Regulation of glycan structures in murine embryonic stem cells: combined transcript profiling of glycan-related genes and glycan structural analysis

Abstract

The abundance and structural diversity of glycans on glycoproteins and glycolipids are highly regulated and play important roles during vertebrate development. Because of the challenges associated with studying glycan regulation in vertebrate embryos, we have chosen to study mouse embryonic stem (ES) cells as they differentiate into embryoid bodies (EBs) or into extraembryonic endodermal (ExE) cells as a model for cellular differentiation. We profiled N- and O-glycan structures isolated from these cell populations and examined transcripts encoding the corresponding enzymatic machinery for glycan biosynthesis in an effort to probe the mechanisms that drive the regulation of glycan diversity. During differentiation from mouse ES cells to either EBs or ExE cells, general trends were detected. The predominance of high mannose N-glycans in ES cells shifted to an equal abundance of complex and high mannose structures, increased sialylation, and increased α-Gal termination in the differentiated cell populations. Whereas core 1 O-glycan structures predominated in all three cell populations, increased sialylation and increased core diversity characterized the O-glycans of both differentiated cell types. Increased polysialylation was also found in both differentiated cell types. Differences between the two differentiated cell types included greater sialylation of N-glycans in EBs, whereas α-Gal-capped structures were more prevalent in ExE cells. Changes in glycan structures generally, but not uniformly, correlated with alterations in transcript abundance for the corresponding biosynthetic enzymes, suggesting that transcriptional regulation contributes significantly to the regulation of glycan expression. Knowledge of glycan structural diversity and transcript regulation should provide greater understanding of the roles of protein glycosylation in vertebrate development.

FIGURE 1.

Relative transcript abundance for N-glycan processing steps involving trimming and branching steps in the endoplasmic reticulum and Golgi complex for mouse ES cells, EBs, and ExE cells. A, schematic representation for N-glycan trimming, branching, and modifications for high mannose, hybrid, and complex N-linked oligosaccharides using the glycan schematic nomenclature indicated in the key and as employed previously (13). Linkages are shown for each step of the biosynthetic pathway, and the numbers in the blue ovals designate the pathway steps in A that link to the transcript abundance data in the corresponding numbered steps in B and C. Relative transcript abundances (C) for the three mouse cell types are shown as a set of grouped histograms plotted on a log₁₀ scale above the corresponding pathway step number (blue numbered oval) and the corresponding gene name(s). Multiple genes for a given pathway step are listed where multiple distinct subunits contribute to catalysis or where several genes within a common family encode enzymes capable of creating the specified linkage. *, statistically significant change (p < 0.05) in relative transcript abundance for either EBs or ExE cells relative to ES cells. The -fold change in relative transcript abundance for each gene is shown in B, plotted as a histogram in linear scale for EBs and ExE cells compared with ES cells. Transcripts present at a higher level in EBs or ExE cells are shown as bars above the axis (positive -fold change), and those with higher levels in ES cells relative to the differentiated cells are shown as bars below the axis (negative -fold change). The horizontal black line at 1-fold indicates no change in transcript abundance between ES cells and EBs or ExE cells. Hash marks are shown where values exceed the axes, and the resulting value is indicated. Error bars in B and C indicate the S.E. for four biological replicates.

FIGURE 2.

-Fold change and prevalence of N-linked glycan classes. The prevalences of individual N-linked glycans were calculated as percentage of total profile (% Total Profile) as described under “Experimental Procedures.” Prevalences for glycans that fall into the indicated structural classes were summed, and classes that exhibited statistically significant differences between EBs, ExE cells, and ES cells are presented (see supplemental Fig. 6 for all glycan classes and for the description of the class abbreviations). A, -fold change in glycan prevalence is shown for EBs and ExE cells compared with ES cells. Glycans present at a higher level in EB or ExE cells are shown as bars above the axis (positive -fold change), and those with higher levels in ES cells relative to the differentiated cells are shown as bars below the axis (negative -fold change). The horizontal black line at 1-fold indicates no change in glycan prevalence between ES cells and EBs or ExE cells. B, glycan prevalences for each cell type are presented. *, statistically significant changes (p < 0.05) for either EBs or ExE cells relative to ES cells. Error bars in A and B indicate the S.E. for three biological replicates.

FIGURE 3.

Relative transcript abundance for biosynthetic pathways leading to the synthesis of O-linked (polypeptide-GalNAc core mucin type) structures in mouse ES cells, EBs, and ExE cells. A, schematic representations for the synthesis of the polypeptide-GalNAc linkages and extension into various branched and extended structures using the glycan schematic nomenclature indicated in the key and as employed previously (13). The relative transcript abundances for O-glycan biosynthetic enzymes from the three mouse cell populations (C) are shown as a set of grouped histograms above the corresponding pathway step number as described in the legend to Fig. 1. The -fold change in relative transcript abundance for each gene is shown in B for EBs and ExE cells compared with ES cells, as described in the legend to Fig. 1. Additional capping and extension reactions for core 2, 3, 4, and 6 O-linked structures are also found in Fig. 4.

FIGURE 4.

Relative transcript abundance for processing steps leading to the extension of complex capping reactions for N-glycan, O-glycan, and glycolipid structures in mouse ES cells, EBs, and ExE cells. A, schematic representation of complex capping reactions for the N-glycan, O-glycan, and glycolipid classes indicated by the boxed structures on the left and the branched scheme for glycan processing to various complex termini are indicated using the glycan schematic nomenclature shown in the key and as employed previously (13). Structures subterminal to the nonreducing terminal GlcNAc residues indicated by a number symbol in the boxed structures are subsequently depicted as R in the remainder of the figure. Individual branches of the complex processing pathways are labeled and shown with colored backgrounds to designate different classes of complex termini. The display of relative transcript abundances for the complex capping reactions from the three mouse cell populations (panels labeled C) is shown as a set of grouped histograms above the corresponding pathway step number as described in the legend to Fig. 1. The -fold change in relative transcript abundance for each gene is shown in the panels labeled B for EBs and ExE cells compared with ES cells as described in the legend to Fig. 1.

FIGURE 5.

-Fold change and prevalence of O-linked glycan classes. The prevalences of individual O-linked glycans were calculated as percentage of total profile (% Total Profile) as described under “Experimental Procedures.” Prevalences for glycans that fall into the indicated structural classes were summed, and classes that exhibited statistically significant differences between EBs, ExE cells, and ES cells are presented (see supplemental Fig. 9 for all glycan classes and for the description of class abbreviations). A, the -fold change in glycan prevalence is shown for EBs and ExE cells compared with ES cells. Glycans present at a higher level in EBs or ExE cells are shown as bars above the axis (positive -fold change), and those with higher levels in ES cells relative to the differentiated cells are shown as bars below the axis (negative -fold change). The horizontal black line at 1-fold indicates no change in glycan prevalence between ES cells and EBs or ExE cells. B, glycan prevalences for each cell type are presented. *, statistically significant changes (p < 0.05) for either EBs or ExE cells relative to ES cells. Error bars in A and B indicate the S.E. for three biological replicates.

FIGURE 6.

Transcript abundance and immunodetection of cell surface polysialic acid glycan structures on ES cells, EBs, and ExE cells. Normalized transcript abundance for St8sia2, St8sia4, and Ncam1 were determined (B), and -fold changes in transcript abundance for EBs and ExE cells relative to ES cells are indicated in A. Polysialyltransferase transcripts for St8sia2, but not St8sia4, were elevated in the differentiated cell populations, whereas Ncam1 transcripts were modestly elevated only in ExE cells. Cell surface staining for polysialic acid was performed using anti-polysialic acid antibodies on fixed preparations of ES cells, EBs, or ExE cells. ES cells and EBs were probed with either OL-28 monoclonal antibody specific for α2,8-polysialic with degree of polymerization >4 (C) or S2-566 monoclonal antibody specifically recognizing Neu5α2,8-Neu5Acα2,3-Gal (D) (46). ES cells exhibited weak background antibody staining, whereas EBs displayed robust cell surface staining with both antibodies. ES cells (E) and ExE cells (F) were probed with the OL-28 antibody (red) and an antibody to the Dab2 protein (green). Dab2 is a clathrin-associated sorting protein that has been shown to be involved in cell positioning and formation of visceral endoderm during mouse embryogenesis (117) and is an established definitive endodermal marker (47). ES cells, indicated by nuclear staining with DAPI (blue), were negative for OL-28 and Dab2 immunoreactivity (individual staining with OL-28, Dab2, and DAPI as indicated; merge of three staining patterns is shown in the top left panel of E). ExE cells were positive for cell surface staining with OL-28 and the intracellular punctate staining with anti-Dab2 (individual staining with OL-28, Dab2, and DAPI as indicated; merge of three staining patterns is shown in the top left panel of F), indicating an up-regulation of cell surface polysialic acid structures in the differentiated cell populations. White bar in each panel, 100 μm.

FIGURE 7.

Relative transcript abundance for biosynthetic pathway steps leading to the synthesis of O-linked (non-polypeptide-GalNAc mucin type and non-polypeptide-Xyl proteoglycan type) structures in mouse ES cells, EBs, and ExE cells. A, schematic representation of the synthesis of various polypeptide-Fuc, polypeptide-Man, polypeptide-Glc, and polypeptide-GlcNAc linkages with extensions into various linear and branched structures using the glycan schematic nomenclature indicated in the key. The display of relative transcript abundances for O-glycan biosynthetic enzymes from the three mouse cell populations (C) is shown as a set of grouped histograms above the corresponding pathway step number as described in the legend to Fig. 1. The -fold change in relative transcript abundance for each gene is shown in B for EBs and ExE cells compared with ES cells, as described in the legend to Fig. 1.

FIGURE 8.

Hierarchical clustering of N- and O-linked glycans and transcripts encoding N-linked glycosylation enzymes in ES cells, EBs, and ExE cells. A, N-linked glycan classes that exhibited statistically significant differences in prevalence between ES cells and either EBs or ExE cells (see Fig. 2) were used to calculate -fold changes relative to the mean prevalence values measured in ES cells. The -fold changes were converted to log₂ values and used to cluster the three biological replicates of each cell type. B, O-linked glycan classes that exhibited statistically significant differences in prevalence between ES cells and either EBs or ExE cells (see Fig. 5) were used to calculate -fold changes relative to the mean prevalence values measured in ES cells. The -fold changes were converted to log₂ values and used to cluster the three biological replicates of each cell type. C, transcript abundances for N- and O-linked biosynthetic genes that exhibited statistically significant differences between ES cells and either EBs or ExE cells were used to calculate -fold changes relative to the mean abundance value measured for ES cells. The -fold changes were converted to log₂ values and used to cluster the four biological replicates in each cell type. The resulting hierarchical clusters for glycans or transcripts resolved the cell populations into ES cell or differentiated cell types.

FIGURE 9.

Pathway diagram for N-glycan biosynthesis incorporating transcript abundance and glycan prevalence. A, -fold changes in glycan prevalence and transcript abundance comparing EBs to ES cells. The color of each circle within the pathway diagram denotes the -fold change in the indicated glycan structure based on the scale of the heat map shown at the bottom right. Glycans at each node are represented by schematics and denoted by numbers that refer to the glycan structures and their prevalences shown in supplemental Figs. 4 and 5. The color of each arrow denotes the -fold change in the indicated transcript based on the same heat map scale at the bottom right. Key processing steps that take high mannose precursors toward complex glycans are diagrammed across the middle of the panel. Biosynthetic steps that branch toward the top of the diagram generate core-fucosylated glycans, and those that branch toward the bottom produce glycans without core Fuc. Interestingly, the changes in the upper and lower branching pathways are not mirror images, indicating that core fucosylation influences or is a consequence of alterations in N-linked glycan processing. The large circles within the grayed areas are pie charts that describe the distribution of complex glycans possessing the indicated structural characteristics in EBs. The individual colors of the pie slices denote the -fold change in the indicated class of glycan between EBs and ES cells, whereas the area of each pie segment reflects the prevalence of that glycan class in EBs. The grayscale pie charts to the right of A present the distribution of prevalences for the complex glycan classes in ES cells for reference. B, same as A except that it compares ExE cells with ES cells. The general trends are similar for EBs and ExE cells, although -fold changes tend to be of greater magnitude for ExE cells. In several instances, the glycan product is decreased for a biosynthetic step catalyzed by an enzyme whose transcript is increased. See “Discussion” for more details. GG, glycans with Gal-Gal termini; Bi, biantennary glycans; Tri, triantennary glycans; Tetra, tetraantennary glycans.