Characterization of the interaction of cystinosin with galectin-3
and vacuolar H+-ATPase
Progress report n° 4 – October 2011
Research project conducted at Inserm U983 (ex-U574) (Necker Hospital, Paris)
Principal investigator : Corinne Antignac
Persons working on the project :
Nathalie Nevo (research assistant, Inserm funded)
Lucie Thomas (research assistant, CRF funded from June 2009 to June 2010 and
since then through the EU-funded Eunefron project)
Background and objectives
The global aim of our research project is to characterize cystinosin cellular trafficking and to
identify functions of cystinosin other than lysosomal export of cystine. The specific aims of
the projects are:
Aim 1: Characterize the interaction of cystinosin and galectin-3,
Aim 2: Characterize the interaction of cystinosin and V-ATPase,
Aim 3: Identify other proteins interacting with the 5th inter-TM loop of cystinosin.
Update on the progress of research plan
Since the last report, most of the effort has been put on the proteomic studies, especially on
the set up of the SILAC technique.
1. Characterization of the interaction of cystinosin and galectin-3
1. Further characterize the interaction
- We had shown that that cystinosin-GFP interacts with galectin-3 through a carbohydratedependent mechanism in MDCK cells. We confirmed this interaction in LLC cells and for HAtagged cystinosin in both LLCCPK and MDCK cells. We also showed that the interaction is
maintained when cystinosin is deleted of the lysosome targeting motifs.
- These results raise the question whether the interaction is true in vivo, which implies that
galectin-3 localized to the lumen of the late endosomes and lysosomes. Galectin-3 is a
soluble, non-glysosylated beta-galactoside binding lectin composed of a single C-terminal
carbohydrate recognition domain linked to a unique N-terminal domain involved in the
oligomerization of the molecule. It is known to have multiple cell and extracellular
localization. It was well-known that it is both a cytoplasmic protein and a secreted protein,
although the mechanism by which it is secreted remains mysterious. It has also recently
been shown that galectin-3 can also be found in recycling endosomes (Schneider et al.,
2010). During this report period, we further expanded the spectrum of galectin-3 subcellular
localization. Indeed, we showed that galectin-3, as cathepsin D (a known intralysosomal
protease) is not degraded by treatment of microsomes by proteinase K, and, even more
convincing, that galectin-3 colocalizes with cystinosin-GFP in lysosomes and late
endosomes.
Immunostaining cystinosin-EGFP MDCK cell lines with anti-galectin-3 antibodies revealed the
presence of galectin-3 inside the vesicles whereas cystinosin-GFP is located at the membrane
of the vesicles.
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- The next step was to try to understand what could be the role of this interaction in vivo.
• One of our major hypothesis was that galectin-3 might have a role in protecting cystinosin
(and potentially other lysosomal membrane proteins) against degradation. To test this
hypothesis, we set up the technique of SILAC (dynamic stable isotope labeling with amino
acids in cell culture) coupled to mass spectrometry to measure the half-life of cystinosinGFP in MDCK cells expressing or not galectin-3. We had previously shown that we could
almost completely knock-down galectin-3 in these cells using sh-RNA directed against
galectin-3. SILAC strategy allows quantification of proteins containing light and heavy amino
acids due to their distinct mass difference. Dynamic SILAC experiments involve heavy (H)
labeling of the cells with 13C6-lys and 13C6-arg which are then grown in medium containing
light (L) lys and arg for different times. The loss of label in time reflects the degradation of the
GFP-Cystinosin while the unlabeled protein corresponds to its synthesis.
Actually, we started this technique more that 18 months ago but have now considerably
improved the setting up. Contrary to what we found in our preliminary results, we did not
observe any difference in the half-life of cystinosin-GFP in MDCK cells vs MDCK cells
depleted of galectin-3 by RNA-interference in a total of 5 independent experiments.
However, as a proof of concept to validate the technique, we have shown that cystinosinGFP in which all the N-glycosylation sites have been mutated to alanine has a drastically
shortened half-life, as expected from the role of sugar moieties on the stabilization of
lysosome membrane proteins, whereas the degradation rate of other proteins did not
change.
We are now checking whether these negative results could be due to the residual galectin-3
after RNA interference, by measuring cystinosin-GFP half-life in fibroblasts of wild-type
mouse vs Gal3-/- KO mice.
2. Assess the phenotype of cystinosin and galectin-3 double knock-out mice (Ctns-/ - ; Gal3 -/-)
in collaboration with S. Cherqui, (Scripps Institute, La Jolla)
The double KO mice are viable and do not have any obvious phenotype until at least 9
months of age. Four groups of mice (of ~4 mice of each genotype per group i.e. wild type,
Ctns-/-, Gal3-/- and Ctns-/-; Gal3-/-) have been generated and have been sacrificed at 9, 12
and 17 months (two groups at 12 months). For each group, blood and urine urea,
creatinine…, cystine content of several organs, histology of the kidney were performed.
Interestingly, although there are large variations within each group and although Ctns-/- ;
Gal3-/- mice accumulate cystine in the kidney to the same extent as Ctns-/- mice, the double
KO group has a less severe phenotype than the Ctns-/- mice, especially at 9 months of age.
However, to verify that all the mice are on a pure C57BL/6J genetic background, we
genotyped mouse DNA on the mouse medium density linkage panel (Illumina) (consisting of
1,449 SNPs, 880 being different between the C57BL/6J and 129S1/SvImJ backgrounds). As
can be seen on the figure below, although the original Gal3-/- mice had been generated by
backcrossing the Gal3-/- allele at least 10 times against the C57BL/6 genetic background, the
Gal3-/- mice and, of course the derived double KO mice, still retain a substantial non C57BL/6
background, even outside of the regions of homologous recombination on chromosomes 14
and 11 for Gal3 and Ctns respectively. As we have shown that the genetic background is
crucial for the development of the cystinotic nephropathy (Nevo et al., 2009), it is likely that
the less severe phenotype of the Ctns-/ - ; Gal3 -/- mice compared to the Ctns-/- mice is due to
the influence of the genetic background and not to the role of the absence of galectin-3.
However, these studies clearly show once again the crucial role of some C57BL/6 alleles in
the setting of nephropathic cystinosis and pave the way for future studies to identify genetic
modifiers that can explain the phenotypic variability in rodents and possibly in humans.
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Schematic representation of SNP genotyping of Ctns+/+, Ctns-/-, Gal3-/- and
Ctns-/-; Gal3-/-mice phenotyped in the study. Each line corresponds to an
informative SNP between the C57BL/6J and 129S1/SvImJ backgrounds. The
SNPs are ordered according to their position on the genome. The 129S1/SvImJ
heterozygous and homozygous alleles are displayed in yellow and red
respectively.
2. nanoLC-MS-MS studies
To confirm the results obtained using MALDI-TOF and to find potential additional partners,
the products of the immunoprecipitation experiments of HA-cystinosin and GFP-cystinosin
either in MDCK or LLCPK cells have been analyzed by nanoLC-MALDI-TOF-TOF (ParisDescartes University proteomics core facility). These analyses confirmed the interaction of
cystinosin with galectin-3 and several subunits of the V-ATPase, and also show the
interaction of cystinosin with various other proteins such as raft proteins (e.g. caveolin-2,
flotillins).
Concerning the interaction of cystinosin with the V-ATPase, thanks to the availability of good
antibodies, we have been able to confirm the interaction by immunoprecipitation with the
endogenous proteins. Interestingly, this interaction is decreased when cystinosin is deleted
of one of its lysosomal targeting signal and completely abolished when cystinosin bore the
N288K mutant, which we found completely abolished cystin transport (Kalatzis et al., 2005).
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