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EXT1 & EXT2 proteins and heparan sulfate biosynthesis
Abstract 2005 MHE Conference
Maria Wilén1, Marta Busse1 and Marion Kusche-Gullberg1,2
1 Department of Medical Biochemistry and Microbiology,
University of Uppsala, Uppsala, Sweden and
2Department of Biomedicine, Division of Physiology,
University of Bergen, Norway
Heparan sulfate is a complex polysaccharide that plays an important role in several cellular processes, including normal fetal
development, wound healing and inflammation. Defects in enzymes involved in heparan sulfate synthesis result in different
abnormalities including abnormal skeletal and kidney development. Heparan sulfate is elongated by the alternating transfer of
glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) units. Concomitant with elongation, the polymer is modified through a
series of reactions that requires the action of several different enzymes. The extent of these reactions varies, giving rise to
heparan sulfate chains with different structural properties.
The chain elongation reaction has been ascribed to a hetero-oligomeric complex of EXT1 and EXT2. Mutations in either EXT1 or
EXT2 have been linked to the human disorder, hereditary multiple exostoses (HME), characterized by the formation of
cartilage-capped bony outgrowths at the end of the long bones.
The individual functions of EXT1 and EXT2 in heparan sulfate chain elongation are currently unknown. EXT1 alone has the
capacity to elongate heparan sulfate chains in vitro. Furthermore, reduced EXT1 expression levels results in the formation of
heparan sulfate chains that are shorter than those normally synthesized. The level of EXT2 protein modifies the catalytic
properties of EXT1 but the role of EXT2 in heparan sulfate chain elongation is not clear.
To evaluate the effect of EXT2-mutations on heparan sulfate structure, we have generated transgenic mice with a general and
constitutive tissue expression of wild-type or mutated EXT2. To understand the individual roles of EXT1 and EXT2, we have
overexpressed the proteins or reduced their levels in mammalian cell systems and studied the effects of these manipulations on
heparan sulfate structure.
Abstract 2005 MHE Conference
Maria Wilén1, Marta Busse1 and Marion Kusche-Gullberg1,2
1 Department of Medical Biochemistry and Microbiology,
University of Uppsala, Uppsala, Sweden and
2Department of Biomedicine, Division of Physiology,
University of Bergen, Norway
Heparan sulfate is a complex polysaccharide that plays an important role in several cellular processes, including normal fetal
development, wound healing and inflammation. Defects in enzymes involved in heparan sulfate synthesis result in different
abnormalities including abnormal skeletal and kidney development. Heparan sulfate is elongated by the alternating transfer of
glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) units. Concomitant with elongation, the polymer is modified through a
series of reactions that requires the action of several different enzymes. The extent of these reactions varies, giving rise to
heparan sulfate chains with different structural properties.
The chain elongation reaction has been ascribed to a hetero-oligomeric complex of EXT1 and EXT2. Mutations in either EXT1 or
EXT2 have been linked to the human disorder, hereditary multiple exostoses (HME), characterized by the formation of
cartilage-capped bony outgrowths at the end of the long bones.
The individual functions of EXT1 and EXT2 in heparan sulfate chain elongation are currently unknown. EXT1 alone has the
capacity to elongate heparan sulfate chains in vitro. Furthermore, reduced EXT1 expression levels results in the formation of
heparan sulfate chains that are shorter than those normally synthesized. The level of EXT2 protein modifies the catalytic
properties of EXT1 but the role of EXT2 in heparan sulfate chain elongation is not clear.
To evaluate the effect of EXT2-mutations on heparan sulfate structure, we have generated transgenic mice with a general and
constitutive tissue expression of wild-type or mutated EXT2. To understand the individual roles of EXT1 and EXT2, we have
overexpressed the proteins or reduced their levels in mammalian cell systems and studied the effects of these manipulations on
heparan sulfate structure.
Research authored by Dr. Kushe-Gullberg
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List of Publications via PubMed
(NIH National Library of Medicine)
List of Publications via PubMed
(NIH National Library of Medicine)
| Dr. Kusche-Gullberg's research |
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2009 Conference abstract
EXT-Dependent Regulation of Heparan Sulfate Structure and Function
Cecilia Österholm1, Marta Busse2, Almir Feta1 and Marion Kusche-Gullberg1
1Department of Biomedicine, University of Bergen, NO-5009, Bergen, Norway. 2Vascular Biology Laboratory, Cancer Research
UK, Lincoln’s Inn Fields Laboratories, London WC 2A 3PX, United Kingdom.
e-mail: marion.kusche@biomed.uib.no
Heparan sulfate is a complex polysaccharide that plays an important role in several cellular processes, including normal fetal
development, wound healing and inflammation. Heparan sulfate chain elongation has been ascribed to a hetero-oligomeric
complex of EXT1 and EXT2. Mutations in either EXT1 or EXT2 have been linked to the human disorder, hereditary multiple
exostoses (HME), characterized by the formation of cartilage-capped bony outgrowths at the end of the long bones.
The individual functions of EXT1 and EXT2 in heparan sulfate chain elongation are currently unknown. EXT1 alone has the
capacity to elongate heparan sulfate chains in vitro. The level of EXT2 protein modifies the catalytic properties of EXT1 but the
role of EXT2 in heparan sulfate chain elongation is not clear. To understand the individual roles of EXT1 and EXT2, we have
overexpressed the proteins or reduced their levels in mammalian cell systems and studied the effects of these manipulations on
heparan sulfate structure. Our findings indicate that the levels of EXT1 and EXT2 influence heparan sulfate chain elongation.
Heparan sulfates are ubiquitously expressed in all tissues, where they function as adhesion molecules and co-receptors. Thus,
they modulate cell-matrix interactions and growth factor signaling. We have previously shown that mouse embryonic fibroblasts
with a gene trap mutation in Ext1 have substantially reduced heparan sulfate chain length. We have now used these fibroblasts
to investigate the functional consequences of the Ext1 mutation for heparan sulfate-dependent growth factor signaling and for
cell interactions with the extracellular matrix. Our results indicate that shorter heparan sulfate chains result in specific growth
factor signaling defects as well as impaired fibroblast-matrix interactions.
EXT-Dependent Regulation of Heparan Sulfate Structure and Function
Cecilia Österholm1, Marta Busse2, Almir Feta1 and Marion Kusche-Gullberg1
1Department of Biomedicine, University of Bergen, NO-5009, Bergen, Norway. 2Vascular Biology Laboratory, Cancer Research
UK, Lincoln’s Inn Fields Laboratories, London WC 2A 3PX, United Kingdom.
e-mail: marion.kusche@biomed.uib.no
Heparan sulfate is a complex polysaccharide that plays an important role in several cellular processes, including normal fetal
development, wound healing and inflammation. Heparan sulfate chain elongation has been ascribed to a hetero-oligomeric
complex of EXT1 and EXT2. Mutations in either EXT1 or EXT2 have been linked to the human disorder, hereditary multiple
exostoses (HME), characterized by the formation of cartilage-capped bony outgrowths at the end of the long bones.
The individual functions of EXT1 and EXT2 in heparan sulfate chain elongation are currently unknown. EXT1 alone has the
capacity to elongate heparan sulfate chains in vitro. The level of EXT2 protein modifies the catalytic properties of EXT1 but the
role of EXT2 in heparan sulfate chain elongation is not clear. To understand the individual roles of EXT1 and EXT2, we have
overexpressed the proteins or reduced their levels in mammalian cell systems and studied the effects of these manipulations on
heparan sulfate structure. Our findings indicate that the levels of EXT1 and EXT2 influence heparan sulfate chain elongation.
Heparan sulfates are ubiquitously expressed in all tissues, where they function as adhesion molecules and co-receptors. Thus,
they modulate cell-matrix interactions and growth factor signaling. We have previously shown that mouse embryonic fibroblasts
with a gene trap mutation in Ext1 have substantially reduced heparan sulfate chain length. We have now used these fibroblasts
to investigate the functional consequences of the Ext1 mutation for heparan sulfate-dependent growth factor signaling and for
cell interactions with the extracellular matrix. Our results indicate that shorter heparan sulfate chains result in specific growth
factor signaling defects as well as impaired fibroblast-matrix interactions.
Mutation in the heparan sulfate biosynthesis enzyme Ext1 influences growth factor signaling and fibroblast
interaction with the extracellular matrix.
Cecilia Österholm, Malgorzata M. Barczyk, Marta Busse, Mona Grønning, Rolf K. Reed, and Marion Kusche-Gullberg
J. Biol. Chem. 2009 284: 34935-34943. First Published on October 22, 2009, doi:10.1074/jbc.M109.005264
Full Text (PDF)
Abstract
Heparan sulfate (HS) chains bind and modulate the signaling efficiency of many ligands, including members of the fibroblast
growth factor (FGF) and platelet-derived growth factor families. We previously reported the structure of HS synthesized by
embryonic fibroblasts from mice with a gene trap mutation of Ext1 that encodes a glycosyltransferase involved in HS chain
elongation. The gene trap mutation results in low expression of Ext1, and, as a consequence, HS chain length is substantially
reduced. In the present study, Ext1 mutant and wild-type mouse embryonic fibroblasts were analyzed for the functional
consequences of the Ext1 mutation for growth factor signaling and interaction with the extracellular matrix. Here, we show that
the phosphorylation of ERK1/2 in response to FGF2 stimulation was markedly decreased in the Ext1 mutant fibroblasts,
whereas neither PDGF-BB nor FGF10 signaling was significantly affected. Furthermore, Ext1 mutants displayed reduced ability
to attach to collagen I and to contract collagen lattices, even though no differences in the expression of collagen-binding
integrins were observed. Reintroduction of Ext1in the Ext1 mutant fibroblasts rescued HS chain length, FGF2 signaling, and the
ability of the fibroblasts to contract collagen. These data suggest that the length of the HS chains is a critical determinant of
HS-protein interactions and emphasize the essential role of EXT1 in providing specific binding sites for growth factors and
extracellular matrix proteins.
interaction with the extracellular matrix.
Cecilia Österholm, Malgorzata M. Barczyk, Marta Busse, Mona Grønning, Rolf K. Reed, and Marion Kusche-Gullberg
J. Biol. Chem. 2009 284: 34935-34943. First Published on October 22, 2009, doi:10.1074/jbc.M109.005264
Full Text (PDF)
Abstract
Heparan sulfate (HS) chains bind and modulate the signaling efficiency of many ligands, including members of the fibroblast
growth factor (FGF) and platelet-derived growth factor families. We previously reported the structure of HS synthesized by
embryonic fibroblasts from mice with a gene trap mutation of Ext1 that encodes a glycosyltransferase involved in HS chain
elongation. The gene trap mutation results in low expression of Ext1, and, as a consequence, HS chain length is substantially
reduced. In the present study, Ext1 mutant and wild-type mouse embryonic fibroblasts were analyzed for the functional
consequences of the Ext1 mutation for growth factor signaling and interaction with the extracellular matrix. Here, we show that
the phosphorylation of ERK1/2 in response to FGF2 stimulation was markedly decreased in the Ext1 mutant fibroblasts,
whereas neither PDGF-BB nor FGF10 signaling was significantly affected. Furthermore, Ext1 mutants displayed reduced ability
to attach to collagen I and to contract collagen lattices, even though no differences in the expression of collagen-binding
integrins were observed. Reintroduction of Ext1in the Ext1 mutant fibroblasts rescued HS chain length, FGF2 signaling, and the
ability of the fibroblasts to contract collagen. These data suggest that the length of the HS chains is a critical determinant of
HS-protein interactions and emphasize the essential role of EXT1 in providing specific binding sites for growth factors and
extracellular matrix proteins.
Press Release 11 / 02 / 07
Contribution of EXT1, EXT2, and EXTL3 to Heparan Sulfate Chain Elongation*
J. Biol. Chem., Vol. 282, Issue 45, 32802-32810, November 9, 2007
To read journal full publication
Marta Busse, Almir Feta, Jenny Presto, Maria Wilén, Mona Grønning, Lena Kjellén, and Marion Kusche-Gullberg
From the Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway and the Department of
Medical Biochemistry and Microbiology, University of Uppsala, BMC Box 582, SE-751 23 Uppsala, Sweden
The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human
members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form
a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the
EXTL proteins exhibit overlapping glycosyl-transferase activities in vitro, so that it is not apparent what reactions they catalyze
in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain
elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into
human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2
siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also
generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length,
which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect
on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a
human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further
investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X,
resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was
not shared by the EXT2-Y419X mutant.
Contribution of EXT1, EXT2, and EXTL3 to Heparan Sulfate Chain Elongation*
J. Biol. Chem., Vol. 282, Issue 45, 32802-32810, November 9, 2007
To read journal full publication
Marta Busse, Almir Feta, Jenny Presto, Maria Wilén, Mona Grønning, Lena Kjellén, and Marion Kusche-Gullberg
From the Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway and the Department of
Medical Biochemistry and Microbiology, University of Uppsala, BMC Box 582, SE-751 23 Uppsala, Sweden
The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human
members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form
a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the
EXTL proteins exhibit overlapping glycosyl-transferase activities in vitro, so that it is not apparent what reactions they catalyze
in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain
elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into
human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2
siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also
generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length,
which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect
on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a
human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further
investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X,
resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was
not shared by the EXT2-Y419X mutant.
| Photo's taken during the Third International MHE Research Conference |
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