The alpha- and beta-subunits of the human UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase [corrected] are encoded by a single cDNA.
Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an alpha2beta2gamma2-subunit structure was proposed. Although cDNA encoding the gamma-subunit has been described, cDNAs for the alpha- and beta-subunits have not. Using partial amino acid sequences from the bovine alpha- and beta-subunits, we have isolated a human cDNA that encodes both the alpha- and beta-subunits. Both subunits contain a single predicted membrane-spanning domain. The alpha- and beta-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the alpha/beta-subunits-precursor cDNA with or without the gamma-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the alpha-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.
Kudo
Mariko
M
Genzyme Corp., Oklahoma City, Oklahoma 73104, USA.
Bao
Ming
M
D'Souza
Anil
A
Ying
Fu
F
Pan
Huaqin
H
Roe
Bruce A
BA
Canfield
William M
WM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D015152:Blotting, Northern; D020134:Catalytic Domain; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008247:Lysosomes / Q000737:chemistry*; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics* / Q000502:physiology*
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D009841:Oligonucleotides; D010455:Peptides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D017351:N-Acetylglucosaminyltransferases; D017855:Transferases (Other Substituted Phosphate Groups); C504791:GNPTG protein, human; D008239:Lysine