CD133 protein N-glycosylation processing contributes to cell surface recognition of the primitive cell marker AC133 epitope

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.

FIGURE 1.

Human cell line stably expressing cell surface AC133. A, an adapted schematic diagram of the MAPLE vector harboring the CD133 (isoform 2) cDNA in-frame with the VA tag and driven by the constitutive human cytomegalovirus (CMV) promoter. B, lysates from the parental HEK293 (untransduced) and HEK293/CD133-VA cell lines were immunoblotted with either FLAG or AC133/1 antibodies. A specific band at ∼130 kDa corresponds to a glycosylated form of CD133-VA. β-Actin was used as a loading control. A representative blot is shown (n = 3). C, FLAG and AC133 immunofluorescence of HEK293/CD133-VA is shown. Scale bars, 25 μm. D, parental HEK293 and HEK293/CD133-VA cell lines were stained with phycoerythrin-labeled AC133 and analyzed by FACS. A representative plot is shown (n = 3).

FIGURE 2.

Schematic of the FACS-based pooled lentiviral shRNA screen used to identify genes involved in AC133 expression. HEK293/AC133 cells were infected with the 54K pooled lentiviral shRNA library, and shRNA-targeted gene knockdown was allowed to occur. shRNA-infected cells were fractionated into HEK293/AC133^low and HEK293/AC133^high populations by FACS. Genomic DNA from fractionated cells was extracted and used as template for PCR amplification of shRNA barcodes. Detection of shRNA barcodes and corresponding gene targets was achieved by hybridization to custom Affymetrix microarrays, and a threshold was created to determine shRNA hits.

FIGURE 3.

N-Glycan biosynthesis pathway contributes to cell surface AC133 expression. Adapted N-glycan biosynthesis pathway map starting in the ER from the KEGG (43) (published with permission) highlights gene hits for cell surface AC133 recognition. Genes highlighted in light green were identified from the primary pooled lentiviral shRNA screen. Genes highlighted in dark green were identified from the primary screen and validated in the secondary esiRNA validation screen.

FIGURE 4.

Tunicamycin treatment down-regulates CD133 N-glycoslyation and stability. A, CD133 levels, as determined by AC133 immunoblotting, were monitored by Western blot analysis for vehicle-only or tunicamycin-treated HEK293/CD133. α-Tubulin was used as a loading control. Representative immunoblots are shown (n = 3). B, HEK293/CD133-VA treated with either vehicle-only control or 1.0 ng/ml tunicamycin was stained with AC133-APC and analyzed by FACS. A representative plot is shown (n = 3).

FIGURE 5.

Single-site N-glycosylation CD133 mutants. A, lysates of single-site N-glycan-deficient CD133 mutants expressed in HEK293 cells were immunoblotted for AC133/1. β-Actin was used as a loading control. The experiment was performed in biological triplicate, and a representative replicate is shown. B, quantitative PCR was used to monitor CD133-YFP transcript of wild-type or single-site N-glycosylation CD133 mutant stables. Transcript levels were normalized to actin and are relative to wild-type CD133. Error bars represent S.D. of three independent replicates. C, representative FACS plot of wild-type CD133 or single-site N-glycosylation CD133 mutants tagged with YFP stably expressed in HEK293 cells stained with AC133-APC is shown (n = 3). Arrows indicate subpopulations that were noticeably different from WT.

FIGURE 6.

CD133 N-glycans determine AC133 cell surface recognition. A, representative histogram from three biological replicates of HEK293 cells expressing either wild-type or mutated CD133 stained with AC133 is shown. B, CD133 levels were monitored by immunoblotting for wild-type or mutant CD133, and β-actin was used as a loading control. A representative set of blots is shown (n = 3). C, quantitative PCR was used to monitor CD133 transcript levels in HEK293 cells stably expressing wild-type or mutant CD133-FLAG. Transcript levels were normalized to actin, and error bars represent S.D. (n = 3). p values were determined by Student's paired t test, with two-tailed distribution. D, FLAG immunofluorescence of HEK293 cells stably expressing wild-type or mutant versions of CD133-FLAG as indicated. Cells were also co-stained for the ER marker CANX. Scale bar, 50 μm.

FIGURE 7.

Complex CD133 N-glycans mediate AC133 recognition. A, HEK293/CD133-VA cells were treated with vehicle or 20 μm swainsonine, and CD133-VA and lysates made from equal amount of cells were FLAG-immunoprecipitated. FLAG-immunoprecipitated CD133-VA were treated with either PNGase F or Endo Hf. Samples were immunoblotted with AC133. Arrow represent the CD133 band with N-glycans that have not undergone complex N-glycosylation. The experiment was performed in biological triplicate, and a representative replicate is shown. B, cell surface AC133 levels of HEK293/CD133-VA cells treated with either vehicle or 20 μm swainsonine were analyzed by flow cytometry, and the mean value of cell surface AC133-APC is represented. Error bars represent S.D. (n = 3). p value was determined by Student's paired t test, with two-tailed distribution. C, cell surface AC133 recognition was monitored by staining HEK293/CD133-VA cells treated with either control shRNA or shMGAT4C with AC133-APC and analyzed by FACS. A representative histogram from three biological replicates in shown. D, lysates from HEK293/CD133-VA cells expressing shMGAT4C were analyzed by Western blotting to monitor CD133 N-glycosylation. Immunoblotting for α-tubulin served as a loading control. Arrow represents the CD133 band with N-glycans that have not undergone complex N-glycosylation. Experiment was performed in biological triplicate, and a representative replicate is shown. E, lysates from HEK293 cells containing an empty MAPLE vector and HEK293/CD133-VA cells were FLAG-immunoprecipitated and analyzed by Western blotting with either the AC133 or MGAT4C antibodies.