Abstract
UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) is an α2β2γ2 heterohexamer that mediates the initial step in the formation of the mannose 6-phosphate recognition signal on lysosomal acid hydrolases. We previously reported that the specificity of the reaction is determined by the ability of the α/β subunits to recognize a conformation-dependent protein determinant present on the acid hydrolases. We now present evidence that the DNA methyltransferase-associated protein (DMAP) interaction domain of the α subunit functions in this recognition process. First, GST-DMAP pulled down several acid hydrolases, but not nonlysosomal glycoproteins. Second, recombinant GlcNAc-1-phosphotransferase containing a missense mutation in the DMAP interaction domain (Lys732Asn) identified in a patient with mucolipidosis II exhibited full activity toward the simple sugar α-methyl d-mannoside but impaired phosphorylation of acid hydrolases. Finally, unlike the WT enzyme, expression of the K732N mutant in a zebrafish model of mucolipidosis II failed to correct the phenotypic abnormalities. These results indicate that the DMAP interaction domain of the α subunit functions in the selective recognition of acid hydrolase substrates and provides an explanation for the impaired phosphorylation of acid hydrolases in a patient with mucolipidosis II.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Abnormalities, Multiple / enzymology
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Abnormalities, Multiple / metabolism*
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Acetylglucosamine / metabolism
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Animals
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Female
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HEK293 Cells
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HeLa Cells
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Humans
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Hydrolases / metabolism
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Lysosomes / enzymology*
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Male
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Mannosephosphates / metabolism
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Mice
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Mucolipidoses / enzymology
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Mucolipidoses / metabolism*
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Mutagenesis, Site-Directed
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Mutation, Missense
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Phosphorylation / physiology
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Protein Structure, Tertiary / physiology
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Protein Subunits / chemistry
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Protein Subunits / genetics
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Protein Subunits / metabolism
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Ribonucleoproteins, Small Nuclear / chemistry
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Ribonucleoproteins, Small Nuclear / genetics
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Ribonucleoproteins, Small Nuclear / metabolism
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Substrate Specificity
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Transferases (Other Substituted Phosphate Groups) / chemistry
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Transferases (Other Substituted Phosphate Groups) / genetics
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Transferases (Other Substituted Phosphate Groups) / metabolism*
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Zebrafish
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Zebrafish Proteins / chemistry
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Zebrafish Proteins / genetics
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Zebrafish Proteins / metabolism*
Substances
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LSM2 protein, human
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Mannosephosphates
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Protein Subunits
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Ribonucleoproteins, Small Nuclear
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Zebrafish Proteins
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mannose-6-phosphate
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Transferases (Other Substituted Phosphate Groups)
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GNPTAB protein, human
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GNPTAB protein, mouse
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UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase
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Hydrolases
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Acetylglucosamine
Supplementary concepts
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Mucolipidosis II Alpha Beta