Proteomic analysis of immature murine melanocytes at different stages of maturation: a crucial role for calreticulin.
We have established two immature melanocyte cell lines from murine neural crest cells. NCC-E3 cells have Stage II melanosomes and express tyrosinase while in NCCmelb4 cells, the melanosomes remain at Stage I and tyrosinase is not expressed. These cell lines may be useful in studying the differentiation of melanocyte precursors. To perform proteomic analysis of the two cell lines to identify proteins related to and possibly responsible for their different maturation stages. Western blotting, two-dimensional differential image gel electrophoresis (2D-DIGE), liquid chromatography-tandem mass spectrometry (LC-MS/MS), real-time PCR analysis and RNA interference using siRNA were employed in this study. Western blotting revealed that the processed form of gp100, which is specific for Stage II melanosomes, is expressed in NCC-E3 cells but not in NCCmelb4 cells. 2D-DIGE identified two protein spots showing 4.06- and 2.22-fold increases in NCC-E3 cells compared to NCCmelb4 cells. Analysis of those proteins by LC-MS/MS revealed that the former was calreticulin and the latter was BiP/GRP78. When calreticulin mRNA expression in NCC-E3 cells was blocked by siRNA, tyrosinase protein was abolished and DOPA-reactivity was decreased, although tyrosinase mRNA was abundantly expressed after the same treatment. Calreticulin, a lectin chaperone, is an essential molecule for the processing of tyrosinase in murine melanocytes. The role of molecular chaperones such as calreticulin should be considered when analyzing the mechanism(s) of melanocyte differentiation.