title: Impact of Cystinosin Glycosylation on Protein Stability by Differential Dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).
abstract: Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by intralysosomal accumulation of cystine. The causative gene for cystinosis isCTNS, which encodes the protein cystinosin, a lysosomal proton-driven cystine transporter. Over 100 mutations have been reported, leading to varying disease severity, often in correlation with residual cystinosin activity as a transporter and with maintenance of its protein-protein interactions. In this study, we focus on the ΔITILELP mutation, the only mutation reported that sometimes leads to severe forms, inconsistent with its residual transported activity. ΔITILELP is a deletion that eliminates a consensus site on N66, one of the protein's seven glycosylation sites. Our hypothesis was that the ΔITILELP mutant is less stable and undergoes faster degradation. Our dynamic stable isotope labeling by amino acids in cell culture (SILAC) study clearly showed that wild-type cystinosin is very stable, whereas ΔITILELP is degraded three times more rapidly. Additional lysosome inhibition experiments confirmed ΔITILELP instability and showed that the degradation was mainly lysosomal. We observed that in the lysosome, ΔITILELP is still capable of interacting with the V-ATPase complex and some members of the mTOR pathway, similar to the wild-type protein. Intriguingly, our interactomic and immunofluorescence studies showed that ΔITILELP is partially retained at the endoplasmic reticulum (ER). We proposed that the ΔITILELP mutation causes protein misfolding, ER retention and inability to be processed in the Golgi apparatus, and we demonstrated that ΔITILELP carries high-mannose glycans on all six of its remaining glycosylation sites. We found that the high turnover of ΔITILELP, because of its immature glycosylation state in combination with low transport activity, might be responsible for the phenotype observed in some patients.
authors.affiliation: From the ‡INSERM U1163, Laboratory of Hereditary Kidney Diseases, Imagine Institute, Paris, France.|‡‡Paris Descartes-Sorbonne Paris Cité University, Paris, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Nevo
authors.affiliation: From the ‡INSERM U1163, Laboratory of Hereditary Kidney Diseases, Imagine Institute, Paris, France.|‡‡Paris Descartes-Sorbonne Paris Cité University, Paris, France.
authors.forename: Lucie
authors.initials: L
authors.lastname: Thomas
authors.affiliation: §Plateforme Protéomique Paris Descartes Necker, PPN, 3P5-Necker, SFR Necker, US24, 75014 Paris, France.|‡‡Paris Descartes-Sorbonne Paris Cité University, Paris, France.
authors.forename: Cerina
authors.initials: C
authors.lastname: Chhuon
authors.affiliation: From the ‡INSERM U1163, Laboratory of Hereditary Kidney Diseases, Imagine Institute, Paris, France.|‡‡Paris Descartes-Sorbonne Paris Cité University, Paris, France.
authors.forename: Zuzanna
authors.initials: Z
authors.lastname: Andrzejewska
authors.affiliation: ¶The CPN Proteomics Facility - 3P5, Center of Psychiatry and Neuroscience, UMR INSERM 894, 75014 Paris, France.|‡‡Paris Descartes-Sorbonne Paris Cité University, Paris, France.
authors.forename: Joanna
authors.initials: J
authors.lastname: Lipecka
authors.affiliation: ‖Plateforme Protéomique Paris Descartes Cochin, 3P5-Cochin, 75014 Paris, France.|‡‡Paris Descartes-Sorbonne Paris Cité University, Paris, France.
authors.forename: François
authors.initials: F
authors.lastname: Guillonneau
authors.affiliation: From the ‡INSERM U1163, Laboratory of Hereditary Kidney Diseases, Imagine Institute, Paris, France.|‡‡Paris Descartes-Sorbonne Paris Cité University, Paris, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Bailleux
authors.affiliation: §Plateforme Protéomique Paris Descartes Necker, PPN, 3P5-Necker, SFR Necker, US24, 75014 Paris, France.|**INSERM U1151, 75015 Paris, France.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Edelman
authors.affiliation: From the ‡INSERM U1163, Laboratory of Hereditary Kidney Diseases, Imagine Institute, Paris, France.|‡‡Paris Descartes-Sorbonne Paris Cité University, Paris, France.|§§Assistance Publique - Hôpitaux de Paris (AP-HP), Department of Genetics, Necker Hospital, Paris, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Antignac
authors.affiliation: §Plateforme Protéomique Paris Descartes Necker, PPN, 3P5-Necker, SFR Necker, US24, 75014 Paris, France; chiara.guerrera@inserm.fr.|‡‡Paris Descartes-Sorbonne Paris Cité University, Paris, France.
authors.forename: Ida Chiara
authors.initials: IC
authors.lastname: Guerrera
mesh_terms: D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D009154:Mutation; D041681:NIH 3T3 Cells; D011134:Polysaccharides / Q000378:metabolism; D017510:Protein Folding; D055550:Protein Stability; D059748:Proteolysis; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism
chemical_list: D026921:Amino Acid Transport Systems, Neutral; C111445:CTNS protein, human; D011134:Polysaccharides; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D025262:Vacuolar Proton-Translocating ATPases