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 122232 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 3 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 3 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, 3-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.
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 1/2 and 3 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.
|Liu, Lin; Lee, Wang-Sik; Doray, Balraj et al. (2016) Role of Spacer-1 in the Maturation and Function of GlcNAc-1-Phosphotransferase. FEBS Lett :|
|van Meel, Eline; Kornfeld, Stuart (2016) Mucolipidosis III GNPTG Missense Mutations Cause Misfolding of the Î³ 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|
|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 167:2844-6|
|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|
|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|
|van Meel, Eline; Qian, Yi; Kornfeld, Stuart A (2014) Mislocalization of phosphotransferase as a cause of mucolipidosis III Î±Î². 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 Î³. PLoS One 9:e109768|
|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|
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