J Biol Chem. 1993 Oct 25;268(30):22627-33.
C-terminal processing of human beta-glucuronidase. The propeptide is required for full expression of catalytic activity, intracellular retention, and proper phosphorylation.
Islam MR, Grubb JH, Sly WS.
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, Missouri.
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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.

From the full text article:

Many lysosomal enzymes undergo several post-translational modifications following their synthesis on membranebound ribosomes and during their transport to lysosomes (reviewed in Ref. 1). The early processing steps include cleavage of the N-terminal signal sequence (2), N-linked glycosylation (3,4), oligomerization, and phosphorylation of mannose residues (5). Late processing steps include trimming of carbohydrate chains (6) and proteolytic processing of N- or C-terminal (7, 8) and/or distinct endoproteolytic cleavages in the polypeptide chain (1). During the late processing, small changes in the size have been reported in several lysosomal enzymes (I) including beta-glucuronidases in porcine (7), human (9), mouse (10, 11), and rat (12) tissues. The 2-3-kDa size changes in beta-glucuronidases were not due to glycosylation and have been ascribed to proteolytic processing near the C terminus of the molecule. This processing event is thought to occur in endosomes or lysosomes after segregation of the secretory and the lysosomal pathways at the trans-Golgi network. However, the processing site and the exact role of the propeptide are not yet clear.

One possible function of the propeptide is to serve as a retention signal. In certain murine tissues, including liver and kidney, a significant amount (40-60%) of beta-glucuronidase is retained in the microsomal compartment via interaction with the accessory protein egasyn, which is now known to be identical with mouse esterase-22 (13, 14). Mutant mice that lack egasyn have no retained microsomal beta-glucuronidase.

The observation that only the precursor form of the enzyme that contains the C-terminal peptide forms a complex with egasyn suggested that egasyn interacts with the C-terminal propeptide (15). This conclusion is supported by inhibition studies using a synthetic peptide covering the C-terminal 30 residues of the precursor (16) and by a naturally occurring mutation in this region (17), which results in decreased levels of microsomal beta-glucuronidase.

Whether this function is important in human liver and kidney is unclear. The presence of egasyn has so far not been demonstrated in human tissues (18). Comparison of the derived amino acid sequences of rat (12), mouse (19, 20), and human (9) beta-glucuronidase showed remarkably less homology (40%) in the propeptide region than in the rest of the sequences (77%). In addition the putative egasyn reactive site (RPFLF), which is conserved in rat and mouse P-glucuronidase and is homologous with the active site of serine protease inhibitors, is only partially conserved in the human enzyme.

In this report, we determined the C-terminal processing site in human placental beta-glucuronidase. In order to evaluate the role of the propeptide removed by this processing step, we used site-directed mutagenesis to create a mutant that deleted the propeptide. Deletion of the propeptide suppressed full realization of catalytic activity. The truncated mutant enzyme achieved only half the expected activity, even though the C terminus is removed from mature enzyme naturally in endosome/lysosome with no loss of activity. Furthermore, deletion of the propeptide decreased the phosphorylation and targeting of the enzyme.

Categories: 1993, Beta-glucuronidase, Post-translational modification, Glycosylation, Phosphorylation