UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an ␣ 2 ␤ 2 ␥ 2 hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the ␥ subunit along with kinetic studies of recombinant ␣ 2 ␤ 2 ␥ 2 and ␣ 2 ␤ 2 forms of the transferase, we have explored the function of the ␣/␤ and ␥ subunits. The findings demonstrate that the ␣/␤ subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the ␣/␤ subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the ␥ subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the ␣/␤ subunits toward a subset of the acid hydrolases, the ␥ subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the ␥ subunit binds and presents the high mannose glycans of the acceptor to the ␣/␤ catalytic site in a favorable manner.In higher eukaryotes, newly synthesized acid hydrolases acquire mannose 6-phosphate (Man-6-P) 3 residues on their N-linked glycans as they traverse the Golgi (1). These residues serve as high affinity ligands for binding to Man-6-P receptors in the trans-Golgi network. The hydrolase-receptor complexes are then packaged into transport carriers for delivery to endosomes and lysosomes. The Man-6-P recognition marker is synthesized in two steps. First, UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) binds to a conformation-dependent protein determinant on the acid hydrolase and transfers GlcNAc-1-P from UDP-GlcNAc to one or two of the mannose residues of the N-linked high mannose oligosaccharide. Second, N-acetylglucosamine 1-phosphodiester ␣-N-acetyglucosaminidase ("uncovering enzyme") excises the N-acetylglucosamine to generate the Man-6-P monoester.GlcNAc-1-phosphotransferase is a heterohexamer composed of three subunits (␣ 2 ␤ 2 ␥ 2 ) (2). The ␣ and ␤ subunits are encoded by a single gene GNPTAB, and the ␥ subunit is encoded by a separate gene GNPTG (3-5). Although it has been established that the ␣/␤ subunits contain the catalytic activity of the enzyme, the possible participation of these subunits in the recognition of the common protein determinant of the acid hydrolases has not been explored. Furthermore, the role(s) of the ␥ subunit is poorly understood. The initial insight into the function of the subunits came from studies of patients with the autosomal recessive lysosomal storage disorders termed mucolipidosis II (I-cell disease) and mucolipidosis III (pseudo-Hurler polydystrophy), the latter being the less severe of the two (6). These disorders arise from mutations in the genes encoding GlcNAc-1-phospho...

Mice deficient in GNPTAB exhibited severe retinal degeneration. Additional features observed in patients with ML II, a lysosomal storage disease, are also present in these mice. Understanding underlying mechanisms of this gene in the eye will increase its therapeutic potential for the treatment of retinal diseases.

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on acid hydrolases. The transferase exists as an ␣ 2 ␤ 2 ␥ 2 hexameric complex with the ␣-and ␤-subunits derived from a single precursor molecule. The catalytic function of the transferase is attributed to the ␣-and ␤-subunits, whereas the ␥-subunit is believed to be involved in the recognition of a conformation-dependent protein determinant common to acid hydrolases. Using knock-out mice with mutations in either the ␣/␤ gene or the ␥ gene, we show that disruption of the ␣/␤ gene completely abolishes phosphorylation of high mannose oligosaccharides on acid hydrolases whereas knock-out of the ␥ gene results in only a partial loss of phosphorylation. These findings demonstrate that the ␣/␤-subunits, in addition to their catalytic function, have some ability to recognize acid hydrolases as specific substrates. This process is enhanced by the ␥-subunit.

N-Acetylglucosamine-1-phosphodiester ␣-N-acetylglucosaminidase, also known as "uncovering" enzyme (UCE), is localized in the trans-Golgi network, where it removes a covering N-acetylglucosamine from the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Here we show that UCE is synthesized as an inactive proenzyme that is activated by the endoprotease furin, which cleaves an RARLPR2D sequence to release a 24-amino acid propiece. As furin is localized in the trans-Golgi network, newly synthesized UCE is inactive until it reaches this terminal Golgi compartment. LoVo cells (derived from a human colon adenocarcinoma) lack furin activity and have extremely low UCE activity. Addition of furin to LoVo cell extracts restores UCE activity to normal levels, demonstrating that the UCE proenzyme is stable in this cell type. LoVo cells secrete acid hydrolases with phosphomannose diesters as a consequence of the deficient UCE activity. This demonstrates for the first time that UCE is the only enzyme in these cells capable of efficiently uncovering phosphomannose diesters. UCE also hydrolyzes UDPGlcNAc, a sugar donor for Golgi N-acetylglucosaminyltransferases. The fact that UCE is not activated until it reaches the trans-Golgi network may ensure that the pool of UDP-GlcNAc in the Golgi stack is not depleted, thereby maintaining proper oligosaccharide assembly.

Gaucher disease is a lysosomal glycolipid storage disorder characterized by defects in acid-β-glucosidase (GlcCerase), the enzyme responsible for the catabolism of glucosylceramide. We recently demonstrated that isofagomine (IFG), an iminosugar that binds to the active site of GlcCerase, enhances the folding, transport and activity of the N370S mutant form of GlcCerase. In this study we compared the effects of IFG on a number of other glucosidases and glucosyltransferases. We report that IFG has little or no inhibitory activity towards intestinal disaccharidase enzymes, ER α-glucosidase II or glucosylceramide synthase at concentrations previously shown to enhance N370S GlcCerase folding and trafficking in Gaucher fibroblasts. Furthermore, treatment of wild type fibroblasts with high doses of IFG did not alter the processing of newly synthesized N-linked oligosaccharides. These findings support further evaluation of IFG as a potential therapeutic agent in the treatment of some forms of Gaucher disease.

Background: DUF2233 domain is present in bacteria and human UCE, which is implicated in lysosomal storage disorders. Results: Functional residues in DUF2233 and UCE identified in the structure of a bacterial DUF2233 domain were investigated. Conclusion: A function for this domain in bacteria is proposed, and functional residues in human UCE were identified. Significance: This is the first structure/function study of this protein domain.

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