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Thomas Dierks - One of the best experts on this subject based on the ideXlab platform.
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expanding the genetic cause of multiple Sulfatase deficiency a novel sumf1 variant in a patient displaying a severe late infantile form of the disease
Molecular Genetics and Metabolism, 2017Co-Authors: Ilona Jaszczuk, Lars Schlotawa, Thomas Dierks, Karthikeyan Radhakrishnan, Andreas Ohlenbusch, Dominique Koppenhofer, Mariusz Babicz, Monika Lejman, Agnieszka ługowskaAbstract:Abstract Multiple Sulfatase deficiency (MSD) is a rare inherited metabolic disease caused by defective cellular Sulfatases. Activity of Sulfatases depends on post-translational modification catalyzed by formylglycine-generating enzyme (FGE), encoded by the SUMF1 gene. SUMF1 pathologic variants cause MSD, a syndrome presenting with a complex phenotype. We describe the first Polish patient with MSD caused by a yet undescribed pathologic variant c.337G > A [p.Glu113Lys] (i.e. p.E113K) in heterozygous combination with the known deletion allele c.519 + 5_519 + 8del [p.Ala149_Ala173del]. The clinical picture of the patient initially suggested late infantile metachromatic leukodystrophy, with developmental delay followed by regression of visual, hearing and motor abilities as the most apparent clinical symptoms. Transient signs of ichthyosis and minor dysmorphic features guided the laboratory workup towards MSD. Since MSD is a rare disease and there is a variable clinical spectrum, we thoroughly describe the clinical outcome of our patient. The FGE-E113K variant, expressed in cell culture, correctly localized to the endoplasmic reticulum but was retained intracellularly in contrast to the wild type FGE. Analysis of FGE-mediated activation of steroid Sulfatase in immortalized MSD cells revealed that FGE-E113K exhibited only approx. 15% of the activity of wild type FGE. Based on the crystal structure we predict that the exchange of glutamate-113 against lysine should induce a strong destabilization of the secondary structure, possibly affecting the folding for correct disulfide bridging between C235-C346 as well as distortion of the active site groove that could affect both the intracellular stability as well as the activity of FGE. Thus, the novel variant of the SUMF1 gene obviously results in functionally impaired FGE protein leading to a severe late infantile type of MSD.
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ArylSulfatase K is the Lysosomal 2-Sulfoglucuronate Sulfatase
ACS Chemical Biology, 2017Co-Authors: Omkar P. Dhamale, Elena M. Wiegmann, Kanar Al-mafraji, Torben Lubke, Roger Lawrence, Bhahwal Ali Shah, William C Lamanna, Thomas Dierks, Geert-jan Boons, Jeffrey D EskoAbstract:The degradation of glycosaminoglycans (GAGs) involves a series of exolytic glycosidases and Sulfatases that act sequentially on the nonreducing end of the polysaccharide chain. Enzymes have been cloned that catalyze all of the known linkages with the exception of the removal of the 2-O-sulfate group from 2-sulfoglucuronate, which is found in heparan sulfate and dermatan sulfate. Here, we show using synthetic disaccharide substrates that arylSulfatase K is the glucuronate-2-Sulfatase. ArylSulfatase K acts selectively on 2-sulfoglucuronate and lacks activity against 2-sulfoiduronate, whereas iduronate-2-Sulfatase (IDS) desulfates synthetic disaccharides containing 2-sulfoiduronate but not 2-sulfoglucuronate. As arylSulfatase K has all of the properties expected of a lysosomal enzyme, we conclude that arylSulfatase K is the long sought lysosomal glucuronate-2-Sulfatase, which we designate GDS.
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eukaryotic formylglycine generating enzyme catalyses a monooxygenase type of reaction
FEBS Journal, 2015Co-Authors: Jianhe Peng, Thomas Dierks, Kurt Von Figura, Karthikeyan Radhakrishnan, Sarfaraz Alam, Malaiyalam Mariappan, M G Rudolph, Caroline May, Bernhard SchmidtAbstract:C α-formylglycine (FGly) is the catalytic residue of Sulfatases in eukaryotes. It is generated by a unique post-translational modification catalysed by the FGly-generating enzyme (FGE) in the endoplasmic reticulum. FGE oxidizes a cysteine residue within the conserved CxPxR sequence motif of nascent Sulfatase polypeptides to FGly. Here we show that this oxidation is strictly dependent on molecular oxygen (O2) and consumes 1 mol O2 per mol FGly formed. For maximal activity FGE requires an O2 concentration of 9% (105 μM). Sustained FGE activity further requires the presence of a thiol-based reductant such as DTT. FGly is also formed in the absence of DTT, but its formation ceases rapidly. Thus inactivated FGE accumulates in which the cysteine pair Cys336/Cys341 in the catalytic site is oxidized to form disulfide bridges between either Cys336 and Cys341 or Cys341 and the CxPxR cysteine of the Sulfatase. These results strongly suggest that the Cys336/Cys341 pair is directly involved in the O2 -dependent conversion of the CxPxR cysteine to FGly. The available data characterize eukaryotic FGE as a monooxygenase, in which Cys336/Cys341 disulfide bridge formation donates the electrons required to reduce one oxygen atom of O2 to water while the other oxygen atom oxidizes the CxPxR cysteine to FGly. Regeneration of a reduced Cys336/Cys341 pair is accomplished in vivo by a yet unknown reductant of the endoplasmic reticulum or in vitro by DTT. Remarkably, this monooxygenase reaction utilizes O2 without involvement of any activating cofactor.
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rapid degradation of an active formylglycine generating enzyme variant leads to a late infantile severe form of multiple Sulfatase deficiency
European Journal of Human Genetics, 2013Co-Authors: Lars Schlotawa, Thomas Dierks, Bernhard Schmidt, Karthikeyan Radhakrishnan, Matthias R Baumgartner, Regula Schmid, Jutta GartnerAbstract:Multiple Sulfatase deficiency (MSD) is a rare inborn error of metabolism affecting posttranslational activation of Sulfatases by the formylglycine generating enzyme (FGE). Due to mutations in the encoding SUMF1 gene, FGE's catalytic capacity is impaired resulting in reduced cellular Sulfatase activities. Both, FGE protein stability and residual activity determine disease severity and have previously been correlated with the clinical MSD phenotype. Here, we report a patient with a late infantile severe course of disease. The patient is compound heterozygous for two so far undescribed SUMF1 mutations, c.156delC (p.C52fsX57) and c.390A>T (p.E130D). In patient fibroblasts, mRNA of the frameshift allele is undetectable. In contrast, the allele encoding FGE-E130D is expressed. FGE-E130D correctly localizes to the endoplasmic reticulum and has a very high residual molecular activity in vitro (55% of wildtype FGE); however, it is rapidly degraded. Thus, despite substantial residual enzyme activity, protein instability determines disease severity, which highlights that potential MSD treatment approaches should target protein folding and stabilization mechanisms.
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sumf1 mutations affecting stability and activity of formylglycine generating enzyme predict clinical outcome in multiple Sulfatase deficiency
European Journal of Human Genetics, 2011Co-Authors: Lars Schlotawa, Thomas Dierks, Bernhard Schmidt, Karthikeyan Radhakrishnan, Eva C Ennemann, Anupam Chakrapani, Hansjurgen Christen, Hugo W Moser, Beat Steinmann, Jutta GartnerAbstract:Multiple Sulfatase Deficiency (MSD) is caused by mutations in the Sulfatase-modifying factor 1 gene encoding the formylglycine-generating enzyme (FGE). FGE post translationally activates all newly synthesized Sulfatases by generating the catalytic residue formylglycine. Impaired FGE function leads to reduced Sulfatase activities. Patients display combined clinical symptoms of single Sulfatase deficiencies. For ten MSD patients, we determined the clinical phenotype, FGE expression, localization and stability, as well as residual FGE and Sulfatase activities. A neonatal, very severe clinical phenotype resulted from a combination of two nonsense mutations leading to almost fully abrogated FGE activity, highly unstable FGE protein and nearly undetectable Sulfatase activities. A late infantile mild phenotype resulted from FGE G263V leading to unstable protein but high residual FGE activity. Other missense mutations resulted in a late infantile severe phenotype because of unstable protein with low residual FGE activity. Patients with identical mutations displayed comparable clinical phenotypes. These data confirm the hypothesis that the phenotypic outcome in MSD depends on both residual FGE activity as well as protein stability. Predicting the clinical course in case of molecularly characterized mutations seems feasible, which will be helpful for genetic counseling and developing therapeutic strategies aiming at enhancement of FGE.
Bernhard Schmidt - One of the best experts on this subject based on the ideXlab platform.
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eukaryotic formylglycine generating enzyme catalyses a monooxygenase type of reaction
FEBS Journal, 2015Co-Authors: Jianhe Peng, Thomas Dierks, Kurt Von Figura, Karthikeyan Radhakrishnan, Sarfaraz Alam, Malaiyalam Mariappan, M G Rudolph, Caroline May, Bernhard SchmidtAbstract:C α-formylglycine (FGly) is the catalytic residue of Sulfatases in eukaryotes. It is generated by a unique post-translational modification catalysed by the FGly-generating enzyme (FGE) in the endoplasmic reticulum. FGE oxidizes a cysteine residue within the conserved CxPxR sequence motif of nascent Sulfatase polypeptides to FGly. Here we show that this oxidation is strictly dependent on molecular oxygen (O2) and consumes 1 mol O2 per mol FGly formed. For maximal activity FGE requires an O2 concentration of 9% (105 μM). Sustained FGE activity further requires the presence of a thiol-based reductant such as DTT. FGly is also formed in the absence of DTT, but its formation ceases rapidly. Thus inactivated FGE accumulates in which the cysteine pair Cys336/Cys341 in the catalytic site is oxidized to form disulfide bridges between either Cys336 and Cys341 or Cys341 and the CxPxR cysteine of the Sulfatase. These results strongly suggest that the Cys336/Cys341 pair is directly involved in the O2 -dependent conversion of the CxPxR cysteine to FGly. The available data characterize eukaryotic FGE as a monooxygenase, in which Cys336/Cys341 disulfide bridge formation donates the electrons required to reduce one oxygen atom of O2 to water while the other oxygen atom oxidizes the CxPxR cysteine to FGly. Regeneration of a reduced Cys336/Cys341 pair is accomplished in vivo by a yet unknown reductant of the endoplasmic reticulum or in vitro by DTT. Remarkably, this monooxygenase reaction utilizes O2 without involvement of any activating cofactor.
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rapid degradation of an active formylglycine generating enzyme variant leads to a late infantile severe form of multiple Sulfatase deficiency
European Journal of Human Genetics, 2013Co-Authors: Lars Schlotawa, Thomas Dierks, Bernhard Schmidt, Karthikeyan Radhakrishnan, Matthias R Baumgartner, Regula Schmid, Jutta GartnerAbstract:Multiple Sulfatase deficiency (MSD) is a rare inborn error of metabolism affecting posttranslational activation of Sulfatases by the formylglycine generating enzyme (FGE). Due to mutations in the encoding SUMF1 gene, FGE's catalytic capacity is impaired resulting in reduced cellular Sulfatase activities. Both, FGE protein stability and residual activity determine disease severity and have previously been correlated with the clinical MSD phenotype. Here, we report a patient with a late infantile severe course of disease. The patient is compound heterozygous for two so far undescribed SUMF1 mutations, c.156delC (p.C52fsX57) and c.390A>T (p.E130D). In patient fibroblasts, mRNA of the frameshift allele is undetectable. In contrast, the allele encoding FGE-E130D is expressed. FGE-E130D correctly localizes to the endoplasmic reticulum and has a very high residual molecular activity in vitro (55% of wildtype FGE); however, it is rapidly degraded. Thus, despite substantial residual enzyme activity, protein instability determines disease severity, which highlights that potential MSD treatment approaches should target protein folding and stabilization mechanisms.
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sumf1 mutations affecting stability and activity of formylglycine generating enzyme predict clinical outcome in multiple Sulfatase deficiency
European Journal of Human Genetics, 2011Co-Authors: Lars Schlotawa, Thomas Dierks, Bernhard Schmidt, Karthikeyan Radhakrishnan, Eva C Ennemann, Anupam Chakrapani, Hansjurgen Christen, Hugo W Moser, Beat Steinmann, Jutta GartnerAbstract:Multiple Sulfatase Deficiency (MSD) is caused by mutations in the Sulfatase-modifying factor 1 gene encoding the formylglycine-generating enzyme (FGE). FGE post translationally activates all newly synthesized Sulfatases by generating the catalytic residue formylglycine. Impaired FGE function leads to reduced Sulfatase activities. Patients display combined clinical symptoms of single Sulfatase deficiencies. For ten MSD patients, we determined the clinical phenotype, FGE expression, localization and stability, as well as residual FGE and Sulfatase activities. A neonatal, very severe clinical phenotype resulted from a combination of two nonsense mutations leading to almost fully abrogated FGE activity, highly unstable FGE protein and nearly undetectable Sulfatase activities. A late infantile mild phenotype resulted from FGE G263V leading to unstable protein but high residual FGE activity. Other missense mutations resulted in a late infantile severe phenotype because of unstable protein with low residual FGE activity. Patients with identical mutations displayed comparable clinical phenotypes. These data confirm the hypothesis that the phenotypic outcome in MSD depends on both residual FGE activity as well as protein stability. Predicting the clinical course in case of molecularly characterized mutations seems feasible, which will be helpful for genetic counseling and developing therapeutic strategies aiming at enhancement of FGE.
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molecular basis of multiple Sulfatase deficiency mucolipidosis ii iii and niemann pick c1 disease lysosomal storage disorders caused by defects of non lysosomal proteins
Biochimica et Biophysica Acta, 2009Co-Authors: Thomas Dierks, Marc André Frese, Lars Schlotawa, Kurt Von Figura, Karthikeyan Radhakrishnan, Bernhard SchmidtAbstract:Multiple Sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire Sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all Sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been addressed. Further, the discovery of FGE as an essential Sulfatase activating enzyme has considerable impact on enzyme replacement or gene therapy of lysosomal storage disorders caused by single Sulfatase deficiencies.
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Molecular basis of multiple Sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease - Lysosomal storage disorders caused by defects of non-lysosomal proteins
Biochimica et Biophysica Acta - Molecular Cell Research, 2009Co-Authors: Thomas Dierks, Marc André Frese, Lars Schlotawa, Krishnan Radhakrishnan, Kurt Von Figura, Bernhard SchmidtAbstract:Multiple Sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire Sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all Sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been addressed. Further, the discovery of FGE as an essential Sulfatase activating enzyme has considerable impact on enzyme replacement or gene therapy of lysosomal storage disorders caused by single Sulfatase deficiencies. © 2008 Elsevier B.V. All rights reserved.
Kurt Von Figura - One of the best experts on this subject based on the ideXlab platform.
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eukaryotic formylglycine generating enzyme catalyses a monooxygenase type of reaction
FEBS Journal, 2015Co-Authors: Jianhe Peng, Thomas Dierks, Kurt Von Figura, Karthikeyan Radhakrishnan, Sarfaraz Alam, Malaiyalam Mariappan, M G Rudolph, Caroline May, Bernhard SchmidtAbstract:C α-formylglycine (FGly) is the catalytic residue of Sulfatases in eukaryotes. It is generated by a unique post-translational modification catalysed by the FGly-generating enzyme (FGE) in the endoplasmic reticulum. FGE oxidizes a cysteine residue within the conserved CxPxR sequence motif of nascent Sulfatase polypeptides to FGly. Here we show that this oxidation is strictly dependent on molecular oxygen (O2) and consumes 1 mol O2 per mol FGly formed. For maximal activity FGE requires an O2 concentration of 9% (105 μM). Sustained FGE activity further requires the presence of a thiol-based reductant such as DTT. FGly is also formed in the absence of DTT, but its formation ceases rapidly. Thus inactivated FGE accumulates in which the cysteine pair Cys336/Cys341 in the catalytic site is oxidized to form disulfide bridges between either Cys336 and Cys341 or Cys341 and the CxPxR cysteine of the Sulfatase. These results strongly suggest that the Cys336/Cys341 pair is directly involved in the O2 -dependent conversion of the CxPxR cysteine to FGly. The available data characterize eukaryotic FGE as a monooxygenase, in which Cys336/Cys341 disulfide bridge formation donates the electrons required to reduce one oxygen atom of O2 to water while the other oxygen atom oxidizes the CxPxR cysteine to FGly. Regeneration of a reduced Cys336/Cys341 pair is accomplished in vivo by a yet unknown reductant of the endoplasmic reticulum or in vitro by DTT. Remarkably, this monooxygenase reaction utilizes O2 without involvement of any activating cofactor.
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molecular basis of multiple Sulfatase deficiency mucolipidosis ii iii and niemann pick c1 disease lysosomal storage disorders caused by defects of non lysosomal proteins
Biochimica et Biophysica Acta, 2009Co-Authors: Thomas Dierks, Marc André Frese, Lars Schlotawa, Kurt Von Figura, Karthikeyan Radhakrishnan, Bernhard SchmidtAbstract:Multiple Sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire Sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all Sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been addressed. Further, the discovery of FGE as an essential Sulfatase activating enzyme has considerable impact on enzyme replacement or gene therapy of lysosomal storage disorders caused by single Sulfatase deficiencies.
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Molecular basis of multiple Sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease - Lysosomal storage disorders caused by defects of non-lysosomal proteins
Biochimica et Biophysica Acta - Molecular Cell Research, 2009Co-Authors: Thomas Dierks, Marc André Frese, Lars Schlotawa, Krishnan Radhakrishnan, Kurt Von Figura, Bernhard SchmidtAbstract:Multiple Sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire Sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all Sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been addressed. Further, the discovery of FGE as an essential Sulfatase activating enzyme has considerable impact on enzyme replacement or gene therapy of lysosomal storage disorders caused by single Sulfatase deficiencies. © 2008 Elsevier B.V. All rights reserved.
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a general binding mechanism for all human Sulfatases by the formylglycine generating enzyme
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Dirk Roeser, Thomas Dierks, Bernhard Schmidt, Kurt Von Figura, Andrea Preusserkunze, Kathrin Gasow, Julia G Wittmann, Markus G RudolphAbstract:The formylglycine (FGly)-generating enzyme (FGE) uses molecular oxygen to oxidize a conserved cysteine residue in all eukaryotic Sulfatases to the catalytically active FGly. Sulfatases degrade and remodel sulfate esters, and inactivity of FGE results in multiple Sulfatase deficiency, a fatal disease. The previously determined FGE crystal structure revealed two crucial cysteine residues in the active site, one of which was thought to be implicated in substrate binding. The other cysteine residue partakes in a novel oxygenase mechanism that does not rely on any cofactors. Here, we present crystal structures of the individual FGE cysteine mutants and employ chemical probing of wild-type FGE, which defined the cysteines to differ strongly in their reactivity. This striking difference in reactivity is explained by the distinct roles of these cysteine residues in the catalytic mechanism. Hitherto, an enzyme-substrate complex as an essential cornerstone for the structural evaluation of the FGly formation mechanism has remained elusive. We also present two FGE-substrate complexes with pentamer and heptamer peptides that mimic Sulfatases. The peptides isolate a small cavity that is a likely binding site for molecular oxygen and could host reactive oxygen intermediates during cysteine oxidation. Importantly, these FGE-peptide complexes directly unveil the molecular bases of FGE substrate binding and specificity. Because of the conserved nature of FGE sequences in other organisms, this binding mechanism is of general validity. Furthermore, several disease-causing mutations in both FGE and Sulfatases are explained by this binding mechanism.
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molecular basis for multiple Sulfatase deficiency and mechanism for formylglycine generation of the human formylglycine generating enzyme
Cell, 2005Co-Authors: Thomas Dierks, Bernhard Schmidt, Kurt Von Figura, Malaiyalam Mariappan, Achim Dickmanns, Andrea Preusserkunze, Ralf Ficner, Markus G RudolphAbstract:Sulfatases are enzymes essential for degradation and remodeling of sulfate esters. Formylglycine (FGly), the key catalytic residue in the active site, is unique to Sulfatases. In higher eukaryotes, FGly is generated from a cysteine precursor by the FGly-generating enzyme (FGE). Inactivity of FGE results in multiple Sulfatase deficiency (MSD), a fatal autosomal recessive syndrome. Based on the crystal structure, we report that FGE is a single-domain monomer with a surprising paucity of secondary structure and adopts a unique fold. The effect of all 18 missense mutations found in MSD patients is explained by the FGE structure, providing a molecular basis of MSD. The catalytic mechanism of FGly generation was elucidated by six high-resolution structures of FGE in different redox environments. The structures allow formulation of a novel oxygenase mechanism whereby FGE utilizes molecular oxygen to generate FGly via a cysteine sulfenic acid intermediate.
Lars Schlotawa - One of the best experts on this subject based on the ideXlab platform.
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expanding the genetic cause of multiple Sulfatase deficiency a novel sumf1 variant in a patient displaying a severe late infantile form of the disease
Molecular Genetics and Metabolism, 2017Co-Authors: Ilona Jaszczuk, Lars Schlotawa, Thomas Dierks, Karthikeyan Radhakrishnan, Andreas Ohlenbusch, Dominique Koppenhofer, Mariusz Babicz, Monika Lejman, Agnieszka ługowskaAbstract:Abstract Multiple Sulfatase deficiency (MSD) is a rare inherited metabolic disease caused by defective cellular Sulfatases. Activity of Sulfatases depends on post-translational modification catalyzed by formylglycine-generating enzyme (FGE), encoded by the SUMF1 gene. SUMF1 pathologic variants cause MSD, a syndrome presenting with a complex phenotype. We describe the first Polish patient with MSD caused by a yet undescribed pathologic variant c.337G > A [p.Glu113Lys] (i.e. p.E113K) in heterozygous combination with the known deletion allele c.519 + 5_519 + 8del [p.Ala149_Ala173del]. The clinical picture of the patient initially suggested late infantile metachromatic leukodystrophy, with developmental delay followed by regression of visual, hearing and motor abilities as the most apparent clinical symptoms. Transient signs of ichthyosis and minor dysmorphic features guided the laboratory workup towards MSD. Since MSD is a rare disease and there is a variable clinical spectrum, we thoroughly describe the clinical outcome of our patient. The FGE-E113K variant, expressed in cell culture, correctly localized to the endoplasmic reticulum but was retained intracellularly in contrast to the wild type FGE. Analysis of FGE-mediated activation of steroid Sulfatase in immortalized MSD cells revealed that FGE-E113K exhibited only approx. 15% of the activity of wild type FGE. Based on the crystal structure we predict that the exchange of glutamate-113 against lysine should induce a strong destabilization of the secondary structure, possibly affecting the folding for correct disulfide bridging between C235-C346 as well as distortion of the active site groove that could affect both the intracellular stability as well as the activity of FGE. Thus, the novel variant of the SUMF1 gene obviously results in functionally impaired FGE protein leading to a severe late infantile type of MSD.
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rapid degradation of an active formylglycine generating enzyme variant leads to a late infantile severe form of multiple Sulfatase deficiency
European Journal of Human Genetics, 2013Co-Authors: Lars Schlotawa, Thomas Dierks, Bernhard Schmidt, Karthikeyan Radhakrishnan, Matthias R Baumgartner, Regula Schmid, Jutta GartnerAbstract:Multiple Sulfatase deficiency (MSD) is a rare inborn error of metabolism affecting posttranslational activation of Sulfatases by the formylglycine generating enzyme (FGE). Due to mutations in the encoding SUMF1 gene, FGE's catalytic capacity is impaired resulting in reduced cellular Sulfatase activities. Both, FGE protein stability and residual activity determine disease severity and have previously been correlated with the clinical MSD phenotype. Here, we report a patient with a late infantile severe course of disease. The patient is compound heterozygous for two so far undescribed SUMF1 mutations, c.156delC (p.C52fsX57) and c.390A>T (p.E130D). In patient fibroblasts, mRNA of the frameshift allele is undetectable. In contrast, the allele encoding FGE-E130D is expressed. FGE-E130D correctly localizes to the endoplasmic reticulum and has a very high residual molecular activity in vitro (55% of wildtype FGE); however, it is rapidly degraded. Thus, despite substantial residual enzyme activity, protein instability determines disease severity, which highlights that potential MSD treatment approaches should target protein folding and stabilization mechanisms.
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sumf1 mutations affecting stability and activity of formylglycine generating enzyme predict clinical outcome in multiple Sulfatase deficiency
European Journal of Human Genetics, 2011Co-Authors: Lars Schlotawa, Thomas Dierks, Bernhard Schmidt, Karthikeyan Radhakrishnan, Eva C Ennemann, Anupam Chakrapani, Hansjurgen Christen, Hugo W Moser, Beat Steinmann, Jutta GartnerAbstract:Multiple Sulfatase Deficiency (MSD) is caused by mutations in the Sulfatase-modifying factor 1 gene encoding the formylglycine-generating enzyme (FGE). FGE post translationally activates all newly synthesized Sulfatases by generating the catalytic residue formylglycine. Impaired FGE function leads to reduced Sulfatase activities. Patients display combined clinical symptoms of single Sulfatase deficiencies. For ten MSD patients, we determined the clinical phenotype, FGE expression, localization and stability, as well as residual FGE and Sulfatase activities. A neonatal, very severe clinical phenotype resulted from a combination of two nonsense mutations leading to almost fully abrogated FGE activity, highly unstable FGE protein and nearly undetectable Sulfatase activities. A late infantile mild phenotype resulted from FGE G263V leading to unstable protein but high residual FGE activity. Other missense mutations resulted in a late infantile severe phenotype because of unstable protein with low residual FGE activity. Patients with identical mutations displayed comparable clinical phenotypes. These data confirm the hypothesis that the phenotypic outcome in MSD depends on both residual FGE activity as well as protein stability. Predicting the clinical course in case of molecularly characterized mutations seems feasible, which will be helpful for genetic counseling and developing therapeutic strategies aiming at enhancement of FGE.
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molecular basis of multiple Sulfatase deficiency mucolipidosis ii iii and niemann pick c1 disease lysosomal storage disorders caused by defects of non lysosomal proteins
Biochimica et Biophysica Acta, 2009Co-Authors: Thomas Dierks, Marc André Frese, Lars Schlotawa, Kurt Von Figura, Karthikeyan Radhakrishnan, Bernhard SchmidtAbstract:Multiple Sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire Sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all Sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been addressed. Further, the discovery of FGE as an essential Sulfatase activating enzyme has considerable impact on enzyme replacement or gene therapy of lysosomal storage disorders caused by single Sulfatase deficiencies.
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Molecular basis of multiple Sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease - Lysosomal storage disorders caused by defects of non-lysosomal proteins
Biochimica et Biophysica Acta - Molecular Cell Research, 2009Co-Authors: Thomas Dierks, Marc André Frese, Lars Schlotawa, Krishnan Radhakrishnan, Kurt Von Figura, Bernhard SchmidtAbstract:Multiple Sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire Sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all Sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been addressed. Further, the discovery of FGE as an essential Sulfatase activating enzyme has considerable impact on enzyme replacement or gene therapy of lysosomal storage disorders caused by single Sulfatase deficiencies. © 2008 Elsevier B.V. All rights reserved.
Andrea Ballabio - One of the best experts on this subject based on the ideXlab platform.
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disease pathogenesis explained by basic science lysosomal storage diseases as autophagocytic disorders
Principles and Practice of Constraint Programming, 2009Co-Authors: Andrea BallabioAbstract:Lysosomal storage diseases (LSDs) are characterized by intra-lysosomal accumulation of undegraded metabolites due to the defective activity of lysosomal enzymes. There is a paucity of data, however, relating to the mechanisms that link this accumulation with disease pathology. Several LSDs can be attributed to deficiencies in the activity of Sulfatase enzymes. The gene responsible for the post-translational modification that activates Sulfatases, Sulfatase modifying factor 1 (SUMF1), is defective in the rare autosomal recessive disorder multiple Sulfatase deficiency (MSD). A mouse model of MSD (Sumfl knockout mouse) exhibits a similar phenotype to patients with MSD, with marked lysosomal storage of undegraded metabolites, and increased expression of inflammatory markers and apoptotic markers. Investigation of disease pathology in mouse models of two LSDs (MSD and mucopolysaccharidosis (MPS) Type IIIA) has revealed an increased number of autophagosomes in these animals compared with wild-type mice. This appears to result from impaired auto-phagosome-lysosome fusion, which may in turn lead to an absence of autophagy. The suggestion that LSDs can be defined as disorders of autophagy implies that there may be some overlap between pathological mechanisms of LSDs and more common neuro-degenerative diseases, and this may help provide direction for future therapeutic strategies.
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proteoglycan desulfation determines the efficiency of chondrocyte autophagy and the extent of fgf signaling during endochondral ossification
Genes & Development, 2008Co-Authors: Carmine Settembre, Andrea Ballabio, Emilio Arteagasolis, Marc D Mckee, Raquel De Pablo, Qais Al Awqati, Gerard KarsentyAbstract:Cartilage extracellular matrix (ECM) contains large amounts of proteoglycans made of a protein core decorated by highly sulfated sugar chains, the glycosaminoglycans (GAGs). GAGs desulfation, a necessary step for their degradation, is exerted by Sulfatases that are activated by another enzyme, Sulfatase-Modifying Factor 1 (SUMF1), whose inactivation in humans leads to severe skeletal abnormalities. We show here that despite being expressed in both osteoblasts and chondrocytes Sumf1 does not affect osteoblast differentiation. Conversely, in chondrocytes it favors ECM production and autophagy and promotes proliferation and differentiation by limiting FGF signaling. Thus, proteoglycan desulfation is a critical regulator of chondrogenesis.
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multistep sequential control of the trafficking and function of the multiple Sulfatase deficiency gene product sumf1 by pdi ergic 53 and erp44
Human Molecular Genetics, 2008Co-Authors: Alessandro Fraldi, Andrea Ballabio, Ester Zito, Fabio Annunziata, Alessia Lombardi, Marianna Cozzolino, Maria Chiara Monti, Carmine Spampanato, Piero Pucci, Roberto SitiaAbstract:Sulfatase modifying factor 1 (SUMF1) encodes for the formylglicine generating enzyme, which activates Sulfatases by modifying a key cysteine residue within their catalytic domains. SUMF1 is mutated in patients affected by multiple Sulfatase deficiency, a rare recessive disorder in which all Sulfatase activities are impaired. Despite the absence of canonical retention/retrieval signals, SUMF1 is largely retained in the endoplasmic reticulum (ER), where it exerts its enzymatic activity on nascent Sulfatases. Part of SUMF1 is secreted and paracrinally taken up by distant cells. Here we show that SUMF1 interacts with protein disulfide isomerase (PDI) and ERp44, two thioredoxin family members residing in the early secretory pathway, and with ERGIC-53, a lectin that shuttles between the ER and the Golgi. Functional assays reveal that these interactions are crucial for controlling SUMF1 traffic and function. PDI couples SUMF1 retention and activation in the ER. ERGIC-53 and ERp44 act downstream, favoring SUMF1 export from and retrieval to the ER, respectively. Silencing ERGIC-53 causes proteasomal degradation of SUMF1, while down-regulating ERp44 promotes its secretion. When over-expressed, each of three interactors favors intracellular accumulation. Our results reveal a multistep control of SUMF1 trafficking, with sequential interactions dynamically determining ER localization, activity and secretion.
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functional correction of cns lesions in an mps iiia mouse model by intracerebral aav mediated delivery of sulfamidase and sumf1 genes
Human Molecular Genetics, 2007Co-Authors: Alessandro Fraldi, Andrea Ballabio, Alessia Lombardi, Kim M Hemsley, Allison C Crawley, Adeline A Lau, Leanne Sutherland, Alberto Auricchio, John J. HopwoodAbstract:Mucopolysaccharidosis type IIIA (MPS-IIIA or Sanfilippo syndrome) is a lysosomal storage disorder caused by the congenital deficiency of sulfamidase (SGSH) enzyme and consequent accumulation of partially degraded heparan sulfate (HS) in lysosomes. The central nervous system (CNS) is the predominant site of tissue damage in MPS-IIIA. Here we describe a gene therapy approach for MPS-IIIA in a mouse model using recombinant adeno-associated virus serotype 5 (AAV2/5) as a vehicle to deliver therapeutic genes to the CNS. SUMF1 (SUIfatase Modifying Factor 1) exhibits an enhancing effect on Sulfatase activity when co-expressed with Sulfatases. Consistent with these findings, we demonstrated that co-delivery of SUMF1 and SGSH (via an AAV2/5-CMV-SGSH-IRES-SUMF1 vector) resulted in a synergistic increase in SGSH activity, both in primary neural cells and in murine brain. A study aimed at testing the therapeutic efficacy of simultaneous brain administration of SUMF1 and SGSH was then performed by injecting the lateral ventricles of newborn MPS-IIIA/normal mice with either AAV2/5-CMV-SGSH-IRES-SUMF1 or AAV2/5-CMV-GFP vectors. Widespread GFP expression was observed within the GFP-injected brain, and a stable and significant increase of SGSH activity was detected in several brain regions following SGSH-IRES-SUMF1 administration. Treatment with AAV2/5-CMV-SGSH-IRES-SUMF1 vectors resulted in a visible reduction in lysosomal storage and inflammatory markers in transduced brain regions. Finally, the MPS-IIIA mice treated with therapeutic genes displayed an improvement in both motor and cognitive functions. Our results suggest that early treatment of CNS lesions by AAV-mediated intraventricular injection of both SGSH and SUMF1 genes may represent a feasible therapy for MPS-IIIA.
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systemic inflammation and neurodegeneration in a mouse model of multiple Sulfatase deficiency
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Carmine Settembre, Maria Pia Cosma, Ida Annunziata, Ester Zito, Carmine Spampanato, Daniela Zarcone, Gilda Cobellis, Edoardo Nusco, Carlo Tacchetti, Andrea BallabioAbstract:Sulfatases are involved in several biological functions such as degradation of macromolecules in the lysosomes. In patients with multiple Sulfatase deficiency, mutations in the SUMF1 gene cause a reduction of Sulfatase activities because of a posttranslational modification defect. We have generated a mouse line carrying a null mutation in the Sumf1 gene. Sulfatase activities are completely absent in Sumf1(-/-) mice, indicating that Sumf1 is indispensable for Sulfatase activation and that mammals, differently from bacteria, have a single Sulfatase modification system. Similarly to multiple Sulfatase deficiency patients, Sumf1(-/-) mice display frequent early mortality, congenital growth retardation, skeletal abnormalities, and neurological defects. All examined tissues showed progressive cell vacuolization and significant lysosomal storage of glycosaminoglycans. Sumf1(-/-) mice showed a generalized inflammatory process characterized by a massive presence of highly vacuolated macrophages, which are the main site of lysosomal storage. Activated microglia were detected in the cerebellum and brain cortex associated with remarkable astroglyosis and neuronal cell loss. Between 4 and 6 months of age, we detected a strong increase in the expression levels of inflammatory cytokines and of apoptotic markers in both the CNS and liver, demonstrating that inflammation and apoptosis occur at the late stage of disease and suggesting that they play an important role in both the systemic and CNS phenotypes observed in lysosomal disorders. This mouse model, in which the function of an entire protein family has been silenced, offers a unique opportunity to study Sulfatase function and the mechanisms underlying lysosomal storage diseases.
