Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.6.1 (sulfatase)
3,205 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mucopolysaccharidosis type IIID (MPS IIID) is a lysosomal storage disorder resulting from lack of activity of the lysosomal hydrolase N-acetylglucosamine 6-sulfatase (6S) (EC 3.1.6.14). The syndrome is associated with systemic and central nervous system (CNS) heparan sulfate glycosaminoglycan (HS-GAG) accumulation, secondary storage of lipids, and severe, progressive dementia. In this investigation, caprine MPS IIID, established as a large animal model for the human disease, was used to evaluate the efficacy of enzyme replacement therapy (ERT). Recombinant caprine 6S (rc6S) (1 mg/kg/dose) was administered intravenously to one MPS IIID goat kid at 2, 3, and 4 wks of age. Five days after the last dose, the uronic acid (UA) content and the composition of uncatabolized HS-GAG fractions in the brain of the ERT-treated MPS IIID kid were similar to those from a control, untreated MPS IIID animal. However, hepatic uronic acid levels in the treated MPS IIID kid were approximately 90% lower than those in the untreated MPS IIID control; whereas the composition of the residual hepatic HS-GAG was identical to that in the untreated animal. Marked reduction of lysosomal storage vacuoles in hepatic cells of the treated MPS IIID kid was observed, but ERT had no effect on CNS lesions. No residual 6S activity was detected in brain or liver. This preliminary investigation indicates that other treatment regimens will be necessary to ameliorate MPS III-related CNS lesions.
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PMID:Caprine mucopolysaccharidosis IIID: a preliminary trial of enzyme replacement therapy. 1130 88

We describe the design and synthesis of substrate and internal standard conjugates for application in profiling enzyme activity of the enzymes alpha-D-2-deoxy-2-N-sulfonamido-glucosamine sulfamidase, alpha-D-2-deoxy-2-N-acetyl-glucosamine hydrolase, acetyl-coenzymeA:alpha-D-2-deoxy-2-amino-glucosamine transferase, and alpha-D-2-deoxy-2-N-acetyl-glucosamine-6-sulfate sulfatase. Deficiency of any one of these enzymes results in a single clinical phenotype known as Sanfilippo syndrome. Such substrates have been proven effective in the confirmation of enzyme deficiency by a combination of affinity chromatography (AC) and electrospray ionization mass spectrometry (ESIMS), which forms the foundation for a new analytical technology (ACESIMS) of general interest and application to clinical and biomedical research.
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PMID:Design and synthesis of substrate and internal standard conjugates for profiling enzyme activity in the Sanfilippo syndrome by affinity chromatography/electrospray ionization mass spectrometry. 1145 66

Here we report the cloning of a full-length cDNA encoding the human ortholog (HSulf-1) of the developmentally regulated putative sulfatases QSulf-1 (Dhoot, G. K., Gustafsson, M. K., Ai, X., Sun, W., Standiford, D. M., and Emerson, C. P., Jr. (2001) Science 293, 1663-1666) and RSulfFP1 (Ohto, T., Uchida, H., Yamazaki, H., Keino-Masu, K., Matsui, A., and Masu, M. (2002) Genes Cells 7, 173-185) as well as a cDNA encoding a closely related protein, designated HSulf-2. We have also obtained cDNAs for the mouse orthologs of both Sulfs. We demonstrate that the proteins encoded by both classes of cDNAs are endoproteolytically processed in the secretory pathway and are released into conditioned medium of transfected CHO cells. We demonstrate that the mammalian Sulfs exhibit arylsulfatase activity with a pH optimum in the neutral range; moreover, they can remove sulfate from the C-6 position of glucosamine within specific subregions of intact heparin. Taken together, our results establish that the mammalian Sulfs are extracellular endosulfatases with strong potential for modulating the interactions of heparan sulfate proteoglycans in the extracellular microenvironment.
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PMID:Cloning and characterization of two extracellular heparin-degrading endosulfatases in mice and humans. 1236 95

In the previous paper (Myette, J. R., Shriver, Z., Claycamp, C., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2003) J. Biol. Chem. 278, 12157-12166), we described the molecular cloning, recombinant expression, and preliminary biochemical characterization of the heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. In this paper, we extend our structure-function investigation of the 2-O-sulfatase. First, we have constructed a homology-based structural model of the enzyme active site, using as a framework the available crystallographic data for three highly related arylsulfatases. In this model, we have identified important structural parameters within the enzyme active site relevant to enzyme function, especially as they relate to its substrate specificity. By docking various disaccharide substrates, we identified potential structural determinants present within these substrates that would complement this unique active site architecture. These determinants included the position and number of sulfates present on the glucosamine, oligosaccharide chain length, the presence of a Delta4,5-unsaturated double bond, and the exolytic versus endolytic potential of the enzyme. The predictions made from our model provided a structural basis of substrate specificity originally interpreted from the biochemical and kinetic data. Our modeling approach was further complemented experimentally using peptide mapping in tandem with mass spectrometry and site-directed mutagenesis to physically demonstrate the presence of a covalently modified cysteine (formylglycine) within the active site. This combinatorial approach of structure modeling and biochemical studies provides insight into the molecular basis of enzyme function.
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PMID:The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. A structural and biochemical study of the enzyme active site and saccharide substrate specificity. 1251 74

The venom of Naja nigricolis was found to contain a high level of the enzyme aryl sulfatase. The enzyme was isolated from the venom of N. nigriclois and purified to electrophoretic homogeneity by gel chromatography on Sephadex G-100, DEAE-cellulose, and phenyl-sepharose columns. The enzyme was optimally active at pH 5 and 40 degrees C. Arrhenius plot for the determination of the activation energy (E(a)) gave the value 25 kJ/mol with a half-life (t(1/2)) of 5 min at 50 degrees C. It was highly activated by Fe(2+) and Ca(2+) and inhibited by Co(2+) and Mn(2+). The enzyme catalyzed the hydrolysis of the fluorescent compound methylumbelliferyl-sulfate (MU-SO(4)). Double reciprocal plots of initial velocity data, using MU-SO(4) as substrate, gave a K(M) value of 110 microM and V(max) of 225 micromol min(-1) x mg(-1). N. nigricolis Aryl sulphatase also hydrolyzed chondroitin-4-sulphate. It was inhibited competitively by N-acetyl glucosamine sulfate (GlcNAc-SO(4)), glucose-6-sulfate (Glc-6-SO(4)), and glucose 1-sulfate (Glc-1-SO(4)). Extrapolated inhibition binding constants (K(i)) gave the values of 3, 25, and 315 microM for GlcNAc-SO(4), Glc-6-SO(4), and Glc-1-SO(4) respectively.
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PMID:Aryl sulfatase from Naja nigricolis venom: characterization and possible contribution in the pathology of snake poisoning. 1261 48

Heparan sulfate d-glucosaminyl 3-O-sulfotransferases (3-OSTs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 3 of the glucosamine residue of heparan sulfate and heparin. A sixth member of the human 3-OST family, named 3-OST-5, was recently reported (Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.-P., Malmstrom, A., Shukla, D., and Liu, J. (2002) J. Biol. Chem. 277, 37912-37919). In the present study, we cloned putative catalytic domain of the human 3-OST-5 and expressed it in insect cells as a soluble enzyme. Recombinant 3-OST-5 only exhibited sulfotransferase activity toward heparan sulfate and heparin. When incubated heparan sulfate with [35S]PAPS, the highest incorporation of35S was observed, and digestion of the product with a mixture of heparin lyases yielded two major35S-labeled disaccharides, which were determined as DeltaHexA-GlcN(NS,3S,6S) and DeltaHexA(2S)-GlcN(NS,3S) by further digestion with 2-sulfatase and degradation with mercuric acetate. However, when used heparin as acceptor, we identified a highly sulfated disaccharide unit as a major product. This had a structure of DeltaHexA(2S)-GlcN(NS,3S,6S). Quantitative real-time PCR analysis revealed that 3-OST-5 was highly expressed in fetal brain, followed by adult brain and spinal cord, and at very low or undetectable levels in the other tissues. Finally, we detected a tetrasulfated disaccharide unit in bovine intestinal heparan sulfate. To our knowledge, this is the first report to describe not only the natural occurrence of tetrasulfated disaccharide unit but also the enzymatic formation of this novel structure.
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PMID:Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide. 1274 Mar 61

Mucopolysaccharidosis IIID (MPS IIID) is a lysosomal storage disease associated with deficient activity of the enzyme N-acetylglucosamine 6-sulfatase (EC 3.1.6.14), a lysosomal hydrolase in the heparan sulfate glycosaminoglycan (HS-GAG) degradation pathway. In caprine MPS IIID, enzyme replacement therapy reversed early postnatal systemic but not primary or secondary central nervous system (CNS) substrate accumulations. The caprine MPS IIID large animal model system was used in this investigation to define the developmental profile of morphological and biochemical perturbations to estimate a time frame for therapeutic intervention. Light and electron microscopy were used to compare the CNS, liver, and kidney of normal +/+, MPS IIID carrier +/-, and MPS IIID-affected -/- goat kids (kids), at 60, 113-114, 128-129, and 135 d gestation (dg) of a 150-d gestational period, at birth, and at 59-64 d of postnatal (d-pn) age. In the CNS of -/- kids, morphological correlations of HS-GAG and glycolipid accumulations were evident in early differentiating neurons at 60 dg. CNS and systemic developmental, regional, and cellular differences in -/- kids at all time points included more prominent and earlier accumulation of lucent, putative HS-GAG substrates in lysosomes of meningeal and perivascular macrophages and hepatic sinusoidal cells than in CNS, hepatic, or renal parenchymal cells. The amounts and compositions of HS-GAG substrates in the brain and liver of +/+, +/-, and -/- kids were determined at 60, 65, 113-114, and 128-135 dg, at birth, and 53-78 d-pn. In the CNS of -/- kids, HS-GAG concentrations were variable and exceeded those of age-matched control tissue samples in the third but not the second trimester. In contrast, hepatic HS-GAG levels in -/- kids exceeded control values at all time points evaluated and paralleled the progressive morphological alterations. CNS and hepatic HS-GAG compositions in -/- kids were similar to each other and were more complex at all pre- and postnatal ages than those from control kids. Based on the time frame of development of CNS lesions and biochemical perturbations, prenatal therapeutic intervention in caprine MPS IIID is likely to be necessary to prevent or ameliorate substantive CNS and systemic lesions.
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PMID:Caprine mucopolysaccharidosis IIID: fetal and neonatal brain and liver glycosaminoglycan and morphological perturbations. 1545 41

Heparan sulfate is a linear glycosaminoglycan with considerable structural diversity that binds a myriad of growth factors and proteins that play pivotal roles in a variety of biological processes. We have investigated the structural complexity of partially degraded fragments of heparan sulfate in mucopolysaccharidosis type IIIA in which there is a defect in heparan sulfate catabolism. Mono- to hexadecasaccharides were isolated from the urine of a mucopolysaccharidosis IIIA patient and shown to have non-reducing end glucosamine N-sulfate residues, reflecting the catabolic deficiency in heparan N-sulfatase (sulfamidase) activity. The use of nitrous acid digestion (pH 1.5) combined with separation by reverse-phase high-performance liquid chromatography and analysis by electrospray ionization-mass spectrometry identified multiple forms of these oligosaccharides with some N-acetylated glucosamine residues and one to three sulfates per disaccharide. Furthermore, we demonstrated that each oligosaccharide existed in multiple sulfated forms. Many structural isomers were present, suggesting a complex mixture of oligosaccharides present in the urine as a consequence of a defect in heparan sulfate degradation.
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PMID:Characterization of sulfated oligosaccharides in mucopolysaccharidosis type IIIA by electrospray ionization mass spectrometry. 1680 63

Sulf-1 and Sulf-2 are novel extracellular sulfatases that act on internal glucosamine 6-O-sulfate modifications within heparan sulfate proteoglycans and regulate their interactions with various signaling molecules, including Wnt ligands. Although the Sulfs are multidomain proteins, there is limited information available about how the subdomains contribute to their enzymatic and signaling activities. In this study, we found that both human Sulfs were synthesized as prepro-enzymes and cleaved by a furin-type proteinase to form disulfide-bond linked heterodimers of 75- and 50-kDa subunits. The mature Sulfs were secreted into conditioned medium, as well as retained on the cell membrane. Although the catalytic center resides in the N-terminal 75-kDa subunit, the C-terminal 50-kDa subunit was indispensable for both arylsufatase and glucosamine 6-O-sulfate-endosulfatase activity. We found that the hydrophilic regions of the Sulfs were essential for endosulfatase activity but not for arylsulfatase activity. Using Edman sequencing, we identified furin-type proteinase cleavage sites in Sulf-1 and Sulf-2. Deletion of these sequences resulted in uncleavable forms of Sulfs. The uncleavable Sulfs retained enzymatic activity. However, they were unable to potentiate Wnt signaling, which may be due to their defective localization into lipid rafts on the plasma membrane.
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PMID:Functional consequences of the subdomain organization of the sulfs. 1952 Aug 66

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.
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PMID:Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity. 1972 71


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