Impact of Cystinosin Glycosylation on Protein Stability by Differential Dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Cerina Chhuon,Ida Chiara Guerrera,François Guillonneau,Aleksander Edelman,Zuzanna Andrzejewska,Lucie Thomas,Corinne Antignac,Anne Bailleux,Joanna Lipecka,Nathalie Nevo

doi:10.1074/mcp.m116.063867

Abstract

Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by intralysosomal accumulation of cystine. The causative gene for cystinosis is CTNS, 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.

Highlights

Cystinosis (OMIM 219800) is a rare autosomal recessive lysosomal storage disorder characterized by intralysosomal accumulation of cystine, leading to impaired function of multiple organs [1]
Detection of EGFP-tagged cystinosin peptides by Mass spectrometry (MS) To analyze the turnover of WT and mutated cystinosin, we transduced 3T3 cells with lentiviral constructs to stably express the cystinosin-EGFP fusion protein and its mutated forms
The schematic representation of WT cystinosin and ΔITILELP can be found in Figure 1A and 1B

Summary

Introduction

Cystinosis (OMIM 219800) is a rare autosomal recessive lysosomal storage disorder characterized by intralysosomal accumulation of cystine, leading to impaired function of multiple organs [1]. To expand our knowledge of the cellular mechanisms involved in the pathogenicity of ΔITILELP, we analyzed its fate in the cell by studying its glycosylation, cellular localization and specific protein partners, as compared to WT cystinosin.

Results

Conclusion