C-terminal processing of human beta-glucuronidase. The propeptide is required for full expression of catalytic activity, intracellular retention, and proper phosphorylation.
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.
Full-textDOI: · Available from: Rafiq Islam, Dec 10, 2014
- SourceAvailable from: Gian Pietro Di Sansebastiano
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- "In addition, the propeptide is required for proper phosphorylation of the glycans, and thus for efficient lyzosomal targeting. Finally, the propeptide is also necessary for the enzyme to achieve maximal activity (Islam et al., 1993) as we also found in tobacco protoplasts. The glycans are also needed for proper folding of the enzyme. "
ABSTRACT: E. coliβ-glucuronidase, a cytosolic enzyme, was found not to be a good reporter enzyme for secretion studies in plants. In this study, we chose to test and adapt an animal β-glucuronidase as a better reporter protein for the secretory pathway of plants. We modified rat β-glucuronidase to obtain secreted and vacuolar variants. Five different C-termini were produced: the original C-terminus of the rat enzyme, a 19 codon deletion (Δ19), a 15 codon deletion (Δ15) and fusions of the Δ19 or Δ15 termini with the last 6 or 7 codons of the vacuolar sorting determinant of tobacco chitinase A, respectively. The signal sequence of the rat β-glucuronidase polypeptide was replaced by the sequence encoding the signal peptide of tobacco chitinase A. In a transient expression system, the best enzymatic activity was found with β-glucuronidase having the 15 codons deletion, therefore Δ15 (secRGUS) and Δ15 + Chi (RGUS-Chi) were further evaluated and their efficiency of secretion or vacuolar targeting were tested under different conditions. To determine the correct targeting of reporter genes, we compared the localization of β-glucuronidase and of an endogenous marker, α-mannosidase. Treating cells with drugs that specifically affect different aspects of the secretory pathway also tested the validity of RGUS-based reporters. A non-specific inhibitor such as cytochalasin D and a wide range inhibitor such as BFA were compared with specific inhibitors such as wortmannin and bafilomycin A1. Finally, monensin and NH4Cl were used to evaluate the role of vacuolar pH in correct RGUS-Chi targeting. The two new reporter proteins proved to be good tools for our studies in the transient expression system in tobacco protoplasts and for further applications.Plant Biosystems 11/2007; 141(3):329-336. DOI:10.1080/11263500701627513 · 1.91 Impact Factor
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- "The low level of fusion protein in the cell layer is predicted based on the intracellular processing pathway of the wild-type GUSB. The enzyme normally undergoes C-terminal cleavage during transit from the trans-Golgi to lysosomes (Islam et al. 1993). Thus, the intracellular lysosomal pool of the genetic fusion protein would be cleaved but the secreted pool would be intact. "
ABSTRACT: The neurotropic atoxic fragment of tetanus toxin has been used as a carrier for transporting macromolecules into neurons but all studies to date have tested cytosolic proteins. In this study we investigated the effect of a genetic addition of the tetanus toxin C fragment sequence to a lysosomal enzyme which contains a signal sequence for insertion into the membrane-bound compartment and must be extensively modified in the endoplasmic reticulum (ER) and Golgi to attain functionality. In-frame fusion constructs between the atoxic C fragment and beta-glucuronidase were compared with the wild-type enzyme for: (i) enzymatic activity; (ii) heat stability; (iii) pH dependence; (iv) specific activity; (v) apparent molecular mass and (vi) receptor-mediated uptake by fibroblasts and neurons. The modified proteins had biochemical properties similar to wild-type enzyme but exhibited different enzyme secretion profiles. Addition of the secreted fusion enzyme to cultures of primary neurons showed significantly increased neuronal uptake of the modified protein compared with the wild-type, demonstrating the bifunctionality of the chimeric molecule.Journal of Neurochemistry 07/2005; 93(5):1334-44. DOI:10.1111/j.1471-4159.2005.03133.x · 4.24 Impact Factor
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- "β-Gluc is a tetrameric glycoprotein with a molecular weight of about 330 kDa and consists of four identical monomeres of 651 amino acids (Jain et al. 1996). During transport from the endoplasmatic reticulum to lysosomes, the 83 to 80 kDa monomers are proteolytically processed to a 79 and 77 kDa polypeptide which in turn can be cleaved to 64 kDa and 18 kDa forms (Tanaka et al. 1992; Islam et al. 1993; Gehrmann et al. 1994). Human β-gluc cDNA as well as the gene and the promoter-containing 5' flanking region have been cloned and characterized (Oshima et al. 1987; Miller et al. 1990; Shipley et al. 1991). "
ABSTRACT: A promising development in tumor therapy is the application of non-toxic prodrugs from which the active cytostatic is released by endogenous enzymes such as beta-glucuronidase (beta-gluc). Regulation of beta-gluc expression is one crucial factor modulating bioactivation of prodrugs. Recent experiments in rats indicate regulation of beta-gluc activity by the calcium channel blocker verapamil. To further explore this phenomenon, we investigated the effect of verapamil on beta-gluc enzyme activity, protein (western blot) and mRNA expression (RT-PCR) as well as the underlying mechanisms (effects of verapamil metabolites; promoter activity) in the human hepatoma cell line HepG2. Treatment of HepG2 cells with verapamil revealed down-regulation of beta-gluc activity, protein, and mRNA level down to 50% of the control with EC(50) values of 25 microM. Effects were similar for both enantiomers. Moreover, it was demonstrated that reduced promoter activity contributes to the observed effects. In summary, our data demonstrate regulation of human beta-glucuronidase expression by verapamil. Based on our findings we hypothesize that coadministration of verapamil may effect cleavage of glucuronides by beta-glucuronidase.Archiv für Experimentelle Pathologie und Pharmakologie 01/2004; 368(6):463-9. DOI:10.1007/s00210-003-0837-x · 2.36 Impact Factor