Abstract

The AC133 epitope expressed on the CD133 glycoprotein has been widely used as a cell surface marker of numerous stem cell and cancer stem cell types. It has been recently proposed that posttranslational modification and regulation of CD133 may govern cell surface AC133 recognition. Therefore, we performed a large scale pooled RNA interference (RNAi) screen to identify genes involved in cell surface AC133 expression. Gene hits could be validated at a rate of 70.5% in a secondary assay using an orthogonal RNAi system, demonstrating that our primary RNAi screen served as a powerful genetic screening approach. Within the list of hits from the primary screen, genes involved in N-glycan biosynthesis were significantly enriched as determined by Ingenuity Canonical Pathway analyses. Indeed, inhibiting biosynthesis of the N-glycan precursor using the small molecule tunicamycin or inhibiting its transfer to CD133 by generating N-glycan-deficient CD133 mutants resulted in undetectable cell surface AC133. Among the screen hits involved in N-glycosylation were genes involved in complex N-glycan processing, including the poorly characterized MGAT4C, which we demonstrate to be a positive regulator of cell surface AC133 expression. Our study identifies a set of genes involved in CD133 N-glycosylation as a direct contributing factor to cell surface AC133 recognition and provides biochemical evidence for the function and structure of CD133 N-glycans.