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Michael H Gelb - One of the best experts on this subject based on the ideXlab platform.
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fluorimetric assays for n acetylgalactosamine 6 sulfatase and arylsulfatase b based on the natural substrates for confirmation of mucopolysaccharidoses types iva and vi
Clinica Chimica Acta, 2015Co-Authors: Arun Babu Kumar, Zdenek Spacil, Farideh Ghomashchi, Sophia Masi, Tomomi Sumida, Frantisek Turecek, Ronald C Scott, Michael H GelbAbstract:Background: Treatments have been developed for mucopolysaccharidoses IVA (MPS IVA) and MPS VI suggesting the need for eventual newborn screening. Biochemical enzyme assays are important for diagnosis. Previously reported fluorimetric assays of the relevant enzymes are based on substrates with poor activity or specificity. Methods: We developed new fluorimetric assays for N-Acetylgalactosamine-6-sulfatase (GALNS) and arylsulfatase B (ARSB) based on the natural substrates, N-Acetylgalactosamine-6-sulfate (and 4-sulfate), which have improved activity and specificity toward the relevant enzymes. The new substrates were tested on dried blood spots on newborn screening cards, and assays showed acceptable linearity in response with the amount of enzyme present (using quality control samples). Results: When tested on dried blood spots from random newborns and affected patients, the assays showed good discrimination between the 2 sample groups. Conclusions: The analytical range of the new fluorimetric assays, defined as the ratio of enzyme-dependent-to-enzyme-independent assay response, is likely to be insufficient to use these assays for newborn screening. Rather, these new fluorimetric assays should be useful in a diagnostic lab to confirm a diagnosis via biochemical enzyme testing. (C) 2015 Elsevier B.V. All rights reserved.
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fluorimetric assays for n acetylgalactosamine 6 sulfatase and arylsulfatase b based on the natural substrates for confirmation of mucopolysaccharidoses types iva and vi
Clinica Chimica Acta, 2015Co-Authors: Arun Babu Kumar, Zdenek Spacil, Farideh Ghomashchi, Sophia Masi, Tomomi Sumida, Frantisek Turecek, Ronald C Scott, Makoto Ito, Michael H GelbAbstract:Abstract Background Treatments have been developed for mucopolysaccharidoses IVA (MPS IVA) and MPS VI suggesting the need for eventual newborn screening. Biochemical enzyme assays are important for diagnosis. Previously reported fluorimetric assays of the relevant enzymes are based on substrates with poor activity or specificity. Methods We developed new fluorimetric assays for N-Acetylgalactosamine-6-sulfatase (GALNS) and arylsulfatase B (ARSB) based on the natural substrates, N-Acetylgalactosamine-6-sulfate (and 4-sulfate), which have improved activity and specificity toward the relevant enzymes. The new substrates were tested on dried blood spots on newborn screening cards, and assays showed acceptable linearity in response with the amount of enzyme present (using quality control samples). Results When tested on dried blood spots from random newborns and affected patients, the assays showed good discrimination between the 2 sample groups. Conclusions The analytical range of the new fluorimetric assays, defined as the ratio of enzyme-dependent-to-enzyme-independent assay response, is likely to be insufficient to use these assays for newborn screening. Rather, these new fluorimetric assays should be useful in a diagnostic lab to confirm a diagnosis via biochemical enzyme testing.
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tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots application to screening newborns for mucopolysaccharidosis ii hunter syndrome
Analytical Chemistry, 2010Co-Authors: Brian J Wolfe, Frantisek Turecek, Ronald C Scott, Sophie Blanchard, Martin Sadilek, Michael H GelbAbstract:We report a new assay of N-Acetylgalactosamine-4-sulfatase (aryl sulfatase B) activity in dried blood spots (DBS) for the early detection of mucopolysaccharidosis VI (Maroteaux−Lamy syndrome) in newborn screening. The assay uses a synthetic substrate consisting of N-Acetylgalactosamine-4-sulfate moiety glycosidically linked to a hydrophobic residue and furnished with a tert-butyloxycarbamido group as a marker for specific mass spectrometric fragmentation. Incubation with aryl sulfatase B present in DBS converts the substrate to a desulfated product which is detected by electrospray tandem mass spectrometry and quantified using a homologous internal standard. Assay and workup procedures were optimized to be compatible with the work flow in newborn screening laboratories. Analysis of DBS from human newborns showed clear distinction of aryl sulfatase B activity from 89 healthy individuals where it ranged between 1.4 and 16.9 μmol/(h L of blood), with an average activity of 7.4 μmol/(h L of blood), and an MPS...
Jacques U Baenziger - One of the best experts on this subject based on the ideXlab platform.
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congenital disorders of glycosylation with emphasis on loss of dermatan 4 sulfotransferase
Progress in Molecular Biology and Translational Science, 2010Co-Authors: Lijuan Zhang, Jacques U Baenziger, Thomas Muller, Andreas R JaneckeAbstract:Abstract The autosomal, recessively inherited, adducted thumb-clubfoot syndrome (ATCS) represents a generalized connective tissue disorder with congenital malformations, contractures of thumbs and feet, and a typical facial appearance. Cognitive development is normal in ATCS patients during childhood. ATCS is caused by homozygous nonsense and missense mutations in CHST14 which encodes an N -acetylgalactosamine 4- O -sulfotransferase 1 (D4ST1) that catalyzes the 4- O -sulfation of N -acetylgalactosamine in the repeating iduronic acid-α-1,3- N -acetylgalactosamine disaccharide sequence to form dermatan sulfate (DS). ATCS mutations lead to either a decrease or a loss of D4ST1 activity, as revealed by absence of DS and an excess of chondroitin sulfate (CS) in patient's fibroblasts. Either of these effects or their combination might cause the observed clinical symptoms by altering the physiological pattern of dermatan and CS chains on their corresponding proteoglycans (PGs). ATCS is the only recognized disorder resulting from a defect that is specific to DS biosynthesis, and thus represents another class of the congenital glycosylation disorders. Congenital disorders of glycosylation (CDG) include all genetic diseases that result from defects in the synthesis of glycans. These disorders cause a wide range of human diseases, with examples emanating from all medical subspecialties. ATCS is the first human disorder that emphasizes a role for DS in human development and extracellular matrix maintenance.
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molecular cloning and characterization of a dermatan specific n acetylgalactosamine 4 o sulfotransferase
Journal of Biological Chemistry, 2001Co-Authors: Matthias R Evers, Hyunggyoo Kang, Jacques U BaenzigerAbstract:Abstract We have identified and characterized anN-Acetylgalactosamine-4-O-sulfotransferase designated dermatan-4-sulfotransferase-1 (D4ST-1) (GenBankTM accession number AF401222) based on its homology to HNK-1 sulfotransferase. The cDNA predicts an open reading frame encoding a type II membrane protein of 376 amino acids with a 43-amino acid cytoplasmic domain and a 316-amino acid luminal domain containing two potential N-linked glycosylation sites. D4ST-1 has significant amino acid identity with HNK-1 sulfotransferase (21.4%),N-Acetylgalactosamine-4-O-sulfotransferase 1 (GalNAc-4-ST1) (24.7%),N-Acetylgalactosamine-4-O-sulfotransferase 2 (GalNAc-4-ST2) (21.0%), chondroitin-4-O-sulfotransferase 1 (27.3%), and chondroitin-4-O-sulfotransferase 2 (22.8%). D4ST-1 transfers sulfate to the C-4 hydroxyl of β1,4-linked GalNAc that is substituted with an α-linked iduronic acid (IdoUA) at the C-3 hydroxyl. D4ST-1 shows a strong preferencein vitro for sulfate transfer to IdoUAα1,3GalNAcβ1,4 that is flanked by GlcUAβ1,3GalNAcβ1,4 as compared with IdoUAα1,3GalNAcβ1,4 flanked by IdoUAα1,3GalNAcβ1,4. The specificity of D4ST-1 when assayed in vitro suggests that the addition of sulfate to GalNAc occurs immediately after epimerization of GlcUA to IdoUA. The open reading frame of D4ST-1 is encoded by a single exon located on human chromosome 15q14. Northern blot analysis reveals a single 2.4-kilobase transcript. D4ST-1 message is expressed in virtually all tissues at some level but is most highly expressed in pituitary, placenta, uterus, and thyroid. The properties of D4ST-1 indicate that sulfation of the GalNAc moieties in dermatan is mediated by a distinct GalNAc-4-O-sulfotransferase and occurs following epimerization of GlcUA to IdoUA.
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molecular cloning and characterization of a dermatan specific n acetylgalactosamine 4 o sulfotransferase
Journal of Biological Chemistry, 2001Co-Authors: Matthias R Evers, Hyunggyoo Kang, Guoqing Xia, Melitta Schachner, Jacques U BaenzigerAbstract:We have identified and characterized an N-Acetylgalactosamine-4-O-sulfotransferase designated dermatan-4-sulfotransferase-1 (D4ST-1) (GenBank(TM) accession number AF401222) based on its homology to HNK-1 sulfotransferase. The cDNA predicts an open reading frame encoding a type II membrane protein of 376 amino acids with a 43-amino acid cytoplasmic domain and a 316-amino acid luminal domain containing two potential N-linked glycosylation sites. D4ST-1 has significant amino acid identity with HNK-1 sulfotransferase (21.4%), N-Acetylgalactosamine-4-O-sulfotransferase 1 (GalNAc-4-ST1) (24.7%), N-Acetylgalactosamine-4-O-sulfotransferase 2 (GalNAc-4-ST2) (21.0%), chondroitin-4-O-sulfotransferase 1 (27.3%), and chondroitin-4-O-sulfotransferase 2 (22.8%). D4ST-1 transfers sulfate to the C-4 hydroxyl of beta1,4-linked GalNAc that is substituted with an alpha-linked iduronic acid (IdoUA) at the C-3 hydroxyl. D4ST-1 shows a strong preference in vitro for sulfate transfer to IdoUAalpha1,3GalNAcbeta1,4 that is flanked by GlcUAbeta1,3GalNAcbeta1,4 as compared with IdoUAalpha1,3GalNAcbeta1,4 flanked by IdoUAalpha1,3GalNAcbeta1,4. The specificity of D4ST-1 when assayed in vitro suggests that the addition of sulfate to GalNAc occurs immediately after epimerization of GlcUA to IdoUA. The open reading frame of D4ST-1 is encoded by a single exon located on human chromosome 15q14. Northern blot analysis reveals a single 2.4-kilobase transcript. D4ST-1 message is expressed in virtually all tissues at some level but is most highly expressed in pituitary, placenta, uterus, and thyroid. The properties of D4ST-1 indicate that sulfation of the GalNAc moieties in dermatan is mediated by a distinct GalNAc-4-O-sulfotransferase and occurs following epimerization of GlcUA to IdoUA.
Osami Habuchi - One of the best experts on this subject based on the ideXlab platform.
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mice deficient in n acetylgalactosamine 4 sulfate 6 o sulfotransferase exhibit enhanced liver fibrosis and delayed recovery from fibrosis in carbon tetrachloride treated mice
Heliyon, 2016Co-Authors: Hiroko Habuchi, Takahiro Ushida, Osami HabuchiAbstract: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.
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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
Journal of Biological Chemistry, 2010Co-Authors: Shiori Ohtakeniimi, Hiroko Habuchi, Saori Kakehi, Sachiko Kondo, Tadayuki Ohta, Koji Kimata, Osami HabuchiAbstract:Abstract Chondroitin sulfate (CS) and dermatan sulfate (DS) containing N-Acetylgalactosamine 4,6-bissulfate (GalNAc(4,6-SO4)) 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-SO4) 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-SO4) residues in CS and DS disappeared completely, indicating that GalNAc4S-6ST should be a sole enzyme responsible for the synthesis of GalNAc(4,6-SO4) residues in both CS and DS. IdoA-GalNAc(4,6-SO4) units that account for ∼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-SO4) 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-SO4) 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.
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human n acetylgalactosamine 4 sulfate 6 o sulfotransferase cdna is related to human b cell recombination activating gene associated gene
Journal of Biological Chemistry, 2001Co-Authors: Shiori Ohtake, Masakazu Fukuta, Osami HabuchiAbstract:Abstract N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3′-phosphoadenosine 5′-phosphosulfate to position 6 ofN-Acetylgalactosamine 4-sulfate (GalNAc(4SO4)) in chondroitin sulfate and dermatan sulfate. We have previously purified the enzyme to apparent homogeneity from the squid cartilage. We report here cloning and characterization of human GalNAc4S-6ST. The strategy for identification of human GalNAc4S-6ST consisted of: 1) determination of the amino acid sequences of peptides derived from the purified squid GalNAc4S-6ST, 2) amplification of squid DNA by polymerase chain reaction, and 3) homology search using the amino acid sequence deduced from the squid DNA. The human GalNAc4S-6ST cDNA contains a single open reading frame that predicts a type II transmembrane protein composed of 561 amino acid residues. The recombinant protein expressed from the human GalNAc4S-6ST cDNA transferred sulfate from 3′-phosphoadenosine 5′-phosphosulfate to position 6 of the nonreducing terminal and internal GalNAc(4SO4) residues contained in chondroitin sulfate A and dermatan sulfate. When a trisaccharide and a pentasaccharide having sulfate groups at position 4 of N-Acetylgalactosamine residues were used as acceptors, only nonreducing terminal GalNAc(4SO4) residues were sulfated. The nucleotide sequence of the human GalNAc4S-6ST cDNA was nearly identical to the sequence of human B cell recombination activating gene-associated gene.
Leo Feferman - One of the best experts on this subject based on the ideXlab platform.
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increased chst15 follows decline in arylsulfatase b arsb and disinhibition of non canonical wnt signaling potential impact on epithelial and mesenchymal identity
Oncotarget, 2020Co-Authors: Sumit Bhattacharyya, Leo Feferman, Xiaorui Han, Ke Xia, Fuming Zhang, Robert J Linhardt, Joanne K TobacmanAbstract:Expression of CHST15 (carbohydrate sulfotransferase 15; chondroitin 4-sulfate-6-sulfotransferase; BRAG), the sulfotransferase enzyme that adds 6-sulfate to chondroitin 4-sulfate (C4S) to make chondroitin 4,6-disulfate (chondroitin sulfate E, CSE), was increased in malignant prostate epithelium obtained by laser capture microdissection and following arylsulfatase B (ARSB; N-Acetylgalactosamine-4-sulfatase) silencing in human prostate epithelial cells. Experiments in normal and malignant human prostate epithelial and stromal cells and tissues, in HepG2 cells, and in the ARSB-null mouse were performed to determine the pathway by which CHST15 expression is up-regulated when ARSB expression is reduced. Effects of Wnt-containing prostate stromal cell spent media and selective inhibitors of WNT, JNK, p38, SHP2, β-catenin, Rho, and Rac-1 signaling pathways were determined. Activation of WNT signaling followed declines in ARSB and Dickkopf WNT Signaling Pathway Inhibitor (DKK)3 and was required for increased CHST15 expression. The increase in expression of CHST15 followed activation of non-canonical WNT signaling and involved Wnt3A, Rac-1 GTPase, phospho-p38 MAPK, and nuclear DNA-bound GATA-3. Inhibition of JNK, Sp1, β-catenin nuclear translocation, or Rho kinase had no effect. Consistent with higher expression of CHST15 in prostate epithelium, disaccharide analysis showed higher levels of CSE and chondroitin 6-sulfate (C6S) disaccharides in prostate epithelial cells. In contrast, chondroitin 4-sulfate (C4S) disaccharides were greater in prostate stromal cells. CSE may contribute to increased C4S in malignant epithelium when GALNS (N-aceytylgalactosamine-6-sulfate sulfatase) is increased and ARSB is reduced. These effects increase chondroitin 4-sulfates and reduce chondroitin 6-sulfates, consistent with enhanced stromal characteristics and epithelial-mesenchymal transition.
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Defining the Role of Arylsulfatase B (N-Acetylgalactosamine 4-Sulfatase) in Cellular Metabolism
The FASEB Journal, 2015Co-Authors: Leo FefermanAbstract:Arylsulfatase B (ARSB; N-Acetylgalactosamine 4-sulfatase) is the lysosomal and membrane enzyme that removes 4-sulfate groups from the non-reducing end of chondroitin 4-sulfate and dermatan sulfate,...
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arylsulfatase b n acetylgalactosamine 4 sulfatase potential role as a biomarker in prostate cancer
Prostate Cancer and Prostatic Diseases, 2013Co-Authors: Leo Feferman, Peter H Gann, Ryan Deaton, Grace Guzman, Andre KajdacsyballaAbstract:Arylsulfatase B (N-Acetylgalactosamine-4-sulfatase): potential role as a biomarker in prostate cancer
Avadhesha Surolia - One of the best experts on this subject based on the ideXlab platform.
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structures of the complexes of peanut lectin with methyl beta galactose and n acetyllactosamine and a comparative study of carbohydrate binding in gal galnac specific legume lectins
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: R Ravishankar, Avadhesha Surolia, K Suguna, M VijayanAbstract:The crystal structures of complexes of peanut lectin with methyl-β-galactose and N-acetyllactosamine have been determined at 2.8 and 2.7 A, respectively. These, and the complexes involving lactose and the T-antigenic disaccharide reported previously, permit a detailed characterization of peanut-lectin–carbohydrate association and the role of water molecules therein. The water molecules in the combining site are substantially conserved in the four complexes. The role of interacting sugar hydroxyl groups, when absent, are often mimicked by ordered water molecules not only at the primary combining site, but also at the site of the second sugar ring. The similarity of peanut-lectin–sugar interactions with those in other galactose/N-Acetylgalactosamine-specific lectins also extend to a substantial degree to water bridges. The comparative study provides a structural explanation for the exclusive specificity of peanut lectin for galactose at the monosaccharide level, compared with that of the other lectins for galactose as well as N-Acetylgalactosamine. The complexes also provide a qualitative structural rationale for differences in the strengths of binding of peanut lectin to different sugars.
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molecular basis of recognition by gal galnac specific legume lectins influence of glu 129 on the specificity of peanut agglutinin pna towards c2 substituents of galactose
Glycobiology, 1998Co-Authors: Vivek Sharma, V R Srinivas, P Adhikari, M Vijayan, Avadhesha SuroliaAbstract:The ability to discriminate between galactose and N-acetylga-lactosamine, observed in some lectins, is crucial for their biological activity as well as their usefulness as tools in biology and medicine. However, the molecular basis of differential binding of lectins to these two sugars is poorly understood. Peanut agglutinin (PNA) is one of the few galactose-specific legume lectins which does not bind N-Acetylgalactosamine at all and is, therefore, ideal for the study of the basis of specificity towards C-2 substituted derivatives of galactopyranosides. Examination of the three-dimensional structure of PNA in complex with lactose revealed the presence of both a longer loop and bulkier residues in the region surrounding the C-2 hydroxyl of the galactopyranoside ring, which can sterically prevent the accommodation of a bulky substituent in this position. One such residue, is a glutamic acid at position 129 which protrudes into the binding site and perhaps directly obstructs any substitution at the C-2 position. Two mutants in bacterially expressed PNA were therefore constructed. These were E129D and E129A, in which Glu129 was replaced by Asp and Ala, respectively. The specificity of the mutants for galactose, galactosamine, and N-Acetylgalactosamine was examined through observing the inhibition of hemagglutination and binding of the lectin to immobilized asialofetuin. The results showed that the affinity of E129A and E129D for C-2-substituted derivatives of the galactose varies. The mutant E129D showed significant binding towards N-Acetylgalactosamine, suggesting that the residue Glu 129 is crucial in imparting exclusive galactose-specificity upon PNA. This study not only attempts to provide an explanation for the inability of PNA to accommodate C-2-substituted derivatives at its primary subsite, but also seeks to present a basis for engineering lectins with altered specificities.
