title: Molecular cloning and functional expression of two splice forms of human N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase.
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.
authors.affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: R
authors.initials: R
authors.lastname: Kornfeld
authors.forename: M
authors.initials: M
authors.lastname: Bao
authors.forename: K
authors.initials: K
authors.lastname: Brewer
authors.forename: C
authors.initials: C
authors.lastname: Noll
authors.forename: W
authors.initials: W
authors.lastname: Canfield
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic; D006031:Glycosylation; D006801:Humans; D007438:Introns; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D010727:Phosphoric Diester Hydrolases; C026645:N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase