We have focused our recent efforts on studying the biosynthesis and targeting of newly synthesized lysosomal enzymes to lysosomes. The asparagine-linked oligosaccharide units of lysosomal enzymes undergo an extensive series of processing reactions as the newly synthesized enzymes move from the rough endoplasmic reticulum, where they are initially glycosylated, to their final destination in lysosomes. The most important modification is the generation of the phosphomannosyl recognition marker, which occurs in two steps. First, N-acetylglucosamine 1-phosphate is transferred to an acceptor mannose by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase, resulting in a phosphate group in diester linkage between outer N-acetylglucosamine and an inner mannose. Next, a phosphodiester glycosidase removes the N-acetylglucosamine, leaving the phosphate in monoester linkage to the underlying mannose residue. This exposed phosphomannosyl residue serves as an essential component of a recognition marker which leads to binding to high-affinity receptors and subsequent translocation to lysosomes. The transferase acts specifically on lysosomal enzymes, thereby catalyzing the initial, determining step by which newly synthesized lysosomal enzymes are distinguished from other newly synthesized glycoproteins and marked for eventual targeting to lysosomes. We have found that deglycosylated lysosomal enzymes are potent inhibitors of the transferase, indicating that these enzymes contain a common protein sequence or conformation that is recognized by the transferase. Our current experiments are directed toward the identification of this recognition signal. Our preliminary results indicate that the conformation of the lysosomal enzymes is important for the expression of the recognition signal. We have also identified a number of murine cell lines that lack the 215 Kda Man-6-P receptor and yet possess high levels of intracellular acid hydrolases which are contained in dense granules characteristic of lysosomes. We have recently identified and isolated a second Man-6-P receptor in these cells. This receptor, a 46 Kda glycoprotein, is widely distributed among cell types. We are trying to determine the physiologic function of each receptor. (A)

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Washington University
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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
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

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