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. Genetic defects in this intracellular protein transport pathway give rise to severe lysosomal storage diseases, illustrating the importance of this system. 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 enzymes N-acetylgucosamine-1- phosphotransferase (Ptase). This cis-Golgi transferase is a type III transmembrane ?2?2?2 hexameric protein encoded by two genes. We established that the two Notch repeat modules and the DNA methyltransferase-associated protein (DMAP) interaction domain of the ? subunit mediate the specific recognition of the common protein determinant of acid hydrolases.
Specific Aim 1 is directed toward defining the role of each domain in this process.
Aim 2 focuses on the role of the N-terminal cytoplasmic tail of Ptase in maintaining proper Golgi localization.
This aim i s derived from our finding that three ML III patient missense mutations in the cytoplasmic N-tail of the ?-subunit result in mislocalization of the mutant Ptase to the endosome/lysosome system. Our hypothesis is that these mutations prevent recycling of this Golgi enzyme due to a failure to be incorporated into COPI vesicles, a required step for the recycling process. In support of this, our preliminary findings show direct binding of the WT N-tail, but not the mutant peptides, to the ? and ? subunits of COPI. The goal is to understand these interactions at the molecular level.
Aim 3 is to determine the generality of the direct interaction of the cytoplasmic N-tails of the large family of Golgi type II transmembrane glycosyltransferases with the COPI subunits. Strong evidence exists that the glycosyltransferases recycle from late to early Golgi cisternae or the ER via COPI vesicles, but how the transferases are packaged into the vesicles is poorly understood.
This aim has the potential to establish a new paradigm for this process.

Public Health Relevance

This work is directly relevant to the molecular understanding of two serious lysosomal storage diseases termed ML II and ML III. Both are caused by mutations in the genes that encode the subunits of Ptase. The work is also relevant to the production of lysosomal enzymes for use in enzyme replacement therapy in the treatment of individuals with other forms of lysosomal storage diseases.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Snyderwine, Elizabeth G
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Washington University
Internal Medicine/Medicine
Schools of Medicine
Saint Louis
United States
Zip Code
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
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
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
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
Doray, Balraj; Misra, Saurav; Qian, Yi et al. (2012) Do GGA adaptors bind internal DXXLL motifs? Traffic 13:1315-25
Govero, Jennifer; Doray, Balraj; Bai, Hongdong et al. (2012) Analysis of Gga null mice demonstrates a non-redundant role for mammalian GGA2 during development. PLoS One 7:e30184
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

Showing the most recent 10 out of 120 publications