Heparan sulfate 6-O-sulfotransferase isoform-dependent regulatory effects of heparin on the activities of various proteases in mast cells and the biosynthesis of 6-O-sulfated heparin.

Heparan sulfate 6-O-sulfotransferase (HS6ST) is an enzyme involved in heparan sulfate (HS) biosynthesis that transfers a sulfate residue to position 6 of the GlcNAc/GlcNSO(3) residues of HS, and it consists of three isoforms. Heparin, the highly sulfated form of HS, resides in connective tissue mast cells and is involved in the storage of mast cell proteases (MCPs). However, it is not well understood which isoform(s) of HS6ST participates in 6-O-sulfation of heparin and how the 6-O-sulfate residues in heparin affect MCPs. To investigate these issues, we prepared fetal skin-derived mast cells (FSMCs) from wild type (WT) and HS6ST-deficient mice (HS6ST-1(-/-), HS6ST-2(-/-), and HS6ST-1(-/-)/HS6ST-2(-/-)) and determined the structure of heparin, the protease activity, and the mRNA expression of each MCP in cultured FSMCs. The activities of tryptase and carboxypeptidase-A were decreased in HS6ST-2(-/-)-FSMCs in which 6-O-sulfation of heparin was decreased at 50% of WT-FSMCs and almost lost in HS6ST-1(-/-)/HS6ST-2(-/-)-FSMCs, which lacked the 6-O-sulfation in heparin nearly completely. In contrast, chymase activity was retained even in HS6ST-1(-/-)/HS6ST-2(-/-)-FSMCs. Each MCP mRNA was not decreased in any of the mutant FSMCs. Western blot analysis showed that tryptase (mMCP-6) was almost absent from HS6ST-1(-/-)/HS6ST-2(-/-)-FSMCs indicating degradation/secretion of the enzyme protein. These observations suggest that both HS6ST-1 and HS6ST-2 are involved in 6-O-sulfation of heparin and that the proper packaging and storage of tryptase, carboxypeptidase-A, and chymase may be regulated differently by the 6-O-sulfate residues in heparin. It is thus likely that 6-O-sulfation of heparin plays important roles in regulating MCP functions.

Heparan sulfate 6-O-sulfotransferase 1, a gene involved in extracellular sugar modifications, is mutated in patients with idiopathic hypogonadotrophic hypogonadism.

Neuronal development is the result of a multitude of neural migrations, which require extensive cell-cell communication. These processes are modulated by extracellular matrix components, such as heparan sulfate (HS) polysaccharides. HS is molecularly complex as a result of nonrandom modifications of the sugar moieties, including sulfations in specific positions. We report here mutations in HS 6-O-sulfotransferase 1 (HS6ST1) in families with idiopathic hypogonadotropic hypogonadism (IHH). IHH manifests as incomplete or absent puberty and infertility as a result of defects in gonadotropin-releasing hormone neuron development or function. IHH-associated HS6ST1 mutations display reduced activity in vitro and in vivo, suggesting that HS6ST1 and the complex modifications of extracellular sugars are critical for normal development in humans. Genetic experiments in Caenorhabditis elegans reveal that HS cell-specifically regulates neural branching in vivo in concert with other IHH-associated genes, including kal-1, the FGF receptor, and FGF. These findings are consistent with a model in which KAL1 can act as a modulatory coligand with FGF to activate the FGF receptor in an HS-dependent manner.

Glycosaminoglycans in the blood of hereditary multiple exostoses patients: Half reduction of heparan sulfate to chondroitin sulfate ratio and the possible diagnostic application.

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder with wide variation in clinical phenotype and is caused by heterogeneous germline mutations in two of the Ext genes, EXT-1 and EXT-2, which encode ubiquitously expressed glycosyltransferases involved in the polymerization of heparan sulfate (HS) chains. To examine whether the Ext mutation could affect HS structures and amounts in HME patients being heterozygous for the Ext genes, we collected blood from patients and healthy individuals, separated it into plasma and cellular fractions and then isolated glycosaminoglycans (GAGs) from those fractions. A newly established method consisting of a combination of selective ethanol precipitation of GAGs, digestion of GAGs recovered on the filter-cup by direct addition of heparitinase or chondroitinase reaction solution and subsequent high-performance liquid chromatography of the unsaturated disaccharide products enabled the analysis using the least amount of blood (200 µL). We found that HS structures of HME patients were almost similar to those of controls in both plasma and cellular fractions. However, interestingly, although both the amounts of HS and chondroitin sulfate (CS) varied depending on the different individuals, the amounts of HS in both the plasma and cellular fractions of HME patient samples were decreased and the ratios of HS to CS (HS/CS) of HME patient samples were almost half those of healthy individuals. The results suggest that HME patients' blood exhibited reduced HS amounts and HS/CS ratios, which could be used as a diagnostic biomarker for HME.

Involvement of heparan sulfate 6-O-sulfation in the regulation of energy metabolism and the alteration of thyroid hormone levels in male mice.

Here, we report that male heparan sulfate 6-O-sulfotransferase-2 (Hs6st2) knockout mice showed increased body weight in an age-dependent manner even when fed with a normal diet and showed a phenotype of impaired glucose metabolism and insulin resistance. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the expression of mitochondrial uncoupling proteins Ucp1 and Ucp3 was reduced in the interscapular brown adipose tissue (BAT) of male Hs6st2 knockout mice, suggesting reduced energy metabolism. The serum level of thyroid-stimulating hormone was significantly higher and that of thyroxine was lower in the knockout mice. When cultures of brown adipocytes from wild-type and Hs6st2 knockout mice isolated and differentiated in vitro were treated with FGF19 (fibroblast growth factor 19) or FGF21 in the presence or the absence of heparitinase I, phosphorylation of p42/p44 mitogen-activated protein (MAP) kinase was reduced. Heparan sulfate (HS) 6-O-sulfation was reduced not only in BAT but also in the thyroid tissue of the knockout mice. Thus, 6-O-sulfation in HS seems to play an important role in mediating energy metabolism by controlling thyroid hormone levels and signals from the FGF19 subfamily proteins, and the alteration of the HS composition may result in metabolic syndrome phenotypes such as altered glucose and insulin tolerance.

Mice deficient in N-acetylgalactosamine 4-sulfate 6-o-sulfotransferase are unable to synthesize chondroitin/dermatan sulfate containing N-acetylgalactosamine 4,6-bissulfate residues and exhibit decreased protease activity in bone marrow-derived mast cells.

Chondroitin sulfate (CS) and dermatan sulfate (DS) containing N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6-SO(4))) show various physiological activities through interacting with numerous functional proteins. N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate in CS or DS to yield GalNAc(4,6-SO(4)) residues. We here report generation of transgenic mice that lack GalNAc4S-6ST. GalNAc4S-6ST-null mice were born normally and fertile. In GalNAc4S-6ST-null mice, GalNAc(4,6-SO(4)) residues in CS and DS disappeared completely, indicating that GalNAc4S-6ST should be a sole enzyme responsible for the synthesis of GalNAc(4,6-SO(4)) residues in both CS and DS. IdoA-GalNAc(4,6-SO(4)) units that account for approximately 40% of total disaccharide units of DS in the liver of the wild-type mice disappeared in the liver DS of GalNAc4S-6ST-null mice without reduction of IdoA content. Bone marrow-derived mast cells (BMMCs) derived from GalNAc4S-6ST-null mice contained CS without GlcA-GalNAc(4,6-SO(4)) units. Tryptase and carboxypeptidase A activities of BMMCs derived from GalNAc4S-6ST-null mice were lower than those activities of BMMCs derived from wild-type mice, although mRNA expression of these mast cell proteases was not altered. Disaccharide compositions of heparan sulfate/heparin contained in the mast cells derived from BMMCs in the presence of stem cell factor were much different from those of heparan sulfate/heparin in BMMCs but did not differ significantly between wild-type mice and GalNAc4S-6ST-null mice. These observations suggest that CS containing GalNAc(4,6-SO(4)) residues in BMMCs may contribute to retain the active proteases in the granules of BMMCs but not for the maturation of BMMCs into connective tissue-type mast cells.

Heparan sulfate 2-O-sulfotransferase is required for triglyceride-rich lipoprotein clearance.

Hepatic clearance of triglyceride-rich lipoproteins depends on heparan sulfate and low density lipoprotein receptors expressed on the basal membrane of hepatocytes. Binding and uptake of the lipoproteins by way of heparan sulfate depends on the degree of sulfation of the chains based on accumulation of plasma triglycerides and delayed clearance of triglyceride-rich lipoproteins in mice bearing a hepatocyte-specific alteration of N-acetylglucosamine (GlcNAc) N-deacetylase-N-sulfotransferase 1 (Ndst1) (MacArthur, J. M., Bishop, J. R., Stanford, K. I., Wang, L., Bensadoun, A., Witztum, J. L., and Esko, J. D. (2007) J. Clin. Invest. 117, 153-164). Inactivation of Ndst1 led to decreased overall sulfation of heparan sulfate due to coupling of uronyl 2-O-sulfation and glucosaminyl 6-O-sulfation to initial N-deacetylation and N-sulfation of GlcNAc residues. To determine whether lipoprotein clearance depends on 2-O-and 6-O-sulfation, we evaluated plasma triglyceride levels in mice containing loxP-flanked conditional alleles of uronyl 2-O-sulfotransferase (Hs2st(f/f)) and glucosaminyl 6-O-sulfotransferase-1 (Hs6st1(f/f)) and the bacterial Cre recombinase expressed in hepatocytes from the rat albumin (Alb) promoter. We show that Hs2st(f/f)AlbCre(+) mice accumulated plasma triglycerides and exhibited delayed clearance of intestinally derived chylomicrons and injected human very low density lipoproteins to the same extent as observed in Ndst1(f/f)AlbCre(+) mice. In contrast, Hs6st1(f/f)AlbCre(+) mice did not exhibit any changes in plasma triglycerides. Chemically modified heparins lacking N-sulfate and 2-O-sulfate groups did not block very low density lipoprotein binding and uptake in isolated hepatocytes, whereas heparin lacking 6-O-sulfate groups was as active as unaltered heparin. Our findings show that plasma lipoprotein clearance depends on specific subclasses of sulfate groups and not on overall charge of the chains.

Specific and flexible roles of heparan sulfate modifications in Drosophila FGF signaling.

Specific sulfation sequence of heparan sulfate (HS) contributes to the selective interaction between HS and various proteins in vitro. To clarify the in vivo importance of HS fine structures, we characterized the functions of the Drosophila HS 2-O and 6-O sulfotransferase (Hs2st and Hs6st) genes in FGF-mediated tracheal formation. We found that mutations in Hs2st or Hs6st had unexpectedly little effect on tracheal morphogenesis. Structural analysis of mutant HS revealed not only a loss of corresponding sulfation, but also a compensatory increase of sulfation at other positions, which maintains the level of HS total charge. The restricted phenotypes of Hsst mutants are ascribed to this compensation because FGF signaling is strongly disrupted by Hs2st; Hs6st double mutation, or by overexpression of 6-O sulfatase, an extracellular enzyme which removes 6-O sulfate groups without increasing 2-O sulfation. These findings suggest that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts.

Bone marrow derived mast cells injected into the osteoarthritic knee joints of mice induced by sodium monoiodoacetate enhanced spontaneous pain through activation of PAR2 and action of extracellular ATP.

Conditions that resemble osteoarthritis (OA) were produced by injection of sodium monoiodoacetate (MIA) into the knee joints of mice. Bone marrow derived mast cells (BMMCs) injected into the OA knee joints enhanced spontaneous pain. Since no spontaneous pain was observed when BMMCs were injected into the knee joints of control mice that had not been treated with MIA, BMMCs should be activated within the OA knee joints and release some pain-inducible factors. Protease activated receptor-2 (PAR2) antagonist (FSLLRY-NH2) almost abolished the pain-enhancing effects of BMMCs injected into the OA knee joints, suggesting that tryptase, a mast cell protease that is capable of activating PAR2, should be released from the injected BMMCs and enhance pain through activation of PAR2. When PAR2 agonist (SLIGKV-NH2) instead of BMMCs was injected into the OA knee joints, it was also enhanced pain. Apyrase, an ATP degrading enzyme, injected into the OA knee joints before BMMCs suppressed the pain enhanced by BMMCs. We showed that purinoceptors (P2X4 and P2X7) were expressed in BMMCs and that extracellular ATP stimulated the release of tryptase from BMMCs. These observations suggest that ATP may stimulate degranulation of BMMCs and thereby enhanced pain. BMMCs injected into the OA knee joints stimulated expression of IL-1ß, IL-6, TNF-α, CCL2, and MMP9 genes in the infrapatellar fat pads, and PAR2 antagonist suppressed the stimulatory effects of BMMCs. Our study suggests that intermittent pain frequently observed in OA knee joints may be due, at least partly, to mast cells through activation of PAR2 and action of ATP, and that intraarticular injection of BMMCs into the OA knee joints may provide a useful experimental system for investigating molecular mechanisms by which pain is induced in OA knee joints.

A novel mice model of acute flares in osteoarthritis elicited by intra-articular injection of cultured mast cells.

PURPOSE: Mast cells are multifunctional in osteoarthritis (OA), and infiltration of activated mast cells likely contributes to disease severity and progression. However, the detailed mechanisms of action are unclear. The purpose of this study was to elucidate the role of mast cell infiltration in OA at histological level using a new mice model and to investigate pharmacological inhibitory effects of existing mast cell stabilizers in this model. METHODS: Mice were injected intra-articularly with monosodium iodoacetate (MIA 0.5 mg) or PBS on day 0, and PBS, with or without mast cells (MC: 1 × 106 cells) on day 14. They were divided into four groups: OA flare (MIA + MC), OA (MIA + PBS), MC non-OA (PBS + MC), and PBS non-OA (PBS + PBS). In OA flare, the MC stabilizer drug (tranilast: 400 mg/kg/day) or PBS was administered intraperitoneally from days 15 to 21. RESULTS: Histologically, modified Mankin score of the OA flare was significantly higher than that of OA (7.0 [1.8] vs. 3.3 [1.3], P < 0.05), and a larger number of mast cells was observed in OA flare than in OA (34.5 [6.3]/mm2 vs. 27.2 [2.3]/mm2, P < 0.05) on day 22. OA flare also showed acute exacerbation of pain and increased gene expression of pro-inflammatory cytokines and aggrecanase compared with OA. Administration of tranilast to OA flare-up provoked significant improvements in term of histological changes, pain, and gene expression at day 22. CONCLUSION: Our novel model possibly mimics OA flare conditions, which may open a new strategy of disease-modifying treatment for OA, focused on controlling the multiple functions of mast cells.

Functional analysis of chick heparan sulfate 6-O-sulfotransferases in limb bud development.

Heparan sulfate (HS) interacts with numerous growth factors, morphogens, receptors, and extracellular matrix proteins. Disruption of HS synthetic enzymes causes perturbation of growth factor signaling and malformation in vertebrate and invertebrate development. Our previous studies show that the O-sulfation patterns of HS are essential for the specific binding of growth factors to HS chains, and that depletion of O-sulfotransferases results in remarkable developmental defects in Drosophila, zebrafish, chick, and mouse. Here, we show that inhibition of chick HS-6-O-sulfotransferases (HS6ST-1 and HS6ST-2) in the prospective limb region by RNA interference (RNAi) resulted in the truncation of limb buds and reduced Fgf-8 and Fgf-10 expressions in the apical ectodermal ridge and in the underlying mesenchyme, respectively. HS6ST-2 RNAi resulted in a higher frequency of limb truncation and a more marked change in both Fgf-8 and Fgf-10 expressions than that achieved with HS6ST-1 RNAi. HS6ST-1 RNAi and HS6ST-2 RNAi caused a significant but distinct reduction in the levels of different 6-O-sulfation in HS, possibly as a result of their different substrate specificities. Our data support a model where proper levels and patterns of 6-O-sulfation of HS play essential roles in chick limb bud development.

Preparation of chondroitin sulfate libraries containing disulfated disaccharide units and inhibition of thrombin by these chondroitin sulfates.

Chondroitin sulfate (CS) containing GlcA-GalNAc(4,6-SO(4)) (E unit) and CS containing GlcA(2SO(4))-GalNAc(6SO(4)) (D unit) have been implicated in various physiological functions. However, it has been poorly understood how the structure and contents of disulfated disaccharide units in CS contribute to these functions. We prepared CS libraries containing E unit or D unit in various proportions by in vitro enzymatic reactions using recombinant GalNAc 4-sulfate 6-O-sulfotransferase and uronosyl 2-O-sulfotransferase, and examined their inhibitory activity toward thrombin. The in vitro sulfated CSs containing disulfated disaccharide units showed concentration-dependent direct inhibition of thrombin when the proportion of E unit or D unit in the CSs was above 15-17%. The CSs containing both E unit and D unit exhibited higher inhibitory activity toward thrombin than the CSs containing either E unit or D unit alone, if the proportion of the total disulfated disaccharide units of these CSs was comparable. The thrombin-catalyzed degradation of fibrinogen, a physiological substrate for thrombin, was also inhibited by the CS containing both E unit and D unit. These observations indicate that the enzymatically prepared CS libraries containing various amounts of disulfated disaccharide units appear to be useful for elucidating the physiological function of disulfated disaccharide units in CS.

Specific inhibition of FGF-2 signaling with 2-O-sulfated octasaccharides of heparan sulfate.

In fibroblast growth factor (FGF)-2 signaling, the formation of a ternary complex of FGF-2, tyrosine-kinase fibroblast growth factor receptor (FGFR)-1, and cell surface heparan sulfate (HS) proteoglycan is known to be critical for the activation of FGFR-1 and downstream signal transduction. Exogenous heparin polymer and some octasaccharides inhibited FGF-2-induced phosphorylation both of FGFR-1 and of extracellular signal-regulated kinase (ERK1/2) in Chinese hamster ovary (CHO)-K1 cells transfected with FGFR-1, which present HS on their cell surface. The inhibitory effect of octasaccharide was dependent on the number of 2-O-sulfate groups within a molecule but independent of the number of 6-O-sulfate groups. Sulfation at the 2-O-position was a prerequisite not only for the binding of HS to FGF-2 but also for regulation of FGF-2 signaling and competitive inhibition with endogenous HS. Interestingly, FGF-4-induced phosphorylation was impeded only by specific octasaccharides containing both 2-O- and 6-O-sulfated groups, which were necessary for binding FGF-4. In CHO-677 cells deficient in HS biosynthesis, heparin enhanced FGF-2-induced phosphorylation of ERK1/2. On the other hand, an FGF-2-binding octasaccharide inhibited the phosphorylation. Our data suggest that the activity of particular heparin-binding factors can be inhibited by distinctive oligosaccharides that can bind the factors but cannot form functional signaling complexes irrespective of whether cells have a normal complement of HS or lack HS.

Determination of substrate specificity of sulfotransferases and glycosyltransferases (proteoglycans).

Proteoglycans have sulfated linear polysaccharide chains, that is, heparan sulfate, heparin, chondroitin sulfates, dermatan sulfate, and keratan sulfate. Many glycosyltransferases and sulfotransferases are involved in biosynthesis of the polysaccharides. Specificities of these enzymes have been mainly determined by evaluating their activities to various acceptor carbohydrates and by analyzing the structure of the products. For the latter purpose, enzymatic hydrolysis using heparitinases, heparinase, and chondroitinases or chemical degradation employing nitrous acid deamination has been effectively used in combination with high-performance liquid chromatography (HPLC) of the degraded products. As examples, we describe methods for assays and product characterization of sulfotransferases involved in biosynthesis of these polysaccharides, namely heparan sulfate 2-sulfotransferase, heparan sulfate 6-sulfotransferases, chondroitin 4-sulfotransferases, chondroitin 6-sulfotransferase, N-acetylgalactosamine 4-sulfate 6-sulfotransferase, and N-acetylglucosamine 6-sulfotransferases.

Mice deficient in N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase exhibit enhanced liver fibrosis and delayed recovery from fibrosis in carbon tetrachloride-treated mice.

BACKGROUND: Chondroitin/dermatan sulfate (CS/DS) rich in N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6SO4)) residues is present as decorin and/or biglycan in mouse liver, and GalNAc(4,6SO4) residues disappeared completely in N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) knockout (KO) mice. The aim of this study was to investigate whether CS/DS rich in GalNAc(4,6SO4) residues participate in the progression or resolution of liver fibrosis. METHODS: Wild type (WT) and GalNAc4S-6ST KO mice were treated with CCl4 for 5 weeks. After discontinuation of CCl4 administration, histochemical and biochemical changes and expression of genes related to matrix components were compared between WT and GalNAc4S-6ST KO mice. RESULTS AND CONCLUSION: On 2 days after cessation of CCl4 administration, higher fibrosis was observed in KO mice than in WT mice by Sirius Red staining. Serum alanine aminotransferase activity was higher in KO mice than in WT mice. Hydroxyproline contents and Sirius Red staining showed that repair of liver fibrosis in the recovery stages appeared to be delayed in KO mice. Expression of mRNA of matrix metalloproteinase (MMP)-2, MMP-13 and versican peaked at 2 days after cessation of CCl4 administration and was higher in KO mice than in WT mice. Expression of MMP-9 in the recovery stage was lower in KO mice than in WT mice. Our findings demonstrate that defect in GalNAc4S-6ST, which resulted in disappearance of CS/DS containing GalNAc(4,6SO4), appear to contribute to progression of liver fibrosis, delayed recovery from fibrosis, and various changes in the expression of proteoglycans and MMPs in carbon tetrachloride-treated mice.

Sulfated glycosaminoglycans control the extracellular trafficking and the activity of the metalloprotease inhibitor TIMP-3.

Tissue inhibitor of metalloproteinase 3 (TIMP-3) is an important regulator of extracellular matrix (ECM) turnover. TIMP-3 binds to sulfated ECM glycosaminoglycans or is endocytosed by cells via low-density lipoprotein receptor-related protein 1 (LRP-1). Here, we report that heparan sulfate (HS) and chondroitin sulfate E (CSE) selectively regulate postsecretory trafficking of TIMP-3 by inhibiting its binding to LRP-1. HS and CSE also increased TIMP-3 affinity for glycan-binding metalloproteinases, such as adamalysin-like metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), by reducing the dissociation rate constants. The sulfation pattern was crucial for these activities because monosulfated or truncated heparin had a reduced ability to bind to TIMP-3 and increase its affinity for ADAMTS-5. Therefore, sulfation of ECM glycans regulates the levels and inhibitory activity of TIMP-3 and modulates ECM turnover, and small mimicries of sulfated glycans may protect the tissue from the excess destruction seen in diseases such as osteoarthritis, cancer, and atherosclerosis.

Mice deficient in heparan sulfate 6-O-sulfotransferase-1.

Heparan sulfate chains are initially synthesized on core proteins as linear polysaccharides composed of glucuronic acid-N-acetylglucosamine repeating units and subjected to marked structural modification by sulfation at various places and epimerization of hexuronic acid residues (C5-epimerase) at the Golgi lumen and further by 6-O-desulfation at the cell surface, which generates their characteristic divergent fine structures. This chapter focuses on the biological and physiological functions of 6-O-sulfation in HS and the characterization of the enzymes catalyzing 6-O-sulfation (HS6ST). HS6STs in mammals such as humans and mice comprise of three isoforms (HS6ST-1, -2, and -3) and one alternatively spliced form of HS6ST-2 (HS6ST-2S). Each of these isoforms has distinct substrate preferences, albeit overlapping each other. These HS6ST isoforms are expressed in a spatiotemporally regulated manner in most organs. HS6ST-1-deficient mice are lethal mostly at later embryonic stages and exhibit abnormal angiogenesis in labyrinthine zone of placenta and aberrant lung morphology similar to pulmonary emphysema. These knockout mice also exhibit retinal axon guidance abnormality at the optic chiasm. Other HS6ST-deficient animals reveal various malformations in muscle development and branching morphology of the caudal vein of zebrafish, in tracheal formation of Drosophila, and in axon guidance of ventral nerve cord interneurons of Caenorhabditis elegans. Mouse embryonic fibroblasts prepared from HS6ST-1/HS6ST-2 double knockout mice did produce HS lacking 6-O-sulfation and responded differently to various FGFs dependent signaling.

6-O-sulfation of heparan sulfate differentially regulates various fibroblast growth factor-dependent signalings in culture.

Heparan sulfate (HS) interacts with diverse heparin-binding growth factors and thereby regulates their bioactivities. These interactions depend on the structures characterized by the sulfation pattern and isomer of uronic acid residues. One of the biosynthetic modifications of HS, namely 6-O-sulfation, is catalyzed by three isoforms of HS6-O-sulfotransferase. We generated HS6ST-1- and/or HS6ST-2-deficient mice (6ST1-KO, 6ST2-KO, and double knock-out (dKO)) that exhibited different phenotypes. We examined the effects of HS 6-O-sulfation in heparin-binding growth factor signaling using fibroblasts derived from these mutant mice. Mouse embryonic fibroblasts (MEF) prepared from E14.5 dKO mice produced HS with little 6-O-sulfate, whereas 2-O-sulfation in HS from dKO-MEF (dKO-HS) was increased by 1.9-fold. HS6-O-sulfotransferase activity in the dKO-MEF was hardly detected, and HS2-O-sulfotransferase activity was 1.5-fold higher than that in wild type (WT)-MEFs. The response of dKO-MEFs to fibroblast growth factors (FGFs) was distinct from that of WT-MEFs; in dKO-MEFs, FGF-4- and FGF-2-dependent signalings were reduced to approximately 30 and 60% of WT-MEFs, respectively, and FGF-1-dependent signaling was moderately reduced compared with that of WT-MEFs but only at the lower FGF-1 concentrations. Analysis with a surface plasmon resonance biosensor demonstrated that the apparent affinity of dKO-HS for FGF-4 was markedly reduced and was also reduced for FGF-1. In contrast, the affinity of dKO-HS for FGF-2 was 2.5-fold higher than that of HS from WT-MEFs. Thus, 6-O-sulfate in HS may regulate the signalings of some of HB-GFs, including FGFs, by inducing different interactions between ligands and their receptors.

Essential role of heparan sulfate 2-O-sulfotransferase in chick limb bud patterning and development.

The interactions of heparan sulfate (HS) with heparin-binding growth factors, such as fibroblast growth factors (FGFs), depend greatly on the chain structures. O-Sulfations at various positions on the chain are major factors determining HS structure; therefore, O-sulfation patterns may play a crucial role in controlling the developmental and morphogenetic processes of various tissues and organs by spatiotemporally regulating the activities of heparin-binding growth factors. In a previous study, we found that HS-2-O-sulfotransferase is strongly expressed throughout the mesoderm of chick limb buds during the early stages of development. Here we show that inhibition of HS-2-O-sulfotransferase in the prospective limb region by small inhibitory RNA resulted in the truncation of limb buds and reduced Fgf-8 expression in the apical ectodermal ridge. The treatment also reduced Fgf-10 expression in the mesenchyme. Moreover 2-O-sulfated HS, normally abundant in the basement membranes and mesoderm under ectoderm in limb buds, was significantly reduced in the treated buds. Phosphorylation levels of ERK and Akt were up-regulated in such truncated buds. Thus, we have shown for the first time that 2-O-sulfation of HS is essential for the FGF signaling required for limb bud development and outgrowth.

Regulation of heparan sulfate 6-O-sulfation by beta-secretase activity.

The enzymes involved in glycosaminoglycan chain biosynthesis are mostly Golgi resident proteins, but some are secreted extracellularly. For example, the activities of heparan sulfate 6-O-sulfotransferase (HS6ST) and heparan sulfate 3-O-sulfotransferase are detected in the serum as well in the medium of cell lines. However, the biological significance of this is largely unknown. Here we have investigated by means of monitoring green fluorescent protein (GFP) fluorescence how C-terminally GFP-tagged HS6STs that are stably expressed in CHO-K1 cell lines are secreted/shed. Brefeldin A and monensin treatments revealed that the N-terminal hydrophobic domain of HS6ST3 is processed in the endoplasmic reticulum or cis/medial Golgi. Treatment of HS6ST3-GFP-expressing cells with various protease inhibitors revealed that the cell-permeable beta-secretase inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal (Z-VLL-CHO) specifically inhibits HS6ST secretion, although this effect was specific for HS6ST3 but not for HS6ST1 and HS6ST2. However, Z-VLL-CHO treatment did not increase the molecular size of the HS6ST3-GFP that accumulated in the cell. Z-VLL-CHO treatment also induced the intracellular accumulation of SP-HS6ST3(-TMD)-GFP, a modified secretory form of HS6ST3 that has the preprotrypsin leader sequence as its N-terminal hydrophobic domain. Diminishment of beta-secretase activity by coexpressing the amyloid precursor protein of a Swedish mutant, a potent beta-secretase substrate, also induced intracellular HS6ST3-GFP accumulation. Moreover, Z-VLL-CHO treatment increased the 6-O-sulfate (6S) levels of HS, especially in the disaccharide unit of hexuronic acid-GlcNS(6S). Thus, the HS6ST3 enzyme in the Golgi apparatus and therefore the 6-O sulfation of heparan sulfates in the cell are at least partly regulated by beta-secretase via an indirect mechanism.