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Abstract
We have isolated and sequenced human cDNA and mouse genomic DNA clones encoding N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (phosphodiester alpha-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 alpha-GlcNAcase and no homology to other known proteins. It predicts a type I membrane-spanning 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 tyrosine-based 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 alpha-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 alpha-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.
Highlights
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
Cloning of the cDNA Encoding Human Phosphodiester ␣GlcNAcase—Affinity purified, homogeneous bovine phosphodiester ␣-GlcNAcase was subjected to amino-terminal amino acid sequencing as described previously [6]
The endogenous COS cell phosphodiester ␣-GlcNAcase activity is predominantly (92%) found in the cell membrane fraction as is that encoded by the full-length human cDNA (67%), but the truncated human phosphodiester ␣-GlcNAcase was predominantly secreted into the medium (75%)
Summary
Materials—The enzymes were obtained from the following suppliers: Taq DNA polymerase, EcoRI, and HindIII from Promega; BamHI from New England Biolabs; and T4 DNA ligase from Life Technologies, Inc. Isolation of cDNAs Encoding Human Phosphodiester ␣-GlcNAcase— The amino acid sequences of the three bovine phosphodiester ␣-GlcNAcase peptides were used to BLAST search the non-redundant GenBankTM EST data base [8], and four clones were found that had sequence homologous to tryptic peptide 3 (see Table I) at their 5Ј ends. Northern Blot—The BamHI-HindIII fragment of clone 6.5, gel-purified and labeled with [␣-32P]dCTP as above, was used to probe a human multiple tissue Northern blot (CLONTECH) using the Express Hyb and protocol supplied by the manufacturer. The ligation was performed with T4 DNA ligase, and the reaction mixture was used to transform DH5␣-competent cells that yielded many well separated ampicillin-resistant colonies of which 10 were cloned and all showed a 520-bp insert after EcoRI/BamHI digestion. The intensity of the bands on the Western blot was quantitated by laser densitometry using the Molecular Dynamics Personal Densitometer ImageQuant system
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