Identification of Glutaminyl Cyclase isoenzyme isoQC as a regulator of SIRPα-CD47 axis

摘要

Genome-wide CRISPR-Cas9 screen identified genes essential for cell surface CD47 expression. HCT116 colon cancer cells were mutagenized with a pooled lentiviral sgRNA library and CD47low cells were enriched by FACS sorting. Fluorescence-activated cell sorting (FACS) analysis of surface CD47 in DLD1 and HEK293T cells stably expressing the indicated sgRNAs. Shown is a representative experiment ( ). Mean fluorescent intensity (MFI) values are shown in . The data were normalized using Cas9 control. Shown is the mean ± SD of three independent experiments. test, **  P d FACS analysis of cell surface CD47 using CC2C6 antibody in HCT116 (left) and HEK293T (right) cells stably expressing the indicated sgRNAs after treatment with the increasing concentrations of isoQC inhibitor (PQ529) for 48 h. The data were normalized using DMSO control. Shown is the mean ± SD of three independent experiments. two-way ANOVA, **  P e FACS analysis of cell surface CD47 expression in isoQC knockout HEK293T cells with the indicated overexpressed-plasmids. Shown is a representative experiment. Data represent three independent experiments with similar results. pGlu modification of human CD47 N-terminal peptide by purified isoQC was monitored by nanoESI-MS mass spectrometry. Co-immunoprecipitation of endogenous CD47 and isoQC in HEK293T cells (IP, CD47 antibody: sc-59079; IB, CD47 antibody: CST 63000). Shown is a representative experiment. Data represent three independent experiments with similar results. FACS analysis of cell surface CD47 using anti-CD47 B6H12 monoclonal antibody (clone B6H12) in the indicated knockout HEK293T cells. The data were normalized using Cas9 control. Shown is the mean ± SD of three independent experiments. test, ***  i FACS analysis of the binding of SIRPα to cell surface in the indicated knockout HEK293T cells. The data were normalized using Cas9 control. Shown is the mean ± SD of three independent experiments. test, **  P j FACS analysis of the interaction between SIRPα and CD47 in indicated knockout HEK293T cells. Cells were treated with increasing concentrations of SIRPα. The data were normalized using Cas9 control. Shown is a mean ± SD of three independent experiments. two-way ANOVA, *  P k, Phagocytosis of knockout DLD1 cells by macrophages was analyzed via FACS. The data were normalized using Cas9 control. Shown is the mean ± SD of three independent experiments ( ). test, *  p + macrophages, expressed as a percentage of the total macrophages, as depicted in the representative FACS shown in ( ). FACS analysis of the phagocytosis of the indicated DLD1 cells treated with isoQC inhibitor (PQ529, 10 μM) or vehicle control for 48 h before co-culture. The data were normalized using vehicle control. Shown is the mean ± SD of five independent experiments. test, **  n Correlation of isoQC expression with prognosis in Acute Myeloid Leukemia patients (  = 58)