Our studies have two basic studies. The first is to understand how the oligosaccharide units of glycoproteins are synthesized and how they function as recognition markers in biologic processes. The second is to understand at the molecular level how newly synthesized lysosomal enzymes are directed to lysosomes. For the first goal, we plan to use lectin- resistant cell lines with specific blocks in oligosaccharide biosynthesis to uncover new pathways for the synthesis of oligosaccharides. Several approaches will be made to understand lysosomal enzyme targeting. First, we plan to identify the common protein recognition marker for lysosomal enzymes required for the generation of phosphomannosyl residues which serve as the recognition signal for targeting proteins to lysosomes. To do this, we will construct chimeric molecules between the secretory protein pepsinogen and the lysosomal enzyme cathepsin D to define the regions on cathepsin D needed for recognition by phosphotransferase and subsequence phosphorylation. We plan to clone phosphotransferase and to use this clone to study the defects in patients with mucolipidosis II and III, two autosomal recessive genetic disorders of phosphotransferase. We also plan to clone alpha N-acetylglucosamine-1-phosphodiester N- acetylglucosaminidase, the second enzyme required for the generation of phosphomannosyl residues. In addition, we plan to mutagenize the cDNA's for the cation-independent and the cation-dependent mannose 6-phosphate receptors to generate receptor molecules with mutations and deletions in their cytoplasmic domains. These cDNA's will be transfected into receptor negative mouse L cells to determine the effects on receptor function in lysosomal enzyme sorting at the Golgi and endocytosis at the cell surface.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37CA008759-27
Application #
3481620
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1979-09-01
Project End
1995-02-28
Budget Start
1993-03-01
Budget End
1994-02-28
Support Year
27
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Washington University
Department
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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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