Following a retrospective view on development of the present concept of microdomain, current views of multiple microdomains are briefly presented. Based on physical properties of membrane components, some lipids are clustered, segregated from other lipids, and organized with cytoplasmic lipophilic signal transducer molecules and with transmembrane receptors with hydrophobic domain, and their associated partners. Typical examples are: (i) glycosphingolipids (GSLs) and sphingomyelin organized with cSrc, Src family kinases, and small G-proteins, collectively termed “glycosignaling domain (GSD)” and involved in GSL-dependent cell adhesion and signaling; (ii) caveolae and caveolar membrane, characterized by high content of cholesterol and caveolin, organized with GPI-anchors and signal transducers, and involved in endocytosis and signal transduction; (iii) integrins and tetraspan membrane proteins (TMP) complexed with some type of GSL; (iv) growth factor receptors with tyrosine kinases complexed with cholesterol-enriched domain, with or without some type of GSL. The defined composition and association of signal transducer molecules in microdomain iii and iv is yet unclear. Relative quantity and proportion of these types of microdomain differs and is characteristic for different types of cells.

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The glycosphingolipid in the cell membrane seems to exist in “rafs”, and the research on the raft to understand its biological meaning has been carried out as described in the minireview of this special issue. Though it is important to clarify the effect of raft formation on the biochemical function, it is also necessary to study the physiochemical factor in the formation of glycolipid domain. In the author's research, the effect of the matrix lipid on recognition function of the glycolipid was examined using the lipid monolayer as a biomembrane model. In addition, the driving force in which glycolipids form cluster structures has been examined by analysis of the surface pressure-molecular area isotherm of an air-water interface monolayer, and by observation of the topology of mixed lipid membranes by atomic force microscope.

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Recent years have been characterized by a huge interest in the structure and function of mammalian cell membrane lipid domains. The interest in this subject grew further, when their participation in important membrane-associated events such as signal transmission, cell adhesion and lipid/protein sorting was postulated. A common feature of cell membrane domains is their peculiar lipid composition, being enriched in glycosphingolipids, sphingomyelin and cholesterol. A series of theoretical considerations and several experimental data suggest that glycosphingolipids play an important role in the formation and function of membrane domains. Within this review, the involvement of glycosphingolipids in the biogenesis, structure and function of domains is discussed in light of their strong amphiphilic nature and of their peculiar chemical features. These features differentiate glycosphingolipids from other lipids in the membrane, allowing either self-interaction or interaction with other membrane components and external ligands. Due to these interactions, glycosphingolipids undergo lateral phase, separation, segregation, and therefore form core domains within the membrane; glycosphingolipid domains constitute the nucleation point that allows co-segregation of other lipids and proteins in a complex domain; finally, glycosphingolipids confer dynamic properties on domains, that are essential to the modulation of cell functions.

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Glycosphingolipid-cholesterol formed as microdomains in cell membranes have been proposed to function as rafts for the attachment of specific proteins including glycosylphosphatidylinositol-anchored proteins, glycoproteins and proteoglycans. The microdomains are postulated to be involved in transmembrane signaling. Here, the functional roles of glycoconjugates in the physical properties of these microdomains, as well as in signal transduction are discussed.

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Ligand- or antibody-mediated aggregation of surface receptors, such as antigen receptors on T cells, B cells and mast cells, induces signaling pathways resulting in diverse functional responses. Cell activation can also be initiated by aggregation of glycoproteins anchored to the plasma membrane via glycosylphosphatidylinositol (GPI), as well as by some glycolipids. Numerous recent data suggest that the signaling capacity of the aggregated membrane glycoconjugates reflects their association with specialized membrane microdomains (lipid rafts), enriched in glycosphingolipids, cholesterol and signal transduction molecules. These domains are relatively resistant to solubilization by nonionic detergents at low temperature, and due to their low density they can be easily isolated by density gradient ultracentrifugation. Under certain conditions the lipid rafts can be observed by microscopy. Although the concept of lipid rafts as hot spots of transmembrane signaling (signalosomes) is an attractive hypothesis, the issue is controversial and little is known about their properties and functions under in vivo conditions. This minireview is focused on recent research on the role of lipid rafts in transmembrane signaling, based mostly on biochemical, morphological and functional studies of mast cells and their derivatives.

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Shiga toxin, an enterotoxin produced from Shigella dysenteriae serotype and enterohemorrhagic Escherichia coli binds specifically to globotriaosylceramide, Gb3, on the cell surface and causes cell death. Stx is shown to induce apoptosis in several cell lines, including human renal tubular cell line ACHN and Burkitt's lymphoma Ramos cells. In order to study the early signal transduction after Stx addition, Gb3-enriched microdomains were prepared from these cells by sucrose density gradient centrifugation of Triton X-100 lysate as buoyant, detergent-insoluble microdomain, rafts. Gb3 was only recovered in rafts and associated with Src family kinase Yes or Lyn. Tyrosine residues of raft proteins were hyperphosphorylated in 10min and turned to resting level in 30min after Stx addition. Yes was thought to be responsible for hyperphosphorylation observed in raft proteins, because Yes activity increased in 3-10min after Stx addition. Unexpectedly, however, all of the Yes activity was obtained in high-density, detergent-soluble fraction, but not in rafts. Stx was known to be internalized as complex with Gb3 via retrograde endocytosis. Therefore, Yes was also thought to enter into intracellular space with Stx/Gb3 complex, where Yes was activated and increased solubility for Triton X-100. Since Stx B subunit could alone induce Yes activation, the binding of Stx to Gb3 might cause temporal activation of Yes in DS in the process of apoptosis.

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Previous studies have suggested that caveolae, glycosphingolipid- and cholesterol-enriched microdomains, or lipid rafts, within the plasma membrane of eukaryotic cells are implicated in many important cellular events, such as polarized sorting of apical membrane proteins in epithelial cells and signal transduction. Until recently, however, the existence of such domains in vivo remained controversial. The past few years have brought compelling evidence that microdomains indeed exist in living cells. These special membrane structures have a very unusual polarity in their enrichment of sphingolipids such as ganglioside GM1 (GM1), sphingomyelin, cholesterol, and signaling molecules such as receptor-type tyrosine kinase, G proteins, glycophosphatidylinositol-anchored proteins such as c-Src. In the nervous system, however, these membrane structures have been scarcely investigated, although the neuron is the most attractive and the greatest challenge to research on membrane traffic and function. An increasing body of evidence has suggested that neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3/4 (NT-3/4) are actually involved in the process of neuronal differentiation, survival, and synaptic plasticity. Recent studies including ours have suggested that the receptors for these neurotrophic factors, Trk-family tyrosine kinase, are present in these special membrane structures and they modulate these Trk-family tyrosine kinase functions. In this review, I will focus on the current understanding of the structure and the function of these special membrane microdomains in neuronal cells, especially their action on the cellular signaling events.

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In the first part of this review, a new and efficient strategy for the synthesis of the sugar chains of glycoproteins is described. For the synthesis of N-linked sugar chains, the core-trisaccharide was synthesized by a chemical method with the aid of lipase. The side chain oligosaccharides were obtained enzymatically, using glycosidases, and the branched oligosaccharide blocks were obtained by regioselective chemical synthesis. Finally, the synthesized core-trisaccharide and side chain oligosaccharide blocks were combined to obtain N-linked sugar chains of arbitral structures.
In the second part of this review, stepwise elongation of sugar chains employing exo-glycosidases to construct O-linked glycopeptides from GalNAc-linked peptides is described. Moreover, one-step syntheses using endo GlcNAc'ase or endo GalNAc'ase were performed for the construction of N-linked or O-linked glycopeptides, respectively. Several examples of the synthesis of glycopeptides are given.

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To translate the emerging insights into the functionality of the sugar code into applications in organic materials science, we have paid special attention to carbohydrate-protein (lectin) interactions. They are increasingly delineated to play an important role in biological recognition systems. Thus, the custom-made design of research tools and the examination of how to practically exploit them are becoming burgeoning research areas for producing new functional materials. We here report preparation and characterization of novel types of neoglycoprotein-liposome conjugates, and indicate applications by studying recognition functions of these tailored carriers with defined sugar part as the recognition function using a model system and in vivo experiments. Various types of neoglycoprotein-liposome conjugates were prepared according to a method including preparation of mixed micelles and then of liposomes, chemical coupling of neoglycoproteins to the characterized liposomes, and further sequential enzymatic glycosylation to refine the glycan part. The assays indicated carbohydrate-specific recognition functions of these neoglycoprotein-liposome conjugates. Monitoring of tissue distribution using Ehrlich solid tumor-bearing mice showed individual response of diverse tissues towards various types of applied neoglycoprotein-liposome conjugates harboring a series of sugar chain ligands including mono-and oligosaccharides. This type of carbohydrate-conjugated material is expected to find applications in basic glycoscientific research as well as in applied areas such as tissue-specific drug targeting material

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