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Essentials of Glycobiology Lecture 8 April 8, 2004 Hud Freeze. Structure, biosynthesis and general biology of Glycophospholipid (GPI) Anchors. CHONDROITIN SULFATE. HYALURONAN. GLYCOSAMINO- GLYCANS. HEPARAN SULFATE. N-LINKED CHAINS. O-LINKED CHAIN. GLYCOPHOSPHO- LIPID ANCHOR. - PowerPoint PPT Presentation
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Essentials of Glycobiology
Lecture 8
April 8, 2004
Hud Freeze
Structure, biosynthesis and general biology of
Glycophospholipid (GPI) Anchors
Major Glycan
Classes in Animal Cells
OSer
OSer/Thr
NAsn
Ser-O-
OUTSIDE
INSIDE
NAsn
S S S
-O-SerS SSS S
EtnP
INOSITOL
P
NH
Ac
P
NS NS
Ac
S
2
P
GlycoproteinGlycoprotein
ProteoglycanProteoglycan
GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID
ANCHORANCHOR
N-LINKED CHAINSN-LINKED CHAINS
O-LINKED O-LINKED CHAINCHAIN
HYALURONANHYALURONAN
GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS HEPARAN SULFATEHEPARAN SULFATE
CHONDROITINCHONDROITIN SULFATESULFATE
Sialic AcidsSialic Acids
GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID
O-LINKED GlcNAcO-LINKED GlcNAc
Basic Glycosylphosphatidylinositol (GPI) AnchorBasic Glycosylphosphatidylinositol (GPI) Anchor
Protein
Glycan
Phospholipid
Lecture Overview
Historical BackgroundDefining the Core Structure Biosynthesis & Transfer of GPI AnchorsThe Signal for Addition of GPI AnchorsOccurrence and Variations in NaturePostulated Biological RolesGenetic DisordersPerspectives & Future Directions
Discovery of GPI-AnchorsDiscovery of GPI-Anchors..
19631963-First data suggests protein-lipid anchors: crude bacterial phospholipase C releases alkaline phosphatase from mammalian cells.
mid-1970’smid-1970’s Hiro Ikezawa in Japan, and Martin Low in the U.S. show that purified bacterial phosphatidylinositol phospholipase C releases some enzymes, e.g.,alkaline phosphatase, from cell surfaces. Propose Inositol-containing phospholipid protein linkage
Alan Williams in U.K. notes that antigen Thy-1 properties of glycolipid and glycoprotein.
However: No structural data!
GPI-anchors? Really?
Discovery of GPI-AnchorsDiscovery of GPI-AnchorsThe C-terminus of Thy-1 glycoprotein found to have both
fatty acids and ethanolamine. In 1981, Tony Holder and George Cross groups showed
that soluble form of the variant surface glycoprotein (sVSG) of African trypanosomes contains an immuno-crossreactive carbohydrate (CRD) attached to its C-terminus via an amide linkage involving ethanolamine.
Mervyn Turner’s group showed that trypanosomes contain an enzyme which rapidly releases the membrane-associated VSG (mfVSG) upon cellular damage. mfVSG becomes water soluble.
sVSG so rapid membrane form is only detected by rapidly boiling trypanosomes in (SDS) prior to electrophoresis.
Discovery of GPI-Anchors. 1985: Hart & Englund groups at Johns Hopkins show
that the lipid-anchor on VSG is added within one minute of the polypeptide’s synthesis in the endoplasmic reticulum (ER). They postulate a pre-assembled membrane anchor is attached en bloc.
1985: Michael Ferguson and colleagues at Oxford publish a tour de force structural analysis of the glycolipid attached to the mfVSG of trypanosomes. These studies structurally define the term ‘glycosyl-phosphatidylinositol’ (GPI).
THE LESSON: SHOW ME THE STRUCTURE!!!THE LESSON: SHOW ME THE STRUCTURE!!!
Basic Glycosylphosphatidylinositol (GPI) AnchorBasic Glycosylphosphatidylinositol (GPI) Anchor
Protein
Glycan
Phospholipid
Examples of GPI-Anchored ProteinsExamples of GPI-Anchored Proteins
Cell surface hydrolasesalkaline phosphataseacetylcholinesterase5’ nucleotidase
Adhesion molecules
neural cell adhesion molecule
heparan sulfate proteoglycan
Others
decay accelerating factor
scrapie prion proteinfolate receptor
Protozoal antigenstrypanosome VSGleishmanial proteaseplasmodium antigens
Mammalian antigens
Thy-1carcinoembryonic antigen
Structure of the Basic GPI Anchor
EtnP
INOSITOL
P
= Mannose (Man)
= Glucosamine
Etn = Ethanolamine
P = Phosphate
NH2
NH2Pig-A
PNH Defect
GPI-Linked Protein
Cell SurfaceMembrane
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.
PLANTS ALSO MAKE GPI-ANCHORS
Casper Vroemen,http://www.dpw.wau.nl/molbi/mediacenter/images/embryo11.jpg
Studying GPI Biosynthesis in vitroStudying GPI Biosynthesis in vitro
cell membranessalts, buffersradiolabeled sugardonor
30 °C
add solventsspin
evaporate
F
O
thin layerchromatography
O F
Structural Analysis Structural Analysis of the GPI Anchorof the GPI Anchor
Enzymatic and Enzymatic and chemical chemical
cleavage sites cleavage sites are useful in are useful in identifying identifying
GPI anchored GPI anchored membrane proteinsmembrane proteins
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 is cleaved by the transpeptidase upon Anchor addition
Rules for C-Terminal Sequences Signaling the Addition of GPI-Anchors
Residue to which anchor is attached (termed site) and residue two amino acids on carboxyl side ( + 2 site) always have small side-chains
+ 1 site can have large side-chains.
+ 2 site followed by 5 to 10 hydrophilic amino acids,
Next, add fifteen to twenty hydrophobic amino acids at or near the carboxy-terminus
GPI Anchor FunctionsGPI Anchor FunctionsDense packing of Proteins on Cell SurfaceIncreased Protein mobility on Cell SurfaceTargeting of proteins to Apical DomainsSpecific release from Cell SurfaceControl of Exit from ER?Developmental regulation of protein
expression?Generation of Protein ComplexitySignal transduction?Toxin BindingParasite Cell structure
Possible Role of the GPI-Anchor in ER ExitPossible Role of the GPI-Anchor in ER Exit
- -
transport vesicles
+
+
transport vesicles
Positive Signal: Anchor is recognized by packaging machinery
Negative Signal: Retention mechanism is displaced by anchor
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.
UPS AND DOWNS OF GPI-LINKED PLACENTAL ALKALINE PHOSPHATASE
Frances J. Sharom www.chembio.uoguelph.ca/ sharom/
Sialic AcidsSialic Acids
GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID Ac
O-LINKED GlcNAcO-LINKED GlcNAcOSer
OSer/Thr
NAsn
Ser-O-
N-LINKED CHAINSN-LINKED CHAINS
O-LINKED O-LINKED CHAINCHAIN
NAsn
S S S
-O-SerS SSS S
GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS
P
NS NS
Ac
S
P
GlycoproteinGlycoprotein
HYALURONANHYALURONAN
HEPARAN SULFATEHEPARAN SULFATE
CHONDROITINCHONDROITIN SULFATESULFATE
Paroxysmal Nocturnal Hemoglobinuria: Somatic Loss of Glycophospholipid
Anchors in Hematopoietic Stem Cells
INSIDE
OUTSIDE
INOSITOL
P
NH2
GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID
ANCHORANCHOR EtnP
Biosynthesis of GPI anchors
• The first step in biosynthesis of the GPI anchor requires at least four genes
• One of them, PIG-A is an X-linked gene
Mutation in PNH
MUTATIONS INDOL-P-MAN SYNAND USE
Paroxysmal Nocturnal Hemoglobinuria
An acquired clonal 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.
Factors that determine why mutant clones
expand have not been determined.
Paroxysmal Nocturnal Hemoglobinuria
Pre existing PNH clones have a conditional growth advantage depending on some factor present in the marrow environment of PNH patients.
However, cells with the PNH phenotype have been found at a frequency of 22 per million in normal individuals. These rare cells were collected by flow sorting and had PIG-A mutations.
Thus, PIG-A gene mutations are not sufficient for the development of clinically evident PNH.
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.
NON-POLITICAL FLIP-FLOP: A GOOD THING
PATIENT HAS MPDU1 DEFICIENCYInefficient use of Dol-P-Man and Dol-P-Glc
PTPATIENT WITH DPM1 DEFICIENCY Decreased Dol-P-Man synthesis
Both patients are blind with severe developmental delay
Pathology may result from impaired N-linked or GPI-anchor synthesis