C-TERMINAL PROCESSING OF HUMAN BETA-GLUCURONIDASE - THE PROPEPTIDE ISREQUIRED FOR FULL EXPRESSION OF CATALYTIC ACTIVITY, INTRACELLULAR RETENTION, AND PROPER PHOSPHORYLATION

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 c odon for Arg634 to a stop codon by site-directed mutagenesis and studi ed expression of the truncated mutant enzyme in COS-7 cells. An increa sed fraction of newly synthesized enzyme from R634Stop cDNA was secret ed. Pulse-chase experiments provided no evidence for increased degrada tion of the intracellular R634Stop enzyme. The total amount of catalyt ic activity expressed from the R634Stop mutant cDNA was only half that seen with the wild type cDNA, and the K(cat) of the mutant enzyme was 52% that of wild type enzyme. These results indicate that the C-termi nal propeptide in the precursor is important for beta-glucuronidase to achieve maximal activity. The truncated enzyme formed hybrid tetramer s in cotransfection experiments with the cDNA for rat beta-glucuronida se. There appeared to be no decrease in stability of the R634Stop enzy me, since chaotropic agents, heat treatment, and pH had similar effect s on the mutant and the wild type enzymes. The uptake rate of the trun cated mutant (R634Stop) enzyme by beta-glucuronidase-deficient human f ibroblast 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 P-32-labeled phosphorylated oligosaccharides revea led that the truncated mutant enzyme was 32-34% less phosphorylated an d appeared to contain proportionately more covered phosphate groups th an the wild type enzyme. These results suggest that the propeptide inf luences the accessibility to both processing enzymes that produce the mannose-6-phosphate recognition marker on beta-glucuronidase.