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 identified two different exonic point mutations causing beta-glucuronidase (beta G1) deficiency in two Japanese patients with mucopolysaccharidosis type VII (MPSVII). Enzyme assay of lysates of the lymphocytes and cultured fibroblasts showed little residual activity. The beta G1-specific mRNA levels were normal, as determined by northern blot analysis. Mutated cDNA clones, including the entire coding sequence, were isolated using the polymerase chain reaction (PCR) products derived from beta G1-deficient fibroblasts. Sequence analysis of the full-length mutated cDNAs showed C----T transitions, which resulted in a single Ala619----Val change (case 1, a 24-year-old male) and a Arg382----Cys change (case 2, a 7-year-old female). The former change was revealed by a loss of the cleavage site for the Fnu4HI in the mutated cDNA. On the basis of the loss of Fnu4HI restriction site, the patient (case 1) was a homozygote with this mutation. The mutational change in patient 2 was confirmed by direct sequencing and by demonstrating heterozygosity for the mutation in her parents. The Ala619----Val and Arg382----Cys mutations each disrupt a different domain which is highly conserved among human, rat, and Escherichia coli beta G1s. Each of these two amino acid changes reduced the beta G1 activity of the corresponding mutant beta G1 expressed following transfection of COS cells with expression vectors harboring the mutated cDNAs.
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PMID:Mucopolysaccharidosis type VII: characterization of mutations and molecular heterogeneity. 170 66

Egasyn (esterase-22), a member of the nonspecific carboxylesterase multigene family (E.C. 3.1.1.1), is the endoplasmic reticulum (ER)-targeting protein of beta-glucuronidase. We utilized the polymerase chain reaction (PCR) in the eventual isolation of murine egasyn cDNAs. PCR primers were based upon: (1) partial amino acid sequences derived from egasyn peptides and (2) a conserved active site region shared by carboxylesterases. The amino acid sequence deduced from the PCR product matched that obtained from egasyn protein. This product was utilized as a probe to screen a cDNA library. Two cDNAs whose composite sequence encoded an open reading frame of 562 amino acids were isolated. A message size of 1700-2000 bp was revealed by RNA blot hybridization analysis. S1 nuclease protection analyses detected mRNA in liver, kidney, lung, and submandibular gland, but not in spleen, brain, and testes. Genetic mapping confirmed the location of an egasyn cDNA fragment in cluster 1 of the esterase region on chromosome 8. Transfection of COS cells with the 2022-bp cDNA resulted in the expression of esterase activity, which comigrated on native gels with liver esterase-22. The features of the deduced amino acid sequence of the egasyn cDNA are compared with previously characterized carboxylesterases and with other lumenal ER proteins.
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PMID:Characterization and functional expression of a cDNA encoding egasyn (esterase-22): the endoplasmic reticulum-targeting protein of beta-glucuronidase. 178 3

We have sequenced 4.2 kb of the 5' flanking region of the human beta-glucuronidase gene, compared this sequence to the 5' upstream sequence reported for the murine gene, determined the transcription start sites of the human gene, and studied expression of human minigene deletion constructs in COS cells. The 200 bp immediately 5' to the translation initiation codon have a high G + C content (72%) and contain no TATA box, two properties commonly associated with "housekeeping genes." The sequence 5' to -200 bp contains seven Alu repetitive elements which account for more than 50% of this flanking sequence. From deletion analysis of minigene constructs, 200 bp of 5' sequence appeared sufficient for maximal expression in transfected COS cells. S1 nuclease protection analysis showed that transcription initiates from a cluster of sites around -30 bp in all tissues examined. In some cases, a low but detectable level of transcription also initiates 126 bp upstream of the ATG. Inspection of the sequence surrounding both start sites revealed some similarity to the recently described "initiator" transcriptional control element (S.T. Smale and D. Baltimore (1989), Cell 57: 103-113). Comparison of the 5'flanking sequence with that available from the murine beta-glucuronidase gene reveals only one 28-bp highly conserved region, which surrounds the -126 start site.
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PMID:Analysis of the 5' flanking region of the human beta-glucuronidase gene. 191 6

Mouse egasyn cDNA was inserted into expression vector pCDpoly and transfected into mammalian cell lines. Transfected human HepG2 cells, monkey COS-1 cells, and mouse L cells expressed egasyn-esterase catalytic activity. Within COS-1 cells, egasyn was localized to the endoplasmic reticulum. Although individual cells produced large amounts of egasyn, no secretion was observed. No beta-glucuronidase-egasyn complexes were formed in transfected HepG2 or COS-1 cells. However, these complexes were readily detected in transfected L cells. Although the signal for retention of egasyn in the endoplasmic reticulum appears to be species independent, the signal for association with beta-glucuronidase is species restricted.
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PMID:Expression of egasyn-esterase in mammalian cells. Sequestration in the endoplasmic reticulum and complexation with beta-glucuronidase. 200 90

We have identified a mutation causing beta-glucuronidase (beta Gl) deficiency in a 6-year-old girl with mucopolysaccharidosis type VII. Enzyme assay of lysates of a girl's lymphocytes or cultured fibroblasts showed little residual activity and a normal beta Gl-specific mRNA level, as revealed by Northern-blot analysis. Sequencing of the full-length mutated cDNA revealed a C----T transition, an event causing a single Ala619----Val change (we designated this variant beta GGifu). This change is detected by loss of the cleavage site for the enzyme Fnu4HI in the mutated cDNA. On the basis of the loss of Fnu4HI restriction site, the patient was shown to be a homozygote with the beta GGifu mutation and her parents and brother were heterozygotes. This mutation disrupts a functional domain consisting of a region of sequence highly conserved among human, rat and bacterial beta Gls, and it reduces the enzyme activity, as tested by transfection of COS cells with expression vectors harboring the mutated cDNA.
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PMID:Molecular basis of mucopolysaccharidosis type VII: replacement of Ala619 in beta-glucuronidase with Val. 211 90

We have identified several mutations causing beta-glucuronidase (beta Gl) deficiency in three cases with mucopolysaccaridosis type VII (MPS VII). Enzyme assay of lysates of these lymphocytes or cultured fibroblasts showed little residual activity and that the beta Gl-specific mRNA levels were normal, as revealed by Northern blot analysis. Mutated cDNA clones including the entire coding sequencing were isolated from a library in case 1 and PCR (polymerase chain reaction) products in case 2 and 3 derived from cultured fibroblasts. Sequencing of the full-length mutated cDNA revealed C----T transitions, an event causing a single Ala619----Val change (cases 1 and 2) and Arg382----Cys and Pro649----Leu changes (case 3). The former change is detected by loss of the cleavage site for the enzyme Fnu 4 HI in the mutated cDNA. On the basis of the loss of Fnu 4 HI restriction site, the patients (cases 1 and 2) were shown to be a homozygote with this mutation and the parents and brother in case 1 were heterozygotes. The Ala619----Val and Arg382----Cys mutations disrupt a functional domain consisting of a region of sequence highly conserved among human, rat and bacterial beta Gl's, and they lower the enzyme activity, as tested by transfection of COS cells with expression vectors harboring the mutated cDNA. However the Pro649----Leu mutation does not lower the enzyme activity.
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PMID:[Molecular basis of mucopolysaccaridosis type VII (beta-glucuronidase deficiency)]. 223 63

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

A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.
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PMID:Rat liver beta-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells. 335 37

We report here the cDNA sequence for human placental beta-glucuronidase (beta-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. We also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH2-terminal amino acid sequence determined for human spleen beta-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human beta-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human beta-glucuronidase, demonstrate the existence of two populations of mRNA for beta-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length.
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PMID:Cloning, sequencing, and expression of cDNA for human beta-glucuronidase. 346 7

We cloned and sequenced the 8767-bp full-length cDNA for the chicken cation-independent mannose-6-phosphate receptor (CI-MPR), of interest because, unlike its mammalian homologs, it does not bind insulin-like growth factor II (IGF-II). The cDNA encodes a protein of 2470 aa that includes a putative signal sequence, an extracytoplasmic domain consisting of 15 homologous repeat sequences, a 23-residue transmembrane sequence, and a 161-residue cytoplasmic sequence. Overall, it shows 60% sequence identity with human and bovine CI-MPR homologs, and all but two of 122 cysteine residues are conserved. However, it shows much less homology in the N-terminal signal sequence, in repeat 11, which is proposed to contain the IGF-II-binding site in mammalian CI-MPR homologs, and in the 14-aa residue segment in the cytoplasmic sequence that has been proposed to mediate G-protein-coupled signal transduction in response to IGF-II binding by the human CI-MPR. Transient expression in COS-7 cells produced a functional CI-MPR which exhibited mannose-6-phosphate-inhibitable binding and mediated endocytosis of recombinant human beta-glucuronidase. Expression of the functional chicken CI-MPR in mice lacking the mammalian CI-MPR should clarify the controversy over the physiological role of the IGF-II-binding site in mammalian CI-MPR homologs.
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PMID:Cloning and expression of the cDNA of chicken cation-independent mannose-6-phosphate receptor. 756 13


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