C-terminal processing of human [beta]-Glucuronidase: The propeptide is required for full expression of catalytic activity, intracellular retention, and proper phosphorylation

Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, Missouri 63104.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/1993; 268(30):22627-33.
Source: PubMed


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.

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Available from: Rafiq Islam, Dec 10, 2014
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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). "
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