Analysis of Mucolipidosis II/III GNPTAB Missense Mutations Identifies Domains of UDP-GlcNAc: lysosomal Enzyme GlcNAc-1-phosphotransferase Involved in Catalytic Function and Lysosomal Enzyme Recognition

作者：Qian Yi; van Meel Eline; Flanagan Steet Heather; Yox Alex; Steet Richard^*; Kornfeld Stuart

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2015, 290(5): 3045-3056.

DOI：10.1074/jbc.M114.612507

• Enzyme
• Genetic Disease
• Golgi
• Lysosomal Storage Disease
• Zebrafish
• GlcNAc-1-phosphotransferase
• Notch Domains
• Mannose 6-phosphate
• Mucolipidosis II
• Mucolipidosis III

Background: Mutations in GNPTAB cause the lysosomal disorders mucolipidosis II and III . Results: All reported missense mutations were studied and showed various consequences on its gene product, GlcNAc-1-phosphotransferase. Conclusion: Domains responsible for catalytic activity and lysosomal hydrolase recognition were identified. Significance: Analysis of patient mutations provided new insight into the functional domains of GlcNAc-1-phosphotransferase.
UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase tags newly synthesized lysosomal enzymes with mannose 6-phosphate recognition markers, which are required for their targeting to the endolysosomal system. GNPTAB encodes the and subunits of GlcNAc-1-phosphotransferase, and mutations in this gene cause the lysosomal storage disorders mucolipidosis II and III . Prior investigation of missense mutations in GNPTAB uncovered amino acids in the N-terminal region and within the DMAP domain involved in Golgi retention of GlcNAc-1-phosphotransferase and its ability to specifically recognize lysosomal hydrolases, respectively. Here, we undertook a comprehensive analysis of the remaining missense mutations in GNPTAB reported in mucolipidosis II and III patients using cell- and zebrafish-based approaches. We show that the Stealth domain harbors the catalytic site, as some mutations in these regions greatly impaired the activity of the enzyme without affecting its Golgi localization and proteolytic processing. We also demonstrate a role for the Notch repeat 1 in lysosomal hydrolase recognition, as missense mutations in conserved cysteine residues in this domain do not affect the catalytic activity but impair mannose phosphorylation of certain lysosomal hydrolases. Rescue experiments using mRNA bearing Notch repeat 1 mutations in GNPTAB-deficient zebrafish revealed selective effects on hydrolase recognition that differ from the DMAP mutation. Finally, the mutant R587P, located in the spacer between Notch 2 and DMAP, was partially rescued by overexpression of the subunit, suggesting a role for this region in subunit binding. These studies provide new insight into the functions of the different domains of the and subunits.

• 出版日期2015-1-30