👤 M Kusche-Gullberg

NDST1 (glucosaminyl N-deacetylase/N-sulfotransferase) is a key enzyme in heparan sulfate (HS) biosynthesis, where it is responsible for HS N-deacetylation and N-sulfation. In addition to the full length human enzyme of 882 amino acids, here designated NDST1A, a shorter form containing 825 amino acids (NDST1B) is synthesized after alternative splicing of the NDST1 mRNA. NDST1B is mostly expressed at a low level, but increased amounts are seen in several types of cancer where it is associated with shorter survival. In this study, we aimed at characterizing the enzymatic properties of NDST1B and its effect on HS biosynthesis. Purified recombinant NDST1B lacked both N-deacetylase and N-sulfotransferase activities. Interestingly, HEK293 cells overexpressing NDST1B synthesized HS with reduced sulfation and altered domain structure. Fluorescence resonance energy transfer-microscopy demonstrated that both NDST1A and NDST1B had the capacity to interact with the HS copolymerase subunits EXT1 and EXT2 and also to form NDST1A/NDST1B dimers. Since lysates from cells overexpressing NDST1B contained less NDST enzyme activity than control cells, we suggest that NDST1B works in a dominant negative manner, tentatively by replacing the active endogenous NDST1 in the enzyme complexes taking part in biosynthesis. Show less

Breast cancer remains a leading cause of cancer-related mortality in women. In recent years, regulation of genes involved in heparan sulphate (HS) biosynthesis have received increased interest as regulators of breast cancer cell adhesion and invasion. The exostosin (EXT) proteins are glycosyltransferases involved in elongation of HS, a regulator of intracellular signaling, cell-cell interactions, and tissue morphogenesis. The EXT family contains five members: EXT1, EXT2, and three EXT-like (EXTL) members: EXTL1, EXTL2, and EXTL3. While the expression levels of these enzymes change in tumor cells, little is known how this changes the structure and function of HS. In the present study, we investigated gene expression profiles of the EXT family members, their glycosyltransferase activities and HS structure in the estrogen receptor (ER), and progesterone receptor (PR) positive MCF7 cells, and the ER, PR, and human epidermal growth factor receptor-2 (HER2) negative MDA-MB-231 and HCC38 epithelial breast carcinoma cell lines. The gene expression profiles for MDA-MB-231 and HCC38 cells were very similar. In both cell lines Show less

Exostosin-1 (EXT1), a member of the EXT protein family, is indispensable for synthesis of heparan sulfate (HS) chains that bind to and modulate the signaling efficiency of numerous growth factor activities. We have previously shown that Ext1 mutated mouse embryonic fibroblasts produce short sulfated HS chains which dramatically influence tumor cell behavior in a 3-dimensional (3D) heterospheroid system composed of tumor cells and fibroblasts. In this study, we have used both 2D co-culture and 3D heterospheroid models, consisting of human A549 carcinoma cells co-cultured with wild-type or Ext1-mutated mouse embryonic fibroblasts. Gene expression profiling of differentially expressed genes in fibroblast/A549 heterospheroids identified P311 as a gene substantially down-regulated in A549 cells co-cultured with Ext1-mutated fibroblasts. In addition, we observed that the Ext1 mutants displayed reduced Tgf-β1 mRNA levels and lower levels of secreted active TGF-β protein. Re-introduction of Ext1 in the Ext1 mutant fibroblasts rescued the levels of Tgf-β1 mRNA, increased the amounts of secreted active TGF-β in these cells, as well as P311 mRNA levels in adjacent A549 cells. Accordingly, small interfering RNAs (siRNAs) against fibroblast Tgf-β1 reduced P311 expression in neighboring A549 tumor cells. Our data raises the possibility that fibroblast Ext1 levels play a role in P311 expression in A549/fibroblast co-culture through TGF-β1. This study considers a possible novel mechanism of Ext1-regulated heparan sulfate structure in modifying tumor-stroma interactions through altering stromal tgf-ß1 expression. Show less

Heparan sulfate proteoglycans are ubiquitously located on cell surfaces and in the extracellular matrices. The negatively charged heparan sulfate chains interact with a multitude of different proteins, thereby influencing a variety of cellular and developmental processes, for example cell adhesion, migration, tissue morphogenesis, and differentiation. The human exostosin (EXT) family of genes contains five members: the heparan sulfate polymerizing enzymes, EXT1 and EXT2, and three EXT-like genes, EXTL1, EXTL2, and EXTL3. EXTL2 has been ascribed activities related to the initiation and termination of heparan sulfate chains. Here we further investigated the role of EXTL2 in heparan sulfate chain elongation by gene silencing and overexpression strategies. We found that siRNA-mediated knockdown of EXTL2 in human embryonic kidney 293 cells resulted in increased chain length, whereas overexpression of EXTL2 in the same cell line had little or no effect on heparan sulfate chain length. To study in more detail the role of EXTL2 in heparan sulfate chain elongation, we tested the ability of the overexpressed protein to catalyze the in vitro incorporation of N-acetylglucosamine and N-acetylgalactosamine to oligosaccharide acceptors resembling unmodified heparan sulfate and chondroitin sulfate precursor molecules. Analysis of the generated products revealed that recombinant EXTL2 showed weak ability to transfer N-acetylgalactosamine to heparan sulfate precursor molecules but also, that EXTL2 exhibited much stronger in vitro N-acetylglucosamine-transferase activity related to elongation of heparan sulfate chains. Show less

Heparan sulfates are complex sulfated molecules found in abundance at cell surfaces and in the extracellular matrix. They bind to and influence the activity of a variety of molecules like growth factors, proteases and morphogens and are thus involved in various cell-cell and cell-matrix interactions. The mammalian EXT proteins have glycosyltransferase activities relevant for HS chain polymerization, however their exact role in this process is still confusing. In this review, we summarize current knowledge about the biochemical activities and some proposed functions of the members of the EXT protein family and their roles in human disease. Show less

Stromal fibroblasts are important determinants of tumor cell behavior. They act to condition the tumor microenvironment, influence tumor growth, support tumor angiogenesis and affect tumor metastasis. Heparan sulfate proteoglycans, present both on tumor and stromal cells, interact with a large number of ligands including growth factors, their receptors, and structural components of the extracellular matrix. Being ubiquitously expressed in the tumor microenvironment heparan sulfate proteoglycans are candidates for playing central roles in tumor-stroma interactions. The objective of this work was to investigate the role of heparan sulfate expressed by stromal fibroblasts in modulating the growth of tumor cells and in controlling the interstitial fluid pressure in a 3-D model. We generated spheroids composed of fibroblasts alone, or composite spheroids, composed of fibroblasts and tumor cells. Here we show that stromal fibroblasts with a mutation in the heparan sulfate elongating enzyme Ext1 and thus a low heparan sulfate content, formed composite fibroblast/tumor cell spheroids with a significant lower interstitial fluid pressure than corresponding wild-type fibroblast/tumor cell composite spheroids. Furthermore, immunohistochemistry of composite spheroids revealed that the cells segregated, so that after 6 days in culture, the wild-type fibroblasts formed an inner core and the tumor cells an outer layer of cells. For composite spheroids containing Ext1-mutated fibroblasts this segregation was less obvious, indicating impaired cell migration. Analysis of tumor cells expressing the firefly luciferase gene revealed that the changes in tumor cell migration in mutant fibroblast/tumor cell composite spheroids coincided with a lower proliferation rate. This is the first demonstration that stromal Ext1-levels modulate tumor cell proliferation and affect the interstitial fluid pressure in a 3-D spheroid model. Learning how structural changes in stromal heparan sulfate influence tumor cells is essential for our understanding how non-malignant cells of the tumor microenvironment influence tumor cell progression. Show less

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. Show less

HS (heparan sulfate) proteoglycans are key regulators of vital processes in the body. HS chains with distinct sequences bind to various protein ligands, such as growth factors and morphogens, and thereby function as important regulators of protein gradient formation and signal transduction. HS is synthesized through the concerted action of many different ER (endoplasmic reticulum) and Golgi-resident enzymes. In higher organisms, many of these enzymes occur in multiple isoforms that differ in substrate specificity and spatial and temporal expression. In order to investigate how the structural complexity of HS has evolved, in the present study we focused on the starlet sea anemone (Nematostella vectensis), which belongs to the Anthozoa, which are considered to have retained many ancestral features. Members of all of the enzyme families involved in the generation and modification of HS were identified in Nematostella. Our results show that the enzymes are highly conserved throughout evolution, but the number of isoforms varies. Furthermore, the HS polymerases [Ext (exostosin) enzymes Ext1, Ext2 and Ext-like3] represent distinct subgroups, indicating that these three genes have already been present in the last common ancestor of Cnidaria and Bilateria. In situ hybridization showed up-regulation of certain enzymes in specific areas of the embryo at different developmental stages. The specific mRNA expression pattern of particular HS enzymes implies that they may play a specific role in HS modifications during larval development. Finally, biochemical analysis of Nematostella HS demonstrates that the sea anemone synthesizes a polysaccharide with a unique structure. Show less

Heparan sulfate (HS) proteoglycans influence embryonic development and adult physiology through interactions with protein ligands. The interactions depend on HS structure, which is determined largely during biosynthesis by Golgi enzymes. How biosynthesis is regulated is more or less unknown. During polymerization of the HS chain, carried out by a complex of the exostosin proteins EXT1 and EXT2, the first modification enzyme, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), introduces N-sulfate groups into the growing polymer. Unexpectedly, we found that the level of expression of EXT1 and EXT2 affected the amount of NDST1 present in the cell, which, in turn, greatly influenced HS structure. Whereas overexpression of EXT2 in HEK 293 cells enhanced NDST1 expression, increased NDST1 N-glycosylation, and resulted in elevated HS sulfation, overexpression of EXT1 had opposite effects. Accordingly, heart tissue from transgenic mice overexpressing EXT2 showed increased NDST activity. Immunoprecipitaion experiments suggested an interaction between EXT2 and NDST1. We speculate that NDST1 competes with EXT1 for binding to EXT2. Increased NDST activity in fibroblasts with a gene trap mutation in EXT1 supports this notion. These results support a model in which the enzymes of HS biosynthesis form a complex, or a GAGosome. Show less

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. Show less

Mutational defects in either EXT1 or EXT2 genes cause multiple exostoses, an autosomal hereditary human disorder. The EXT1 and EXT2 genes encode glycosyltransferases that play an essential role in heparan sulfate chain elongation. In this study, we have analyzed heparan sulfate synthesized by primary fibroblast cell cultures established from mice with a gene trap mutation in Ext1. The gene trap mutation results in embryonic lethality, and homozygous mice die around embryonic day 14. Metabolic labeling and immunohistochemistry revealed that Ext1 mutant fibroblasts still produced small amounts of heparan sulfate. The domain structure of the mutant heparan sulfate was conserved, and the disaccharide composition was similar to that of wild type heparan sulfate. However, a dramatic difference was seen in the polysaccharide chain length. The average molecular sizes of the heparan sulfate chains from wild type and Ext1 mutant embryonic fibroblasts were estimated to be around 70 and 20 kDa, respectively. These data suggest that not only the sulfation pattern but also the length of the heparan sulfate chains is a critical determinant of normal mouse development. Show less

Multiple exosotoses is a dominantly inherited bone disorder caused by defects in EXT1 and EXT2, genes encoding glycosyltransferases involved in heparan sulfate chain elongation. Heparan sulfate polymerization occurs by the alternating addition of glucuronic acid and N-acetylglucosamine units to the nonreducing end of the polysaccharide. EXT1 and EXT2 are suggested to be dual glucuronyl/N-acetylglucosaminyltransferases, and a heterooligomeric complex of EXT1 and EXT2 (EXT1/2) is considered to be the biological functional polymerization unit. Here, we have investigated the in vitro polymerization capacities of recombinant soluble EXT1, EXT2, and EXT1/2 complex on exogenous oligosaccharide acceptors derived from Escherichia coli K5 capsular polysaccharide. Incubations of recombinant EXT1 or EXT1/2 complex with 3H-labeled oligosaccharide acceptors and the appropriate nucleotide sugars resulted in conversion of the acceptors to higher molecular weight compounds but with different efficacies for EXT1 and EXT1/2. In contrast, incubations with recombinant EXT2 resulted in the addition of a single glucuronic acid but no further polymerization. These results indicate that EXT1 alone and the EXT1/2 heterocomplex can act as heparan sulfate polymerases in vitro without the addition of additional auxiliary proteins. Show less

The tumor suppressors EXT1 and EXT2 are associated with hereditary multiple exostoses and encode bifunctional glycosyltransferases essential for chain polymerization of heparan sulfate (HS) and its analog, heparin (Hep). Three highly homologous EXT-like genes, EXTL1-EXTL3, have been cloned, and EXTL2 is an alpha1,4-GlcNAc transferase I, the key enzyme that initiates the HS/Hep synthesis. In the present study, truncated forms of EXTL1 and EXTL3, lacking the putative NH2-terminal transmembrane and cytoplasmic domains, were transiently expressed in COS-1 cells and found to harbor alpha-GlcNAc transferase activity. EXTL3 used not only N-acetylheparosan oligosaccharides that represent growing HS chains but also GlcAbeta1-3Galbeta1-O-C2H4NH-benzyloxycarbonyl (Cbz), a synthetic substrate for alpha-GlcNAc transferase I that determines and initiates HS/Hep synthesis. In contrast, EXTL1 used only the former acceptor. Neither EXTL1 nor EXTL3 showed any glucuronyltransferase activity as examined with N-acetylheparosan oligosaccharides. Heparitinase I digestion of each transferase-reaction product showed that GlcNAc had been transferred exclusively through an alpha1,4-configuration. Hence, EXTL3 most likely is involved in both chain initiation and elongation, whereas EXTL1 possibly is involved only in the chain elongation of HS and, maybe, Hep as well. Thus, their acceptor specificities of the five family members are overlapping but distinct from each other, except for EXT1 and EXT2 with the same specificity. It now has been clarified that all of the five cloned human EXT gene family proteins harbor glycosyltransferase activities, which probably contribute to the synthesis of HS and Hep. Show less

The proteins encoded by the EXT1, EXT2, and EXTL2 genes, members of the hereditary multiple exostoses gene family of tumor suppressors, are glycosyltransferases required for the heparan sulfate biosynthesis. Only two homologous genes, rib-1 and rib-2, of the mammalian EXT genes were identified in the Caenorhabditis elegans genome. Although heparan sulfate is found in C. elegans, the involvement of the rib-1 and rib-2 proteins in heparan sulfate biosynthesis remains unclear. In the present study, the substrate specificity of a soluble recombinant form of the rib-2 protein was determined and compared with those of the recombinant forms of the mammalian EXT1, EXT2, and EXTL2 proteins. The present findings revealed that the rib-2 protein was a unique alpha1,4-N-acetylglucosaminyltransferase involved in the biosynthetic initiation and elongation of heparan sulfate. In contrast, the findings confirmed the previous observations that both the EXT1 and EXT2 proteins were heparan sulfate copolymerases with both alpha1,4-N-acetylglucosaminyltransferase and beta1,4-glucuronyltransferase activities, which are involved only in the elongation step of the heparan sulfate chain, and that the EXTL2 protein was an alpha1,4-N-acetylglucosaminyltransferase involved only in the initiation of heparan sulfate synthesis. These findings suggest that the biosynthetic mechanism of heparan sulfate in C. elegans is distinct from that reported for the mammalian system. Show less

The D-glucuronyltransferase and N-acetyl-D-glucosaminyltransferase reactions in heparan sulfate biosynthesis have been associated with two genes, EXT1 and EXT2, which are also implicated in the inherited bone disorder, multiple exostoses. Since the cell systems used to express recombinant EXT proteins synthesize endogenous heparan sulfate, and the EXT proteins tend to associate, it has not been possible to define the functional roles of the individual protein species. We therefore expressed EXT1 and EXT2 in yeast, which does not synthesize heparan sulfate. The recombinant EXT1 and EXT2 were both found to catalyze both glycosyltransferase reactions in vitro. Coexpression of the two proteins, but not mixing of separately expressed recombinant EXT1 and EXT2, yields hetero-oligomeric complexes in yeast and mammalian cells, with augmented glycosyltransferase activities. This stimulation does not depend on the membrane-bound state of the proteins. Show less