M. Kusche-Gullberg | University of Bergen (original) (raw)

Papers by M. Kusche-Gullberg

Research paper thumbnail of Novel Drosophila laminin A chain reveals structural relationships between laminin subunits

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1993

Laminins are trimeric glycoproteins composed of A, B1, and B2 chains that play important roles in... more Laminins are trimeric glycoproteins composed of A, B1, and B2 chains that play important roles in cell adhesion and differentiation and in the assembly of basement membranes. There is evidence that multiple independent gene products can be used to supply the A, B1, and B2-like chains, thereby generating diverse laminin molecules. The set of A chain-like polypeptides are the most divergent. The amino-terminal portion of each chain, called the short arm, is composed of alternating cysteine-rich and globular domains. The amino-terminal two-thirds of the newly characterized Drosophila laminin A chain appears to encode a novel structure, but the analysis presented here shows that substantial portions of its amino acid sequence are related to sequences found in other A, B1, and B2 chains. A portion of the Drosophila A chain is composed of sequences like those found in B2 chains. These sequences parallel a portion of the vertebrate A chains, suggesting a structure for a shared ancestral A ...

Research paper thumbnail of Phenotypic characterization of the human mast-cell line HMC-1

Scandinavian journal of immunology, 1994

The cell line HMC-1, derived from a patient with mast cell leukaemia, is the only established cel... more The cell line HMC-1, derived from a patient with mast cell leukaemia, is the only established cell line exhibiting a phenotype similar to that of human mast cells. This paper reports on a detailed characterization of the expression of a panel of markers for various types of immature and mature haematopoietic cells in the HMC-1. We also studied the potential of HMC-1 to differentiate upon treatment with conditioned media from the human T-cell line Mo, retinoic acid or DMSO. HMC-1 was found to express several mast cell-related markers. A high expression of Kit, the receptor for stem-cell factor, was detected. The majority of the cells were stained with a MoAb against the mast cell-specific serine protease tryptase. Of particular interest was the finding that beta-tryptase mRNA, but not alpha-tryptase mRNA, was expressed in HMC-1. Using enzyme-histochemistry we were able to show that the beta-tryptase was enzymatically active, indicating that tryptase can form active homotetramers. Bot...

Research paper thumbnail of Laminin A chain: expression during Drosophila development and genomic sequence

The EMBO journal, 1992

A Drosophila laminin A chain gene was characterized as a 14 kb genomic nucleotide sequence which ... more A Drosophila laminin A chain gene was characterized as a 14 kb genomic nucleotide sequence which encodes an open reading frame of 3712 amino acids in 15 exons. Overall, this A chain is similar to its vertebrate counterparts, especially in its N- and C-terminal globular domains, but the sequence that forms the laminin A short arm is quite different and larger. Laminin messages appear in newly formed mesoderm and are later prominently expressed in hemocytes, which also synthesize basement membrane collagen IV. The composition of Drosophila basement membranes changes with development. A novel method of tandemly fused RNA probes showed that developmental increases of laminin mRNAs were primarily associated with periods of morphogenesis, and preceded those of collagen IV, a protein strongly expressed during growth. The ratio of A:B1:B2 mRNAs varied little during embryogenesis, with less mRNA for A than B chains. Staining of embryos with antibodies confirmed and extended the information p...

Research paper thumbnail of Phenotypic characterization of mice lacking genes coding for NDSTs, heparan sulfate glucos-aminyl N-deacetylase/N-sulfo-transferase

Research paper thumbnail of Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation

Proceedings of the National Academy of Sciences, 2008

Research paper thumbnail of Biosynthesis of Hyaluronan: DIRECTION OF CHAIN ELONGATION

Journal of Biological Chemistry, 2005

Hyaluronan (HA), a functionally essential glycosaminoglycan in vertebrate tissues and a putative ... more Hyaluronan (HA), a functionally essential glycosaminoglycan in vertebrate tissues and a putative virulence factor in certain pathogenic bacteria, is an extended linear polymer composed of alternating units of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). Uncertainty regarding the mechanism of HA biosynthesis has included the directionality of chain elongation, i.e. whether addition of monosaccharide units occurs at the reducing or non-reducing terminus of nascent chains. We have investigated this problem using yeast-derived recombinant HA synthases from Xenopus laevis (xlHAS1) and from Streptococcus pyogenes (spHAS). The enzymes were incubated with UDP-[3H]GlcUA and UDP-[14C]GlcNAc, under experimental conditions designed to yield HA chains with differentially labeled reducing-terminal and non-reducing terminal domains. Digestion of the products with a mixture of beta-glucuronidase and beta-N-acetylglucosaminidase exoenzymes resulted in truncation of the HA chain strictly from the non-reducing end and release of labeled monosaccharides. The change in 3H/14C ratio of the monosaccharide fraction, during the course of exoglycosidase digestion, was interpreted to indicate whether sugar units had been added at the reducing or non-reducing end. The results demonstrate that the vertebrate xlHAS1 and the bacterial spHAS extend HA in opposite directions. Chain elongation catalyzed by xlHAS1 occurs at the non-reducing end of the HA chain, whereas elongation catalyzed by spHAS occurs at the reducing end. The spHAS is the first glycosyltransferase that has been unanimously demonstrated to function at the reducing end of a growing glycosaminoglycan chain.

Research paper thumbnail of Heparan Sulfate Synthesized by Mouse Embryonic Stem Cells Deficient in NDST1 and NDST2 Is 6-O-Sulfated but Contains No N-Sulfate Groups

Journal of Biological Chemistry, 2004

Research paper thumbnail of Biosynthetic Oligosaccharide Libraries for Identification of Protein-binding Heparan Sulfate Motifs. EXPLORING THE STRUCTURAL DIVERSITY BY SCREENING FOR FIBROBLAST GROWTH FACTOR (FGF) 1 AND FGF2 BINDING

Journal of Biological Chemistry, 2002

Heparan sulfate is crucial for vital reactions in the body because of its ability to bind various... more Heparan sulfate is crucial for vital reactions in the body because of its ability to bind various proteins. The identification of protein-binding heparan sulfate sequences is essential to our understanding of heparan sulfate biology and raises the possibility to develop drugs against diseases such as cancer and inflammatory conditions. We present proof-of-principle that in vitro generated heparan sulfate oligosaccharide libraries can be used to explore interactions between heparan sulfate and proteins, and that the libraries expand the available heparan sulfate sequence space. Oligosaccharide libraries mimicking highly 6-O-sulfated domains of heparan sulfate were constructed by enzymatic O-sulfation of O-desulfated, end-group (3)H-labeled heparin octasaccharides. Acceptor oligosaccharides that were 6-O-desulfated but only partially 2-O-desulfated yielded oligosaccharide arrays with increased ratio of iduronyl 2-O-sulfate/glucosaminyl 6-O-sulfate. The products were probed by affinity chromatography on immobilized growth factors, fibroblast growth factor-1 (FGF1) and FGF2, followed by sequence analysis of trapped oligosaccharides. An N-sulfated octasaccharide, devoid of 2-O-sulfate but with three 6-O-sulfate groups, was unexpectedly found to bind FGF1 as well as FGF2 at physiological ionic strength. However, a single 2-O-sulfate group in the absence of 6-O-sulfation gave higher affinity for FGF2. FGF1 binding was also augmented by 2-O-sulfation, preferentially in combination with an adjacent upstream 6-O-sulfate group. These results demonstrate the potential of the enzymatically generated oligosaccharide libraries.

Research paper thumbnail of Drosophila Heparan Sulfate, a Novel Design

Journal of Biological Chemistry, 2012

Research paper thumbnail of Regulated Diversity of Heparan Sulfate

Journal of Biological Chemistry, 1998

... replaces the N-bound acetyl group of the GlcNAc residue with a sulfate group, presents at ...... more ... replaces the N-bound acetyl group of the GlcNAc residue with a sulfate group, presents at ... the NDST reaction as well as further downstream modifications, hence the structural diversity of HS ... biological functions of HS but also of its biosynthesis, in particular the roles of enzyme ...

Research paper thumbnail of rib-2, a Caenorhabditis elegans Homolog of the Human Tumor Suppressor EXT Genes Encodes a Novel alpha 1,4-N-Acetylglucosaminyltransferase Involved in the Biosynthetic Initiation and Elongation of Heparan Sulfate

Journal of Biological Chemistry, 2001

Research paper thumbnail of Heparan Sulfate: Biosynthesis, Structure, and Function

International Review of Cell and Molecular Biology, 2016

Research paper thumbnail of Presence of N-Unsubstituted Glucosamine Units in Native Heparan Sulfate Revealed by a Monoclonal Antibody

Journal of Biological Chemistry, 1995

Research paper thumbnail of Enzymatically Active N-Deacetylase/N-Sulfotransferase-2 Is Present in Liver but Does Not Contribute to Heparan Sulfate N-Sulfation

Journal of Biological Chemistry, 2006

Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with grow... more Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1-/- mice was drastically lowered, liver HS from wild-type mice, from NDST1+/-, NDST2-/-, and NDST1+/- / NDST2-/- mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1+/- / NDST2-/- embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1-/-, and NDST1+/- embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (approximately 40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1-/- embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.

Research paper thumbnail of Mutation in the Heparan Sulfate Biosynthesis Enzyme EXT1 Influences Growth Factor Signaling and Fibroblast Interactions with the Extracellular Matrix

Journal of Biological Chemistry, 2009

Heparan sulfate (HS) chains bind and modulate the signaling efficiency of many ligands, including... more 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.

Research paper thumbnail of Contribution of EXT1, EXT2, and EXTL3 to Heparan Sulfate Chain Elongation

Journal of Biological Chemistry, 2007

The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate bios... more 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.

Research paper thumbnail of Substrate Specificity and Domain Functions of Extracellular Heparan Sulfate 6-O-Endosulfatases, QSulf1 and QSulf2

Journal of Biological Chemistry, 2006

Research paper thumbnail of Stem cell factor-dependent human cord blood derived mast cells express α- and β-tryptase, heparin and chondroitin sulphate

Research paper thumbnail of cDNA cloning and expression of UDP-glucose dehydrogenase from bovine kidney

Glycobiology, 1999

We have isolated a cDNA encoding UDP-glucose dehydrogenase from a bovine kidney cDNA-library, the... more We have isolated a cDNA encoding UDP-glucose dehydrogenase from a bovine kidney cDNA-library, the first mammalian cDNA clone published. [After submission of the manuscript, a study appeared describing the molecular cloning and characterization of the human and mouse UDP-glucose dehydrogenase genes (Spicer et al., 1998).] The enzyme catalyzes the conversion of UDP-glucose to UDP-glucuronic acid, an essential precursor in glycosaminoglycan biosynthesis. The cDNA has an open reading frame of 1482 nucleotides coding for a 55 kDa protein. Expression of the enzyme in COS-7 cells showed a 3-fold increase in UDP-glucose dehydrogenase activity; also, the C-terminal 23 amino acids was shown not to be necessary for enzyme activity. Northern blots from human and mouse tissues reveal high expression in liver and low in skeletal muscle. Human tissues have a major transcript size of 3.2 kilobases and a minor of 2.6 whereas mouse tissues have a single 2.6 kilobase transcript. We have also developed a sensitive and direct assay using UDP-[14C]Glc as a substrate for detection of small amounts of UDPGDH activity.

Research paper thumbnail of Target selection of heparan sulfate hexuronic acid 2-O-sulfotransferase

Glycobiology, 2010

The signaling of various molecules involved in development and regulation of cell growth are regu... more The signaling of various molecules involved in development and regulation of cell growth are regulated by heparan sulfate (HS). Specific binding of HS to ligand proteins depends on the HS sulfation pattern, where the spacing and number of O-sulfate groups are of special importance. HS 2-O-sulfotransferase catalyzes 2-O-sulfation of glucuronic and iduronic acid residues with a 5-fold higher preference for iduronic acid, as inferred from previously determined kinetic parameters. To study in more detail the regulation of HS hexuronic acid 2-O-sulfation, we tested the ability of the enzyme to catalyze glucuronic acid 2-O-sulfation in polysaccharide mixtures with different glucuronic acid/iduronic acid ratios, using 3'-phosphoadenosine 5'-phospho[(35)S]sulfate as sulfate donor. The 2-O-sulfotransferase revealed a more pronounced preference for 2-O-sulfation of iduronic acid than predicted. Even incubations with a 99:1 ratio of glucuronic acid to iduronic acid resulted in almost exclusive iduronic acid 2-O-sulfation. Unexpectedly, when the 2-O-sulfotransferase was co-immunoprecipitated with the glucuronyl C5-epimerase (that converts glucuronic acid to iduronic acid), both glucuronic acid and iduronic acid residues were sulfated to the same extent when a polysaccharide containing only glucuronic acid was used as a substrate. Attempting to understand the mechanism by which extended regions of iduronic acid 2-O-sulfation are formed during HS biosynthesis, a (3)H-labeled N-sulfated iduronic acid containing octasaccharide substrate was incubated with the 2-O-sulfotransferase and 3'-phosphoadenosine 5'-phosphosulfate. The 2-O-sulfotransferase showed a preference for mono-2-O-sulfated substrates as compared with octasaccharides with no 2-O-sulfate group.

Research paper thumbnail of Novel Drosophila laminin A chain reveals structural relationships between laminin subunits

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1993

Laminins are trimeric glycoproteins composed of A, B1, and B2 chains that play important roles in... more Laminins are trimeric glycoproteins composed of A, B1, and B2 chains that play important roles in cell adhesion and differentiation and in the assembly of basement membranes. There is evidence that multiple independent gene products can be used to supply the A, B1, and B2-like chains, thereby generating diverse laminin molecules. The set of A chain-like polypeptides are the most divergent. The amino-terminal portion of each chain, called the short arm, is composed of alternating cysteine-rich and globular domains. The amino-terminal two-thirds of the newly characterized Drosophila laminin A chain appears to encode a novel structure, but the analysis presented here shows that substantial portions of its amino acid sequence are related to sequences found in other A, B1, and B2 chains. A portion of the Drosophila A chain is composed of sequences like those found in B2 chains. These sequences parallel a portion of the vertebrate A chains, suggesting a structure for a shared ancestral A ...

Research paper thumbnail of Phenotypic characterization of the human mast-cell line HMC-1

Scandinavian journal of immunology, 1994

The cell line HMC-1, derived from a patient with mast cell leukaemia, is the only established cel... more The cell line HMC-1, derived from a patient with mast cell leukaemia, is the only established cell line exhibiting a phenotype similar to that of human mast cells. This paper reports on a detailed characterization of the expression of a panel of markers for various types of immature and mature haematopoietic cells in the HMC-1. We also studied the potential of HMC-1 to differentiate upon treatment with conditioned media from the human T-cell line Mo, retinoic acid or DMSO. HMC-1 was found to express several mast cell-related markers. A high expression of Kit, the receptor for stem-cell factor, was detected. The majority of the cells were stained with a MoAb against the mast cell-specific serine protease tryptase. Of particular interest was the finding that beta-tryptase mRNA, but not alpha-tryptase mRNA, was expressed in HMC-1. Using enzyme-histochemistry we were able to show that the beta-tryptase was enzymatically active, indicating that tryptase can form active homotetramers. Bot...

Research paper thumbnail of Laminin A chain: expression during Drosophila development and genomic sequence

The EMBO journal, 1992

A Drosophila laminin A chain gene was characterized as a 14 kb genomic nucleotide sequence which ... more A Drosophila laminin A chain gene was characterized as a 14 kb genomic nucleotide sequence which encodes an open reading frame of 3712 amino acids in 15 exons. Overall, this A chain is similar to its vertebrate counterparts, especially in its N- and C-terminal globular domains, but the sequence that forms the laminin A short arm is quite different and larger. Laminin messages appear in newly formed mesoderm and are later prominently expressed in hemocytes, which also synthesize basement membrane collagen IV. The composition of Drosophila basement membranes changes with development. A novel method of tandemly fused RNA probes showed that developmental increases of laminin mRNAs were primarily associated with periods of morphogenesis, and preceded those of collagen IV, a protein strongly expressed during growth. The ratio of A:B1:B2 mRNAs varied little during embryogenesis, with less mRNA for A than B chains. Staining of embryos with antibodies confirmed and extended the information p...

Research paper thumbnail of Phenotypic characterization of mice lacking genes coding for NDSTs, heparan sulfate glucos-aminyl N-deacetylase/N-sulfo-transferase

Research paper thumbnail of Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation

Proceedings of the National Academy of Sciences, 2008

Research paper thumbnail of Biosynthesis of Hyaluronan: DIRECTION OF CHAIN ELONGATION

Journal of Biological Chemistry, 2005

Hyaluronan (HA), a functionally essential glycosaminoglycan in vertebrate tissues and a putative ... more Hyaluronan (HA), a functionally essential glycosaminoglycan in vertebrate tissues and a putative virulence factor in certain pathogenic bacteria, is an extended linear polymer composed of alternating units of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). Uncertainty regarding the mechanism of HA biosynthesis has included the directionality of chain elongation, i.e. whether addition of monosaccharide units occurs at the reducing or non-reducing terminus of nascent chains. We have investigated this problem using yeast-derived recombinant HA synthases from Xenopus laevis (xlHAS1) and from Streptococcus pyogenes (spHAS). The enzymes were incubated with UDP-[3H]GlcUA and UDP-[14C]GlcNAc, under experimental conditions designed to yield HA chains with differentially labeled reducing-terminal and non-reducing terminal domains. Digestion of the products with a mixture of beta-glucuronidase and beta-N-acetylglucosaminidase exoenzymes resulted in truncation of the HA chain strictly from the non-reducing end and release of labeled monosaccharides. The change in 3H/14C ratio of the monosaccharide fraction, during the course of exoglycosidase digestion, was interpreted to indicate whether sugar units had been added at the reducing or non-reducing end. The results demonstrate that the vertebrate xlHAS1 and the bacterial spHAS extend HA in opposite directions. Chain elongation catalyzed by xlHAS1 occurs at the non-reducing end of the HA chain, whereas elongation catalyzed by spHAS occurs at the reducing end. The spHAS is the first glycosyltransferase that has been unanimously demonstrated to function at the reducing end of a growing glycosaminoglycan chain.

Research paper thumbnail of Heparan Sulfate Synthesized by Mouse Embryonic Stem Cells Deficient in NDST1 and NDST2 Is 6-O-Sulfated but Contains No N-Sulfate Groups

Journal of Biological Chemistry, 2004

Research paper thumbnail of Biosynthetic Oligosaccharide Libraries for Identification of Protein-binding Heparan Sulfate Motifs. EXPLORING THE STRUCTURAL DIVERSITY BY SCREENING FOR FIBROBLAST GROWTH FACTOR (FGF) 1 AND FGF2 BINDING

Journal of Biological Chemistry, 2002

Heparan sulfate is crucial for vital reactions in the body because of its ability to bind various... more Heparan sulfate is crucial for vital reactions in the body because of its ability to bind various proteins. The identification of protein-binding heparan sulfate sequences is essential to our understanding of heparan sulfate biology and raises the possibility to develop drugs against diseases such as cancer and inflammatory conditions. We present proof-of-principle that in vitro generated heparan sulfate oligosaccharide libraries can be used to explore interactions between heparan sulfate and proteins, and that the libraries expand the available heparan sulfate sequence space. Oligosaccharide libraries mimicking highly 6-O-sulfated domains of heparan sulfate were constructed by enzymatic O-sulfation of O-desulfated, end-group (3)H-labeled heparin octasaccharides. Acceptor oligosaccharides that were 6-O-desulfated but only partially 2-O-desulfated yielded oligosaccharide arrays with increased ratio of iduronyl 2-O-sulfate/glucosaminyl 6-O-sulfate. The products were probed by affinity chromatography on immobilized growth factors, fibroblast growth factor-1 (FGF1) and FGF2, followed by sequence analysis of trapped oligosaccharides. An N-sulfated octasaccharide, devoid of 2-O-sulfate but with three 6-O-sulfate groups, was unexpectedly found to bind FGF1 as well as FGF2 at physiological ionic strength. However, a single 2-O-sulfate group in the absence of 6-O-sulfation gave higher affinity for FGF2. FGF1 binding was also augmented by 2-O-sulfation, preferentially in combination with an adjacent upstream 6-O-sulfate group. These results demonstrate the potential of the enzymatically generated oligosaccharide libraries.

Research paper thumbnail of Drosophila Heparan Sulfate, a Novel Design

Journal of Biological Chemistry, 2012

Research paper thumbnail of Regulated Diversity of Heparan Sulfate

Journal of Biological Chemistry, 1998

... replaces the N-bound acetyl group of the GlcNAc residue with a sulfate group, presents at ...... more ... replaces the N-bound acetyl group of the GlcNAc residue with a sulfate group, presents at ... the NDST reaction as well as further downstream modifications, hence the structural diversity of HS ... biological functions of HS but also of its biosynthesis, in particular the roles of enzyme ...

Research paper thumbnail of rib-2, a Caenorhabditis elegans Homolog of the Human Tumor Suppressor EXT Genes Encodes a Novel alpha 1,4-N-Acetylglucosaminyltransferase Involved in the Biosynthetic Initiation and Elongation of Heparan Sulfate

Journal of Biological Chemistry, 2001

Research paper thumbnail of Heparan Sulfate: Biosynthesis, Structure, and Function

International Review of Cell and Molecular Biology, 2016

Research paper thumbnail of Presence of N-Unsubstituted Glucosamine Units in Native Heparan Sulfate Revealed by a Monoclonal Antibody

Journal of Biological Chemistry, 1995

Research paper thumbnail of Enzymatically Active N-Deacetylase/N-Sulfotransferase-2 Is Present in Liver but Does Not Contribute to Heparan Sulfate N-Sulfation

Journal of Biological Chemistry, 2006

Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with grow... more Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1-/- mice was drastically lowered, liver HS from wild-type mice, from NDST1+/-, NDST2-/-, and NDST1+/- / NDST2-/- mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1+/- / NDST2-/- embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1-/-, and NDST1+/- embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (approximately 40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1-/- embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.

Research paper thumbnail of Mutation in the Heparan Sulfate Biosynthesis Enzyme EXT1 Influences Growth Factor Signaling and Fibroblast Interactions with the Extracellular Matrix

Journal of Biological Chemistry, 2009

Heparan sulfate (HS) chains bind and modulate the signaling efficiency of many ligands, including... more 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.

Research paper thumbnail of Contribution of EXT1, EXT2, and EXTL3 to Heparan Sulfate Chain Elongation

Journal of Biological Chemistry, 2007

The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate bios... more 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.

Research paper thumbnail of Substrate Specificity and Domain Functions of Extracellular Heparan Sulfate 6-O-Endosulfatases, QSulf1 and QSulf2

Journal of Biological Chemistry, 2006

Research paper thumbnail of Stem cell factor-dependent human cord blood derived mast cells express α- and β-tryptase, heparin and chondroitin sulphate

Research paper thumbnail of cDNA cloning and expression of UDP-glucose dehydrogenase from bovine kidney

Glycobiology, 1999

We have isolated a cDNA encoding UDP-glucose dehydrogenase from a bovine kidney cDNA-library, the... more We have isolated a cDNA encoding UDP-glucose dehydrogenase from a bovine kidney cDNA-library, the first mammalian cDNA clone published. [After submission of the manuscript, a study appeared describing the molecular cloning and characterization of the human and mouse UDP-glucose dehydrogenase genes (Spicer et al., 1998).] The enzyme catalyzes the conversion of UDP-glucose to UDP-glucuronic acid, an essential precursor in glycosaminoglycan biosynthesis. The cDNA has an open reading frame of 1482 nucleotides coding for a 55 kDa protein. Expression of the enzyme in COS-7 cells showed a 3-fold increase in UDP-glucose dehydrogenase activity; also, the C-terminal 23 amino acids was shown not to be necessary for enzyme activity. Northern blots from human and mouse tissues reveal high expression in liver and low in skeletal muscle. Human tissues have a major transcript size of 3.2 kilobases and a minor of 2.6 whereas mouse tissues have a single 2.6 kilobase transcript. We have also developed a sensitive and direct assay using UDP-[14C]Glc as a substrate for detection of small amounts of UDPGDH activity.

Research paper thumbnail of Target selection of heparan sulfate hexuronic acid 2-O-sulfotransferase

Glycobiology, 2010

The signaling of various molecules involved in development and regulation of cell growth are regu... more The signaling of various molecules involved in development and regulation of cell growth are regulated by heparan sulfate (HS). Specific binding of HS to ligand proteins depends on the HS sulfation pattern, where the spacing and number of O-sulfate groups are of special importance. HS 2-O-sulfotransferase catalyzes 2-O-sulfation of glucuronic and iduronic acid residues with a 5-fold higher preference for iduronic acid, as inferred from previously determined kinetic parameters. To study in more detail the regulation of HS hexuronic acid 2-O-sulfation, we tested the ability of the enzyme to catalyze glucuronic acid 2-O-sulfation in polysaccharide mixtures with different glucuronic acid/iduronic acid ratios, using 3'-phosphoadenosine 5'-phospho[(35)S]sulfate as sulfate donor. The 2-O-sulfotransferase revealed a more pronounced preference for 2-O-sulfation of iduronic acid than predicted. Even incubations with a 99:1 ratio of glucuronic acid to iduronic acid resulted in almost exclusive iduronic acid 2-O-sulfation. Unexpectedly, when the 2-O-sulfotransferase was co-immunoprecipitated with the glucuronyl C5-epimerase (that converts glucuronic acid to iduronic acid), both glucuronic acid and iduronic acid residues were sulfated to the same extent when a polysaccharide containing only glucuronic acid was used as a substrate. Attempting to understand the mechanism by which extended regions of iduronic acid 2-O-sulfation are formed during HS biosynthesis, a (3)H-labeled N-sulfated iduronic acid containing octasaccharide substrate was incubated with the 2-O-sulfotransferase and 3'-phosphoadenosine 5'-phosphosulfate. The 2-O-sulfotransferase showed a preference for mono-2-O-sulfated substrates as compared with octasaccharides with no 2-O-sulfate group.