Extensive determination of glycan heterogeneity reveals an unusual abundance of high mannose glycans in enriched plasma membranes of human embryonic stem cells

Abstract

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation.

Fig. 1.

Representative MALDI-FT-ICR mass spectra of N-glycans found in hESC membranes in the positive detection ion mode. Glycans eluted from SPE fractions (10, 20, and 40% ACN) were combined prior to MS analysis. Glycan mass profiles of HSF-6 hESC (A) and H1 hESC (B) membranes are shown. The structures are putative and are based on accurate masses and tandem mass spectrometry.

Fig. 2.

Quantitation and isomer separation of N-glycans on cell membranes. The combined SPE fraction (10, 20, and 40%) was analyzed by nanoLC-Chip/TOF. Shown are representative base peak chromatograms of N-glycans found on four different cell membranes: HSF-6 hESC (A), H1 hESC (B), IMR-90 fibroblast (C), and normal breast cell line, MCF10A (D). Proposed structures corresponding to the most abundant glycan at that retention time are provided. An asterisk represents non-glycan peaks.

Fig. 3.

Extracted ion chromatogram of high mannose type glycans found in HSF-6 hESC. The left panel shows Man7 isomers, and the right panel shows Man8 isomers.

Fig. 4.

Glycan quantitation using peak areas of chromatograms from nanoLC-Chip/TOF MS. A, overall abundances based on N-glycan types. High mannose glycans are the most abundant in both H1 hESC and HSF-6 hESC membranes, whereas complex/hybrid glycans are in abundance in IMR-90 fibroblast and normal breast cell line, MCF 10A. B, glycan abundance profile of high mannose and complex/hybrid type glycans in H1 hESC and HSF-6 hESC membranes, respectively. Man8 and Man9 are the most abundant glycans in both hESC. The complex/hybrid type glycans were further sorted into four groups based on glycan composition and the number of fucose (n = 1–3). Sialic acid represents sialylated (but nonfucoyslated) glycans, and Hex:HexNAc represents nonsialylated and nonfucosylated glycans. The complex/hybrid type glycans are generally fucosylated in both hESC membranes.

Fig. 5.

Validation of glycan expression using lectins. A and B, fixed hESC colonies that were stained with Hoescht dye (nuclear stain), antibody against SSEA-4 with secondary antibody (depicted by AlexaFluor 594), and Con A-FITC and GNA-FITC (20 μg/ml). All of the colonies were positively stained with Hoescht and expressed SSEA-4. A, cells bound to Con A compared with colonies that were labeled with Con A inhibitory control (Con A+I). B, GNA-FITC also bound to cells, compared with the inhibitory control (GNA+I). All of the images were taken with 10× magnification. Scale bar, 20 μm. C and D, flow cytometry was used to quantify amount of lectin binding on live hESCs. C, this dot plot shows the populations of cells (black) that were double-labeled with both lectin (Con A-FITC) and SSEA-4 positive (APC) compared with the unstained population (gray) and inhibitory control (blue). D, these histograms represent the distribution of live hESCs depicted in C that were stained with Con A-FITC and GNA-FITC (40 μg/ml) compared with unstained (gray fill) and inhibitory controls (blue).