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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

DISEASE(S): Disease Free

SUBMITTER: Shiva Ahmadi

LAB HEAD: Dominic Winter

PROVIDER: PXD007546 | Pride | 2018-06-06

REPOSITORIES: Pride

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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 | PXD009621 | 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.

2022-09-15 | PXD029609 | Pride

The human disease gene TMEM251 is essential for lysosomal transport and viral infection

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.

2022-09-09 | PXD029746 | Pride

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

Project description:Mucolipidosis type III is a rare disease caused by mutations in the GNPTG gene, encoding the gamma-subunit of GlcNAc-1-phosphotransferase. Since osteoarthritis and joint stiffness are typical symptoms of the disease, we aimed to investigate the role of the gamma-subunit on cartilage homeostasis. We used microarray analysis to compare the global gene expression in the articular cartilage of Gnptg-KO and wild-type mice.

2020-10-14 | GSE157180 | GEO

Targeting of non-phosphorylated lysosomal enzymes involves secretion and Lrp1-mediated recapture

Project description:Most lysosomal enzymes require mannose 6-phosphate (M6P) residues for efficient receptor-mediated lysosomal targeting. Although the lack of M6P results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types suggesting the existence of M6P-independent transport routes. SILAC-based analysis of purified lysosomes from M6P-deficient mouse fibroblasts (PTki) revealed unchanged amounts of 11 lysosomal enzymes, including cathepsin D and B (CtsD, CtsB), while demonstrating mossiorting of a variety of soluble lysosomal enzymes

2015-05-01 | PXD001221 | Pride

Targeted Quantification of the Lysosomal Proteome in Complex Samples

Project description:The lysosome, as the main degradative organelle of eukaryotic cells, is involved in numerous cellular processes. A defect in one of its proteins often results in lysosomal storage diseases (LSDs). For the study of lysosomal proteins, mass spectrometry (MS) has emerged as the method of choice. Lysosomal proteins are, however, low-abundant, restricting the analysis of lysosomal proteins in unbiased approaches to the investigation of lysosome-enriched fractions. The use of targeted MS provides an attractive alternative to analyze lysosomal proteins in a complex background. In this study, the two targeted MS approaches data-independent acquisition (DIA) and parallel reaction monitoring (PRM) were compared with regard to their ability to analyse lysosomal proteins. These experiments were conducted with samples of different complexity: low-complex lysosome-enriched fractions, medium-complex mouse embryonic fibroblasts (MEF), and high-complex liver whole tissue lysate. While both MS approaches were able to identify and quantify lysosomal proteins, PRM outperformed DIA, especially in high-complex samples.

2021-03-08 | PXD023278 | Pride

Anp32e is a histone chaperone that removes H2A.Z from chromatin

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2014-01-22 | E-GEOD-51579 | biostudies-arrayexpress

β2-adrenergic agonists rescue lysosome acidification and function in PSEN1 deficiency by reversing defective ER to lysosome delivery of ClC-7

Project description:Lysosomal dysfunction is considered pathogenic in Alzheimer Disease (AD). Loss of Presenilin-1(PSEN1) function causing early onset AD impedes acidification via defective vATPase V0a1 subunit delivery to lysosomes. We report that isoproterenol and related β2-adrenergic agonists re-acidify lysosomes in PSEN1 KO cells and fibroblasts from PSEN1 familial AD(FAD) patients, restores lysosomal calcium homeostasis and proteolysis, and reverses impaired autophagy flux. We identify a novel rescue mechanism involving PKA-mediated facilitated delivery of ClC-7 to lysosomes, which stimulates chloride influx and reverses markedly lowered Cl- content of PSEN1 KO lysosomes. Notably, PSEN1 loss-of-function impedes ER-to-lysosome delivery of ClC-7, thus accounting for lysosomal Cl- deficits that compound pH deficits due to deficient vATPase function. Transcriptomics of PSEN1-deficient cells reveal strongly down-regulated ER-to-lysosome transport pathways and reversibility by isoproterenol. Our findings uncover a broadened PSEN1 role in lysosomal ion homeostasis and novel pH modulation of lysosomes through β-adrenergic regulation of ClC-7, which can be therapeutically modulated.

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GSE1 is a prominent interactor of HDAC1

Project description:We analysed the composition of histone acetylase 1 (HDAC1)-associated proteins in different cellular backgrounds using quantitative mass spectrometry (MS). First, we affinity purified (AP) C-terminally FLAG-tagged HDAC1 from the nearly haploid human cell line HAP1 (Andersson et al, 1987). To examine cross-species conservation in the composition of HDAC1 complexes we additionally analysed HDAC1-FLAG purifications from mouse embryonic fibroblasts (MEFs). Genetic suppressor element 1 (GSE1) was identified as a prominent interaction partner of HDAC1. Hence, we purified V5-tagged GSE1 from HAP1 cells and analysed co-precipitated proteins.

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