Download Essentials of Glycobiology Lecture 6 (7) April 7th. (9) 1998 Ajit Varki

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
CHONDROITIN
SULFATE
HYALURONAN
P
GLYCOSAMINOGLYCANS
HEPARAN SULFATE
S
S
S
S
S
NS
NS
-O-Ser
Proteoglycan
N-LINKED CHAINS
Ac
O-LINKED
CHAIN
GLYCOPHOSPHOLIPID
ANCHOR
P
S
O
Ser/Thr
GLYCOSPHINGOLIPID
N
Asn
N
Asn
INOSITOL
Glycoprotein
Ac
Sialic Acids
INSIDE
O
Ser
Etn
P
NH 2
OUTSIDE
O-LINKED GlcNAc
S
S
Ser-O-
P
S
Basic Glycosylphosphatidylinositol (GPI) Anchor
Phospholipid
Lecture Overview
Historical Background
Defining the Core Structure
Biosynthesis & Transfer of GPI Anchors
The Signal for Addition of GPI Anchors
Occurrence and Variations in Nature
Postulated Biological Roles
Genetic Disorders
Perspectives & Future Directions
Discovery of GPI-Anchors.
 1963-First data suggests protein-lipid anchors: crude
bacterial phospholipase C releases alkaline phosphatase
from mammalian cells.
 mid-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 Inositolcontaining 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-Anchors
 The 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 immunocrossreactive carbohydrate (CRD) attached to its Cterminus via an amide linkage involving ethanolamine.
 Mervyn Turner’s group showed that trypanosomes
contain an enzyme which rapidly releases the membraneassociated 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 ‘glycosylphosphatidylinositol’ (GPI).
THE LESSON: SHOW ME THE STRUCTURE!!!
Basic Glycosylphosphatidylinositol (GPI) Anchor
Phospholipid
Examples of GPI-Anchored Proteins
Cell surface hydrolases
alkaline phosphatase
acetylcholinesterase
5’ nucleotidase
Adhesion molecules
neural cell adhesion molecule
heparan sulfate proteoglycan
Protozoal antigens
trypanosome VSG
leishmanial protease
plasmodium antigens
Mammalian antigens
carcinoembryonic antigen
Thy-1
Others
scrapie prion protein
folate receptor
decay accelerating factor
Structure of the Basic GPI Anchor
GPI-Linked Protein
= Mannose (Man)
NH 2 = Glucosamine
Etn
P
= Ethanolamine
Etn
P
= Phosphate
PNH Defect
NH 2
Pig-A
INOSITOL
P
Cell Surface
Membrane
PLANTS ALSO MAKE GPI-ANCHORS
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Casper Vroemen,http://www.dpw.wau.nl/molbi/mediacenter/images/embryo11.jpg
Studying GPI Biosynthesis in vitro
thin layer
chromatography
F
30 °C
O
cell membranes
salts, buffers
radiolabeled
sugardonor
add solvents
spin
evaporate
O
F
Structural Analysis
of the GPI Anchor
Enzymatic and
chemical
cleavage sites
are useful in
identifying
GPI anchored
membrane proteins
Proposed
branched
pathway for
biosynthesis of
mammalian GPI
anchors
Examples of C-Terminal Sequences Signaling
the Addition of GPI-Anchors
Protein
GPI-Signal Sequence
Acetylcholinesterase (Torpedo)
NQFLPKLLNATAC DGELSSSGTSSSKGIIFYVLFS ILYLIFY
Alkaline Phosphatase (placenta)
TACDLAPPAGTTD AAHPGRSVVPALLPLLAGTLLLLETATAP
Decay Accelerating Factor
HETTPNKGSGTTS GTTRLLSGHTCFTLTGLLGTLVTMGLLT
PARP (T. Brucei)
EPEPEPEPEPEP G AATLKSVALPFAIAAAALVAAF
Prion Protein (hamster)
QKESQAYYDGRRS SAVLFSSPP VILLISFLIFLMVG
Thy-1 (rat)
Variant Surface Glycoprotein (T. Brucei)
KTINVIRDKLVKC GGISLLVQNTSWLLLLLLSLSFLQATDFISI
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 w
site) and residue two amino acids on carboxyl side
(w + 2 site) always have small side-chains
 w + 1 site can have large side-chains.
w + 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 Functions
Dense packing of Proteins on Cell Surface
Increased Protein mobility on Cell Surface
Targeting of proteins to Apical Domains
Specific release from Cell Surface
Control of Exit from ER?
Developmental regulation of protein
expression?
Generation of Protein Complexity
Signal transduction?
Toxin Binding
Parasite Cell structure
Possible Role of the GPI-Anchor in ER Exit
Negative Signal: Retention mechanism is displaced by anchor
transport vesicles
-
-
Positive Signal: Anchor is recognized by packaging machinery
transport vesicles
+
+
UPS AND DOWNS OF GPI-LINKED PLACENTAL ALKALINE PHOSPHATASE
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Frances J. Sharom
www.chembio.uoguelph.ca/ sharom/
Sean Munro, Cell, 115, 377-388, Nov 2003
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CHONDROITIN
SULFATE
HYALURONAN
P
GLYCOSAMINOGLYCANS
S
S
Ser-O-
HEPARAN SULFATE
Paroxysmal Nocturnal
Hemoglobinuria:
Somatic Loss of Glycophospholipid
Anchors in Hematopoietic
Stem Cells
N-LINKED CHAINS
S
S
S
S
S
NS
NS
Ac
O-LINKED
CHAIN
GLYCOPHOSPHOLIPID
ANCHOR
P
S
O
Ser/Thr
GLYCOSPHINGOLIPID
-O-Ser
N
Asn
N
Asn
NH 2
INOSITOL
Glycoprotein
Ac
OUTSIDE
Sialic Acids
INSIDE
O-LINKED GlcNAc
O
Ser
Etn
P
P
S
Taroh Kinoshita
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• 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 IN
DOL-P-MAN SYN
AND USE
Biosynthesis
of GPI anchors
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.
NON-POLITICAL FLIP-FLOP: A GOOD THING
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Dol
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CDG-If
PTPATIENT WITH DPM1 DEFICIENCY
Decreased Dol-P-Man synthesis
PATIENT HAS MPDU1 DEFICIENCY
Inefficient use of Dol-P-Man and Dol-P-Glc
Both patients are blind with severe developmental delay
Pathology may result from impaired N-linked or GPI-anchor synthesis
FUTURE PERSPECTIVES
THE FUNCTION OF GPI-ANCHORS IS STILL UNRESOLVED
LIKE THE FUNCTIONS OF GLYCOYSLATION
MAYBE ALL THE THEORIES ARE CORRECT