We have isolated and sequenced human cDNA and mouse genomic DNA clones encoding N-acetylglucosamine-1-phosphodiester ␣-N-acetylglucosaminidase (phosphodiester ␣-GlcNAcase) which catalyzes the second step in the synthesis of the mannose 6-phosphate recognition signal on lysosomal enzymes. The gene is organized into 10 exons. The protein sequence encoded by the clones shows 80% identity between human and mouse phosphodiester ␣-GlcNAcase and no homology to other known proteins. It predicts a type I membranespanning glycoprotein of 514 amino acids containing a 24-amino acid signal sequence, a luminal domain of 422 residues with six potential N-linked glycosylation sites, a single 27-residue transmembrane region, and a 41-residue cytoplasmic tail that contains both a tyrosinebased and an NPF internalization motif. Human brain expressed sequence tags lack a 102-base pair region present in human liver cDNA that corresponds to exon 8 in the genomic DNA and probably arises via alternative splicing. COS cells transfected with the human cDNA expressed 50 -100-fold increases in phosphodiester ␣-GlcNAcase activity proving that the cDNA encodes the subunits of the tetrameric enzyme. Transfection with cDNA lacking the 102-base pair region also gave active enzyme. The complete genomic sequence of human phosphodiester ␣-GlcNAcase was recently deposited in the data base. It showed that our cDNA clone was missing only the 5 -untranslated region and initiator methionine and revealed that the human genomic DNA has the same exon organization as the mouse gene.The biosynthesis of the mannose 6-phosphate recognition signal on the oligosaccharides of lysosomal acid hydrolases occurs in the Golgi apparatus and is catalyzed by the sequential action of two enzymes. The first step is the addition of N-acetylglucosamine-1-P to the C-6 hydroxyl group on selected mannose residues in the high mannose oligosaccharides of lysosomal enzymes that serve as substrates for UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. The second step is catalyzed by N-acetylglucosamine-1-phosphodiester ␣-N-acetylglucosaminidase (phosphodiester ␣-GlcNAcase), 1 which removes the covering GlcNAc to expose the mannose 6-phosphate recognition signal on the lysosomal acid hydrolases (1). These lysosomal enzymes can then bind to one of the two mannose 6-phosphate receptors in the trans-Golgi network (TGN) and be transferred to endosomes and subsequently to lysosomes (2, 3). The phosphodiester ␣-GlcNAcase plays an important role in lysosomal enzyme targeting because the mannose 6-phosphate receptors do not bind GlcNAc-P-Man. We have studied the kinetics, substrate specificity, and hydrodynamic properties of phosphodiester ␣-GlcNAcase from bovine liver (4, 5) and recently purified it over 600,000-fold to homogeneity using a two-step immunoaffinity purification procedure (6). The native membrane-associated enzyme exists as a tetramer (272 kDa) composed of two dimers (136 kDa) each containing a pair of disulfide-linked monomers (68 kDa). The ...
N-Acetylglucosamine-1-phosphodiester ␣-N-Acetylglucosaminidase (EC 3.1.4.45; phosphodiester ␣-GlcNAcase) catalyzes the second step in the synthesis of the mannose 6-phosphate determinant required for efficient intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. A partially purified preparation of phosphodiester ␣-GlcNAcase from bovine pancreas was used to generate a panel of murine monoclonal antibodies. The anti-phosphodiester ␣-GlcNAcase monoclonal antibody UC1 was coupled to a solid support and used to immunopurify the bovine liver enzyme 670,000-fold in two steps to apparent homogeneity with an overall yield of 14%. The purified phosphodiester ␣-GlcNAcase has a specific activity of 498 mol of The subunit structure of the enzyme was determined using a combination of analytical gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and amino-terminal sequencing. The data indicate that bovine phosphodiester ␣-GlcNAcase is a 272,000-Da complex of four identical 68,000-Da glycoprotein subunits arranged as two disulfide-linked homodimers. A soluble form of the enzyme, isolated from fetal bovine serum, showed the same subunit structure. Both forms of the enzyme reacted with a rabbit antibody raised to the amino-terminal peptide of the liver enzyme, suggesting that phosphodiester ␣-GlcNAcase is a type I membranespanning glycoprotein with its amino terminus in the lumen of the Golgi apparatus.The trafficking of most lysosomal hydrolases in higher eucaryotes is mediated by a mannose 6-phosphate-dependent pathway (1). Two enzymes sequentially modify asparagine-linked oligosaccharides on newly synthesized lysosomal hydrolases to generate a mannose 6-phosphate recognition marker. The first step is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (EC 2.7.8.17; GlcNAc-phosphotransferase).
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