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)

beta-Glucuronidase is retained within the endoplasmic reticulum (ER) via complex formation with esterase-22 (egasyn), which in turn has a COOH-terminal HTEL ER retention sequence. To identify the regions of glucuronidase that interact with egasyn, complex formation was assayed in COS cells cotransfected with egasyn cDNA and with either deletion constructs of glucuronidase or with constructs containing specific glucuronidase propeptide sequences appended to the carboxyl terminus of a rat secretory protein alpha 1-acid glycoprotein. The region of glucuronidase essential for complex formation is a linear octamer sequence at the COOH terminus of the propeptide. A portion of this octamer is similar to a sequence near the reactive site of serpins. This and associated data indicate that an interaction related to that between serine proteinases and their serpin inhibitors retains beta-glucuronidase within the ER. Further, attachment of this octamer sequence provides an alternative method of targeting proteins to the ER lumen of any cell that contains egasyn. These and related results demonstrate that complex formation with esterases/proteinases within the ER is important in the subcellular targeting and/or processing of certain proteins.
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PMID:The beta-glucuronidase propeptide contains a serpin-related octamer necessary for complex formation with egasyn esterase and for retention within the endoplasmic reticulum. 774 42

Commercially available Wistar rats are genetically heterogeneous with respect to acid beta-D-glucuronidase (EC 3.2.1.31) in liver tissue: Of 43 rats studied, 27 animals exhibited only approximately 40% catalytic activity of the remaining group. Analysis by subcellular and density gradient fractionation, and polyacrylamide gel electrophoresis techniques showed that livers with high activity exhibit a dual enzyme distribution in lysosomes (app. 76%) and endoplasmic reticulum (named "microsomal enzyme form"; app. 24%), whereas those with low activity not only lack the microsomal enzyme form - presumably due to the absence of egasyn - but also display reduced lysosomal enzyme activity.
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PMID:Heterogeneity of Wistar rats with respect to acid beta-D-glucuronidase (EC 3.2.1.31) in liver. 795 Oct 44

We have investigated targeting to the endoplasmic reticulum (ER) of wild-type GUS and a modified form (GUS S358) by making an N-terminal fusion of the beta-glucuronidase (GUS) enzyme with the wheat alpha-amylase signal peptide. In vitro studies demonstrated that the modified GUS (S358) lacked the glycosylation site present within the wild-type enzyme. Analysis of transgenic tobacco plants revealed that the modified GUS enzyme retained activity upon passage to the ER. When further experiments were carried out to determine the cellular location of the modified GUS enzyme, it was found that (contrary to expectation) the majority of GUS activity was retained within the cell and was not secreted to the cell surface via the default pathway. The data indicated that the modified GUS enzyme is an unsuitable reporter enzyme for studying protein secretion.
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PMID:Endoplasmic reticulum targeting of active modified beta-glucuronidase (GUS) in transgenic tobacco plants. 795 35

The 5' regulatory region and putative signal sequence of a rice alpha-amylase gene, alpha Amy8, was fused to a bacterial gene encoding beta-glucuronidase (GUS) and introduced into rice, tobacco, and potato via Agrobacterium-mediated transformation systems. Expression of this chimeric gene in suspension cells of transgenic plants was suppressed by the presence of sucrose in the medium and induced by its absence. Induction or suppression of GUS expression in transgenic rice could be reversed by the deprivation or replenishment, respectively, of sucrose in the medium. The expressed GUS fusion protein was translocated to the endoplasmic reticulum, modified by glycosylation, and secreted into the culture medium of transgenic cells. In the presence of a glycosylation inhibitor, tunicamycin, the enzymatically active form of GUS was assembled in the endoplasmic reticulum. The yield of GUS secreted by transgenic cells was estimated to be as high as 40% of total secreted proteins. The reversible induction of the alpha-amylase promoter in culture cells by sugar level in the medium provides an excellent inducible expression system for basic research in plant science. Combination of the alpha-amylase promoter and signal sequence also offers a novel approach for large scale production of low cost, easily purified, secreted recombinant proteins.
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PMID:Novel gene expression system for plant cells based on induction of alpha-amylase promoter by carbohydrate starvation. 802 Dec 73

Nodulin-24 is a nodule-specific protein of the peribacteroid membrane (PBM) in soybean. It has an apparent molecular mass of 33 kDa while its full-length cDNA encodes a polypeptide of only 24 kDa. In vitro transcription of nodulin-24 cDNA followed by translation resulted in a peptide translocated into microsomal membranes with cleavage of a signal sequence. The cleavage site of the signal sequence in nodulin-24 was determined to be between Ala (A25) and Arg (R26) by microsequencing of the [3H]leucine-labeled processed peptide. Fusion of the signal sequence of nodulin-24 with the beta-glucuronidase peptide prevented co-translational cleavage of the signal sequence although the translocation of the fused protein into microsomes occurred co-translationally. Trypsin treatment of membrane-translocated nodulin-24 did not result in any alteration in size suggesting that the newly synthesized peptide is fully protected in the membrane vesicle. Fusion of nodulin-24 with beta-glucuronidase also showed no change in size following trypsin treatment, suggesting that nodulin-24 has no membrane-spanning region. In addition, in vitro synthesized nodulin-24 was present in the supernatant fraction after sonication of microsomal membranes. Mature nodulin-24, on the other hand, is not solubilized from PBM by sodium carbonate (pH 11) or EGTA and is soluble only in detergent. These data suggest that nodulin-24 is synthesized as a lumenal protein in the endoplasmic reticulum and post-translationally attached to the membranes en route to the PBM. This processing results in a significant increase in the apparent molecular mass of nodulin-24 which may be due to the attachment of membrane lipids as this protein shares characteristics with membrane lipoproteins of many pathogenic bacteria.
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PMID:Nodulin-24 follows a novel pathway for integration into the peribacteroid membrane in soybean root nodules. 812 12

We have determined the effects of the Niemann-Pick type C (NPC) lesion, which impairs transport of cholesterol from lysosomes, on the androgenic status of male NPC mice. The mice have low serum testosterone levels resulting from decreased testosterone secretion. Testosterone secretion is reduced in NPC mouse testes incubated with 8-bromo-cAMP, 20 alpha-hydroxycholesterol, and pregnenolone compared to testosterone release by normal mouse testes under identical conditions. Ultrastructural examination of testes revealed a paucity of lipid droplets, extensive accumulation of inclusion bodies, and distorted endoplasmic reticulum in Leydig cells of adult NPC mice. The hypoandrogenemia caused systemic deficiencies in NPC mice. Seminal vesicles, a testosterone-responsive tissue, were underdeveloped in NPC male mice. The testosterone-responsive kidney beta-glucuronidase activity was also underexpressed. Seminal vesicle mass and beta-glucuronidase activity were increased by testosterone treatment of NPC mice. Many hepatic proteins, identified by microsequencing, were also deficient in NPC male mice. Levels of alpha 2-mu-globulin, glutathione S-transferase-pi, carbonic anhydrase-III, and selenium-binding protein increased in normal male mice during puberty, but did not increase in the NPC male mice. Based on the increases in protein expression during puberty, differential expression in males and females, and the reported involvement of androgens in regulating expression of some of these proteins, deficient expression of most of these proteins in male NPC mice appears to result from low testosterone levels. We conclude that a defect in testicular testosterone production in NPC male mice causes a pleiotropic deficiency in androgen-sensitive expression of proteins in various organs.
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PMID:The murine Niemann-Pick type C lesion affects testosterone production. 824 19

Egasyn is localized within the lumen of the endoplasmic reticulum (ER) where it complexes with and thus causes sequestration of a considerable portion of beta-glucuronidase. Egasyn has an HTEL sequence at its carboxyl terminus rather than the KDEL sequence that serves as a retention signal for many ER lumenal proteins. To determine whether the HTEL sequence acts as an ER retention signal and/or functions in complex formation, HTEL-deleted egasyn was expressed in mammalian cell lines. The majority of HTEL-deleted egasyn was secreted, while wild type egasyn was retained in the ER. Furthermore, the egasyn HTEL sequence, when added to the carboxyl termini of two secretory proteins, mouse esterase, Es-N, and rat alpha 1-acid glycoprotein (AGP), caused retention of both proteins within the ER, demonstrating that the HTEL sequence is both necessary and sufficient for retention of egasyn and, by extension, the egasyn-glucuronidase complex within the ER. Other carboxyl terminal tetrapeptides including HIEL and HVEL, naturally occurring in other ER lumenal proteins, were also sufficient for ER retention of AGP, while HTEHT and HTEHK were inefficient in ER retention. The HTEL sequence, in contrast, is not required for egasyn-glucuronidase complex formation. Further, the complex is apparently unstable outside the ER since it was not visible in the medium of cells transfected with egasyn lacking the HTEL sequence despite abundant secretion of this egasyn. These results show that it is possible to localize proteins within the lumen of the ER if they form complexes with ER lumenal proteins containing an intrinsic ER retention sequence.
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PMID:The signal for retention of the egasyn-glucuronidase complex within the endoplasmic reticulum. 834 16

We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes.
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PMID:Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins. 870 Sep 11

Vesicle traffic between the endoplasmic reticulum and the Golgi apparatus in mammals requires the small GTP-binding protein Rab2, but Saccharomyces cerevisiae appears not to have a Rab2 homolog. Here it is shown that the higher plant, Arabidopsis thaliana, contains a gene, At-RAB2, whose predicted product shares 79% identity with human Rab2 protein. Transgenic plants containing fusions between beta-glucuronidase and sequences upstream of At-RAB2 demonstrated histochemical staining predominantly in maturing pollen and rapidly growing organs of germinating seedlings. beta-glucuronidase activity in pollen is first detectable at microspore mitosis and increases thereafter. In this respect, the promoter of At-RAB2 behaves like those of class II pollen-specific genes, whose products are often required after germination for pollen tube growth. Seedling germination and pollen tube growth are notable for their unusually high rates of cell wall and membrane biosynthesis. These results are consistent with a role for At-RAB2 in secretory activity.
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PMID:A homolog of the mammalian GTPase Rab2 is present in Arabidopsis and is expressed predominantly in pollen grains and seedlings. 901 59

An Arabidopsis oleosin was used as a model to study oleosin topology and targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteinase K mapping of the membrane topology. Oleosin maintains a conformation with a membrane-integrated hydrophobic domain flanked by N- and C-terminal domains located on the outer microsome surface. This is a unique membrane topology on the endoplasmic reticulum (ER). Three universally conserved proline residues within the "proline knot" motif of the oleosin hydrophobic domain were substituted by leucine residues. After in vitro translation, only minor differences in proteinase K protection could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on the ER was observed between the two oleosin forms. However, as an oleosin-beta-glucuronidase translational fusion, the proline knot variant failed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expressing oleosin-beta-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native form. Therefore, the proline knot motif is not important for ER integration and the determination of topology but is required for oil body targeting. The loss of the proline knot results in an intrinsic instability in the oleosin polypeptide during trafficking.
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PMID:Role of the proline knot motif in oleosin endoplasmic reticulum topology and oil body targeting. 928 16


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