Purpose: To investigate intracellular trafficking of a signal sequence variant of precursor cystatin C with biochemical properties intermediary between those of the wild–type and of AMD–associated variant cystatin C. Methods: The penultimate amino acid of the wild–type cystatin C signal sequence (Ala) was mutated to serine by in vitro mutagenesis using the QuikChange Site–directed mutagenesis kit. The resulting construct (Ala25Ser cysC–EGFP) encoded the full–length mutated pre cystatin C fused in–frame to the N–terminus of pEGFP–N3 vector. Primary RPE cells in culture and HeLa cells were transiently transfected with an endotoxin–free plasmid preparation of the construct using FUGENE 6 transfection reagent. The localization and trafficking of the newly synthesized Ala25Ser pre cystatin C were studied in living cells by confocal microscopy. MitoTracker Red CM–H2Xros was used to stain active mitochondria. Bleaching experiments of various sub–cellular organelles and regions of the cells were performed in order to assess the specificity of the localization. In addition, precursor cystatin C was analyzed qualitatively and semi–quantitatively in cell lysates and conditioned media of transfected cells by Western blotting. Results: Ala25Ser cysC–EGFP was distributed between a perinuclear localization identified as ER/Golgi, and the mitochondria, the latter indicated by co–localization with MitoTracker Red. Some diffuse staining was observed in the nucleus but not in the nucleolus, and in the cytoplasm of transiently transfected cells. Following bleaching, rapid re–fluorescence of the ER/Golgi body was observed while bleaching of the nucleus removed the minimal nuclear fluorescence which was not restored readily. Endogenous cystatin C and cystatin C–EGFP fusion protein were detected in similar proportions in lysates of cells transfected with wild–type and mutant precursor cystatin C constructs. Secretion of the Ala25Ser cystatin C fusion protein was however reduced compared with the wild–type cystatin C fusion protein while secretion of endogenous cystatin C was not affected. Conclusions: Variant cystatin C investigated displays an intermediary phenotype which helps elucidate the mechanism of intracellular mis–localization previously determined for the AMD–associated variant Ala25Thr cystatin C. The intracellular pattern of localization, together with the partial secretion impairment suggests that the mutant precursor cystatin C fails to be properly translocated to the ER.
|Journal||Investigative Ophthalmology & Visual Science|
|Publication status||Published - 1 May 2005|