Open AccessEFFECT OF EDEM1 OVEREXPRESSION ON THE GENERATION AND ASSEMBLY OF MAJOR HISTOCOMPATIBILITY COMPLEXES
Author(s) -
Alexandra Cîrciumaru,
Gabriela Chiriţoiu,
Livia Elena Sima,
Mihai Bojinca,
Ștefana M. Petrescu
Publication year - 2016
Publication title -
romanian journal of rheumatology
Language(s) - English
Resource type - Journals
eISSN - 2069-6086
pISSN - 1843-0791
DOI - 10.37897/rjr.2016.4.2
Subject(s) - transfection , flow cytometry , hek 293 cells , microbiology and biotechnology , western blot , cell culture , endoplasmic reticulum associated protein degradation , biology , major histocompatibility complex , endoplasmic reticulum , chemistry , antigen , immunology , unfolded protein response , biochemistry , genetics , gene
Background. A better understanding of the role of endoplasmic reticulum degradation-enhancing alpha-mannosidase – like protein 1 (EDEM1) in endoplasmic reticulum associated degradation (ERAD) may open new therapeutic approaches in autoimmune diseases. Aim. To study ERAD and EDEM1 in the generation and assembly of MHC I and the potential role in the pathophysiology of autoimmune diseases. Materials and methods. HEK293T cell line (human embrionic kidney cells), A375 cell line (amelanotic melanoma cells) and THP-1 cell line (leukemic monocytes used both as undifferentiated and differentiated) underwent transient transfection with EDEM1 and mock transfection with pTriEx. Western blot experiments assessed the total cellular MHC I levels in cell lysates, while expression on the cellular surface was quantified by flow cytometry of fixed cells. Results were analysed using the FACS Calibur and CellQuest Pro dedicated software. Experiments were done twice with duplicate probes for the Western blot assay and triplicate probes were used for flow cytometry. GraphPad Prism was used for data analysis. Results. MHC I plasma membrane routing and expression was similar in HEK293T and A375 both in mock transfected and non-transfected cells. Western blot assay for EDEM1 transfected cells showed bands corresponding to the total MHC I that migrated at 42kDa mass in non-transfected and mock transfected Hek293T, A375 and undifferentiated THP-1 cells. Mock transfected differentiated THP-1 cells showed a reduction of total MHC I. EDEM1 transfected Hek293T, A375 and undifferentiated THP-1 cells displayed higher levels of total MHC I, while differentiated THP-1 cells showed a marked reduction. Flow cytometry assay showed significantly reduced cell surface MHC I levels in Hek293T cell line. We observed a modest reduction of MHC I complexes on the cellular surface in undifferentiated THP-1 EDEM1 transfected cells, while there was no significant change in the A375 EDEM1 transfected cell line, as well as the differentiated THP-1 EDEM1 transfected cells. Conclusion. The impact of ERAD’s EDEM1 in MHC I reduction may have an important role in autoimmune disease, making ERAD an interesting therapeutic target.