Ubiquitination is a fundamental cellular function that regulates the degradation and transport of proteins. The Membrane-Associated Ring CH (MARCH) proteins are a family of membrane-bound E3 ubiquitin ligases that are able to ubiquitinate substrate proteins through the transmembrane domain. MARCH-1 is known to regulate endocytosis of MHC-II and CD86 on the plasma membrane of dendritic cells (DCs) and B cells, and is crucial for antigen presentation on these immune cells. However it is unknown if MARCH-1 interacts with other substrates, and if it is involved in other biological or immunological functions.
To identify novel substrate targets of MARCH-1, we utilized mass spectrometry-derived proteomic analysis to identify differences in surface molecule expression of immune cells. Peptide sequences detected on the plasma membrane of wildtype B cells is compared with B cells derived from MARCH-1 deficient mice, and potential substrates are identified based on protein ratio and peptide intensity of their unique peptides from the proteomic profiles.
Interestingly, one of the differentially detected substrates was Complement Component 3 (C3). The increased expression of surface C3 on B cells and DCs from MARCH-1-KO mice was verified through FACS and microscopy. Mixed bone marrow chimera experiments indicated that MARCH-1-KO-derived B cells and DCs retain uniquely increased expression of surface C3, even when residing in a wildtype host. Curiously, the increased surface C3 expression declined under in vitro conditions, suggesting that MARCH-1-KO-derived B cells and DCs may have increased capacity to acquired surface C3 exogenously, through an unelucidated cell-intrinsic mechanism that is regulated by MARCH-1.