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: Yi Qian, Heather Flanagan-Steet, Eline van Meel, Richard Steet, Stuart A Kornfeld
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 110 Issue 25 Pg. 10246-51 (Jun 18 2013) ISSN: 1091-6490 [Electronic] United States
• PMID: 23733939 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• LSM2 protein, human
• Mannosephosphates
• Protein Subunits
• Ribonucleoproteins, Small Nuclear
• Zebrafish Proteins
• mannose-6-phosphate
• Transferases (Other Substituted Phosphate Groups)
• GNPTAB protein, human
• GNPTAB protein, mouse
• UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase
• Hydrolases
• Acetylglucosamine

Topics

• Abnormalities, Multiple (enzymology, metabolism)
• Acetylglucosamine (metabolism)
• Animals
• Female
• HEK293 Cells
• HeLa Cells
• Humans
• Hydrolases (metabolism)
• Lysosomes (enzymology)
• Male
• Mannosephosphates (metabolism)
• Mice
• Mucolipidoses (enzymology, metabolism)
• Mutagenesis, Site-Directed
• Mutation, Missense
• Phosphorylation (physiology)
• Protein Structure, Tertiary (physiology)
• Protein Subunits (chemistry, genetics, metabolism)
• Ribonucleoproteins, Small Nuclear (chemistry, genetics, metabolism)
• Substrate Specificity
• Transferases (Other Substituted Phosphate Groups) (chemistry, genetics, metabolism)
• Zebrafish
• Zebrafish Proteins (chemistry, genetics, metabolism)