RESUMO
Chondroitin sulfate (CS) and dermatan sulfate (DS) containing GalNAc4,6-disulfate (GalNAc4S6S) were initially discovered in marine animals. Following the discovery, these glycosaminoglycans have been found in various animals including human. In the biosynthesis of CS/DS containing GalNAc4S6S, 3 groups of sulfotransferases are involved; chondroitin 4-sulfotransferases (C4STs), dermatan 4-sulfotransferase-1 (D4ST-1), and GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST). GalNAc4S-6ST and its products have been shown to play important roles in the abnormal pathological conditions such as central nervous system injury, cancer development, abnormal tissue fibrosis, development of osteoporosis, and infection with viruses or nematodes. CS/DS containing GalNAc4S6S has been shown to increase with the functional differentiation of mast cells, macrophages, and neutrophils. Genetic approaches using knockout or knockdown of GalNAc4S-6ST, blocking of the epitopes containing GalNAc4S6S by specific antibodies and chemical technology that enabled the synthesis of oligosaccharides with defined sulfation patterns, have been applied successfully to these investigations. These studies contributed significantly to the basic understanding of the functional roles of CS/DS containing GalNAc4S6S in various abnormal conditions and appear to provide promising clues to the development of possible measures to treat them.
Assuntos
Sulfatos de Condroitina , Dermatan Sulfato , Animais , Glicosaminoglicanos , Humanos , Oligossacarídeos , Sulfotransferases/genéticaRESUMO
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.
RESUMO
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.
Assuntos
Trifosfato de Adenosina/metabolismo , Artrite Experimental/terapia , Dor Crônica/patologia , Articulação do Joelho/patologia , Mastócitos/transplante , Receptor PAR-2/metabolismo , Trifosfato de Adenosina/análise , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/patologia , Células da Medula Óssea/citologia , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/toxicidade , Dor Crônica/etiologia , Modelos Animais de Doenças , Articulação do Joelho/metabolismo , Masculino , Mastócitos/citologia , Mastócitos/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligopeptídeos/administração & dosagem , Receptor PAR-2/agonistas , Receptor PAR-2/antagonistas & inibidores , Receptores Purinérgicos/metabolismo , Líquido Sinovial/metabolismoRESUMO
The chondroitin sulfate (CS)-rich dense extracellular matrix surrounding neuron cell bodies and proximal dendrites in a mesh-like structure is called a perineuronal net (PNN). CS chains in PNNs control neuronal plasticity by binding to PNN effectors, semaphorin-3A (Sema3A) and orthodenticle homeobox 2. Sema3A recognizes CS-containing type-E disaccharide units (sulfated at O-4 and O-6 of N-acetylgalactosamine). Type-E disaccharide units are synthesized by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST). In this study, we demonstrated that Sema3A accumulates in the PNNs surrounding parvalbumin cells, even in mice deficient in GalNAc4S-6ST. In addition, there were no differences in the number and structure of PNNs visualized by Cat316 antibody and Wisteria floribunda lectin, which recognize CS chains, between wild type and GalNAc4S-6ST knockout mice. Therefore, we re-examined the Sema3A binding motif found in CS chains using chemically synthesized CS tetrasaccharides. As a result, we found that non-sulfated GalNAc residues at the non-reducing termini of CS chains are required for the binding of Sema3A.
Assuntos
Glicoproteínas de Membrana/genética , Neurônios/metabolismo , Fatores de Transcrição Otx/genética , Semaforina-3A/genética , Sulfotransferases/genética , Animais , Sulfatos de Condroitina/genética , Sulfatos de Condroitina/metabolismo , Dendritos/genética , Dendritos/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Plasticidade Neuronal/genética , Ligação Proteica/genéticaRESUMO
Chondroitin sulfate E (CS-E) plays a crucial role in diverse processes ranging from viral infection to neuroregeneration. Its regiospecific sulfation pattern, generated by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), is the main structural determinant of its biological activity. Inhibitors of GalNAc4S-6ST can serve as powerful tools for understanding physiological functions of CS-E and its potential therapeutic leads for human diseases. A family of new 4-acylamino-ß-GalNAc derivatives and 4-azido-ß-GalNAc derivatives were synthesized for their potential application as inhibitors of GalNAc4S-6ST. The target compounds were evaluated for their inhibitory activities against GalNAc4S-6ST. The results revealed that 4-pivaloylamino- and 4-azido-ß-GalNAc derivatives displayed evident activities against GalNAc4S-6ST with IC50 value ranging from 0.800 to 0.828 mM. They showed higher activities than benzyl D-GalNAc4S that was used as control.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Galactosamina/síntese química , Galactosamina/farmacologia , Sulfotransferases/antagonistas & inibidores , Amidas/química , Animais , Inibidores Enzimáticos/química , Galactosamina/química , Humanos , Sulfotransferases/metabolismoRESUMO
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.
RESUMO
Osteoporosis is an age-related disorder of bone remodeling in which bone resorption outstrips bone matrix deposition. Although anticatabolic agents are frequently used as first-line therapies for osteoporosis, alternative anabolic strategies that can enhance anabolic, osteogenic potential are actively sought. Sex steroid hormones, particularly estrogens, are bidirectional regulators for bone homeostasis; therefore, estrogen-mediated events are important potential targets for such anabolic therapies. Here, we show that estrogen-induced, osteoanabolic effects were mediated via enhanced production of chondroitin sulfate-E (CS-E), which could act as an osteogenic stimulant in our cell-based system. Conversely, estrogen deficiency caused reduced expression of CS-E-synthesizing enzymes, including GalNAc4S-6ST, and led to decreased CS-E production in cultures of bone marrow cells derived from ovariectomized mice. Moreover, Galnac4s6st-deficient mice had abnormally low bone mass that resulted from impaired osteoblast differentiation. These results indicated that strategies aimed at boosting CS-E biosynthesis are promising alternative therapies for osteoporosis.
Assuntos
Sulfatos de Condroitina/metabolismo , Estrogênios/metabolismo , Osteogênese , Animais , Remodelação Óssea , Osso e Ossos/metabolismo , Células Cultivadas , Sulfatos de Condroitina/farmacologia , Estrogênios/farmacologia , Feminino , Camundongos , Camundongos Knockout , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Fenótipo , Sulfotransferases/genética , Sulfotransferases/metabolismoRESUMO
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.
Assuntos
Sulfatos de Condroitina/metabolismo , Heparitina Sulfato/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Proteínas ADAM/química , Proteínas ADAM/metabolismo , Animais , Cartilagem Articular/metabolismo , Sulfatos de Condroitina/química , Endocitose , Matriz Extracelular/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/química , Humanos , Cinética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Ligação Proteica , Inibidor Tecidual de Metaloproteinase-3/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-3/genéticaRESUMO
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.
Assuntos
Quimases/metabolismo , Heparina/metabolismo , Mastócitos/enzimologia , Pele/enzimologia , Sulfotransferases/metabolismo , Animais , Quimases/genética , Heparina/genética , Isoenzimas , Mastócitos/citologia , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pele/citologia , Sulfotransferases/genéticaRESUMO
Chondroitin sulfate proteoglycans (CSPGs) represent a major barrier to regenerating axons in the central nervous system (CNS), but the structural diversity of their polysaccharides has hampered efforts to dissect the structure-activity relationships underlying their physiological activity. By taking advantage of our ability to chemically synthesize specific oligosaccharides, we demonstrate that a sugar epitope on CSPGs, chondroitin sulfate-E (CS-E), potently inhibits axon growth. Removal of the CS-E motif significantly attenuates the inhibitory activity of CSPGs on axon growth. Furthermore, CS-E functions as a protein recognition element to engage receptors including the transmembrane protein tyrosine phosphatase PTPσ, thereby triggering downstream pathways that inhibit axon growth. Finally, masking the CS-E motif using a CS-E-specific antibody reversed the inhibitory activity of CSPGs and stimulated axon regeneration in vivo. These results demonstrate that a specific sugar epitope within chondroitin sulfate polysaccharides can direct important physiological processes and provide new therapeutic strategies to regenerate axons after CNS injury.
Assuntos
Axônios/patologia , Axônios/fisiologia , Proteoglicanas de Sulfatos de Condroitina/imunologia , Epitopos/imunologia , Regeneração Nervosa/fisiologia , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Axônios/efeitos dos fármacos , Configuração de Carboidratos , Galinhas , Proteoglicanas de Sulfatos de Condroitina/química , Sulfatos de Condroitina/química , Sulfatos de Condroitina/imunologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/metabolismo , Cones de Crescimento/patologia , Camundongos , Neuritos/enzimologia , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Acetilgalactosamina/análogos & derivados , Medula Óssea/enzimologia , Sulfatos de Condroitina/biossíntese , Dermatan Sulfato/biossíntese , Glicosaminoglicanos/biossíntese , Peptídeo Hidrolases/metabolismo , RNA Mensageiro/genética , Sulfotransferases/deficiência , Acetilgalactosamina/biossíntese , Acetilgalactosamina/química , Animais , Medula Óssea/ultraestrutura , Sulfatos de Condroitina/química , DNA/genética , Primers do DNA , Dermatan Sulfato/química , Dissacarídeos/análise , Éxons/genética , Vetores Genéticos , Mastócitos/enzimologia , Mastócitos/ultraestrutura , Camundongos , Microscopia Eletrônica , Reação em Cadeia da Polimerase , Baço/enzimologia , Sulfotransferases/genéticaRESUMO
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.
Assuntos
Antitrombinas/química , Antitrombinas/farmacologia , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacologia , Dissacarídeos/química , Trombina/antagonistas & inibidores , Animais , Antitrombinas/metabolismo , Sulfatos de Condroitina/biossíntese , Relação Dose-Resposta a Droga , Fator X/antagonistas & inibidores , Fibrinogênio/metabolismo , Humanos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfotransferases/isolamento & purificação , Sulfotransferases/metabolismo , Trombina/metabolismoRESUMO
N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO4) residues of chondroitin sulfate to yield chondroitin sulfate E (CS-E). We have previously demonstrated that phenyl-beta-D-GalNAc(4SO4) could serve as an acceptor for GalNAc4S-6ST, thereby inhibiting GalNAc4S-6ST competitively. In this paper we compared the inhibitory effects of various glycosides in which various hydrophobic aglycons were attached to D-GalNAc(4SO4) via ss anomeric configuration. p-Nitrophenyl-beta-D-GalNAc(4SO4) and p-chlorophenyl-beta-D-GalNAc(4SO4) were stronger inhibitors than phenyl-beta-D-GalNAc(4SO4). Among inhibitors examined here, 3-estradiol-beta-D-GalNAc(4SO4) was the strongest inhibitor; the Ki of 3-estradiol-beta-D-GalNAc(4SO4) for the competitive inhibition was 0.008 mM, which was much lower than the Ki of phenyl-beta-D-GalNAc(4SO4), 0.98 mM. In contrast, 7-estradiol-beta-D-GalNAc(4SO4) showed only weak inhibition to GalNAc4S-6ST. 3-Estradiol-beta-D-GalNAc(4SO4) did not inhibit chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase under the concentration where GalNAc4S-6ST was inhibited by 90%. When 3-estradiol-beta-D-GalNAc(4SO4) was added to the culture medium of chondrosarcoma cells expressing human GalNAc4S-6ST, a significant, albeit small, reduction in the cellular synthesis of CS-E was observed. These results suggest that estradiol group of 3-estradiol-beta-D-GalNAc(4SO4) may enhance the inhibitory activity of the glycoside through increasing the affinity to the enzyme and may allow the glycosides to diffuse at a low efficiency into the cells to inhibit cellular synthesis of CS-E.
Assuntos
Acetilgalactosamina/química , Acetilgalactosamina/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Sulfotransferases/antagonistas & inibidores , Configuração de Carboidratos , Linhagem Celular Tumoral , Sulfatos de Condroitina/biossíntese , Glicosídeos/química , Glicosídeos/farmacologia , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Cinética , Enxofre/metabolismoRESUMO
Bone marrow-derived mast cells (BMMCs) contain chondroitin sulfate (CS)-E comprised of GlcA-GalNAc(4SO4) units and GlcA-GalNAc(4,6-SO4) units. GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO4) residues of CS. On the basis of the specificity of GalNAc4S-6ST, it is thought that CS-E is synthesized in BMMC through the sequential sulfation by chondroitin 4-sulfotransferase (C4ST)-1 and GalNAc4S-6ST. In this paper, we investigated whether GalNAc4S-6ST and C4ST-1 are actually expressed in BMMCs in which CS-E is actively synthesized. As the bone marrow cells differentiate to BMMCs, level of C4ST-1 and GalNAc4S-6ST messages increased, whereas chondroitin 6-sulfotransferase (C6ST)-1 message decreased. In the extract of BMMCs, activity of GalNAc4S-6ST and C4ST but not C6ST were detected. The recombinant mouse GalNAc4S-6ST transferred sulfate to both nonreducing terminal and internal GalNAc(4SO4) residues; the activity toward nonreducing terminal GalNAc(4SO4) was increased with increasing pH. When CS-E synthesized by BMMCs was metabolically labeled with 35SO4 in the presence of bafilomycin A, chloroquine or NH4Cl, the proportion of the nonreducing terminal GalNAc(4,6-SO4) was increased compared with the control, suggesting that GalNAc4S-6ST in BMMC may elaborate CS-E in the intracellular compartment with relatively low pH where sulfation of the internal GalNAc(4SO4) by GalNAc4S-6ST preferentially occurs.
Assuntos
Células da Medula Óssea/metabolismo , Sulfatos de Condroitina/biossíntese , Mastócitos/metabolismo , Sulfotransferases/metabolismo , Cloreto de Amônio/farmacologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células Cultivadas , Cloroquina/farmacologia , Sulfatos de Condroitina/química , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dissacarídeos/análise , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Macrolídeos/farmacologia , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfotransferases/genéticaRESUMO
N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO(4)) residues in chondroitin sulfate (CS). We previously purified squid GalNAc4S-6ST and cloned a cDNA encoding the partial sequence of squid GalNAc4S-6ST. In this paper, we cloned squid GalNAc4S-6ST cDNA containing a full open reading frame and characterized the recombinant squid GalNAc4S-6ST. The cDNA predicts a Type II transmembrane protein composed of 425 amino acid residues. The recombinant squid GalNAc4S-6ST transferred sulfate preferentially to the internal GalNAc(4SO(4)) residues of chondroitin sulfate A (CS-A); nevertheless, the nonreducing terminal GalNAc(4SO(4)) could be sulfated efficiently when the GalNAc(4SO(4)) residue was included in the unique nonreducing terminal structure, GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), which was previously found in CS-A. Shark cartilage chondroitin sulfate C (CS-C) and chondroitin sulfate D (CS-D), poor acceptors for human GalNAc4S-6ST, served as the good acceptors for the recombinant squid GalNAc4S-6ST. Analysis of the sulfated products formed from CS-C and CS-D revealed that GalNAc(4SO(4)) residues included in a tetrasaccharide sequence, GlcA-GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), were sulfated efficiently by squid GalNAc4S-6ST, and the E-D hybrid tetrasaccharide sequence, GlcA-GalNAc(4,6-SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)) was generated in the resulting sulfated glycosaminoglycans. These observations indicate that the recombinant squid GalNAc4S-6ST is a useful enzyme for preparing a unique chondroitin sulfate containing the E-D hybrid tetrasaccharide structure.
Assuntos
Sulfatos de Condroitina/química , Enzimas/química , Polissacarídeos/química , Sulfotransferases/biossíntese , Sulfotransferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Cromatografia/métodos , Clonagem Molecular , Decapodiformes , Glicosaminoglicanos/química , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Sulfotransferases/químicaRESUMO
N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) is a sulfotransferase responsible for biosynthesis of chondroitin sulfate E (CS-E). CS-E plays important roles in numerous biological events, such as neurite outgrowth. However, the role of GalNAc4S-6ST in tumor progression remains unknown. In the present study, we analyzed expression of GalNAc4S-6ST mRNA in colorectal cancer by combining real-time RT-PCR with in situ hybridization (ISH) using archived formalin-fixed and paraffin-embedded tissue sections. In 57.5% of 40 patients, expression of GalNAc4S-6ST mRNA was increased in cancer tissues compared with paired normal mucosa. ISH using an RNA probe specific for GalNAc4S-6ST revealed that it was expressed in colorectal cancer cells. Analysis of the relationship between expression of GalNAc4S-6ST as determined by real-time RT-PCR assay and various clinicopathological variables revealed that GalNAc4S-6ST was associated with vessel invasion, although a statistically significant difference was not seen (P=0.125 for lymphatic vessel invasion and P=0.242 for venous invasion). Taken together, we show that real-time RT-PCR combined with ISH is useful to investigate quantitatively GalNAc4S-6ST mRNA expression in formalin-fixed and paraffin-embedded tissue sections, and that GalNAc4S-6ST expressed by colorectal cancer cells plays a minor role in tumor progression.
RESUMO
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.
Assuntos
Glicosiltransferases/fisiologia , Proteoglicanas/biossíntese , Sulfotransferases/fisiologia , Desaminação , Hidrólise , Ácido Nitroso/química , Especificidade por SubstratoRESUMO
We have shown previously that purified chondroitin 6-sulfotransferase-1 (C6ST-1) was a glycoprotein abundant in N-linked oligosaccharides and could sulfate both chondroitin (C6ST activity) and keratan sulfate (KSST activity); however, functional roles of the N-glycans have remained unclear. In the present study, we show essential roles of N-glycans attached to C6ST-1 in the generation of the active enzyme and in its KSST activity. Treatment with tunicamycin of COS-7 cells transfected with C6ST-1 cDNA totally abolished production of the active C6ST-1. A nearly complete removal of N-glycans of the recombinant C6ST-1 by peptide N-glycosidase F increased the C6ST activity but decreased the KSST activity. Among six potential N-glycosylation sites, deletion of the fourth or sixth site from the amino terminus inhibited production of the active C6ST-1, whereas deletion of the fifth site resulted in a marked loss of the KSST activity. Wild-type recombinant C6ST-1 showed a typical Golgi localization, whereas M-4 recombinant C6ST-1, in which the fourth N-glycosylation site was deleted, colocalized with calnexin, an endoplasmic reticulum-resident protein. Unlike wildtype recombinant C6ST-1, M-4 recombinant C6ST-1 showed a weak affinity toward wheat germ agglutinin and was converted completely to the nonglycosylated form by endoglycosidase H. These observations suggest that N-glycan attached to the fourth N-glycosylation site may function in the proper processing of N-glycans required for the Golgi localization, thereby causing the production of the active C6ST-1, and that N-glycan attached to the fifth N-glycosylation site may contribute to the KSST activity of C6ST-1.
Assuntos
Complexo de Golgi/enzimologia , Sulfato de Queratano/metabolismo , Oligossacarídeos/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência de Carboidratos , Códon , Primers do DNA , Retículo Endoplasmático/enzimologia , Cinética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligossacarídeos/química , Fases de Leitura Aberta , Polissacarídeos/metabolismo , Transporte Proteico , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tunicamicina/farmacologia , Carboidrato SulfotransferasesRESUMO
We have shown previously that a highly sulfated sequence, GalNAc(4,6-SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), is present at the nonreducing terminal of chondroitin sulfate (CS), and this structure was synthesized from a unique sequence, GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), by sulfation with N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase. Uronosyl 2-O-sulfotrasferase (2OST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 2 of the GlcA residue of CS, is expected to be involved in synthesis of these structures; however, the specificity of 2OST concerning recognition of the sulfation pattern of the acceptor has largely remained unclear. In the present study, we examined the specificity of 2OST in terms of recognition of the sulfation pattern around the targeting GlcA residue. The recombinant 2OST could sulfate CS-A, CS-C, and desulfated dermatan sulfate. When [(35)S]glycosaminoglycans formed from CS-A after the reaction with the recombinant 2OST and [(35)S]PAPS were subjected to limited digestion with chondroitinase ACII, a radioactive tetrasaccharide (Tetra A) was obtained as a sole intermediate product. The sequence of Tetra A was found to be DeltaHexA-GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)) by enzymatic and chemical reactions. These observations indicate that 2OST transfers sulfate preferentially to the GlcA residue located in a unique sequence, -GalNAc(4SO(4))-GlcA-GalNAc(6SO(4))-. When oligosaccharides with different sulfation patterns were used as the acceptor, GalNAc(4SO(4))-GlcA-GalNAc(6SO(4)) and GlcA-GalNAc(4SO(4))-GlcA-GalNAc(6SO(4)) were the best acceptors for 2OST among trisaccharides and tetrasaccharides, respectively. These results suggest that 2OST may be involved in the synthesis of the highly sulfated structure found in CS-A.
Assuntos
Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Sulfotransferases/metabolismo , Animais , Sequência de Bases , Sequência de Carboidratos , Condroitina Liases/metabolismo , DNA Complementar/genética , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Humanos , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Estrutura Molecular , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Oligossacarídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Sulfatos/química , Sulfotransferases/genéticaRESUMO
We have previously cloned N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the C-6 hydroxyl group of the GalNAc 4-sulfate residue of chondroitin sulfate A and forms chondroitin sulfate E containing GlcA-GalNAc(4,6-SO(4)) repeating units. To investigate the function of chondroitin sulfate E, the development of specific inhibitors of GalNAc4S-6ST is important. Because GalNAc4S-6ST requires a sulfate group attached to the C-4 hydroxyl group of the GalNAc residue as the acceptor, the sulfated GalNAc residue is expected to interact with GalNAc4S-6ST and affect its activity. In this study, we synthesized phenyl alpha- or -beta-2-acetamido-2-deoxy-beta-D-galactopyranosides containing a sulfate group at the C-3, C-4, or C-6 hydroxyl groups and examined their inhibitory activity against recombinant GalNAc4S-6ST. We found that phenyl beta-GalNAc(4SO(4)) inhibits GalNAc4S-6ST competitively and also serves as an acceptor. The sulfated product derived from phenyl beta-GalNAc(4SO(4)) was identical to phenyl beta-GalNAc(4,6-SO(4)). These observations indicate that derivatives of beta-D-GalNAc(4SO(4)) are possible specific inhibitors of GalNAc4S-6ST.