Raft domains have been drawing extensive attention as a platform for signaling in cell plasma membranes (PMs). Gangliosides, glycosphingolipids containing one or more sialic acids, are representative raft markers and play a variety of important physiological roles such as invasion of microbial toxins, viruses, and bacteria into cells, regulation of receptor activities, glycosynapse formation1). So far, for immune-fluorescence imagining and immune-electron microscopy to observe the distribution of gangliosides in living cell PMs, the binding proteins such as the antibodies and cholera toxin subunit B (CTxB) have been used. However, since these binding proteins are multivalent and gangliosides were crosslinked, the distribution, dynamics and curvature would be changed even after chemical fixation with 4% paraformaldehyde and 0.2% glutaraldehyde2). Furthermore, due to lack of fluorescent ganglioside probes that faithfully mimic native ganglioside behaviors, the lateral distribution and dynamic behaviors of gangliosides in living cell PMs have been scarcely investigated.Figure 1. Chemical structures of fluorescent GM1, GM2, GM3, and GD1b analoguesDevelopment of ganglioside probesTo solve this issue, in collaboration with Ando and Kiso groups, we tried to develop the subfamilies of fluorescent ganglioside probes which faithfully mimic the native ganglioside behavior3). GM1, GM2, GM3 and GD1b probes were synthesized and conjugated with fluorescent dyes at the C9 position of the sialic acid (S9 type for GM1, GM2 and GM3), C6 position of the terminal galactose (termG6 type for GM1 and GD1b), or C6 position of the GalNAc (GN6 type for GM2) (Figure 1). All these ganglioside probes preferentially partitioned into detergent resistant membrane (DRM) fraction and liquid ordered (Lo) phase in giant plasma membrane vesicles (GPMVs). Furthermore, the GM1 probe bound with CTxB at the same affinity as unlabeled GM1, and the GM3 probe associated with WGA at the same affinity as unlabeled GM33). These results show that the ganglioside probes should meet the following requirements to make sure that they behave like their parental molecules4,5) : 1) gangliosides should be conjugated with hydrophilic dyes such as ATTO488, ATTO594 and fluorescein; and 2) the fluorescent dyes should be conjugated at the terminal 47Unraveling of regulation mechanisms ofreceptor activity by gangliosidesKenichi G. N. SuzukiGifu University, Institute for Glyco-core Research
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