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 VI (MPS VI) is an autosomal recessive disease caused by a deficiency of N-acetylgalactosamine 4-sulfatase (4S) leading to the lysosomal accumulation and urinary excretion of dermatan sulfate. MPS VI has also been described in the Siamese cat. As an initial step toward enzyme replacement therapy with recombinant feline 4S (rf4S) in MPS VI cats, the feline 4S cDNA was isolated and expressed in CHO-KI cells and rf4S was immunopurified from the culture medium. SDS-polyacrylamide gel electrophoresis analysis showed that the precursor form of immunopurified rf4S was a 66-kDa polypeptide that underwent maturation to a 43-44-kDa polypeptide. Endocytosis of rf4S by cultured feline MPS VI myoblasts was predominantly mediated by a mannose 6-phosphate receptor and resulted in the correction of dermatan sulfate storage. The mutation causing feline MPS VI was identified as a base substitution at codon 476, altering a leucine codon to a proline (L476P). The L476P allele displayed no detectable 4S activity when expressed in CHO-KI cells and was observed only as a "precursor" polypeptide that was not secreted into the medium. Identification of the mutation has allowed the development of a rapid PCR-based screening method to genotype individuals within the cat colony.
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PMID:Feline mucopolysaccharidosis type VI. Characterization of recombinant N-acetylgalactosamine 4-sulfatase and identification of a mutation causing the disease. 891 Feb 99

X-linked ichthyosis is the result of steroid sulfatase (STS) deficiency. While most affected individuals have extensive deletions of the STS gene, point mutations have been reported in three patients (1). In this study, we identify an additional three point mutations and characterize the effects of all six mutations on STS activity and expression. All six are unique single base pair substitutions. The mutations are located in a 105-amino acid region of the C-terminal half of the polypeptide. Five of the six mutations involve the substitutions of Pro or Arg for Trp372, Arg for His444, Tyr for Cys446, or Leu for Cys341. The other mutation is in a splice junction and results in a frameshift causing premature termination of the polypeptide at residue 427. All the affected residues are conserved to some degree within the sulfatase family. The six mutations were reproduced in normal STS cDNA and transiently expressed in STS-deficient cells. All six mutant vectors direct the expression of STS protein that lacks enzymatic activity. The mutant polypeptides show a shift in mobility on SDS-PAGE and resistance to proteinase K digestion when translated in the presence of dog pancreas microsomes, indicating glycosylation and normal translocation.
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PMID:Characterization of point mutations in patients with X-linked ichthyosis. Effects on the structure and function of the steroid sulfatase protein. 925 98

In sulfatases a Calpha-formylglycine residue is found at a position where their cDNA sequences predict a cysteine residue. In multiple sulfatase deficiency, an inherited lysosomal storage disorder, catalytically inactive sulfatases are synthesized which retain the cysteine residue, indicating that the Calpha-formylglycine residue is required for sulfatase activity. Using in vitro translation in the absence or presence of transport competent microsomes we found that newly synthesized sulfatase polypeptides carry a cysteine residue and that the oxidation of its thiol group to an aldehyde is catalyzed in the endoplasmic reticulum. A linear sequence of 16 residues surrounding the Cys-69 in arylsulfatase A is sufficient to direct the oxidation. This novel protein modification occurs after or at a late stage of cotranslational protein translocation into the endoplasmic reticulum when the polypeptide is not yet folded to its native structure.
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PMID:Conversion of cysteine to formylglycine: a protein modification in the endoplasmic reticulum. 934 45

In multiple sulfatase deficiency, a rare human lysosomal storage disorder, all known sulfatases are synthesized as catalytically poorly active polypeptides. Analysis of the latter has shown that they lack a protein modification that was detected in all members of the sulfatase family. This novel protein modification generates a 2-amino-3-oxopropanoic acid (C alpha-formylglycine) residue by oxidation of the thiol group of a cysteine that is conserved among all eukaryotic sulfatases. The oxidation occurs in the endoplasmic reticulum at a stage when the nascent polypeptide is not yet folded. The aldehyde is part of the catalytic site and is likely to act as an aldehyde hydrate. One of the geminal hydroxyl groups accepts the sulfate during sulfate ester cleavage leading to the formation of a covalently sulfated enzyme intermediate. The other hydroxyl is required for the subsequent elimination of the sulfate and regeneration of the aldehyde group. In some prokaryotic members of the sulfatase gene family, the DNA sequence predicts a serine residue, and not a cysteine. Analysis of one of these prokaryotic sulfatases, however, revealed the presence of the C alpha-formylglycine indicating that the aldehyde group is essential for all members of the sulfatase family and that it can be generated from either cysteine or serine.
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PMID:A novel protein modification generating an aldehyde group in sulfatases: its role in catalysis and disease. 969 62

As a preliminary step toward muscle-mediated gene therapy in the mucopolysaccharidosis (MPS) type VI cat, we have analyzed the transcriptional regulation of feline N-acetylgalactosamine 4-sulfatase (f4S) gene expression from various retroviral constructs in primary cultures of muscle cells. Two retroviral constructs were made containing the f4S cDNA under the transcriptional control of the human polypeptide chain-elongation factor 1alpha (EF1alpha) gene promoter or the cytomegalovirus (CMV) immediate-early promoter. Two further retroviral constructs were made with the murine muscle creatine kinase (mck) enhancer sequence upstream of the internal promoter. Virus made from each construct was used to transduce feline MPS VI myoblasts. The mck enhancer significantly upregulated f4S gene expression from both the EF1alpha promoter and the CMV promoter in transduced myoblasts and in differentiated myofibers. The highest level of 4S activity was observed in myoblasts and myofibers transduced with the retroviral construct Lmckcmv4S, in which the f4S gene is under the transcriptional regulation of the mck enhancer and CMV immediate-early promoter. Lmckcmv4S-transduced myofibers demonstrated correction of glycosaminoglycan storage and contained a 58-fold elevated level of 4S activity compared with normal myofibers. Recombinant f4S secreted from Lmckcmv4S-transduced myofibers was endocytosed by feline MPS VI myofibers, leading to correction of the biochemical storage phenotype.
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PMID:Regulation of N-acetylgalactosamine 4-sulfatase expression in retrovirus-transduced feline mucopolysaccharidosis type VI muscle cells. 1009

Sulfatases carry at their catalytic site a unique post-translational modification, an alpha-formylglycine residue that is essential for enzyme activity. Formylglycine is generated by oxidation of a conserved cysteine or, in some prokaryotic sulfatases, serine residue. In eukaryotes, this oxidation occurs in the endoplasmic reticulum during or shortly after import of the nascent sulfatase polypeptide. The modification of arylsulfatase A was studied in vitro and was found to be directed by a short linear sequence, CTPSR, starting with the cysteine to be modified. Mutational analyses showed that the cysteine, proline and arginine are the key residues within this motif, whereas formylglycine formation tolerated the individual, but not the simultaneous substitution of the threonine or serine. The CTPSR motif was transferred to a heterologous protein leading to low-efficient formylglycine formation. The efficiency reached control values when seven additional residues (AALLTGR) directly following the CTPSR motif in arylsulfatase A were present. Mutating up to four residues simultaneously within this heptamer sequence inhibited the modification only moderately. AALLTGR may, therefore, have an auxiliary function in presenting the core motif to the modifying enzyme. Within the two motifs, the key residues are fully, and other residues are highly conserved among all known members of the sulfatase family.
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PMID:Sequence determinants directing conversion of cysteine to formylglycine in eukaryotic sulfatases. 1020 63

We have identified two novel periplasmic/cell wall polypeptides that specifically accumulate during sulfur limitation of Chlamydomonas reinhardtii. These polypeptides, present at high levels in the extracellular polypeptide fraction from a sulfur-deprived, cell wall-minus C. reinhardtii strain, have apparent molecular masses of 76 and 88 kD and are designated Ecp76 and Ecp88. N-terminal sequences of these polypeptides facilitated the isolation of full-length Ecp76 and Ecp88 cDNAs. Ecp76 and Ecp88 polypeptides are deduced to be 583 and 595 amino acids, respectively. Their amino acid sequences are similar to each other, with features characteristic of cell wall-localized hydroxyproline-rich glycoproteins; the N terminus of each polypeptide contains a predicted signal sequence, whereas the C terminus is rich in proline, alanine, and serine. Ecp76 and Ecp88 have either no (Ecp88) or one (Ecp76) sulfur-containing amino acid and transcripts encoding these polypeptides are not detected in cultures maintained on complete medium, but accumulate when cells are deprived of sulfur. This accumulation is temporally delayed relative to the accumulation of sulfur stress-induced arylsulfatase and ATP sulfurylase transcripts. The addition of sulfate back to sulfur-starved cultures caused a rapid decline in Ecp76 and Ecp88 mRNAs (half lives < 10 min). Furthermore, the C. reinhardtii sac1 mutant, which lacks a regulatory protein critical for acclimation to sulfur limitation, does not accumulate Ecp76 or Ecp88 transcripts. These results suggest that the Ecp76 and Ecp88 genes are under SacI control, and that restructuring of the C. reinhardtii cell wall during sulfur limitation may be important for redistribution of internal and efficient utilization of environmental sulfur-containing molecules.
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PMID:Sulfur economy and cell wall biosynthesis during sulfur limitation of Chlamydomonas reinhardtii. 1159 40

C(alpha)-formylglycine is the key catalytic residue in the active site of sulfatases. In eukaryotes formylglycine is generated during or immediately after sulfatase translocation into the endoplasmic reticulum by oxidation of a specific cysteine residue. We established an in vitro assay that allowed us to measure formylglycine modification independent of protein translocation. The modifying enzyme was recovered in a microsomal detergent extract. As a substrate we used ribosome-associated nascent chain complexes comprising in vitro synthesized sulfatase fragments that were released from the ribosomes by puromycin. Formylglycine modification was highly efficient and did not require a signal sequence in the substrate polypeptide. Ribosome association helped to maintain the modification competence of nascent chains but only after their release efficient modification occurred. The modifying machinery consists of soluble components of the endoplasmic reticulum lumen, as shown by differential extraction of microsomes. The in vitro assay can be performed under kinetically controlled conditions. The activation energy for formylglycine formation is 61 kJ/mol, and the pH optimum is approximately 10. The activity is sensitive to the SH/SS equilibrium and is stimulated by Ca(2+). Formylglycine formation is efficiently inhibited by a synthetic sulfatase peptide representing the sequence directing formylglycine modification. The established assay system should make possible the biochemical identification of the modifying enzyme.
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PMID:Characterization of posttranslational formylglycine formation by luminal components of the endoplasmic reticulum. 1160 May 3

The sulfur regulatory system of Neurospora crassa consists of a group of sulfur-regulated structural genes (e.g., arylsulfatase) that are under coordinate control of the CYS3 positive regulator and sulfur controller (SCON) negative regulators. Here we report on the cloning of scon-3(+), which encodes a polypeptide of 171 amino acids and is a Skp1 family homolog. Repeat-induced point mutation of scon-3(+) resulted in a phenotype of constitutive expression of arylsulfatase, a phenotype consistent with other sulfur controller mutants. Northern analysis indicated that, unlike other members of the sulfur regulatory system, expression of scon-3(+) is not under the direct control of the CYS3 transcriptional activator. In particular, scon-3(+) mRNA was detectable under sulfur repressing or derepressing conditions in a Deltacys-3 mutant. In yeast, Skp1p and an F-box protein binding partner are core constituents of a class of E3 ubiquitin ligases known as SCF complexes. The N. crassa negative regulator SCON2 contains an F-box motif essential for the operation of the sulfur regulatory system and suggests a role for an SCF complex in the N. crassa sulfur regulatory system. A crucial set of experiments, by using a yeast two-hybrid approach with confirming coimmunoprecipitation assays, demonstrated that SCON3 interacts with SCON2 in a manner dependent upon the F-box motif of SCON2. The protein-protein interaction detected between SCON2 and SCON3 represents the initial demonstration in a filamentous fungus of functional interaction between putative core components of a SCF complex.
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PMID:Cloning and characterization of scon-3+, a new member of the Neurospora crassa sulfur regulatory system. 1247 88

Variant late infantile neuronal ceroid lipofuscinosis, a lysosomal storage disorder characterized by progressive mental deterioration and blindness, is caused by mutations in a polytopic membrane protein (CLN6) with unknown intracellular localization and function. In this study, transient transfection of BHK21 cells with CLN6 cDNA and immunoblot analysis using peptide-specific CLN6 antibodies demonstrated the expression of a approximately 27-kDa protein that does not undergo proteolytic processing. Cross-linking experiments revealed the presence of CLN6 dimers. Using double immunofluorescence microscopy, epitope-tagged CLN6 was shown to be retained in the endoplasmic reticulum (ER) with no colocalization with the cis-Golgi or lysosomal markers. The translocation into the ER and proper folding were confirmed by the N-linked glycosylation of a mutant CLN6 polypeptide. Pulse-chase labeling of fibroblasts from CLN6 patients and from sheep (OCL6) and mouse (nclf) models of the disease followed by immunoprecipitation of cathepsin D indicated that neither the synthesis, sorting nor the proteolytic processing of this lysosomal enzyme was affected in CLN6-defective cells. However, the degradation of the endocytosed index protein arylsulfatase A was strongly reduced in all of the mutant CLN6 cell lines compared with controls. These data suggest that defects in the ER-resident CLN6 protein lead to lysosomal dysfunctions, which may result in lysosomal accumulation of storage material.
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PMID:Defective endoplasmic reticulum-resident membrane protein CLN6 affects lysosomal degradation of endocytosed arylsulfatase A. 1501 Apr 53


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