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William S. Sly - One of the best experts on this subject based on the ideXlab platform.
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new strategies for enzyme replacement therapy for lysosomal storage diseases
Rejuvenation Research, 2010Co-Authors: Jeffrey H. Grubb, Carole Vogler, William S. SlyAbstract:Enzyme replacement therapy is an established means of treating lysosomal storage diseases. Infused enzymes are normally targeted to the lysosomes of affected cells by interactions with cell-surface receptors that recognize carbohydrate moieties such as mannose and mannose 6-phosphate on the enzymes. Therefore, we have investigated alternative strategies to deliver the lysosomal enzyme Beta-Glucuronidase in the enzyme-deficient mucopolysaccharidosis type VII mouse model. Here we summarize our recent efforts to use nontraditional ways to deliver Beta-Glucuronidase. First, we used a chimeric protein of the insulin-like growth factor II (IGF-II) fused to Beta-Glucuronidase to deliver enzyme via the IGF-II binding site on the bifunctional IGF-II/mannose 6-phosphate receptor. Second, we used the 11-amino-acid human immunodeficiency virus (HIV) Tat domain fused to Beta-Glucuronidase to mediate uptake by absorptive endocytosis. Interaction with heparan sulfate on the cell surface internalizes and delivers the Tat-tagged enzyme to the lysosome via plasma membrane recycling. Third, we created a chimeric Beta-Glucuronidase fused to the Fc portion of human immunoglobulin G (IgG) Fc, which was transported by the neonatal Fc receptor from the maternal circulation across the placenta to sites of storage in fetal tissues. Finally, periodate treatment was used to eliminate interaction with carbohydrate receptors, creating an enzyme with increased plasma half-life, resulting in transport across the blood-brain barrier and clearance of storage in neurons. These strategies for delivering lysosomal enzymes could also be used to target nonlysosomal proteins or enzymes identified for bioremediation of other conditions.
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overexpression rescues the mutant phenotype of l176f mutation causing beta glucuronidase deficiency mucopolysaccharidosis in two mennonite siblings
Journal of Biological Chemistry, 1994Co-Authors: Shunji Tomatsu, Seiji Fukuda, Kazuko Sukegawa, Tadao Orii, William S. SlyAbstract:Abstract Two Beta-Glucuronidase-deficient Mennonite siblings were found to be homozygous for a mutation in exon 3 of the Beta-Glucuronidase gene that produces a Leu-->Phe substitution (L176F). The siblings also have the previously described benign polymorphism, P649L. Although their cultured fibroblasts contained 1.5-2.2% of normal Beta-Glucuronidase activity, transient expression of the L176F/P649L cDNA in COS cells produced nearly as much enzyme activity as the wild-type control cDNA. The L176F/P649L enzyme was as stable as wild-type enzyme following endocytosis by fibroblasts and delivery to lysosomes, but was more labile to heat inactivation at 65 degrees C. To study the mutant enzyme at lower levels of expression, we stably transfected mouse mucopolysaccharidosis type VII cells with the L176F/P649L cDNA and selected single-copy cell lines. Metabolic labeling with [35S]methionine revealed that cell lines expressing the mutant enzyme activity at low levels (7-10% of the wild type) actually produced the same amount of enzyme protein as the cell lines expressing the more active wild-type enzyme. However, the cell lines expressing four times this much mutant enzyme protein produced 150-200% as much enzyme activity as the cell line expressing the single-copy wild-type cDNA. These data suggest that overexpression can drive the folding reaction or the self-association of mutant monomers to form active tetramers and, at least partially, correct the Beta-Glucuronidase deficiency seen at low levels of expression with certain missense mutations.
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c terminal processing of human beta glucuronidase the propeptide is required for full expression of catalytic activity intracellular retention and proper phosphorylation
Journal of Biological Chemistry, 1993Co-Authors: M R Islam, Jeffrey H. Grubb, William S. SlyAbstract:Abstract Beta-Glucuronidase undergoes proteolytic C-terminal processing during or after its transport to lysosomes or endosomes. We determined the C-terminal processing site for human placental Beta-Glucuronidase to be the peptide bond between Thr633-Arg634. To evaluate the role of the 18-amino acid peptide removed in C-terminal processing, we changed the codon for Arg634 to a stop codon by site-directed mutagenesis and studied expression of the truncated mutant enzyme in COS-7 cells. An increased fraction of newly synthesized enzyme from R634Stop cDNA was secreted. Pulse-chase experiments provided no evidence for increased degradation of the intracellular R634Stop enzyme. The total amount of catalytic activity expressed from the R634Stop mutant cDNA was only half that seen with the wild type cDNA, and the Kcat of the mutant enzyme was 52% that of wild type enzyme. These results indicate that the C-terminal propeptide in the precursor is important for Beta-Glucuronidase to achieve maximal activity. The truncated enzyme formed hybrid tetramers in cotransfection experiments with the cDNA for rat Beta-Glucuronidase. There appeared to be no decrease in stability of the R634Stop enzyme, since chaotropic agents, heat treatment, and pH had similar effects on the mutant and the wild type enzymes. The uptake rate of the truncated mutant (R634Stop) enzyme by Beta-Glucuronidase-deficient human fibroblast cells was only 55-60% that of the wild type enzyme. Binding to the immobilized cation-independent mannose-6-phosphate receptor and measurement of the 32P-labeled phosphorylated oligosaccharides revealed that the truncated mutant enzyme was 32-34% less phosphorylated and appeared to contain proportionately more covered phosphate groups than the wild type enzyme. These results suggest that the propeptide influences the accessibility to both processing enzymes that produce the mannose-6-phosphate recognition marker on Beta-Glucuronidase.
W M De Vos - One of the best experts on this subject based on the ideXlab platform.
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controlled gene expression systems for lactococcus lactis with the food grade inducer nisin
Applied and Environmental Microbiology, 1996Co-Authors: P G G A De Ruyter, Oscar P Kuipers, W M De VosAbstract:The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes. In the nisin-producing L. lactis strain NZ9700, the specific Beta-Glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached. Expression of the gusA gene was also studied in L. lactis NZ9800, an NZ9700 derivative carrying a deletion in the structural nisA gene that abolishes nisin production, and in L. lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome. In both strains, Beta-Glucuronidase activity was linearly dependent on the amount of nisin added to the medium. Without nisin, no Beta-Glucuronidase production was observed. To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of the nisA gene. Use of the translational fusion vector yielded up to six times more Beta-Glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin. In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold. The Beta-Glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800. This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein. These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins.
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use of the escherichia coli beta glucuronidase gusa gene as a reporter gene for analyzing promoters in lactic acid bacteria
Applied and Environmental Microbiology, 1994Co-Authors: Christ Platteeuw, G Simons, W M De VosAbstract:A transcriptional fusion vector, designated pNZ272, based on the promoterless Beta-Glucuronidase gene (gusA) of Escherichia coli as a reporter gene, has been constructed for lactic acid bacteria. The replicon of pNZ272 was derived from the Lactococcus lactis plasmid pSH71, allowing replication in a wide range of gram-positive bacteria and E. coli. The applicability of pNZ272 and the expression of the gusA gene in L. lactis was demonstrated in shotgun cloning experiments with lactococcal chromosomal and bacteriophage DNA. In addition, three defined lactococcal promoters were inserted in pNZ272: the plasmid-derived lacA promoter, the chromosomal usp45 promoter, and a promoter from bacteriophage phi SK11G. The three resulting plasmids showed Beta-Glucuronidase activity in a gusA-deficient E. coli strain and in four species of lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, and Leuconostoc. The copy numbers of the gusA-expressing plasmids were similar within a single species of lactic acid bacteria. However, the specific Beta-Glucuronidase activity and the gusA mRNA levels varied considerably both within a single species and among different species of lactic acid bacteria. The transcriptional start site of all three promoters was determined and found to be identical in the different species. The results of this comparative promoter analysis indicate that the requirements for efficient transcription initiation differ among the lactic acid bacteria studied.
G Simons - One of the best experts on this subject based on the ideXlab platform.
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use of the escherichia coli beta glucuronidase gusa gene as a reporter gene for analyzing promoters in lactic acid bacteria
Applied and Environmental Microbiology, 1994Co-Authors: Christ Platteeuw, G Simons, W M De VosAbstract:A transcriptional fusion vector, designated pNZ272, based on the promoterless Beta-Glucuronidase gene (gusA) of Escherichia coli as a reporter gene, has been constructed for lactic acid bacteria. The replicon of pNZ272 was derived from the Lactococcus lactis plasmid pSH71, allowing replication in a wide range of gram-positive bacteria and E. coli. The applicability of pNZ272 and the expression of the gusA gene in L. lactis was demonstrated in shotgun cloning experiments with lactococcal chromosomal and bacteriophage DNA. In addition, three defined lactococcal promoters were inserted in pNZ272: the plasmid-derived lacA promoter, the chromosomal usp45 promoter, and a promoter from bacteriophage phi SK11G. The three resulting plasmids showed Beta-Glucuronidase activity in a gusA-deficient E. coli strain and in four species of lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, and Leuconostoc. The copy numbers of the gusA-expressing plasmids were similar within a single species of lactic acid bacteria. However, the specific Beta-Glucuronidase activity and the gusA mRNA levels varied considerably both within a single species and among different species of lactic acid bacteria. The transcriptional start site of all three promoters was determined and found to be identical in the different species. The results of this comparative promoter analysis indicate that the requirements for efficient transcription initiation differ among the lactic acid bacteria studied.
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use of the escherichia coli beta glucuronidase gusa gene as a reporter gene for analyzing promoters in lactic acid bacteria
Applied and Environmental Microbiology, 1994Co-Authors: Christ Platteeuw, G SimonsAbstract:A transcriptional fusion vector, designated pNZ272, based on the promoterless Beta-Glucuronidase gene (gusA) of Escherichia coli as a reporter gene, has been constructed for lactic acid bacteria. The replicon of pNZ272 was derived from the Lactococcus lactis plasmid pSH71, allowing replication in a wide range of gram-positive bacteria and E. coli. The applicability of pNZ272 and the expression of the gusA gene in L. lactis was demonstrated in shotgun cloning experiments with lactococcal chromosomal and bacteriophage DNA. In addition, three defined lactococcal promoters were inserted in pNZ272: the plasmid-derived lacA promoter, the chromosomal usp45 promoter, and a promoter from bacteriophage phi SK11G. The three resulting plasmids showed Beta-Glucuronidase activity in a gusA-deficient E. coli strain and in four species of lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, and Leuconostoc. The copy numbers of the gusA-expressing plasmids were similar within a single species of lactic acid bacteria. However, the specific Beta-Glucuronidase activity and the gusA mRNA levels varied considerably both within a single species and among different species of lactic acid bacteria. The transcriptional start site of all three promoters was determined and found to be identical in the different species. The results of this comparative promoter analysis indicate that the requirements for efficient transcription initiation differ among the lactic acid bacteria studied.
Jeffrey H. Grubb - One of the best experts on this subject based on the ideXlab platform.
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new strategies for enzyme replacement therapy for lysosomal storage diseases
Rejuvenation Research, 2010Co-Authors: Jeffrey H. Grubb, Carole Vogler, William S. SlyAbstract:Enzyme replacement therapy is an established means of treating lysosomal storage diseases. Infused enzymes are normally targeted to the lysosomes of affected cells by interactions with cell-surface receptors that recognize carbohydrate moieties such as mannose and mannose 6-phosphate on the enzymes. Therefore, we have investigated alternative strategies to deliver the lysosomal enzyme Beta-Glucuronidase in the enzyme-deficient mucopolysaccharidosis type VII mouse model. Here we summarize our recent efforts to use nontraditional ways to deliver Beta-Glucuronidase. First, we used a chimeric protein of the insulin-like growth factor II (IGF-II) fused to Beta-Glucuronidase to deliver enzyme via the IGF-II binding site on the bifunctional IGF-II/mannose 6-phosphate receptor. Second, we used the 11-amino-acid human immunodeficiency virus (HIV) Tat domain fused to Beta-Glucuronidase to mediate uptake by absorptive endocytosis. Interaction with heparan sulfate on the cell surface internalizes and delivers the Tat-tagged enzyme to the lysosome via plasma membrane recycling. Third, we created a chimeric Beta-Glucuronidase fused to the Fc portion of human immunoglobulin G (IgG) Fc, which was transported by the neonatal Fc receptor from the maternal circulation across the placenta to sites of storage in fetal tissues. Finally, periodate treatment was used to eliminate interaction with carbohydrate receptors, creating an enzyme with increased plasma half-life, resulting in transport across the blood-brain barrier and clearance of storage in neurons. These strategies for delivering lysosomal enzymes could also be used to target nonlysosomal proteins or enzymes identified for bioremediation of other conditions.
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c terminal processing of human beta glucuronidase the propeptide is required for full expression of catalytic activity intracellular retention and proper phosphorylation
Journal of Biological Chemistry, 1993Co-Authors: M R Islam, Jeffrey H. Grubb, William S. SlyAbstract:Abstract Beta-Glucuronidase undergoes proteolytic C-terminal processing during or after its transport to lysosomes or endosomes. We determined the C-terminal processing site for human placental Beta-Glucuronidase to be the peptide bond between Thr633-Arg634. To evaluate the role of the 18-amino acid peptide removed in C-terminal processing, we changed the codon for Arg634 to a stop codon by site-directed mutagenesis and studied expression of the truncated mutant enzyme in COS-7 cells. An increased fraction of newly synthesized enzyme from R634Stop cDNA was secreted. Pulse-chase experiments provided no evidence for increased degradation of the intracellular R634Stop enzyme. The total amount of catalytic activity expressed from the R634Stop mutant cDNA was only half that seen with the wild type cDNA, and the Kcat of the mutant enzyme was 52% that of wild type enzyme. These results indicate that the C-terminal propeptide in the precursor is important for Beta-Glucuronidase to achieve maximal activity. The truncated enzyme formed hybrid tetramers in cotransfection experiments with the cDNA for rat Beta-Glucuronidase. There appeared to be no decrease in stability of the R634Stop enzyme, since chaotropic agents, heat treatment, and pH had similar effects on the mutant and the wild type enzymes. The uptake rate of the truncated mutant (R634Stop) enzyme by Beta-Glucuronidase-deficient human fibroblast cells was only 55-60% that of the wild type enzyme. Binding to the immobilized cation-independent mannose-6-phosphate receptor and measurement of the 32P-labeled phosphorylated oligosaccharides revealed that the truncated mutant enzyme was 32-34% less phosphorylated and appeared to contain proportionately more covered phosphate groups than the wild type enzyme. These results suggest that the propeptide influences the accessibility to both processing enzymes that produce the mannose-6-phosphate recognition marker on Beta-Glucuronidase.
Christ Platteeuw - One of the best experts on this subject based on the ideXlab platform.
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use of the escherichia coli beta glucuronidase gusa gene as a reporter gene for analyzing promoters in lactic acid bacteria
Applied and Environmental Microbiology, 1994Co-Authors: Christ Platteeuw, G Simons, W M De VosAbstract:A transcriptional fusion vector, designated pNZ272, based on the promoterless Beta-Glucuronidase gene (gusA) of Escherichia coli as a reporter gene, has been constructed for lactic acid bacteria. The replicon of pNZ272 was derived from the Lactococcus lactis plasmid pSH71, allowing replication in a wide range of gram-positive bacteria and E. coli. The applicability of pNZ272 and the expression of the gusA gene in L. lactis was demonstrated in shotgun cloning experiments with lactococcal chromosomal and bacteriophage DNA. In addition, three defined lactococcal promoters were inserted in pNZ272: the plasmid-derived lacA promoter, the chromosomal usp45 promoter, and a promoter from bacteriophage phi SK11G. The three resulting plasmids showed Beta-Glucuronidase activity in a gusA-deficient E. coli strain and in four species of lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, and Leuconostoc. The copy numbers of the gusA-expressing plasmids were similar within a single species of lactic acid bacteria. However, the specific Beta-Glucuronidase activity and the gusA mRNA levels varied considerably both within a single species and among different species of lactic acid bacteria. The transcriptional start site of all three promoters was determined and found to be identical in the different species. The results of this comparative promoter analysis indicate that the requirements for efficient transcription initiation differ among the lactic acid bacteria studied.
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use of the escherichia coli beta glucuronidase gusa gene as a reporter gene for analyzing promoters in lactic acid bacteria
Applied and Environmental Microbiology, 1994Co-Authors: Christ Platteeuw, G SimonsAbstract:A transcriptional fusion vector, designated pNZ272, based on the promoterless Beta-Glucuronidase gene (gusA) of Escherichia coli as a reporter gene, has been constructed for lactic acid bacteria. The replicon of pNZ272 was derived from the Lactococcus lactis plasmid pSH71, allowing replication in a wide range of gram-positive bacteria and E. coli. The applicability of pNZ272 and the expression of the gusA gene in L. lactis was demonstrated in shotgun cloning experiments with lactococcal chromosomal and bacteriophage DNA. In addition, three defined lactococcal promoters were inserted in pNZ272: the plasmid-derived lacA promoter, the chromosomal usp45 promoter, and a promoter from bacteriophage phi SK11G. The three resulting plasmids showed Beta-Glucuronidase activity in a gusA-deficient E. coli strain and in four species of lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, and Leuconostoc. The copy numbers of the gusA-expressing plasmids were similar within a single species of lactic acid bacteria. However, the specific Beta-Glucuronidase activity and the gusA mRNA levels varied considerably both within a single species and among different species of lactic acid bacteria. The transcriptional start site of all three promoters was determined and found to be identical in the different species. The results of this comparative promoter analysis indicate that the requirements for efficient transcription initiation differ among the lactic acid bacteria studied.