Development of Fluorescently Labeled SSEA-3, SSEA-4, and Globo-H Glycosphingolipids for Elucidating Molecular Interactions in the Cell Membrane

Abstract

Glycosphingolipids (GSLs), such as the globo-series GSLs stage-specific embryonic antigen 3 (SSEA-3), SSEA-4, and Globo-H, are specifically expressed on pluripotent stem cells and cancer cells, and are known to be associated with various biological processes such as cell recognition, cell adhesion, and signal transduction. However, the behavior and biological roles of these GSLs are still unclear. In our previous study, we observed the interactions between the lipid raft and GSLs in real-time using single-molecule imaging, where we successfully synthesized various fluorescent analogs of GSLs (e.g., GM1 and GM3). Here, we have developed fluorescent analogs of SSEA-3, SSEA-4, and Globo-H using chemical synthesis. The biophysical properties of these analogs as raft markers were examined by partitioning giant plasma membrane vesicles from RBL-2H3 cells into detergent-resistant membrane fractions and liquid-ordered/liquid-disordered phases. The results indicated that the analogs were equivalent to native-type GSLs. The analogs could be used to observe the behavior of globo-series GSLs for detailing the structure and biological roles of lipid rafts and GSL-enriched nanodomains during cell differentiation and cell malignancy.

Glycosphingolipid-Glycan Signatures of Acute Myeloid Leukemia Cell Lines Reflect Hematopoietic Differentiation

“… 39 Accordingly, one may speculate about the potential immunosuppressive roles of (neo)lacto-series GSLs in AML. Meanwhile, both globosides and SSEA-3, which is a surface marker of human embryonic stem cells 40 and human-induced pluripotent stem cells, 41 showed positive correlations with A4GALT which encodes the corresponding GT responsible for the biosynthesis of globoside glycans. Similarly, the expression of B4GALNT1 encoding for a key enzyme in ganglioside biosynthesis positively associated with ganglioside levels.…”

Development of lacto-series ganglioside fluorescent probe using late-stage sialylation and behavior analysis with single-molecule imaging

“…To afford amino NeuAcLc 4 Cer 3, we designed bicyclic sialyl donor 4, where C9-OH is replaced by the NHTFAc group, with the intention of later converting it into an NH 2 group by global deprotection. 8 As a coupling partner of 4, we envisioned the Lc 4 Cer acceptor 5 with p-tert -butylbenzoyl (TBBz) groups, 24 which would improve the solubility of the glycolipids at low temperatures. Furthermore, a 3,4-diol of the non-reducing terminal Gal was designed to increase C3-OH reactivity.…”

Advanced Chemical Methods for Stereoselective Sialylation and Their Applications in Sialoglycan Syntheses

“…To date, in our synthesis of gangliosides, the α‐stereoselective outcome of sialylation depends on the influence of the 5 N ‐substituent (mainly Troc, TFAc). However, after their development, macrobicyclic sialyl donors have been extensively used for sialylation in ganglioside synthesis, as exemplified by the very robust and straightforward synthesis of stage‐specific embryonic antigen 3 (SSEA‐3), SSEA‐4, and Globo‐H [58a] . In particular, for the synthesis of SSEA‐4, tetrasaccharide acceptor 168 was glycosylated using a macrobicyclic sialyl donor 167 to exclusively produce pentasaccharide 169 in 75 % yield (Scheme 40).…”

“…A macrobicyclic donor 93 bearing dibenzylphosphate as a leaving group could be used for C-glycoside formation. Furthermore, our sialylation method was used in some of the most challenging syntheses of sialoglycans such as gangliosides [58] and α(2,8)-linked sialic acid pentamers. These results demonstrate the high reliability and utility of our method for the synthesis of diverse sialic acid-containing compounds.…”