Functional Study of MUDENG using ScFv Antibody and Its Knockout Cells

Abstract

MUDENG (Mu-2-related death-inducing gene, MuD) is revealed to be involved in cell death singling. MUDENG can be an attractive target for cancer therapeutic intervention. However its function(s) has not been fully discovered yet. It had been first suggested that MuD was involved in the death-inducing mechanism as assumed from the nomenclature of this gene although a recent report claimed that MuD might function as an anti-apoptotic gene. Therefore, the real function of this gene has still remained to be elucidated. Here the function(s) of MuD has been demonstrated by using anti-MuD scFv (single chain fragment variable) and its knockout cells. It has been supposed that MuD encodes two different isoforms, alpha and beta. The alpha form has 103 extra amino acid residues at the N-terminus compared to the beta isoform. The remaining 387 amino acid residues are the same as each other. Two different scFvs (scFv - C22B3 and scFv - M3H9) against MuD were constructed using anti-MuD monoclonal antibodies C22B3 and M3H9. The scFv - C22B3 and scFv-M3H9 recognized amino terminal domain of MuD and middle region of MuD respectively. That is, the scFv - C22B3 targets only alpha isoform and scFv-M3H9, both alpha and beta isoforms. Each scFv was ectopically expressed in U251-MG cells. The cells expressing anti - MuD-scFv - C22B3 showed resistance to TNF - related apoptosis-inducing ligand (TRAIL), on the contrary, scFv-M3H9 neither U251-MG cells made sensitized nor resistant to TRAIL treatment significantly. In order to confirm these results, two kinds of MuD knock out cells using the CRIPR/Cas9 genome editing system were made: the alpha KO cells that knock out (KO) for only alpha MuD isoform and the beta KO cells which target both the alpha and beta isoforms of MsuD. TRAIL induced cytotoxicity was not altered in the alpha mutant cells, while beta mutants were significantly sensitized to TRAIL treatment, indicating that deletion of both isoforms enhances the cytotoxic effect of TRAIL treatment. Taken together, this study indicates an anti-apoptotic function of MuD during the TRAIL mediated death signaling in glioblastoma multiform (GBM).