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.
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.
|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|
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|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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