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Impact of cystinosin glycosylation on protein stability by differential dynamic SILAC

  • Organism: Human
  • Instrument: Qexactive plus
  • SpikeIn: No
Cystinosis is a rare autosomal recessive lysosomal storage disorder, characterized by an intra-lysosomal accumulation of cystine. The causative gene for cystinosis is CTNS, which encodes the protein cystinosin, a lysosomal proton-driven cystine transporter. Over 100 mutations are reported, leading to different severity of the disease, often in correlation with cystinosin residual activity as a transporter and with maintenance of its protein-protein interactions. In this study we focus on ΔITILELP the only mutation reported, in some cases, to lead to severe forms, inconsistently with its residual transported activity. ΔITILELP is a deletion that eliminates a consensus site on N66, one of the 7 glycosylation sites of the protein. Our hypothesis is that ΔITILELP mutant is less stable and undergoes faster degradation. Our dynamic SILAC study clearly shows that wild-type cystinosin is very stable while ΔITILELP is degraded three times faster than the wild-type protein. Additional lysosome inhibition experiments confirm ΔITILELP instability and show that the degradation is mainly lysosomal. We can observe that at the lysosome, ΔITILELP is still capable of interacting with the V-ATPase complex and some members of the mTOR pathway like the wild-type protein. Intriguingly, our interactomic and immunofluorescence studies show that ΔITILELP is partially retained at the ER. We propose that ΔITILELP mutation causes protein misfolding, ER retention and incapability to be processed in the Golgi, as we demonstrate that ΔITILELP carries high mannose glycans on all its 6 remaining glycosylation sites. Altogether, we show that the high turnover of ΔITILELP, due to its immature glycosylation state in combination with low transport activity, might be responsible for the phenotype observed in some patients.
Experiment Description
For turnover studies, immunoprecipitated protein samples were resolved by SDS-PAGE on 10% gel and large protein bands corresponding to cystinosin were excised. In-gel tryspin digestion was performed as described before (5). Nano-LC-MS/MS analysis of in-gel digested samples was performed on an Ultimate 3000 Rapid Separation Liquid Chromatography (RSLC) system coupled to a Q-Exactive Plus mass spectrometer (Thermo Scientific, Waltham, MA, USA). Extracted peptides were resuspended in 0.1% (v/v) trifluoroacetic acid, 10% acetonitrile, and were loaded onto a µ-precolumn (Acclaim PepMap 100 C18, cartridge, 300 µm i.d.×5 mm, 5 µm, Dionex), followed by separation on the analytical 50 cm nano column (0.075 mm ID, Acclaim PepMap 100, C18, 2 µm, Dionex). Chromatography solvents were (A) 0.1% formic acid in water, and (B) 80% acetonitrile, 0.08% formic acid. Peptides were eluted from the column using a gradient from 5% to 40% B over 38 min and were analyzed by data dependent MS/MS, using top-10 acquisition method. Briefly, the instrument settings were as follows: resolution was set to 70,000 for MS scans and 17,500 for the data dependent MS/MS scans in order to increase speed. The MS AGC target was set to 3.106 counts with a maximum injection time of 200 ms, while MS/MS AGC target was set to 1.105 with a maximum injection time of 120 ms. Dynamic exclusion was set to 30 sec. Raw files were processed using the Proteome Discoverer software (ThermoScientific, San Jose, CA) and searched against the Mus musculus Uniprot KB/Swiss-Prot database v.5/7/2012 (16331 entries) integrated with the sequences of the EGFP cystinosin WT and EGFP cystinosin mutants. Search parameters included fixed modification: Carbamidomethyl (C), and variable modifications: Oxidation (M), Label:13C(6) (K), Label:13C(6) (R), and two missed cleavages. Enzyme was trypsin, monoisotopic peptide mass tolerance was ± 2 ppm, and fragment mass tolerance was ± 0.05 Da. The identification validation was performed using percolator, allowing between 1% and 5% of target FDR for peptide based on q-Value and determined by target-decoy approach.
Sample Description
To generate NIH/3T3 fibroblast cell lines stably expressing the human protein cystinosin-EGFP, the construct pCTNS-EGFP (3) was sub-cloned into the lentiviral pRRL.SIN.cPPT.PGK/WPRE vector (15). The cystinosin mutants ΔITILELP, N288K, N323K (5) and N1N7 (the seven putative N-glycosylated sites mutated in alanine) were obtained by the modification of the pRRL.SIN.cPPT.PGK/WPRE-CTNS-EGFP construct with specific mutagenic primers using the Stratagene Quick Change Site-Directed Mutagenesis Kit according to the manufacturer's recommendations. Lentiviruses were produced in HEK293T cells as described before (5). NIH/3T3 cells were transduced by lentiviral particles containing CTNS-EGFP or its mutated forms at an MOI of 7 in the presence of 8 µg/mL polybrene. Generation of EGFP fusion constructs and transfection of NIH/3T3 fibroblasts The construct pCTNS-EGFP and its mutant form ΔITILELP were already described (3). For the mutant N66A (the N66 glycosylation site was mutated in alanine), the modification of the pCTNS-EGFP construct (3) was carried out using the Stratagene Quick Change Site-Directed Mutagenesis Kit according to the manufacturer’s recommendations. NIH/3T3 fibroblasts were transfected with 2 µg of the different constructs using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Cells were lysed 48 h after transfection for western blotting.
Created on 11/16/16 9:15 AM