carcinoembryonic antigen
After Hart, G
Structure of the Basic GPI Anchor
E t nP
INOSITOL
P
= Mannose (Man)
= Glucosamine
Etn = Ethanolamine
P = Phosphate
N H2
N H2Pig-A
PNH Defect
GPI-Linked Protein
Cell SurfaceMembrane
After Hart, G
Structural AnalysisStructural Analysis
of the GPI Anchorof the GPI Anchor
Enzymatic and chemical
cleavage sites
are useful in identifying
GPI anchored
membrane proteins
After Hart, G
After Freeze, H
Biosynthesis
of GPI
anchors
• The first step in biosynthesis of the GPIanchor requires at least four genes
• One of them, PIG-A is an X-linked gene
Mutation in PNH
MUTATIONS INDOL-P-MAN SYNAND USE
After Freeze, H
Paroxysmal Nocturnal Hemoglobinuria
"A hematopoietic stem cell disorder characterized by
intravascular hemolytic anemia. Abnormal blood
cells lack GPI-anchored proteins due to a mutation
in the PIG-A gene.
"Lack of GPI-anchored complement regulatory
proteins, such as decay-accelerating factor (DAF)
and CD59, results in complement-mediated
hemolysis and hemoglobinuria.
Examples of C-Terminal Sequences Signaling
the Addition of GPI-Anchors
Protein GPI-Signal Sequence
Acetylcholinesterase (Torpedo) NQFLPKLLNATAC DGELSSSGTSSSKGIIFYVLFSILYLIFY
Alkaline Phosphatase (placenta) TACDLAPPAGTTD AAHPGRSVVPALLPLLAGTLLLLETATAP
Decay Accelerating Factor HETTPNKGSGTTS GTTRLLSGHTCFTLTGLLGTLVTMGLLT
PARP (T. Brucei) EPEPEPEPEPEPG AATLKSVALPFAIAAAALVAAF
Prion Protein (hamster) QKESQAYYDGRRS SAVLFSSPPVILLISFLIFLMVG
Thy-1 (rat) KTINVIRDKLVKC GGISLLVQNTSWLLLLLLSLSFLQATDFISI
Variant Surface Glycoprotein (T. Brucei) ESNCKWENNACKD SSILVTKKFALTVVSAAFVALLF
Bold AA is site of GPI attachment Sequence to right iscleaved by the transpeptidase upon Anchor addition
After Hart, G
5-10 hydrophilic, 15-20 hydrophobic
ER glycolipid synthesis
Dolichol-P-X
Glycosyl phosphatidylinositol (GPI)
Glycosphingolipids (GSL)
Minimal Defining Structure of a
Glycosphingolipid
Glycan-O-Ceramide
1'O-CH 2-CH-CH=CH-(CH 2)12- C H 3
NH OH
O=CH-(CH 2)x-CH 3
Hexose !1-
Ceramide (Cer)
Sphingosine
Fatty Acyl group
Glycan *
*Glucose (All animals) Galactose (?Vertebrates only) Inositol-P (fungi)
After Varki, A
Biosynthesis of Ceramide and Glucosylceramide
Golgi processing of GlycosphingolipidsMajor Classes of Glycosphingolipids
Series Designation Core Structure
Lacto (LcOSe4) Gal"3GlcNAc"3GalGal""44GlcGlc""1Ceramide1Ceramide
Lactoneo (LcnOSe4) Gal"4GlcNAc"3GalGal""44GlcGlc""1Ceramide1Ceramide
Globo (GbOSe4) GalNAc"3Gal!4GalGal""44GlcGlc""1Ceramide1Ceramide
Isoglobo (GbiOSe4) GalNAc"3Gal!3GalGal""44GlcGlc""1Ceramide1Ceramide
Ganglio (GgOSe4) Gal"3GalNAc"4GalGal""44GlcGlc""1Ceramide1Ceramide
Muco (MucOSe4) Gal"3Gal"3GalGal""44GlcGlc""1Ceramide1Ceramide
Gala (GalOSe2) Gal!4GalGal""1Ceramide1Ceramide
Sulfatides 3-0-Sulfo-GalGal""1Ceramide1Ceramide
Different Core structures generate unique shapesand are expressed in a cell-type specific manner
After Varki, A
Nomenclature Issues
Glycosphingolipid (GSL) = Glycan + Sphingolipid
(named after the Egyptian Sphinx)
Glycosphingolipids often just referred to as “Glycolipids”.
“Ganglioside": a GSL one or more sialic acid residues
Example of nomenclature:
Neu5Ac!3Gal"3GalNAc"4Gal"4Glc"1Cer
= GM1b in the Svennerholm nomenclature
OR
II4Neu5Ac-GgOSe4-Cer
in the official IUPAC-IUB designation
After Varki, A
Pathways for Ganglio-series Glycosphingolipid biosynthesis
!4 "3 "8 "8
GM 3 GD 3 GT 3
GM 2 GD 2 GT 2
!4!4 !4!4
GM 1 a GD1b GT1c
!3 !3 !3 !3
GD1a GT1b GQ1c
GA2
GA1
"3 "3"3 "3
GA1
GT1a GQ1b GP 1 cGD1c
"8 "8 "8 "8
A series B series C seriesO series
GalNAc
T rans fe rase
S ia ly l -
Transferase IIS ia ly l -
Transferase I
S ia ly l -
Transferase III
S ia ly l -
Transferase V
Gal
Transferase II
S ia ly l -
Transferase IV
OAc-G D 3 OAc-G T 3
OAc-G D 3
OAc-G Q1b
OAc-G T1b
OAc-G Q 1 b ( ? )
Gm1b
After Varki, A
Turnover and Degradation of Glycosphingolipids
• Internalized from plasma membrane via endocytosis
• Pass through endosomes (some remodelling possible?)
• Terminal degradation in lysosomes - stepwise reactionsby specific enzymes.
• Some final steps involve cleavages close to the cellmembrane, and require facilitation by specificsphingolipid activator proteins (SAPs).
• Individual components, available for re-utilization invarious pathways.
• At least some of glucosylceramide may remain intact andbe recycled
• Human diseases in which specific enzymes or SAPs aregenetically deficient (storage diseases)
Biological Roles of Glycosphingolipids
• Thought to be critical components of the epidermal (skin)
permeability barrier
• Organizing role in cell membrane. Thought to associate with
GPI anchors in the trans-Golgi, forming “rafts” which target to
apical domains of polarized epithelial cells
• May also be in glycosphingolipid enriched domains (“GEMs”)
which are associated with cytosolic oncogenes and signalling
molecules
• Physical protection against hostile environnments
• Binding sites for the adhesion of symbiont bacteria.
• Highly specific receptor targets for a variety of bacteria, toxins
and viruses.
Biological Roles of Glycosphingolipids
• Specific association of certain glycosphingolipids with
certain membrane receptors.
• Can mediate low-affinity but high specificity
carbohydrate-carbohydrate interactions between different
cell types.
• Targets for autoimmune antibodies in Guillian-Barre and
Miller-Fisher syndromes following Campylobacter
infections and in some patients with human myeloma
• Shed in large amounts by certain cancers - these are found
to have a strong immunosuppressive effects, via as yet
unknown mechanisms
Metabolic relationships in the biosynthesis and
turnover of Sphingolipids
Gal-Ceramide
CeramideGlc-Ceramide
Sphingosine
Ceramide-1-phosphate
Sphingosine-1-phosphate
Sphingomyelin
Glycosphingolipids
Di-methyl-sphingosine
Tri-methyl-sphingosine
Lysogalactosphingolipids
Sphingo-
myelinase
PDMP
NB-DJGFumonisin B1
de novo synthesis
Fumonisin B1
D-MAPP
Plasmalolysogalactosphingolipids
PC
DAG
Sulfatides
After Varki, A
Ganglioside synthetic enzyme KOs
–GlcCer synthase (GlcT): Early embryonic lethal,
differentiation and germ layer formation okay
–LacCer synthase (GalT): ??
–GM3 synthase (SiaTI): Increased sensitivity to insulin
(skeletal muscle receptor phosphorylation)
–GM2 synthase (GalNAcT): Male sterility, demyelinating
disorder
–GD2 synthase (SiaTII): Ok
–GM2 synthase/GD2 synthase double KO: sudden death,
auditory seizures, demyelination
General Principles
–The sialic acids are a family of structurally related
compounds that can bear a variety of post-synthetic
modifications
–Glycan synthesis is compartmentalized within cells
–Precursors begin as lipid-linked species on the cytoplasmic
face of the ER, requiring that substrates be flipped for
further processsing
–Donor substrates contribute to more than one class of
glycoconjugate
–The assembly-line model for glycan extension in the Golgi
apparatus may not be as applicable to glycolipid synthesis
as it is to glycoprotein glycosylation
–Some precursors and intermediates in glycolipid synthesis
are also utilized in signaling pathways