Naoko Komura

Raft-based interactions of gangliosides with a GPI-anchored receptor

Gangliosides, glycosphingolipids containing one or more sialic acid(s) in the glyco-chain, are involved in various important physiological and pathological processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or preparations. Single-fluorescent-molecule imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59s transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside molecules were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities.

The First Total Synthesis of Ganglioside GalNAc‐GD1a, a Target Molecule for Autoantibodies in Guillain–Barré Syndrome

The first synthesis of ganglioside GalNAc-GD1a, featuring efficient glycan assembly and a cyclic glucosyl ceramide as a versatile unit for ganglioside synthesis is described. Although ganglioside GalNAc-GD1a was first found as a brain ganglioside, IgG autoantibodies to GalNAc-GD1a were subsequently found to be closely related to a human peripheral-nerve disorder, Guillain-Barré syndrome, which is the commonest cause of acute flaccid paralysis worldwide. In this study, the characteristic hexasaccharide part carrying two sialic acid residues was synthesized efficiently by use of a readily accessible GM2-core unit as a common unit. The potentially difficult coupling of the oligosaccharide and ceramide moieties was carried out by using a cyclic glucosyl ceramide as a coupling partner for the hexasaccharide part, thereby successfully providing the framework of the target compound. Global deprotection delivered the homogenous ganglioside GalNAc-GD1a. An enzyme-linked immunosorbent assay showed that sera from patients with Guillain-Barré syndrome reacted both with natural and with synthetic GalNAc-GD1a.

Expanded potential of seleno-carbohydrates as a molecular tool for X-ray structural determination of a carbohydrate–protein complex with single/multi-wavelength anomalous dispersion phasing

Seleno-lactoses have been successfully synthesized as candidates for mimicking carbohydrate ligands for human galectin-9 N-terminal carbohydrate recognition domain (NCRD). Selenium was introduced into the mono- or di-saccharides using p-methylselenobenzoic anhydride (Tol2Se) as a novel selenating reagent. The TolSe-substituted monosaccharides were converted into selenoglycosyl donors or acceptors, which were reacted with coupling partners to afford seleno-lactoses. The seleno-lactoses were converted to the target compounds. The structure of human galectin-9 NCRD co-crystallized with 6-MeSe-lactose was determined with single/multi-wavelength anomalous dispersion (SAD/MAD) phasing and was similar to that of the co-crystal with natural lactose.

Development of new ganglioside probes and unraveling of raft domain structure by single-molecule imaging

Gangliosides are involved in a variety of biological roles and are a component of lipid rafts found in cell plasma membranes (PMs). Gangliosides are especially abundant in neuronal PMs and are essential to their physiological functions. However, the dynamic behaviors of gangliosides have not been investigated in living cells due to a lack of fluorescent probes that behave like their parental molecules. We have recently developed, using an entirely chemical method, four new ganglioside probes (GM1, GM2, GM3, and GD1b) that act similarly to their parental molecules in terms of raft partitioning and binding affinity. Using single fluorescent-molecule imaging, we have found that ganglioside probes dynamically enter and leave rafts featuring CD59, a GPI-anchored protein. This occurs both before and after stimulation. The residency time of our ganglioside probes in rafts with CD59 oligomers was 48ms, after stimulation. The residency times in CD59 homodimer and monomer rafts were 40ms and 12ms, respectively. In this review, we introduce an entirely chemical-based ganglioside analog synthesis method and describe its application in single-molecule imaging and for the study of the dynamic behavior of gangliosides in cell PMs. Finally, we discuss how raft domains are formed, both before and after receptor engagement. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.

Syntheses of Fluorescent Gangliosides for the Studies of Raft Domains

Gangliosides, glycosphingolipids containing one or more sialic acids in the glycan chain, are involved in various important biological processes in cell plasma membranes (PMs). However, the behaviors and functions of gangliosides are poorly understood, primarily because of the lack of fluorescent analogs that are equivalent to native gangliosides that can be used as chemical and physical probes. In this study, we developed entirely chemical methods to synthesize fluorescent gangliosides (GM3, GM2, GM1, and GD1b) in which the glycan components are site-specifically labeled with various fluorescent dyes. The functional evaluations of the synthesized fluorescent gangliosides demonstrated the great influence of fluorescent dye on the physical properties of gangliosides in PMs and revealed the fluorescent ganglioside analogs which show similar behaviors to the native gangliosides.

Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs

Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking. Single-molecule tracking of fluorescent GM1 and GM3 revealed that these molecules are transiently and dynamically recruited to monomers (monomer-associated rafts) and homodimer rafts of the raftophilic GPI-anchored protein CD59 in quiescent cells, with exponential residency times of 12 and 40ms, respectively, in a manner dependent on raft-lipid interactions. Upon CD59 stimulation, which induces CD59-cluster signaling rafts, the fluorescent GM1 and GM3 analogs were recruited to the signaling rafts, with a lifetime of 48ms. These results represent the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they show that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner, with much higher frequency than expected previously. Such studies would not have been possible without fluorescent ganglioside probes, which exhibit native-like behavior and single-molecule tracking. In this chapter, we review the methods for single-molecule tracking of fluorescent ganglioside analogs and the results obtained by applying these methods.