A dominant negative splice variant of the heparan sulfate biosynthesis enzyme NDST1 reduces heparan sulfate sulfation.

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.

Endothelial heparan sulfate deficiency reduces inflammation and fibrosis in murine diabetic nephropathy.

Inflammation plays a vital role in the development of diabetic nephropathy, but the underlying regulatory mechanisms are only partially understood. Our previous studies demonstrated that, during acute inflammation, endothelial heparan sulfate (HS) contributes to the adhesion and transendothelial migration of leukocytes into perivascular tissues by direct interaction with L-selectin and the presentation of bound chemokines. In the current study, we aimed to assess the role of endothelial HS on chronic renal inflammation and fibrosis in a diabetic nephropathy mouse model. To reduce sulfation of HS specifically in the endothelium, we generated Ndst1 f/f Tie2Cre + mice in which N-deacetylase/N-sulfotransferase-1 (Ndst1), the gene that initiates HS sulfation modifications in HS biosynthesis, was expressly ablated in endothelium. To induce diabetes, age-matched male Ndst1 f/f Tie2Cre - (wild type) and Ndst1 f/f Tie2Cre + mice on a C57Bl/6J background were injected intraperitoneally with streptozotocin (STZ) (50 mg/kg) on five consecutive days (N = 10-11/group). Urine and plasma were collected. Four weeks after diabetes induction the animals were sacrificed and kidneys were analyzed by immunohistochemistry and qRT-PCR. Compared to healthy controls, diabetic Ndst1 f/f Tie2Cre - mice showed increased glomerular macrophage infiltration, mannose binding lectin complement deposition and glomerulosclerosis, whereas these pathological reactions were prevented significantly in the diabetic Ndst1 f/f Tie2Cre + animals (all three p < 0.01). In addition, the expression of the podocyte damage marker desmin was significantly higher in the Ndst1 f/f Tie2Cre - group compared to the Ndst1 f/f Tie2Cre + animals (p < 0.001), although both groups had comparable numbers of podocytes. In the cortical tubulo-interstitium, similar analyses show decreased interstitial macrophage accumulation in the diabetic Ndst1 f/f Tie2Cre + animals compared to the diabetic Ndst1 f/f Tie2Cre - mice (p < 0.05). Diabetic Ndst1 f/f Tie2Cre + animals also showed reduced interstitial fibrosis as evidenced by reduced density of αSMA-positive myofibroblasts (p < 0.01), diminished collagen III deposition (p < 0.001) and reduced mRNA expression of collagen I (p < 0.001) and fibronectin (p < 0.001). Our studies indicate a pivotal role of endothelial HS in the development of renal inflammation and fibrosis in diabetic nephropathy in mice. These results suggest that HS is a possible target for therapy in diabetic nephropathy.

Potential role for Ext1-dependent heparan sulfate in regulating P311 gene expression in A549 carcinoma cells.

BACKGROUND: 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. METHODS: 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. RESULTS AND CONCLUSIONS: 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-ß1 mRNA levels and lower levels of secreted active TGF-ß protein. Re-introduction of Ext1 in the Ext1 mutant fibroblasts rescued the levels of Tgf-ß1 mRNA, increased the amounts of secreted active TGF-ß in these cells, as well as P311 mRNA levels in adjacent A549 cells. Accordingly, small interfering RNAs (siRNAs) against fibroblast Tgf-ß1 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-ß1. GENERAL SIGNIFICANCE: This study considers a possible novel mechanism of Ext1-regulated heparan sulfate structure in modifying tumor-stroma interactions through altering stromal tgf-ß1 expression.

Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases, in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains.

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.

Role of 6-O-sulfated heparan sulfate in chronic renal fibrosis.

Heparan sulfate (HS) plays a crucial role in the fibrosis associated with chronic allograft dysfunction by binding and presenting cytokines and growth factors to their receptors. These interactions critically depend on the distribution of 6-O-sulfated glucosamine residues, which is generated by glucosaminyl-6-O-sulfotransferases (HS6STs) and selectively removed by cell surface HS-6-O-endosulfatases (SULFs). Using human renal allografts we found increased expression of 6-O-sulfated HS domains in tubular epithelial cells during chronic rejection as compared with the controls. Stimulation of renal epithelial cells with TGF-ß induced SULF2 expression. To examine the role of 6-O-sulfated HS in the development of fibrosis, we generated stable HS6ST1 and SULF2 overexpressing renal epithelial cells. Compared with mock transfectants, the HS6ST1 transfectants showed significantly increased binding of FGF2 (p = 0.0086) and pERK activation. HS6ST1 transfectants displayed a relative increase in mono-6-O-sulfated disaccharides accompanied by a decrease in iduronic acid 2-O-sulfated disaccharide structures. In contrast, SULF2 transfectants showed significantly reduced FGF2 binding and phosphorylation of ERK. Structural analysis of HS showed about 40% down-regulation in 6-O-sulfation with a parallel increase in iduronic acid mono-2-O-sulfated disaccharides. To assess the relevance of these data in vivo we established a murine model of fibrosis (unilateral ureteric obstruction (UUO)). HS-specific phage display antibodies (HS3A8 and RB4EA12) showed significant increase in 6-O-sulfation in fibrotic kidney compared with the control. These results suggest an important role of 6-O-sulfation in the pathogenesis of fibrosis associated with chronic rejection.

Drosophila heparan sulfate, a novel design.

Heparan sulfate (HS) proteoglycans play critical roles in a wide variety of biological processes such as growth factor signaling, cell adhesion, wound healing, and tumor metastasis. Functionally important interactions between HS and a variety of proteins depend on specific structural features within the HS chains. The fruit fly (Drosophila melanogaster) is frequently applied as a model organism to study HS function in development. Previous structural studies of Drosophila HS have been restricted to disaccharide composition, without regard to the arrangement of saccharide domains typically found in vertebrate HS. Here, we biochemically characterized Drosophila HS by selective depolymerization with nitrous acid. Analysis of the generated saccharide products revealed a novel HS design, involving a peripheral, extended, presumably single, N-sulfated domain linked to an N-acetylated sequence contiguous with the linkage to core protein. The N-sulfated domain may be envisaged as a heparin structure of unusually low O-sulfate content.

Novel roles for cooperating collagen receptor families in fibrotic niches.

Recent data indicate that integrin and non-integrin collagen receptors cooperate in the fibrosis-specific microenvironment (i.e., the fibrotic niche). In certain tumor types, DDR1 can regulate the interaction with collagen III to regulate dormancy and metastasis, whereas in other tumor types, DDR1 can be shed and used to reorganize collagen. DDR1 expressed on tumor cells, together with DDR2 and α11ß1 integrin expressed on cancer-associated fibroblasts, can increase tumor tissue stiffness. Integrin α1ß1 and α2ß1 are present on immune cells where they together with the immunosuppressive collagen receptor LAIR-1 can mediate binding to intratumor collagens. In summary, collagen-binding integrins together with DDRs, can create fibrillar collagen niches that act as traps to hinder immune cell trafficking into the tumor cell mass. Binding of collagens via LAIR-1 on immune cells in turn results in CD8+T-cell exhaustion. Continued studies of these complex interactions are needed for successful new stroma-based therapeutic interventions. In the current review, we will summarize recent data on collagen receptors with a special focus on their potential role in tumor fibrosis and highlight their collaborative roles in tumor fibrotic niches.

Heparan sulfate expression is affected by inflammatory stimuli in primary human endothelial cells.

In diabetes the endothelium is either chronically or transiently exposed to hyperglycemic conditions. In addition, endothelial dysfunction in diabetes is related to changes in the inflammatory response and the turnover of extracellular matrix. This study was undertaken to study the effects of inflammatory stimuli on one particular matrix component, the heparan sulfate (HS) proteoglycans (PGs) synthesized by primary human umbilical cord vein endothelial cells (HUVEC). Such cells were cultured in vitro in 5 mM and 25 mM glucose. The latter concentration was used to mimic hyperglycemic conditions in short-term experiments. HUVEC were also cultured in the presence of the inflammatory agents tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), interleukin 1ß (IL-1ß) and transforming growth factor ß (TGF-ß). The cells were labeled with (35)S-sulfate and (35)S-PGs were recovered for further analyses. The major part of the (35)S-PGs was secreted to the medium, irrespective of type of stimuli. Secreted (35)S-PGs were therefore isolated and subjected to further analyses. TNF-α and IL-1α slightly increased the release of (35)S-PGs to the culture medium, whereas IL-1ß treatment gave a significant increase. The different treatments neither changed the ratio of (35)S-HS and (35)S-chondroitin sulfate (CS) nor the macromolecular properties of the (35)S-PGs. However, the (35)S-HS chains were slightly increased in size after TNF-α treatment, and slightly decreased after TGF-ß treatment, but not affected by the other treatments. Compositional analysis of labeled disaccharides showed changes in the amount of 6-O-sulfated glucosamine residues after treatment with TNF-α, IL-1α and IL-1ß. Western immunoblotting showed that major HSPGs recovered from these cells were collagen XVIII, perlecan and agrin, and that secretion of these distinct PGs was increased after IL-1ß stimulation. Hence, short term inflammatory stimuli increased the release of HSPGs in HUVEC and affected both the size and sulfation pattern of HS, depending on type of stimuli.

Integrin α11ß1 in tumor fibrosis: more than just another cancer-associated fibroblast biomarker?

There is currently an increased interest in understanding the role of the tumor microenvironment (TME) in tumor growth and progression. In this context the role of integrins in cancer-associated fibroblasts (CAFs) will need to be carefully re-evaluated. Fibroblast-derived cells are not only in the focus in tumors, but also in tissue fibrosis as well as in inflammatory conditions. The recent transcriptional profiling of what has been called "the pan-fibroblast cell lineage" in mouse and human tissues has identified novel transcriptional biomarker mRNAs encoding the secreted ECM proteins dermatopontin and collagen XV as well as the phosphatidylinositol-anchored membrane protein Pi16. Some of the genes identified in these fibroblasts scRNA-seq datasets will be useful for rigorous comparative characterizations of fibroblast-derived cell subpopulations. At the same time, it will be a challenge in the coming years to validate these transcriptional mRNA datasets at the protein-(expression) and at tissue-(distribution) levels and to find useful protein biomarker reagents that will facilitate fibroblast profiling at the cell level. In the current review we will focus on the role of the collagen-binding integrin α11ß1 in CAFs, summarizing our own work as well as published datasets with information on α11 mRNA expression in selected tumors. Our experimental data suggest that α11ß1 is more than just another biomarker and that it as a functional collagen receptor in the TME is playing a central role in regulating collagen assembly and matrix remodeling, which in turn impact tumor growth and metastasis.

Heparan sulfate dependent binding of plasmatic von Willebrand factor to blood circulating melanoma cells attenuates metastasis.

Heparan sulfate (HS), a highly negatively charged glycosaminoglycan, is ubiquitously present in all tissues and also exposed on the surface of mammalian cells. A plethora of molecules such as growth factors, cytokines or coagulation factors bear HS binding sites. Accordingly, HS controls the communication of cells with their environment and therefore numerous physiological and pathophysiological processes such as cell adhesion, migration, and cancer cell metastasis. In the present work, we found that HS exposed by blood circulating melanoma cells recruited considerable amounts of plasmatic von Willebrand factor (vWF) to the cellular surface. Analyses assisted by super-resolution microscopy indicated that HS and vWF formed a tight molecular complex. Enzymatic removal of HS or genetic engineering of the HS biosynthesis showed that a reduced length of the HS chains or complete lack of HS was associated with significantly reduced vWF encapsulation. In microfluidic experiments, mimicking a tumor-activated vascular system, we found that vWF-HS complexes prevented vascular adhesion. In line with this, single molecular force spectroscopy suggested that the vWF-HS complex promoted the repulsion of circulating cancer cells from the blood vessel wall to counteract metastasis. Experiments in wild type and vWF knockout mice confirmed that the HS-vWF complex at the melanoma cell surface attenuated hematogenous metastasis, whereas melanoma cells lacking HS evade the anti-metastatic recognition by vWF. Analysis of tissue samples obtained from melanoma patients validated that metastatic melanoma cells produce less HS. Transcriptome data further suggest that attenuated expression of HS-related genes correlate with metastases and reduced patients' survival. In conclusion, we showed that HS-mediated binding of plasmatic vWF to the cellular surface can reduce the hematogenous spread of melanoma. Cancer cells with low HS levels evade vWF recognition and are thus prone to form metastases. Therefore, therapeutic expansion of the cancer cell exposed HS may prevent tumor progression.

Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation.

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.

Mutation in the heparan sulfate biosynthesis enzyme EXT1 influences growth factor signaling and fibroblast interactions with the extracellular matrix.

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.

Target selection of heparan sulfate hexuronic acid 2-O-sulfotransferase.

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.

Molecular analysis of heparan sulfate biosynthetic enzyme machinery and characterization of heparan sulfate structure in Nematostella vectensis.

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.

QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling.

The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state "catch or present" model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS-Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.

Heparan sulfate in chronic kidney diseases: Exploring the role of 3-O-sulfation.

One of the main feature of chronic kidney disease is the development of renal fibrosis. Heparan Sulfate (HS) is involved in disease development by modifying the function of growth factors and cytokines and creating chemokine gradients. In this context, we aimed to understand the function of HS sulfation in renal fibrosis. Using a mouse model of renal fibrosis, we found that total HS 2-O-sulfation was increased in damaged kidneys, whilst, tubular staining of HS 3-O-sulfation was decreased. The expression of HS modifying enzymes significantly correlated with the development of fibrosis with HS3ST1 demonstrating the strongest correlation. The pro-fibrotic factors TGFß1 and TGFß2/IL1ß significantly downregulated HS3ST1 expression in both renal epithelial cells and renal fibroblasts. To determine the implication of HS3ST1 in growth factor binding and signalling, we generated an in vitro model of renal epithelial cells overexpressing HS3ST1 (HKC8-HS3ST1). Heparin Binding EGF like growth factor (HB-EGF) induced rapid, transient STAT3 phosphorylation in control HKC8 cells. In contrast, a prolonged response was demonstrated in HKC8-HS3ST1 cells. Finally, we showed that both HS 3-O-sulfation and HB-EGF tubular staining were decreased with the development of fibrosis. Taken together, these data suggest that HS 3-O-sulfation is modified in fibrosis and highlight HS3ST1 as an attractive biomarker of fibrosis progression with a potential role in HB-EGF signalling.

Sulfs are regulators of growth factor signaling for satellite cell differentiation and muscle regeneration.

Heparan sulfate proteoglycans (HSPGs) are required during muscle regeneration for regulating extracellular signaling pathways. HSPGs interact with growth factors and receptors through heparan sulfate (HS) chains. However, the regulatory mechanisms that control HS sulfation to affect the growth factor-dependent proliferation and differentiation of satellite cells are yet unknown. Here we report the essential functions of extracellular HS 6-O-endosulfatases (Sulfs) during muscle regeneration. We show that quiescent and activated satellite cells differentially express mouse Sulf1 (MSulf1) and MSulf2. MSulfs are not required for the formation of skeletal muscles and satellite cells, but they have redundant, essential roles to promote muscle regeneration, as MSulf double mutant mice exhibit delayed myogenic differentiation and prolonged Pax7 expression after cardiotoxin-induced skeletal muscle injury, while single MSulf knockouts regenerate normally. HS structural analysis demonstrates that Sulfs are regulatory HS-modifying enzymes that control HS 6-O-desulfation of activated satellite cells. Mechanistically, we show that MSulfs repress FGF2 signaling in activated satellite cells, leading us to propose that MSulfs are growth factor signaling sensors to control the proliferation to differentiation switch of satellite cells to initiate differentiation during regeneration. Our results establish Sulfs as essential regulators of HS-dependent growth factor signaling in the adult muscle stem cell niche.

The exostosin family of glycosyltransferases: mRNA expression profiles and heparan sulphate structure in human breast carcinoma cell lines.

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 EXTL2 was found to be up-regulated whereas EXT2 was down-regulated. Interestingly, despite having similar expression of HS elongation enzymes the two cell lines synthesized HS chains of significantly different lengths. Furthermore, both MDA-MB-231 and HCC38 exhibited markedly decreased levels of HS 6-O-sulphated disaccharides. Although the gene expression profiles of the elongation enzymes did not correlate with the length of HS chains, our results indicated specific differences in EXT enzyme levels and HS fine structure characteristic of the carcinogenic properties of the breast carcinoma cells.

Sulfotransferases in glycosaminoglycan biosynthesis.

Most of the sulfotransferases participating in glycosaminoglycan biosynthesis have now been identified. Their essential role in generating binding sites for proteins interacting with glycosaminoglycans is apparent. These interactions may influence important biological processes such as growth control, signal transduction, cell adhesion and lipid metabolism. Gene targeting in mice as well as studies in Drosophila melanogaster and Caenorhabditis elegans have shown that dysfunction or lack of glycosaminoglycan sulfotransferases may result in severely disturbed embryonic development.

Purification of a 75 kDa protein from the organelle matrix of human neutrophils and identification as N-acetylglucosamine-6-sulphatase.

A 75 kDa protein was purified to homogeneity from granule extracts of normal human granulocytes using Sephadex G-75 chromatography, Mono-S cation exchange chromatography and chromatofocusing. The protein consisted of one chain with a molecular mass of 75 kDa, as determined by SDS/PAGE. Tryptic peptide analysis by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS and sequence analysis by MS/MS identified the protein to be N-acetylglucosamine-6-sulphatase (EC 3.1.6.14). The identity of the protein was confirmed by demostrating enzymatic activity towards the substrate N-acetylglucosamine 6-sulphate. The enzyme was active over a broad pH range with an optimum of pH 7.0, and showed a K(m) value of 13.0 mM and a V(max) value of approximately 1.8 microM/min per mg. The enzyme also showed O-desulphation activity towards heparan sulphate-derived saccharides. Subcellular fractionation of neutrophil organelles showed the presence of enzymatic activity mainly in the same fractions as primary granules. Furthermore, PMA treatment of the neutrophils induced release of the enzyme, indicating its matrix protein nature. The presence of N-acetylglucosamine-6-sulphatase in human neutrophils implies that neutrophils may play a role in the modulation of cell surface molecules and extracellular matrix by O-desulphation.