J Biol Chem. 1993 Jun 5;268(16):12193-8.
The role of glycosylation and phosphorylation in the expression of active human beta-glucuronidase.
Shipley JM, Grubb JH, Sly WS.
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri.
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Phosphorylation of mannose residues on N-linked oligosaccharide side chains of lysosomal enzymes targets them to lysosomes. We used site-directed mutagenesis to observe the effect of eliminating selective glycosylation sites from human beta-glucuronidase on enzyme sorting. Expression studies allowed us to determine which of four potential sites were glycosylated, preferentially phosphorylated, and required for catalytic activity. All four sites of the human enzyme were glycosylated, whereas in the mouse and rat enzymes, only three of four sites are used. Sites 2 and 3 were preferentially phosphorylated. Elimination of sites 2 and 3 in combination markedly decreased sorting to lysosomes and increased enzyme secretion. Each of the four glycosylation sites could be eliminated individually without drastic reduction in catalytic activity. Activity was progressively lost as combinations of two, three, and four sites were eliminated. Wild-type enzyme produced in the presence of tunicamycin was also inactive, indicating that glycosylation is required for realization of enzyme activity. However, active enzyme could be deglycosylated with only minimal loss of activity. Mutant enzyme completely lacking glycosylation did not form tetramers. Partial restoration of tetramerization was achieved by the co-expression of normal rat enzyme, provided that the normal rat enzyme supplied at least two subunits to the tetramer.

From the full text article:

Beta-glucuronidase (EC 3.2.1.31) is a lysosomal enzyme composed of four identical subunits which plays a role in the stepwise degradation of glucuronic acid-containing glycosaminoglycans (heparan sulfate, dermatan sulfate, and chondroitin sulfate). Deficiency of the enzyme in humans results in the clinical genetic disorder mucopolysaccharidosis type VI1 (MPS VI1 or Sly Syndrome) which is characterized by an accumulation in lysosomes of glycosaminoglycans containing terminal glucuronic acid residues (Hall et al., 1973; Sly et al., 1973).

In higher eukaryotes, the enzyme undergoes a series of posttranslational modifications en route to lysosomes. Individual subunits are synthesized on membrane-bound ribosomes and translocated into the lumen of the ER,¹ whereupon several events occur (Rosenfeld et al., 1982). These include signal sequence cleavage, N-linked glycosylation, disulfide bond formation, and tetramerization. A fraction of the enzyme in rodents is retained in the ER through its association with the protein egasyn (Lusis et al., 1976). This ER retention has not been demonstrated for human beta-glucuronidase, and its functional significance in mice and rats is not clear.

During transit to the Golgi, a phosphotransferase catalyzes the transfer of N-acetylglucosamine-1-PO, from UDPGlcNAc to particular mannose residues on the oligosaccharide side chains of lysosomal enzymes (Reitman and Kornfeld, 1981a, 1981b; Waheed et al., 1982). This enzyme recognizes a protein conformation shared by lysosomal enzymes that is not present in other secretory proteins (Lang et al., 1984, Baranski et al., 1990). In the Golgi, a second enzyme removes the covering N-acetylglucosamine, exposing mannose-6-P04 residues on lysosomal enzymes (Waheed et al., 1981; Varki and Kornfeld, 1981). The mannose-6-P04 residues are recognized in the trans-Golgi network by specific receptors which transport lysosomal enzymes to lysosomes. In addition, beta-glucuronidase undergoes a 2-3-kDa processing event at its C terminus (Erickson and Blobel, 1983) in endosomes (Gabel and Foster, 1987) during transport to lysosomes.

The aim of this study was to use site-directed mutagenesis to eliminate each of the four potential glycosylation sites of human beta-glucuronidase either individually or in combination to determine which sites were used and which, if any, were preferentially phosphorylated. We have found that all four sites are glycosylated, but that two sites are preferentially phosphorylated. In addition, we have shown that glycosylation is required for expression of catalytic activity, but that beta-glucuronidase can be deglycosylated enzymatically after the enzyme has properly folded with only minimal loss of activity.

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