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:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have cloned and sequenced the full-length cDNA for the human cation-independent mannose 6-phosphate receptor from four overlapping clones. The 9104-nucleotide sequence contains 7473 nucleotides which encode a protein of 2491 amino acids. The amino acid sequence includes a putative signal sequence of 40 amino acids, an extracytoplasmic domain consisting of 15 homologous repeat sequences of 134-167 amino acids, a transmembrane region of 23 amino acids, and a cytoplasmic domain of 164 amino acids. The predicted molecular size is greater than 270 kDa. Repeats 7-15 of the extracytoplasmic domain of the human receptor are highly homologous with the sequence recently reported for the partial cDNA for the bovine receptor (Lobel, P., Dahms, N. M., Breitmeyer, J., Chirgwin, J. M., and Kornfeld, S. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2233-2237). The nucleotide sequence for the full-length cDNA and the deduced amino acid sequence for the cation-independent mannose 6-phosphate receptor, which are reported here, are strikingly similar (99.8% identical at the nucleotide level and 99.4% identical at the amino acid level) to those recently reported for the human insulin-like growth factor II receptor from HepG2 hepatoma cells (Morgan, D. O., Edman, J.D., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). These findings support the suggestion that the cation-independent mannose 6-phosphate receptor for lysosomal enzymes is a multifunctional binding protein which is identical with the insulin-like growth factor II receptor. A cDNA construct containing the full coding sequence for the cation-independent mannose 6-phosphate receptor in the expression vector pSVL was used to transfect COS cells. Expression of the cDNA in transfected COS cells produced a cell-surface protein which co-migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with authentic human receptor, bound to an affinity column and was specifically eluted with mannose 6-phosphate, mediated cell-surface binding and endocytosis of beta-glucuronidase, and targeted the endocytosed enzyme to lysosomes.
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PMID:The human cation-independent mannose 6-phosphate receptor. Cloning and sequence of the full-length cDNA and expression of functional receptor in COS cells. 296 3

We recently reported the cDNA cloning, sequence, and expression of the human cation-independent mannose 6-phosphate receptor (hCI-MPR) (Oshima, A., Nolan, C. M., Kyle, J. W., Grubb, J. H., and Sly, W. S. (1988) J. Biol. Chem. 263, 2553-2562). The sequence of the hCI-MPR was virtually identical to that of the human insulin-like growth factor II receptor cDNA (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). To test the role of the putative bifunctional receptor in intracellular sorting of acid hydrolases, we studied its effect on lysosomal enzyme transport following gene transfer to receptor-negative cells. Receptor-negative mouse P388D1 cells were transfected with a cDNA construct containing the entire coding sequence of hCI-MPR under the control of the mouse metallothionine I promoter. Stable transformants were isolated and characterized. The expressed hCI-MPR was localized in membranes including the plasma membrane, bound mannose 6-phosphate containing ligands, and mediated endocytosis which could be specifically blocked by mannose 6-phosphate. We next measured the effect of the expressed hCI-MPR on intracellular and secreted acid hydrolases. The intracellular activity of the lysosomal marker enzymes beta-glucuronidase and beta-hexosaminidase increased up to 2-fold following transformation. In addition, expression of the receptor greatly reduced the fraction of acid hydrolases secreted. These phenotypic changes in the transformed cell lines support the proposed role of the cation-independent mannose 6-phosphate receptor in intracellular sorting and targeting of lysosomal enzymes.
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PMID:Expression of human cation-independent mannose 6-phosphate receptor cDNA in receptor-negative mouse P388D1 cells following gene transfer. 297 5

The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622). Cells were grown in the presence of [3H]mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography. About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15%. The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity. Intracellular levels of these enzymes were reduced in the mutants. The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments. [35S]methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate. Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor.
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PMID:The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors. 627 Jan 23

The murine plasma cell line MOPC 315 efficiently targets newly synthesized acid hydrolases to lysosomes in spite of a marked deficiency in the level of the mannose 6-phosphate receptor (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). To better understand the routing of lysosomal enzymes in this cell line, pulse-chase experiments were performed with [2-3H]mannose and [35S]methionine followed by immunoprecipitation of beta-glucuronidase and IgA. By 3 h of chase, essentially all of the newly synthesized beta-glucuronidase had undergone proteolytic processing, suggesting that the molecules had reached lysosomes. At this time 30% of the pulse-labeled IgA was still intracellular. The oligosaccharides on the intracellular IgA were of the high mannose-type, while the secreted IgA contained processed, complex-type oligosaccharides. This indicates that the intracellular IgA was still in the endoplasmic reticulum or an early region of the Golgi complex when all of the beta-glucuronidase had reached lysosomes. Therefore, beta-glucuronidase and IgA must exit from the endoplasmic reticulum or the early Golgi complex at different rates, a finding that is inconsistent with bulk phase movement of these proteins from the endoplasmic reticulum to the trans Golgi complex. The addition of the ionophore monensin greatly slows the rate of IgA secretion from MOPC 315 cells and the molecules secreted have incompletely processed oligosaccharides. In contrast, monensin only slightly delays the transport of newly synthesized beta-glucuronidase to lysosomes and causes no significant alteration in the extent of oligosaccharide phosphorylation, a process that appears to occur in the early (cis) Golgi complex. However, the labeled beta-glucuronidase was deficient in sialylated, phosphorylated hybrid oligosaccharides whose biosynthesis requires the action of late stage oligosaccharide processing enzymes assumed to be localized in the trans Golgi complex.
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PMID:Targeting of beta-glucuronidase to lysosomes in mannose 6-phosphate receptor-deficient MOPC 315 cells. 633 Jan 27

The expression of various components of the lysosomal and ubiquitin-dependent degradative pathways was characterized in an in vitro model of differentiating enterocytes, the human colon adenocarcinoma Caco-2 cell line. The activities of the cell-associated lysosomal enzymes alpha-D-mannosidase, beta-hexosaminidase, beta-glucuronidase, and beta-galactosidase increased approximately 2- to 4-fold as differentiation proceeded. In contrast, the protein levels of the two mannose 6-phosphate receptors (MPRs), the insulin-like growth factor II/cation-independent MPR (IGF-II/CI-MPR) and the cation-dependent MPR (CD-MPR), did not change significantly during Caco-2 differentiation. In addition, quantitative Western blot analyses revealed that on a molar basis the CD-MPR is 3.5 times more abundant than the IGF-II/CI-MPR in Caco-2 cells. Since only limited secretion of lysosomal enzymes was observed throughout differentiation, the level of expression of the MPRs was sufficient to target the increased levels of lysosomal enzymes to the lysosome. Unlike the expression of lysosomal enzymes, Western blot analysis demonstrated an approximately 40% and approximately 30% decrease, respectively, in the steady-state levels of free and conjugated ubiquitin during Caco-2 differentiation. Taken together, these results show that the ubiquitin-dependent proteolytic pathway is regulated differently than the lysosomal degradative pathway during Caco-2 differentiation.
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PMID:Regulation of lysosomal and ubiquitin degradative pathways in differentiating human intestinal Caco-2 cells. 754 43

The prion encephalopathies are characterized by accumulation in the brain of the abnormal form PrPsc of a normal host gene product PrPc. The mechanism and site of formation of PrPsc from PrPc are currently unknown. In this study, ME7 scrapie-infected mouse brain was used to show, both biochemically and by double-labelled immunogold electron microscopy, that proteinase K-resistant PrPsc is enriched in subcellular structures which contain the cation-independent mannose 6-phosphate receptor, ubiquitin-protein conjugates, beta-glucuronidase, and cathepsin B, termed late endosome-like organelles. The glycosylinositol phospholipid membrane-anchored PrPc will enter such compartment for normal degradation and the organelles may therefore act as chambers for the conversion of PrPc into infectious PrPsc in this murine model of scrapie.
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PMID:The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain. 756 56

beta-Glucuronidase injected i.v. into newborn mucopolysaccharidosis VII mice was cleared from the circulation in less than 1 h and taken up by tissues in a distribution corresponding to the location of the mannose 6-phosphate receptor. One h after a 3.5-mg/kg beta-glucuronidase injection, beta-glucuronidase levels were equal to or greater than normal in every organ examined with the exception of the brain, where 31% normal activity was present. Enzyme was detectable histochemically in the major sites of pathology for mucopolysaccharidosis VII including bone, brain, heart, and fixed tissue macrophages. The half-life of recombinant beta-glucuronidase activity in various organs of injected mucopolysaccharidosis VII mice was 1.5 to 4.5 d. These studies show that recombinant beta-glucuronidase administered to newborn mice reaches the sites of clinically important storage in murine mucopolysaccharidosis VII.
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PMID:Enzyme replacement with recombinant beta-glucuronidase in the newborn mucopolysaccharidosis type VII mouse. 810 4

The direct transfer of certain lysosomal enzymes during cell-to-cell contact between normal lymphocytes and enzyme-deficient recipient cells has previously been reported in vitro and may play an important role in the correction of lysosomal storage diseases by bone marrow transplantation in vivo. In the present study we have used a number of different T, B, and plasma cell lines to examine the expression and immunological specificity of the transfer of the lysosomal enzyme, beta-glucuronidase (Gus). Each of these groups of cell had differing intracellular and secreted levels of Gus activity, which were nevertheless similar within each group. Dermal fibroblasts deficient in the Gus enzyme acquired substantial amounts of additional activity when they were cultured together with the T cells, the B cells, or the plasma cells. This occurred by the direct transfer of Gus from all three types of cell. In addition, with plasma cells, which had very high intracellular enzyme activity and also secreted high levels of Gus into their culture medium, the secreted enzyme was readily internalized by the fibroblasts via the mannose 6-phosphate receptor (MPR). It was notable that the purified endogenous enzymes from plasma cells as well as from B cells, but not from T cells, were also endocytosed by the fibroblasts utilizing this receptor-mediated process. Although the Gus activity from all the cell lines examined had the same molecular size, polyacrylamide electrophoresis and isoelectric focusing patterns showed that the immunologically distinct types of lymphoid cell have characteristic, unique pathways of post-translational lysosomal enzyme processing. These results show that the transfer of lysosomal enzymes from lymphoid cells can occur by two distinct mechanisms, both likely to have important roles in enzyme replacement therapy.
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PMID:Lysosomal enzyme transfer from different types of lymphoid cell. 822 97

The effect of wortmannin on the trafficking of the mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF-II receptor) and its ligand beta-glucuronidase has been determined in murine L cells and normal rat kidney cells. The drug induced a 90% decrease in the steady-state level of the Man-6-P/IGF-II receptor at the plasma membrane without affecting the rate of internalization, indicating that the return of receptor from endosomes to the plasma membrane is retarded. Wortmannin also slowed the movement of receptor from endosomes to the trans-Golgi network by about 60%. Such a kinetic block would dramatically reduce the number of Man-6-P/IGF-II receptors in the trans-Golgi network, which could account for the previously described hypersecretion of procathepsin D induced by wortmannin. In addition, the drug slowed delivery of endocytosed beta-glucuronidase from endosomes to dense lysosomes. These data, taken together with the published reports of others, indicate that wortmannin inhibits membrane trafficking out of multiple compartments of the endosomal system and suggest a role for phosphatidylinositol 3-kinase in regulating these processes.
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PMID:Wortmannin retards the movement of the mannose 6-phosphate/insulin-like growth factor II receptor and its ligand out of endosomes. 946 65

The two mannose 6-phosphate (Man-6-P) binding sites of the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) have been localized to domains 1-3 and 7-9, and studies have shown that Arg435 in domain 3 and Arg 1334 in domain 9 are essential for Man-6-P binding. To determine whether the IGF-II/MPR containing a single Man-6-P binding site is functional, clonal mouse L cell lines stably transfected with either mutant bovine IGF-II/MPR cDNA, containing substitutions at position 435 and/or 1334, or the wild type receptor cDNA were assayed for their ability to sort lysosomal enzymes to the lysosome. Mutant receptors containing a single Man-6-P binding site were approximately 50% less efficient than the wild type receptor in the overall targeting of lysosomal enzymes to the lysosome. Mutant receptors containing a substitution at Arg1334 (Dom9(Ala)), in contrast to those containing a substitution at Arg435 (Dom3(Ala)), were unable to target cathepsin D and beta-hexosaminidase to the lysosome. Equilibrium binding assays using 125I-labeled beta-glucuronidase demonstrated that Dom3(Ala) and Dom9(Ala) had a Kd of 2.0 and 4.3 nM, respectively. In addition, Dom3(Ala), unlike Dom9(Ala), was unable to completely dissociate from ligand under acidic pH conditions. These data indicate that the two Man-6-P binding sites of the IGF-II/MPR are not functionally equivalent.
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PMID:The two mannose 6-phosphate binding sites of the insulin-like growth factor-II/mannose 6-phosphate receptor display different ligand binding properties. 971 56


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