Background: Membrane-associated RING-CHs (MARCHs) are E3 ubiquitin ligases. Numerous studies showed MARCH 1 play a role in regulation of membrane protein trafficking, which implicates that other MARCH family members might be associated with immunoregulation of plasma membrane protein turn-over and expression. Nevertheless identification of these immunoregulated-relvant substrates remains unmet challenge. To identify these substrates, we developed a procedure to enrich membrane proteins from primary antigen presenting cells (dendritic cells (DCs)) in vivo in the presence and absence of MARCHs, and utilize label-free proteomic analysis for substrate identification and comparative quantification.
Methods: DCs were obtained by standard laboratory procedures. Purified DCs were then incubated with membrane-specific antibodies conjugated with FITC. After incubation, the cells were homogenized. The homogeneous were incubated with anti-FITC magnetic micro-beads, and subsequently went through LS column. Non-plasma membrane (PM) fraction was eluted and PM microsomes were collected after final high-pH solution elution. The enriched PM microsomes were trypsin digested and analyzed by LC-MS/MS on WATERS nanoAcquity LC coupled to a THERMO-FISHERQ-EXACTIVE.
Result: From western blots and MS analyses, we demonstrated and confirmed the enhancement of PM proteins from primary cells in wild type and knock out mice. Total of 78 potential substrates were identified. Many of these proteins have been not previously reported in association with MARCH 1 substrates, and might have a functional role in immune regulation. Previously known MARCH 1 substrates, MHC class II and CD86 were also confirmed in our study and validated by western blot and flow cytometer. Identification of substrates recognized by MARCHs and understanding the regulatory roles of MARCHs in immunity will provide fundamental new knowledge about the role of ubiquitination during membrane proteostasis, and potentially lead to clinical applications for the treatment of tumours and immune diseases.