Essentials of Glycobiology Lecture 8 April 8, 2004 Hud Freeze Structure, biosynthesis and general biology of Glycophospholipid (GPI) Anchors.
Download ReportTranscript Essentials of Glycobiology Lecture 8 April 8, 2004 Hud Freeze Structure, biosynthesis and general biology of Glycophospholipid (GPI) Anchors.
Slide 1
Essentials of Glycobiology
Lecture 8
April 8, 2004
Hud Freeze
Structure, biosynthesis and
general biology of
Glycophospholipid (GPI) Anchors
Slide 2
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
Slide 3
Basic Glycosylphosphatidylinositol (GPI) Anchor
Phospholipid
Slide 4
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
Slide 5
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?
Slide 6
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.
Slide 7
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!!!
Slide 8
Basic Glycosylphosphatidylinositol (GPI) Anchor
Phospholipid
Slide 9
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
Slide 10
Slide 11
Structure of the Basic GPI Anchor
GPI-Li nked Protei n
= Mannose (Man)
NH 2 = Gl ucosami ne
Etn
= Ethanolami ne
P
= Phosphate
Etn
P
PNH Defect
NH 2
Pig-A
INOSITOL
P
Cell Sur face
Membr ane
Slide 12
PLANTS ALSO MAKE GPI-ANCHORS
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this pi cture.
Casper Vroemen,http://www.dpw.wau.nl/molbi/mediacenter/images/embryo11.jpg
Slide 13
Studying GPI Biosynthesis in vitro
thin layer
chromatography
F
30 °C
O
cell membranes
salts, buffers
radiolabeled
sugardonor
add solvents
spin
evaporate
O
F
Slide 14
Structural Analysis
of the GPI Anchor
Enzymatic and
chemical
cleavage sites
are useful in
identifying
GPI anchored
membrane proteins
Slide 15
Slide 16
Slide 17
Proposed
branched
pathway for
biosynthesis of
mammalian GPI
anchors
Slide 18
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 GTTRLLSGHT CFT LTGLLGTLVTMGLLT
PARP (T. Brucei)
EPEPEPEPEPEPG AATLKSVALPFAIAAAALVAAF
Prion Protein (hamster)
QKESQAYYDGRRS SAVLFSSPPVILLISFLIFLMVG
Thy-1 (rat)
Variant Surface Glycoprotein (T . Brucei)
KTINVIRDKLVKC GGISLLVQNT SWLLLLLLSLSFLQATDFISI
ESNCKWENNACKD SSILVTKKFALTVVSAAFVALLF
Bold AA is site of GPI attachment Sequence to
right is cleaved by the transpeptidase
upon Anchor addition
Slide 19
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
Slide 20
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
Slide 21
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
+
+
Slide 22
UPS AND DOWNS OF GPI-LINKED PLACENTAL ALKALINE PHOSPHATASE
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Frances J. Sharom
www.chembio.uoguelph.ca/ sharom/
Slide 23
Sean Munro, Cell, 115, 377-388, Nov 2003
Slide 24
QuickTi me™ and a T IFF (Uncompressed) decompressor are needed to see this picture.
Slide 25
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
Slide 26
Taroh Kinoshita
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Slide 27
• 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
Slide 28
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.
Slide 29
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.
Slide 30
NON-POLITICAL FLIP-FLOP: A GOOD THING
Quic kT ime™ and a T IFF (Unc ompres sed) dec ompres sor are needed to s ee this picture.
Slide 31
Dol
Quic kT ime™ and a T IFF (Unc ompres sed) dec ompres sor are needed to s ee this picture.
Slide 32
CDG-If
Slide 33
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
Slide 34
FUTURE PERSPECTIVES
THE FUNCTION OF GPI-ANCHORS IS STILL UNRESOLVED
LIKE THE FUNCTIONS OF GLYCOYSLATION
MAYBE ALL THE THEORIES ARE CORRECT
Essentials of Glycobiology
Lecture 8
April 8, 2004
Hud Freeze
Structure, biosynthesis and
general biology of
Glycophospholipid (GPI) Anchors
Slide 2
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
Slide 3
Basic Glycosylphosphatidylinositol (GPI) Anchor
Phospholipid
Slide 4
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
Slide 5
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?
Slide 6
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.
Slide 7
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!!!
Slide 8
Basic Glycosylphosphatidylinositol (GPI) Anchor
Phospholipid
Slide 9
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
Slide 10
Slide 11
Structure of the Basic GPI Anchor
GPI-Li nked Protei n
= Mannose (Man)
NH 2 = Gl ucosami ne
Etn
= Ethanolami ne
P
= Phosphate
Etn
P
PNH Defect
NH 2
Pig-A
INOSITOL
P
Cell Sur face
Membr ane
Slide 12
PLANTS ALSO MAKE GPI-ANCHORS
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this pi cture.
Casper Vroemen,http://www.dpw.wau.nl/molbi/mediacenter/images/embryo11.jpg
Slide 13
Studying GPI Biosynthesis in vitro
thin layer
chromatography
F
30 °C
O
cell membranes
salts, buffers
radiolabeled
sugardonor
add solvents
spin
evaporate
O
F
Slide 14
Structural Analysis
of the GPI Anchor
Enzymatic and
chemical
cleavage sites
are useful in
identifying
GPI anchored
membrane proteins
Slide 15
Slide 16
Slide 17
Proposed
branched
pathway for
biosynthesis of
mammalian GPI
anchors
Slide 18
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 GTTRLLSGHT CFT LTGLLGTLVTMGLLT
PARP (T. Brucei)
EPEPEPEPEPEPG AATLKSVALPFAIAAAALVAAF
Prion Protein (hamster)
QKESQAYYDGRRS SAVLFSSPPVILLISFLIFLMVG
Thy-1 (rat)
Variant Surface Glycoprotein (T . Brucei)
KTINVIRDKLVKC GGISLLVQNT SWLLLLLLSLSFLQATDFISI
ESNCKWENNACKD SSILVTKKFALTVVSAAFVALLF
Bold AA is site of GPI attachment Sequence to
right is cleaved by the transpeptidase
upon Anchor addition
Slide 19
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
Slide 20
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
Slide 21
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
+
+
Slide 22
UPS AND DOWNS OF GPI-LINKED PLACENTAL ALKALINE PHOSPHATASE
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Frances J. Sharom
www.chembio.uoguelph.ca/ sharom/
Slide 23
Sean Munro, Cell, 115, 377-388, Nov 2003
Slide 24
QuickTi me™ and a T IFF (Uncompressed) decompressor are needed to see this picture.
Slide 25
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
Slide 26
Taroh Kinoshita
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Slide 27
• 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
Slide 28
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.
Slide 29
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.
Slide 30
NON-POLITICAL FLIP-FLOP: A GOOD THING
Quic kT ime™ and a T IFF (Unc ompres sed) dec ompres sor are needed to s ee this picture.
Slide 31
Dol
Quic kT ime™ and a T IFF (Unc ompres sed) dec ompres sor are needed to s ee this picture.
Slide 32
CDG-If
Slide 33
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
Slide 34
FUTURE PERSPECTIVES
THE FUNCTION OF GPI-ANCHORS IS STILL UNRESOLVED
LIKE THE FUNCTIONS OF GLYCOYSLATION
MAYBE ALL THE THEORIES ARE CORRECT