Use of lectins for probing differentiated human embryonic stem cells for carbohydrates

Abstract

The carbohydrates present on the surface of differentiated human embryonic stem cells (hESCs) are not yet well established. Here, we have employed a panel of lectins and several anti-carbohydrate antibodies to determine the carbohydrates that are present at day 12 of hESC differentiation as embryoid bodies (EBs). On the basis of staining with fluorescein-labeled lectins, we have determined the presence of both terminal and internally linked alpha-d-mannopyranosyl groups, poly-N-acetyllactosaminyl chains, both alpha2,3- and alpha2,6-linked N-acetylneuraminic acid (Neu5Ac), alpha1,6-linked l-fucosyl, and beta-D-galactosyl groups, and more specifically, the T, Tn, and sialyl-Tn antigens. However, no alpha1,2-linked l-fucosyl, terminal nonreducing alpha-D-galactosyl, N-acetyl-beta-D-glucosaminyl, nor N-acetyl-alpha-D-galactosaminyl groups were found by this approach. We also established the presence of Neu5Acalpha2,3/2,6-Galbeta1,4 GlcNAc-terminated chains on the surfaces of 12-day-old EBs, as indicated by the great enhancement of staining by Erythrina cristagalli agglutinin (ECA) after treatment with neuraminidase. In each case, inhibition of binding by a haptenic sugar or treatment with neuraminidase was used to eliminate the possibility of nonspecific binding of the lectins. A comparison with undifferentiated cell staining revealed an increase in alpha2,3-linked Neu5Ac as well as a change to exclusively alpha1,6-linked l-fucose upon differentiation.

Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells

“…Furthermore, we quantitate the relative contribution of each glycan types as well as specific isomers on the cell surface. These experiments differ from those by Wearne et al (12) and Satomaa et al (25) in that those were performed on whole cell lysates. The cell surface glycosylation were inferred indirectly using lectins, which are not quantitative when comparing different glycan types.…”

Targeting Aberrant Sialylation in Cancer Cells Using a Fluorinated Sialic Acid Analog Impairs Adhesion, Migration, and In Vivo Tumor Growth

“…a2,3-linked sialic acids were detected using MALII, a2,6-linked sialic acids by SNA-I, exposure of terminal (b-)galactose/T antigen by PNA, exposure of terminal (a-)galactose and (a-)GalNAc by GSL-I, (poly) GlcNAc (chitobiose) by WGA, and (a-)fucose by AAL (30)(31)(32). Free lectin was removed by washing the cells in carbofree blocking solution, and cell-bound biotinylated lectin was conjugated with 2 mg/mL streptavidin-phycoerythrin for 15 minutes (33).…”

Glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage

“…The majority of the studies performed to date have been carried out by using different glycan binding proteins, e.g. lectins or antibodies against glycan epitopes [22][23][24]. Of these binders, antibodies against SSEA-3, SSEA-4, Tra-1-60, Tra-1-81 [25,26] have been generally accepted as tools for embryonic stem cell validation [27].…”