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 alpha2 beta2 gamma2 hexameric protein.
Specific Aim 1 is directed toward identifying which subunits of Ptase mediate the recognition of the common protein determinant of acid hydrolases, a process that is essential for the selective phosphorylation of this class of enzymes.
Aim 2 seeks to identify the function of the Man-6-P receptor homology (MRH)-domain of the 3 subunit. We will test the ability of recombinant Ptase with and without its gamma subunit, or with mutations in the MRH domain, to phosphorylate acid hydrolases in in vitro assays and to bind directly to immobilized acid hydrolases using surface plasmon resonance. These studies will be complemented by analyzing the ability of fibroblasts expressing wild type or gamma deficient Ptase to phosphorylate a panel of acid hydrolases transfected into the cells.
Aim 3 is to determine how Ptase is localized to the cis-subcompartment of the Golgi.
Aim 4 is to define the role of the GGA (for Golgi-localized, gamma-ear containing, ARF-binding) proteins in the packaging and transport of the Man-6-P receptors with bound acid hydrolases at the trans-Golgi network. These studies will utilize mice with disruptions of the genes encoding GGA1 and GGA3.
This aim i s designed to establish whether the GGAs are redundant in intact animals and whether there are tissue specific requirements for specific 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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37CA008759-47
Application #
8444665
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Snyderwine, Elizabeth G
Project Start
1979-09-01
Project End
2014-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
47
Fiscal Year
2013
Total Cost
$702,600
Indirect Cost
$240,363
Name
Washington University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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
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
Qian, Yi; Flanagan-Steet, Heather; van Meel, Eline et al. (2013) The DMAP interaction domain of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is a substrate recognition module. Proc Natl Acad Sci U S A 110:10246-51
van Meel, Eline; Wegner, Daniel J; Cliften, Paul et al. (2013) Rare recessive loss-of-function methionyl-tRNA synthetase mutations presenting as a multi-organ phenotype. BMC Med Genet 14:106
Lee, Wang-Sik; Kang, Changsoo; Drayna, Dennis et al. (2011) Analysis of mannose 6-phosphate uncovering enzyme mutations associated with persistent stuttering. J Biol Chem 286:39786-93
Boonen, Marielle; van Meel, Eline; Oorschot, Viola et al. (2011) Vacuolization of mucolipidosis type II mouse exocrine gland cells represents accumulation of autolysosomes. Mol Biol Cell 22:1135-47
van Meel, Eline; Boonen, Marielle; Zhao, Haibo et al. (2011) Disruption of the Man-6-P targeting pathway in mice impairs osteoclast secretory lysosome biogenesis. Traffic 12:912-24
Qian, Yi; Lee, Intaek; Lee, Wang-Sik et al. (2010) Functions of the alpha, beta, and gamma subunits of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. J Biol Chem 285:3360-70
Lendrihas, Thomas; Hunter, Gregory A; Ferreira, Gloria C (2010) Serine 254 enhances an induced fit mechanism in murine 5-aminolevulinate synthase. J Biol Chem 285:3351-9

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