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LC-MS/MS analysis of Mannose 6-phosphate containing proteins

ABSTRACT: Mucolipidosis III gamma (MLIII) is clinically characterized by onset of first symptoms at an average of 5 years such as stiffness of hands and shoulders, claw hand deformities, scoliosis and progressive destruction of hip joints. The disease is caused by mutations in GNPTG encoding the gamma-subunit of the GlcNAc-1-phosphotransferase complex. This enzyme is responsible for the generation of mannose 6-phosphate (M6P) targeting signals on 70 soluble lysosomal enzymes that are required for their efficient receptor-mediated transport to lysosomes. In fibroblasts from Gnptg-KO mice the GlcNAc-1-phosphotransferase activity is reduced leading to low amounts of M6P-containing proteins. M6P affinity chromatography-assisted and mass spectrometry-based secretome analysis of Gnptg-KO fibroblasts revealed a distinct set of lysosomal enzymes modified with M6P residues.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Fibroblast

SUBMITTER: Shiva Ahmadi

LAB HEAD: Dominic Winter

PROVIDER: PXD009621 | Pride | 2018-06-06

REPOSITORIES: Pride

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	MaxQuant180mingradient.zip	Other
	MaxQuant60mingradient.zip	Other
	MaxQuantTxtFiles-180mingradient.zip	Other
	MaxQuantTxtFiles-60mingradient.zip	Other
	SA_HH_Bead1.raw	Raw

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LC-MS/MS analysis of Mannose 6-phosphate containing proteins

Project description:Mucolipidosis III gamma (MLIII) is clinically characterized by onset of first symptoms at an average of 5 years such as stiffness of hands and shoulders, claw hand deformities, scoliosis and progressive destruction of hip joints. The disease is caused by mutations in GNPTG encoding the gamma-subunit of the GlcNAc-1-phosphotransferase complex. This enzyme is responsible for the generation of mannose 6-phosphate (M6P) targeting signals on 70 soluble lysosomal enzymes that are required for their efficient receptor-mediated transport to lysosomes. In fibroblasts from Gnptg-KO mice the GlcNAc-1-phosphotransferase activity is reduced leading to low amounts of M6P-containing proteins. M6P affinity chromatography-assisted and mass spectrometry-based secretome analysis of Gnptg-KO fibroblasts revealed a distinct set of lysosomal enzymes modified with M6P residues.

2018-06-06 | PXD007546 | Pride

LC-MS/MS analysis of soluble lysosomal enzymes

Project description:Mucolipidosis III gamma (MLIII) is clinically characterized by onset of first symptoms at an average of 5 years such as stiffness of hands and shoulders, claw hand deformities, scoliosis and progressive destruction of hip joints. The disease is caused by mutations in GNPTG encoding the gamma-subunit of the GlcNAc-1-phosphotransferase complex. This enzyme is responsible for the generation of mannose 6-phosphate (M6P) targeting signals on 70 soluble lysosomal enzymes that are required for their efficient receptor-mediated transport to lysosomes. Complementary SILAC-based lysosomal proteomics revealed decreased amounts of several lysosomal enzymes in Gnptg-KO fibroblasts involved in the degradation of lipids, glycans and proteins.

2018-06-07 | PXD007547 | Pride

The Human Disease Gene LYSET is Essential for Lysosomal Enzyme Transport and Viral Infection

Project description:Lysosomes are key degradative compartments of the cell. Transport to lysosomes ismediated by tagging soluble enzymes with mannose 6-phosphate (M6P) by GlcNAc-1-phosphotransferase whose deficiency leads to the severe lysosomal storage disorder mucolipidosisII (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thusdepend on functional GlcNAc-1-phosphotransferase. Using genome-scale CRISPR screens, weidentify LYSET as essential for infection by cathepsin-dependent viruses including SARS-CoV-2. We show that LYSET deficiency results in global loss of M6P tagging and mislocalization ofGlcNAc-1-phosphotransferase to lysosomes. Lyset knockout mice exhibit MLII-like phenotypesand human pathogenic LYSET alleles fail to restore lysosomal sorting defects. Thus, we uncoverLYSET as an indispensable component of the M6P trafficking machinery and reveal thebiochemical basis of an inherited disease caused by mutations in LYSET.

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Project description:After gaining access to the endo-lysosomal pathway, several viruses depend on lysosomal cathepsin proteases to cleave their structural proteins triggering productive entry. Targeting of cathepsins and other luminal lysosomal proteins to lysosomes requires their modification with mannose 6-phosphate (M6P) signals. Key to M6P tagging is N-acetylglucosamine (GlcNAc)-1-phosphotransferase whose deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Here, using genome-scale CRISPR screens, we identify the transmembrane protein TMEM251 as critically important for viral infection by cathepsin-dependent viruses including reovirus, Ebola virus, and SARS-CoV-2. We demonstrate that Golgi-resident TMEM251 is essential for lysosomal sorting and activity of cathepsins. Mechanistically, we show that TMEM251 deficiency in human cells results in global loss of M6P on luminal lysosomal proteins by destabilizing GlcNAc-1-phosphotransferase. Tmem251 knockout mice reveal characteristics typical of MLII including hypersecretion of lysosomal enzymes and accumulation of lysosomal storage material in isolated fibroblasts. Finally, we demonstrate that human pathogenic TMEM251 alleles fail to rescue lysosomal sorting defects in knockout cells. Our results uncover a crucial role of TMEM251 in M6P-dependent lysosomal transport and viral infection. Overall, work here reveals the biochemical basis of an inherited MLII-like disease caused by mutations in TMEM251 and provides insights into infection mechanisms of a broad class of medically important viruses.

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Gene expression in the femur hip joint articular cartilage of 12-week-old Gnptg-KO mice

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