Comparative glycoproteomics of stem cells identifies new players in ricin toxicity

Abstract

Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification. Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes. Despite the importance of glycosylation in biology, the identification and functional validation of complex glycoproteins has remained largely unexplored. Here we develop a novel quantitative approach to identify intact glycopeptides from comparative proteomic data sets, allowing us not only to infer complex glycan structures but also to directly map them to sites within the associated proteins at the proteome scale. We apply this method to human and mouse embryonic stem cells to illuminate the stem cell glycoproteome. This analysis nearly doubles the number of experimentally confirmed glycoproteins, identifies previously unknown glycosylation sites and multiple glycosylated stemness factors, and uncovers evolutionarily conserved as well as species-specific glycoproteins in embryonic stem cells. The specificity of our method is confirmed using sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Ablation of fucosylation confers resistance to the bioweapon ricin, and we discover proteins that carry a fucosylation-dependent sugar code for ricin toxicity. Mutations disrupting a subset of these proteins render cells ricin resistant, revealing new players that orchestrate ricin toxicity. Our comparative glycoproteomics platform, SugarQb, enables genome-wide insights into protein glycosylation and glycan modifications in complex biological systems.

Extended Data Figure 1. Identification of N-glycopeptides and N-glycans.

(a) Glycopeptide MS/MS raw-data were pre-processed and identified using different MS/MS search engines: Mascot (orange), SEQUEST-HT (blue) and X!tandem (green). The glycopeptide sequences identified by these algorithms were highly similar, both with respect to identities and numbers. (b) The N-glycosylation sites identified in our study (orange) were mapped to published annotated N-glycosylation sites in Uniprot (grey) and those identified by Wollscheid et al. (green). (c) Negative-mode MALDI-TOF analysis of 2-AA labelled N-glycans from mESCs directly correlated with the N-glycan profiles identified using our glycoproteomics approach.

Extended Data Figure 2. Quantitative proteomics.

Comparative proteomics of (a) Slc35c1 knock-out (KO) and (b) Fut9 KO versus their respective genetically repaired wild-type (WT) sister murine embryonic stem cells (mESCs) did not show significant changes in the overall protein expression as determined by quantitative proteomics. Differential peptide abundances are represented as scatter-plots of the corresponding TMT-reporter ion intensities. (c) Coverage of the predicted (RNA-seq) mESCs proteome by the quantitative proteomics data set.

Extended Data Figure 3. Stemness of knock-out mESC lines.

The parental control mESC line AN3.12, as well as mutant Hs2st1, Igf2r, Itgb1, Lamp1, Ly75, Slc39a14 mESCs lines were stained for the prototypic mouse embryonic stem cell markers Oct4, SSEA-1 and alkaline phosphatase. DAPI is shown to visualize nuclei. Of note, no obvious growth defects or morphological phenotypes were observed. All mESCs were diploid as determined by Hoechst staining (not shown). Scale bars are indicated. The experiments were repeated three times. Each image is representative of five images.

Figure 1. Glycoproteomics.

(a) Glycoproteomic workflow combining proteomics platforms with a new algorithm for the identification of intact glycopeptides from complex biological samples. (b) MS/MS raw-data pre-processing by charge deconvolution and precursor mass correction allows for the automated identification of glycopeptides, based on low-abundant peptide fragment ions in the lower mass-range of glycopeptide MS/MS spectra. (c) Coverage of experimentally identified glycoproteins (orange-filled circle; Supplementary Table 10) among all predicted mouse glycoproteins (Uniprot, white) and the glycoproteins expressed (based on RNAseq, grey) in mESC. (d) Transcript abundance of all glycoproteins expressed (RNA-seq) in our mESC clone (grey), compared to the glycoproteome experimentally detected (orange).

Figure 2. The glycoproteomes of human and mouse embryonic stem cells.

Glycan-mass histogram of all glyco-peptide spectrum matches (PSMs) identified (FDR < 1 %) from (a) murine and (c) human ESCs. Bin-width 1 Da. Indicated glycoproteins are species-specific or evolutionarily conserved (bold) and carry stemness-related glycans (labelled by their mass). (b) Deamidation of mESC derived glycopeptides upon PNGaseF treatment (blue) confirmed the identity of the N-glycopeptide sequences identified by our approach (orange). Chemical peptide deamidation was observed before PNGaseF treatment (grey). (d) Coverage of experimentally identified glycoproteins (orange) among all hypothetical (Uniprot) human glycoproteins (grey). (e) Cross-species comparison of glycoproteins identified in human (blue) and murine ESCs (green). (f and g) Comparison of glycan profiles at orthologous N-glycosylation sites of mouse Lamp1 and human LAMP1. Glycan-mass histograms of all glyco-PSMs (FDR < 1 %) specific for the N-glycosylation sites are indicated. Bin-width 1 Da. In all figures, glycan structures were inferred from their mono-saccharide compositions, based on known glycobiological rules and are represented according to the consensus pictogram symbols for different mono-saccharides.

Figure 3. Detection of specific fucosylated proteins.

(a) Slc35c1 transports GDP-fucose from the cytosol into the Golgi. Fut9 transfers fucose in α-1,3 linkage to LacNAc structures. Deletion of Slc35c1 abolishes fucosylation of secretory proteins in general. Ablation of Fut9 specifically affects synthesis of the Lewis X glyco-epitope. (b) Quantitative proteomics workflow for intact glycopeptides from Slc35c1 and Fut9 mutant (KO) and their genetically repaired (WT) mESC sister clones. (c) Quantitative glycoproteomics detects the reduction of all fucosylated glycopeptides (red) and the reciprocal increase of non-fucosylated glycopeptides (green) in Slc35c1 KO vs WT mESCs. Ablation of Fut9 affects a specific subset of fucosylated glycopeptides. Oligo-mannosidic type N-glycopeptides (blue) were not affected. Differential glycopeptide expression is shown by scatter-plotting TMT-reporter ion intensities for Slc35c1 KO (upper panel) and Fut9 KO (lower panel) versus WT cells. Data-points of Igf2r glycopeptide (ASIN¹⁵³²ASYSEK) glycoforms are indicated by circles. (d) Site-specific comparison of Igf2r glycopeptides. Differential glycopeptide expression is shown as the ratio of TMT-reporter ion intensities in Slc35c1 and Fut9 KO versus WT cells. The number of PSMs (n) considered for ratio calculations and box-plot representations, are indicated.

Figure 4. Validation of fucosylated proteins involved in ricin toxicity.

(a) Mixed populations of mutant (KO, GFP⁺) and their repaired respective wild-type (WT, mCherryCre⁺) cells were subjected to ricin [4ng/ml] for three days or left untreated (-) and analyzed for viability. The ratios of green to red cells (KO/WT) are shown for each candidate gene. Sister clones for the known ricin sensitivity gene B4Galt1 are shown as positive control and mixtures of GFP and mCherryCre control cells as negative control. (b) HEK cells, harboring CRISPR/Cas9-induced mutations in the indicated genes (2 different sgRNAs each) and control cells (scrambed sgRNAs) were subjected to ricin or left untreated. Cell survival was monitored using Alamar Blue cell viability assay. (c) Igf2r KO murine SCC-VII cells and reconstituted IGF2R wildtype and IGF2R* M6P-binding-domain mutant SCC-VII were subjected to ricin. Cell survival was monitored as above. (d, e) Mixed populations of Igf2r KO and IGF2R/IGF2R* expressing SCC-VII cells were subjected to ricin and the relative amount of IGF2R expressing cells was determined using flow cytometry (d) and confocal microscopy (e). Scale bar 50 μm. Each image is representative of six images of three independent experiments. Data in a, b, c, d are shown as mean ± SD of triplicate cultures. Experiments were repeated three times with similar results. *P<0.05, **P<0.01, ***P<0.001 (two-tailed Student’s t-test).