title: The DMAP interaction domain of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is a substrate recognition module.
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.
authors.affiliation: Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Qian
authors.forename: Heather
authors.initials: H
authors.lastname: Flanagan-Steet
authors.forename: Eline
authors.initials: E
authors.lastname: van Meel
authors.forename: Richard
authors.initials: R
authors.lastname: Steet
authors.forename: Stuart A
authors.initials: SA
authors.lastname: Kornfeld
mesh_terms: D000015:Abnormalities, Multiple / Q000201:enzymology / Q000378:metabolism*; D000117:Acetylglucosamine / Q000378:metabolism; D000818:Animals; D005260:Female; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D008360:Mannosephosphates / Q000378:metabolism; D051379:Mice; D009081:Mucolipidoses / Q000201:enzymology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C105367:LSM2 protein, human; D008360:Mannosephosphates; D021122:Protein Subunits; D017411:Ribonucleoproteins, Small Nuclear; D029961:Zebrafish Proteins; C027693:mannose-6-phosphate; D017855:Transferases (Other Substituted Phosphate Groups); C511050:GNPTAB protein, human; C548056:GNPTAB protein, mouse; C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase; D006867:Hydrolases; D000117:Acetylglucosamine