The objective of this research proposal is to obtain a molecular understanding of the phosphomannosyl targeting system which functions In the delivery of newly synthesized acid hydrolases to lysosomes. Defects in this intracellular protein transport pathway give rise to severe lysosomal storage diseases. A key step in this pathway is the selective phosphorylation of mannose residues on the high mannose glycans of the acid hydrolases by UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (Ptase). This transferase is an alpha2betagamma2 hexameric protein encoded by two genes. We established that the alpha/beta subunits recognize the common protein determinant of the acid hydrolases as well as mediating the catalytic function of the enzyme. The y subunit enhances the activity of the alpha/beta subunits toward a subset of the acid hydrolases.
Specific Aim 1 is directed toward identifying the domain of the alpha/beta subunits that binds the protein determinant of the acid hydrolases.
Aim 2 focuses on the role of the Man-6-P receptor homology (MRH)-domain of the gamma subunits in enhancing the activity of the a/p toward selected hydrolases.
Aim 3 seeks to understand the physiologic basis for the development of neurodegeneration in alpha/beta null mice.
Aim 4 is to determine how Ptase is localized to the cis-Golgi subcompartment.
Aim 5 is to define the essential role of the GGA2 (for Golgi-localized, y-ear containing, ARF-binding) coat protein in maintaining neonatal viability. This function cannot be replaced by GGA1 and GGA3, establishing that the GGAs are not fully redundant. These studies will utilize mice with disruption of the genes encoding the three GGAs.

Public Health Relevance

This research is directly relevant to the understanding of two serious lysosomal storage diseases termed MLII and MLIII. Both are caused by mutations in the alpha/beta and gamma genes of Ptase. The work is also relevant to the production of lysosomal enzymes used for enzyme replacement therapy in the treatment of individuals with lysosomal storage diseases such as Fabry and Pompe disease.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Method to Extend Research in Time (MERIT) Award (R37)
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No Study Section (in-house review) (NSS)
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Snyderwine, Elizabeth G
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Washington University
Internal Medicine/Medicine
Schools of Medicine
Saint Louis
United States
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Liu, Lin; Lee, Wang-Sik; Doray, Balraj et al. (2017) Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy. Mol Ther Methods Clin Dev 5:59-65
Liu, Lin; Lee, Wang-Sik; Doray, Balraj et al. (2017) Role of spacer-1 in the maturation and function of GlcNAc-1-phosphotransferase. FEBS Lett 591:47-55
van Meel, Eline; Kornfeld, Stuart (2016) Mucolipidosis III GNPTG Missense Mutations Cause Misfolding of the ? Subunit of GlcNAc-1-Phosphotransferase. Hum Mutat 37:623-6
van Meel, Eline; Lee, Wang-Sik; Liu, Lin et al. (2016) Multiple Domains of GlcNAc-1-phosphotransferase Mediate Recognition of Lysosomal Enzymes. J Biol Chem 291:8295-307
Qian, Yi; van Meel, Eline; Flanagan-Steet, Heather et al. (2015) 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. J Biol Chem 290:3045-56
Hasanagic, Medina; van Meel, Eline; Luan, Shan et al. (2015) The lysosomal enzyme receptor protein (LERP) is not essential, but is implicated in lysosomal function in Drosophila melanogaster. Biol Open 4:1316-25
Barea, Jaime J; van Meel, Eline; Kornfeld, Stuart et al. (2015) Tuberous sclerosis, polycystic kidney disease and mucolipidosis III gamma caused by a microdeletion unmasking a recessive mutation. Am J Med Genet A 167A:2844-6
Doray, Balraj; Govero, Jennifer; Kornfeld, Stuart (2014) Impact of genetic background on neonatal lethality of Gga2 gene-trap mice. G3 (Bethesda) 4:885-90
Idol, Rachel A; Wozniak, David F; Fujiwara, Hideji et al. (2014) Neurologic abnormalities in mouse models of the lysosomal storage disorders mucolipidosis II and mucolipidosis III ?. PLoS One 9:e109768
van Meel, Eline; Qian, Yi; Kornfeld, Stuart A (2014) Mislocalization of phosphotransferase as a cause of mucolipidosis III ??. Proc Natl Acad Sci U S A 111:3532-7

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