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BBA - General Subjects (v.1780, #3)

Editorial Board (pp. ii).

Introduction to the special issue on glycobiology and sphingobiology by Carl G. Gahmberg; Roger A. Laine (pp. 323-324).

Structure and function of glycosphingolipids and sphingolipids: Recollections and future trends by Sen-itiroh Hakomori (pp. 325-346).

Based on development of various methodologies for isolation and characterization of glycosphingolipids (GSLs), we have identified a number of GSLs with globo-series or lacto-series structure. Many of them are tumor-associated or developmentally regulated antigens. The major question arose, what are their functions in cells and tissues? Various approaches to answer this question were undertaken. While the method is different for each approach, we have continuously studied GSL or glycosyl epitope interaction with functional membrane components, which include tetraspanins, growth factor receptors, integrins, and signal transducer molecules. Often, GSLs were found to interact with other carbohydrates within a specific membrane microdomain termed “glycosynapse”, which mediates cell adhesion with concurrent signal transduction. Future trends in GSL and glycosyl epitope research are considered, including stem cell biology and epithelial–mesenchymal transition (EMT) process.

Keywords: Abbreviations; CCI; carbohydrate-to-carbohydrate interaction; Csk; C-terminal Src kinase; DMS; d; -; erythro; -; N; ,; N; -dimethyl-Sph; EGF; epidermal growth factor; EGFR; epidermal growth factor receptor; EMT; epithelial–mesenchymal transition; FGF; fibroblast growth factor; FGFR; fibroblast growth factor receptor; FN; fibronectin; GEM; glycolipid-enriched microdomain; GSL; glycosphingolipid; LN; laminin; MS; mass spectrometry; PDGF; platelet-derived growth factor; PDGFR; platelet-derived growth factor receptor; PKC; protein kinase C; Sph; sphingosine; Sph-1-P; sphingosine-1-phosphate; SSEA; stage-specific embryonic antigen; TMS; d; -; erythro; -; N; ,; N; ,; N; -trimethyl-Sph; TSP; tetraspanin; Glycosphingolipids are abbreviated as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (; Biochem J; 171: 21–35, 1978, Table I) however, the suffix -Ose or -OseCer is omitted. Ganglio-series gangliosides are abbreviated according to the extended version of Svennerholm's list (e.g., Holmgren et al.,; Proc Natl Acad Sci USA; 77: 1947–1950, 1980)Glycosyl epitope; Glycosphingolipid; Ganglio-series; Globo-series; Lacto-series; Tumor-associated antigen; Developmentally regulated antigen; Contact inhibition; Cell growth; Cell motility; Cell adhesion; Signal transduction; Carbohydrate–carbohydrate interaction; Microdomain; Glycosynapse; Sphingosine-dependent kinase; Protein kinase Cδ; Tetraspanin; Growth factor receptor; Integrin; Epigenetic regulation; Stem cell; Niche; Epithelial–mesenchymal transition (EMT)

The medium is the message: Glycosphingolipids and their soluble analogues by M. De Rosa; H.-J. Park; M. Mylvaganum; B. Binnington; N. Lund; D.R. Branch; C.A. Lingwood (pp. 347-352).

We have made adamantylGSLs by substituting the fatty acids of primarily, globotriaosyl ceramide(Gb₃) and sulfogalactosyl ceramide(SGC), with the rigid α-adamantane hydrocarbon frame. These analogues have proven to be remarkably water-soluble but retain the receptor function of the parent membrane GSL. AdaGb₃ prevents the binding of verotoxins to target cells but increased pathology in vivo, likely due to the partitioning into receptor negative target cells to provide pseudo-receptors. Preincubation of HIV with adaGb₃ prevents cellular infection in vitro and viral-host cell fusion. Cellular accumulation of Gb₃ reduces HIV susceptibility in vitro, whereas lack of Gb₃ promotes infection, suggesting that Gb₃ expression could be a novel risk factor for HIV susceptibility. AdaGb₃ has proven to be a new inhibitor for the MDR1 drug pump (P-glycoprotein) and can reverse drug resistance in cell culture. AdaSGC is bound by hsp70/hsc70 within the N-terminal ATPase domain and inhibits chaperone function. When added to cells transfected with the ΔF508 CFTR mutant, adaSGC was able to decrease ER degradation of this mutant protein, an hsc70 dependent process. Our finding that ΔF508 CFTR expressing cells show reduced SGC biosynthesis suggests that SGC could be an additional natural regulator of the hsp70 chaperone ATPase cycle.

Keywords: Globotriaosyl ceramide; Sulfatide; Verotoxin; HIV; hsp70; Cystic fibrosis

Effect of structural modifications of ganglioside GM2 on intra-molecular carbohydrate-to-carbohydrate interaction and enzymatic susceptibility by Yu-Teh Li; Su-Chen Li; Makoto Kiso; Hideharu Ishida; Laura Mauri; Laura Raimondi; Anna Bernardi; Sandro Sonnino (pp. 353-361).

The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcβ1→4(Neu5Acα2→3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to β-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcβ1→4Gal linkage in GM2 was converted to the GalNAcβ1→6Gal, both the GalNAc and the Neu5Ac became susceptible to β-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates.

Keywords: GM2; Tauro-GM2; GM2-analogs; GM2-activator protein; Tay–Sachs disease; Intra-molecular carbohydrate-to-carbohydrate interaction

Trypanosomatid and fungal glycolipids and sphingolipids as infectivity factors and potential targets for development of new therapeutic strategies by Erika Suzuki; Ameria K. Tanaka; Marcos S. Toledo; Steven B. Levery; Anita H. Straus; Helio K. Takahashi (pp. 362-369).

Several (glyco)(sphingo)lipids from different human pathogens have been characterized, and frequently many of these molecules are participating in host–pathogen interaction. In Leishmania (Leishmania) amazonensis, for example, amastigotes present on their surface glycosphingolipids (GSLs) with the structure Galβ1–3Galα, which is recognized by 30 kDa receptor of macrophages. Furthermore, other Leishmania species, such as Leishmania (Leishmania) major and Leishmania (Viannia) braziliensis present glycosylinositolphospholipids (GIPLs) which are involved in Leishmania–macrophage interaction. It is worth to mention that these antigens are not expressed in mammalian cells. Leishmania promastigotes also present inositol phosphorylceramide (IPC), a unique sphingolipid characteristic of fungi and plants. It was observed that IPC synthesis is essential for parasite division, since Aureobasidin A, an inhibitor of IPC synthase, inhibited significantly promastigote and amastigote growths. Recently, it was also demonstrated that GIPLs, IPC and sterols are preferentially present in the parasite membrane microdomains resistant to Triton X-100 at 4 °C. The disruption of these microdomains by incubating parasites with methyl-β-cyclodextrin inhibited significantly macrophage infectivity by Leishmania. Other pathogens, such as fungi, also present unique glycolipids which may have an important role for the fungal development and/or disease establishment. Taking together these results, this review will discuss different biological roles for (glyco)(sphingo)lipids of different pathogens.

Keywords: Abbreviations; AbA; Aureobasidin A; Cer; Ceramide; CMH; Monohexosylceramide; DRM; Detergent resistant membrane; Gal; Galactose; GalCer; Galactosylceramide; Gal; f; Galactofuranose; Gal; p; Galactopyranose; GIPC; Glycoinositol phosphorylceramide; GIPL; Glycoinositolphospholipid; GlcCer; Glucosylceramide; GlcN; Glucosamine; Glc; p; Glucopyranose; GPI; Glycosylphosphatidylinositol; GSL; Glycosphingolipid; HPTLC; High performance thin layer chromatography; INS; Inositol; IP; Inositol phosphate; IPC; Inositol phosphorylceramide; LPG; Lipophosphoglycan; mAb; Monoclonal antibody; Man; Mannose; MTT; 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; MβCD; Methyl-β-cyclodextrin; PC; Phosphatidylcholine; PE; Phosphatidylethanolamine; PG; Phosphoglycan; PI; Phosphatidylinositol; PPG; Proteophosphoglycan; PS; Phosphatidylserine; SL; Sphingolipid Leishmania; Pathogenic fungi; Glycosylinositol phosphorylceramide; Glycosphingolipid; Inositol phosphorylceramide; Lipid raft

The Yin and Yang of lactosylceramide metabolism: Implications in cell function by Subroto Chatterjee; Ambarish Pandey (pp. 370-382).

Although lactosylceramide (LacCer) plays a pivotal role in the biosynthesis of nearly all the major glycosphingolipids, its function in regulating cellular function has begun to emerge only recently. Our current opinion is that several physiologically critical molecules such as modified/oxidized LDL, growth factors, pro-inflammatory cytokines and fluid shear stress converge upon and activate lactosylceramide synthase to generate LacCer. In turn, LacCer activates an “oxygen-sensitive” signaling pathway involving superoxides, nitric oxide, p21 Ras GTP loading, kinase cascade, PI3kinase/Akt activation, nuclear factor up-regulation ultimately contributing to phenotypic changes such as cell proliferation, adhesion, migration and angiogenesis. Since dys-regulation of such phenotypic changes constitute a hallmark in several diseases of the cardiovascular system, proliferative disorders such as cancer, polycystic kidney disease and inflammatory diseases, LacCer synthase and LacCer provide novel targets for the development of therapeutics aimed at these health conditions.

Keywords: Lactosylceramide; Glycosphingolipids; Gangliosides; Apoptosis; Signal transduction; Cell proliferation; Cell adhesion; Angiogenesis

Role of glycosphingolipid-enriched microdomains in innate immunity: Microdomain-dependent phagocytic cell functions by Fumiko Yoshizaki; Hitoshi Nakayama; Chihiro Iwahara; Kenji Takamori; Hideoki Ogawa; Kazuhisa Iwabuchi (pp. 383-392).

The innate immune system is the first line of defense against pathogenic microorganisms, such as bacteria, fungi, and viruses. Phagocytes, such as neutrophils and macrophages, play an important role in the innate immune system by recognizing, engulfing, and eliminating pathogens. It has been suggested that lipid membrane microdomains/rafts of phagocytes are involved in these innate immune responses, including superoxide generation, cell migration, and phagocytosis. Lactosylceramide (LacCer), a neutral glycosphingolipid, forms glycosphingolipid-enriched microdomains together with the Src family kinase, Lyn, on the neutrophil plasma membrane. LacCer-enriched microdomains have been suggested to play important roles in innate immune function of neutrophils. However, the molecular mechanisms underlying these phenomena remain largely unknown. Recent proteomic analyses of microdomains from phagocytes have provided insight into membrane microdomain-mediated functions in the processes of phagocytosis. In this review, we discuss the membrane microdomain-associated immune functions of phagocytes, focusing on those functions of LacCer-enriched microdomains and recent proteomic approaches to determine the molecular mechanisms underlying these functions.

Keywords: Abbreviations; DRM; detergent-resistant membrane; PAMPs; pathogen-associated molecular patterns; PRR; pattern recognition receptors; GPI; glycosylphosphatidylinositol; β-glucans; polysaccharide β-1,3-D-glucans; CSBG; Candida; soluble β-D-glucan; GalCer; galactosyl ceramide; GlcCer; glucosyl ceramide; Gb3; Galα1-4Galβ1-4Glc-cer; Gb4; GalNacβ1-3Galα1-4Galβ1-4Glc-cer; GM3; NeuAcα2-3Galβ1-4Glcβ1-1′-Cer; GM2; GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glcβ1-1′-Cer; GM1; Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glcβ1-1′-Cer; LacCer; lactosylceramide (Galβ1-4Glc-ceramide)Glycosphingolipid; Membrane microdomains; Lipid rafts; Innate immunity; Phagocytes; Phagocytosis; Lactosylceramide; Interdigitation; Infection; Escape; Cholesterol; Pattern recognition receptors; β-glucan; Phagosome formation; Proteomics

Effect of lipid mimetics of GM3 and lyso-GM3 dimer on EGF receptor tyrosine kinase and EGF-induced signal transduction by Yoshimi Haga; Kenichi Hatanaka; Sen-itiroh Hakomori (pp. 393-404).

The tyrosine kinase activity associated with epidermal growth factor receptor (EGFR) has been a target in studies of pharmacological reagents to inhibit growth of cancer cells, which are mostly of epidermal origin. Lyso-GM3 dimer showed stronger inhibitory effect on the tyrosine kinase of EGFR than GM3, with minimal cytotoxicity [Y. Murozuka, et al. Lyso-GM3, its dimer, and multimer: their synthesis, and their effect on epidermal growth factor-induced receptor tyrosine kinase. Glycoconj. J. 24 (2007) 551–563]. Synthesis of lipids with sphingosine requires many steps, and the yield is low. A biocombinatory approach overcame this difficulty; however, products required a C₁₂ aliphatic chain, rather than the sphingosine head group [Y. Murozuka, et al. Efficient sialylation on azidododecyl lactosides by using B16 melanoma cells. Chemistry & Biodiversity 2 (2005) 1063–1078]. The present study was to clarify the effects of these lipid mimetics of GM3 and lyso-GM3 dimer on EGFR tyrosine kinase activity, and consequent changes of the A431 cell phenotype, as follows. (i) A lipid mimetic of lyso-GM3 dimer showed similar strong inhibitory effect on EGF-induced EGFR tyrosine kinase activity, and similar low cytotoxicity, as the authentic lyso-GM3 dimer. (ii) A lipid mimetic of lyso-GM3 dimer inhibited tyrosine phosphorylation of EGFR or its dimer to a level similar to that of the authentic lyso-GM3 dimer, but more strongly than GM3 or a lipid mimetic of GM3. (iii) Associated with the inhibitory effect of a lipid mimetic of lyso-GM3 dimer on EGF-induced EGFR kinase activity, only Akt kinase activity was significantly inhibited, but kinases associated with other signal transducers were not affected. (iv) The cell cycle of A431 cells, and the effects of GM3 and a lipid mimetic of lyso-GM3 dimer, were studied by flow cytometry, measuring the rate of DNA synthesis with propidium iodide. Fetal bovine serum greatly enhanced S phase and G₂/M phase. Enhanced G₂/M phase was selectively inhibited by pre-incubation of A431 cells with a lipid mimetic of lyso-GM3 dimer, whereas GM3 had only a minimal effect.

Keywords: Abbreviations; Lipid mimetic of GM3; GM3 ganglioside with ceramide-like lipid lacking sphingosine; Lyso-GM3; sialyllactosyl sphingosine; Lipid mimetic of lyso-GM3 (LM-GM3); sialyllactosyl-2-amino-dodecanol; Lipid mimetic of lyso-GM3 dimer (LM-dimer); sialyllactosyl-2-amino-dodecanol linked together through glutamic acid dimer; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serum; sf; serum-freeGM3 ganglioside; Lipid mimetic; EGF receptor; Lyso-GM3 dimer

Phosphatidylglucoside: A new marker for lipid rafts by Yasuko Nagatsuka; Yoshio Hirabayashi (pp. 405-409).

Lipid rafts are functional microdomains enriched with sphingolipids and cholesterol. The fatty acyl chain composition of sphingolipids is a critical factor in the localization of lipids in lipid rafts. The recent studies suggest that lipid rafts are more heterogeneous than previously thought. In addition, our discovery of a new glycolipid, phosphatidylglucoside (PtdGlc), also supports the notion of raft heterogeneity. The complete structural characterization of PtdGlc shows that it consists solely of saturated fatty acyl chains: C18:0 at the sn-1 and C20:0 at the sn-2 positions of the glycerol backbone. This unique fatty acyl composition comprising a single molecular species rarely occurs in known mammalian lipids. Although the structure of PtdGlc is similar to that of phosphatidylinositol, PtdGlc localizes to the outer leaflet of the plasma membrane and is possibly involved in cell–cell interaction signaling in the central nervous system.

Keywords: Lipid raft; Phosphatidylglucoside; Phosphatidylinositol; Glycosphingolipid; Glucosylceramide

Lipid remodeling of GPI-anchored proteins and its function by Morihisa Fujita; Yoshifumi Jigami (pp. 410-420).

Many proteins are attached to the cell surface via a conserved post-stranslational modification, the glycosylphosphatidylinositol (GPI) anchor. GPI-anchored proteins are functionally diverse, but one of their most striking features is their association with lipid microdomains, which consist mainly of sphingolipids and sterols. GPI-anchored proteins modulate various biological functions when they are incorporated into these specialized domains. The biosynthesis of GPI and its attachment to proteins occurs in the endoplasmic reticulum. The lipid moieties of GPI-anchored proteins are further modified during their transport to the cell surface, and these remodeling processes are essential for the association of proteins with lipid microdomains. Recently, several genes required for GPI lipid remodeling have been identified in yeast and mammalian cells. In this review, we describe the pathways for lipid remodeling of GPI-anchored proteins in yeast and mammalian cells, and discuss how lipid remodeling affects the association of GPI-anchored proteins with microdomains in cellular events.

Keywords: Glycosylphosphatidylinositol; GPI-anchored proteins; Lipid remodeling; Microdomain; Lipid rafts

Functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains by Adriane Regina Todeschini; Sen-itiroh Hakomori (pp. 421-433).

At cell surface microdomains, glycosyl epitopes, carried either by glycosphingolipids, N- or O-linked oligosaccharides, are recognized by carbohydrate-binding proteins or complementary carbohydrates. In both cases, the carbohydrate epitopes may be clustered with specific signal transducers, tetraspanins, adhesion receptors or growth factor receptors. Through this framework, carbohydrates can mediate cell signaling leading to changes in cellular phenotype. Microdomains involved in carbohydrate-dependent cell adhesion inducing cell activation, motility, and growth are termed “glycosynapse”. In this review a historical synopsis of glycosphingolipids-enriched microdomains study leading to the concept of glycosynapse is presented. Examples of glycosynapse as signaling unit controlling the tumor cell phenotype are discussed in three contexts: (i) Cell-to-cell adhesion mediated by glycosphingolipids-to-glycosphingolipids interaction between interfacing glycosynaptic domains, through head-to-head ( trans) carbohydrate-to-carbohydrate interaction. (ii) Functional role of GM3 complexed with tetraspanin CD9, and interaction of such complex with integrins, or with fibroblast growth factor receptor, to control tumor cell phenotype and its reversion to normal cell phenotype. (iii) Inhibition of integrin-dependent Met kinase activity by GM2/tetraspanin CD82 complex in glycosynaptic microdomain. Data present here suggest that the organizational status of glycosynapse strongly affects cellular phenotype influencing tumor cell malignancy.

Keywords: Abbreviations; GFR; growth factor receptor tyrosine kinase; GPI; glycosylphosphatidylinositol; GSL; glycosphingolipids; CCI; carbohydrate-to-carbohydrate interaction; Ecad; Ecadherin; EGFR; epidermal growth factor receptor; ECM; extra cellular matrix; FGFR; fibroblast growth factor receptor; GEM; GSL-enriched microdomain; HGF; hepatocyte growth factor; LN; lamin; Le; Lewis; Neu3; ganglioside-specific sialidase; NMR; nuclear magnetic resonance; nOe; nuclear Overhauser effect; TD; signal transducers; TSPs; tetraspaninsGlycosynapse; Glycosphingolipids microdomain; Glycosylation; Tetraspanin; Ganglioside; Integrin

Sulfogalactosylceramides in motor and psycho-cognitive adult metachromatic leukodystrophy: relations between clinical, biochemical analysis and molecular aspects by Benoit Colsch; Carlos Afonso; Jean-Claude Turpin; Jacques Portoukalian; Jean-Claude Tabet; Nicole Baumann (pp. 434-440).

Metachromatic leukodystrophy (MLD) is a human autosomal recessive lysosomal neurodegenerative disorder that results from the accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). During adolescence and/or adulthood, there are 2 clinical presentations. It may be that of a degenerative disease of the central nervous system with mainly spastic manifestations or a spino-cerebellar ataxia, or that of a psychosis. As several lines of evidence indicate that the psychotic form of MLD could be a model of psychosis, we decided to do a pluridisciplinary study on 11 psycho-cognitive cases involving mental and psychiatric testing, in comparison with 5 adult motor cases, a biochemical study with enzyme assays and quantitative mass spectrometry of urinary sulfatides, so as to determine whether there were biochemical particularities related to the psychotic forms. For quantitative mass spectrometry (MS), a non physiological sulfatide with C17:0 fatty acid was synthesized. The major sulfatide isoforms were present in the 2 clinical forms with the following fatty acids and sphingoid bases: C22:1/d18:1, and /or C22:0/d18:2 ( m/ z 862.5), C22:0 (OH)/d18:1 ( m/ z 878,5), C24:0/d18:1 and / or C24:0/C23:1(OH)/d18:2 ( m/ z 890,3), C24:0 (OH)/d18:1( m/ z 906.5). We had shown previously that there were different ASA mutations in the psychiatric adult form (heterozygous I179S) versus the adult motor form (homozygous P426L). We show here that there were no relations with the level of ASA and with the mass spectrometric study of the sulfatide isoforms which were identical in the 2 clinical forms.

Keywords: Sulfatides; Metachromatic leukodystrophy; Schizophrenia; Mass spectrometry; Urine

Antibodies against gangliosides and ganglioside complexes in Guillain–Barré syndrome: New aspects of research by Susumu Kusunoki; Ken-ichi Kaida; Masami Ueda (pp. 441-444).

Guillain–Barré syndrome (GBS) is an acute autoimmune neuropathy, often preceded by an infection. Serum anti-ganglioside antibodies are frequently elevated in titer. Those antibodies are useful for diagnosis. Some of them also may be directly involved in the pathogenetic mechanisms by binding to the regions where the respective target ganglioside is specifically localized. We have recently found the presence of the antibody that specifically recognizes a new conformational epitope formed by two gangliosides (ganglioside complex) in the acute-phase sera of some GBS patients. In particular, the antibodies against GD1a/GD1b and/or GD1b/GT1b complexes are associated with severe GBS requiring artificial ventilation. Some patients with Miller Fisher syndrome also have antibodies against ganglioside complexes including GQ1b; such as GQ1b/GM1 and GQ1b/GD1a. Gangliosides along with other components as cholesterol are known to form lipid rafts, in which the carbohydrate portions of two different gangliosides may form a new conformational epitope. Within the rafts, gangliosides are considered to interact with important receptors or signal transducers. The antibodies against ganglioside complexes may therefore directly cause nerve conduction failure and severe disability in GBS. More study is needed to elucidate the roles of the antibodies against ganglioside complexes.

Keywords: Ganglioside; Peripheral nerve; Guillain–Barré syndrome; Raft; Glycolipid; Autoantibody

Myelin glycosphingolipids, galactosylceramide and sulfatide, participate in carbohydrate–carbohydrate interactions between apposed membranes and may form glycosynapses between oligodendrocyte and/or myelin membranes by Joan M. Boggs; Wen Gao; Yukie Hirahara (pp. 445-455).

Glycosphingolipids (GSLs) can interact with each other by homotypic or heterotypic trans carbohydrate–carbohydrate interactions across apposed membranes, resulting in cell–cell adhesion. This interaction can also provide an extracellular signal which is transmitted to the cytosolic side, thus forming a glycosynapse between two cells. The two major GSLs of myelin, galactosylceramide (GalC) and its sulfated form, galactosylceramide I³-sulfate (SGC), are an example of a pair of GSLs which can participate in these trans carbohydrate–carbohydrate interactions and trigger transmembrane signaling. These GSLs could interact across apposed oligodendrocyte membranes at high cell density or when a membranous process of a cell contacts itself as it wraps around the axon. GalC and SGC also face each other in the apposed extracellular surfaces of the multilayered myelin sheath. Communication between the myelin sheath and the axon regulates both axonal and myelin function and is necessary to prevent neurodegeneration. Participation of transient GalC and SGC interactions in glycosynapses between the apposed extracellular surfaces of mature myelin might allow transmission of signals throughout the myelin sheath and thus facilitate myelin-axonal communication.

Keywords: Abbreviations; Abs; Antibodies; Akt; protein kinase B; Caspr; contactin-associated proteins; CGT; UDP-galactose:ceramide galactosyltransferase; CNS; central nervous system; CREB; cAMP-response element binding protein; CRMP2; collapsin response mediator protein 2; CST; galactosylceramide 3′-sulfotransferase; DIGs; detergent-insoluble glycosphingolipid-enriched membrane domains; F3; contactin; Gal-BSA; galactose conjugated to bovine serum albumin; GalC; galactosylceramide; GEMs; glycosphingolipid-enriched microdomains; GluC; glucosylceramide; GPI; glycosylphosphatidylinositol; GSK-3β; glycogen synthase kinase-3β; GSLs; glycosphingolipids; LacC; lactosylceramide; Lim-K; Lim kinase; MAP1B; microtubule-associated protein-1B; MAPK; mitogen activated protein kinase (p42 and p44, 42 and 44 kDa isoforms); MBP; myelin basic protein; MAG; myelin associated glycoprotein; MEK; MAPK kinase; MEKK; MEK kinase; MLC; myosin light chain; MLCP; myosin light chain phosphatase; MOG; myelin/oligodendrocyte glycoprotein; NCAM; neural cell adhesion molecule; NF-155; neurofascin-155 kDa; PDK; phosphoinositide-dependent kinase; PIP; ₂; phosphatidylinositol-4,5-bisphosphate; PI; ₃; K; phosphatidylinositol 3-kinase; PLP; proteolipid protein; PS; phosphatidylserine; OL; oligodendrocyte; RhoK; Rho kinase; SGC; sulfated form of GalC, cerebroside sulfate; SM; sphingomyelinGlycoconjugate; Lipid raft; Signaling; Cytoskeleton; Kinase

Importance of molecular studies on major blood groups—Intercellular adhesion molecule-4, a blood group antigen involved in multiple cellular interactions by Anne Toivanen; Eveliina Ihanus; Minna Mattila; Hans U. Lutz; Carl G. Gahmberg (pp. 456-466).

Several blood groups, including the LW-blood group were discovered in the first part of last century, but their biochemical characteristics and cellular functions have only more recently been elucidated. The LW-blood group, renamed ICAM-4 (CD242), is red cell specific and belongs to the intercellular adhesion molecule family. ICAM-4 binds to several integrin receptors on blood and endothelial cells and is thus able to form large cellular complexes containing red cells. Its physiological function(s) has remained incompletely understood, but recent work shows that macrophage integrins can bind red cells through this ligand. In this article we discuss molecular properties of major blood group antigens, describe ICAM-4 in more detail, and show that phagocytosis of senescent red cells is in part ICAM-4/β₂-integrin dependent.

Keywords: Abbreviations; LW blood group; ICAM-4, CD242; ICAM; Intercellular adhesion molecule; LFA-1; Leukocyte function associated antigen, α; _L; β; ₂; , CD11a/CD18; Mac-1; α; _M; β; ₂; , CD11b/CD18; α; _X; β; ₂; CD11c/CD18; GPA; Glycophorin A; PS; Phosphatidyl serineICAM-4; LW blood group; Rh-antigen; CD11c/CD18; Erythrophagocytosis

Incomplete synthesis of the Sd^a/Cad blood group carbohydrate in gastrointestinal cancer by Taeko Dohi; Yuki I. Kawamura (pp. 467-471).

Cancer-associated changes in cell surface carbohydrates, including incomplete synthesis of normal carbohydrate epitopes, strongly affect malignant and metastatic potential. Here, we report that compensating for the cancer-associated loss of a single glycosyltransferase, β1,4 N-acetylgalactosaminyltransferese T2, dramatically decreased cell surface expression of both E-selectin ligands (sialyl Lewis^x and sialyl Lewis^a). This modification was associated with elevated expression of the Sd^a carbohydrate determinant, which is expressed in normal gastrointestinal mucosa and is strikingly downregulated in cancer tissues. Loss of E-selectin ligands resulted in decreased adhesion of cancer cells to activated human endothelial cells in vitro and eventually suppressed metastatic potential in vivo.

Keywords: Gastric cancer; Colon cancer; Sd; ^a; Cad; Glycosyltransferase

The N-Linked sugar chains of human immunoglobulin G: Their unique pattern, and their functional roles by Akira Kobata (pp. 472-478).

In contrast to other serum glycoproteins, the majority of the N-linked sugar chains of human serum IgG are not sialylated. In addition, extremely high micro-heterogeneity occurs in the serum IgG sugar chains. This micro-heterogeneity is mainly produced by the presence or absence of the two galactoses, the bisecting GlcNAc, and the fucose residue. Interesting evidence is that the molar ratio of each sugar chain of the serum IgG samples is quite constant in healthy individuals. By adding the information of the characteristic feature of the sugar patterns of myeloma IgG samples and glycosylated Bence Jones proteins, which are the products of monoclonal B-cells, it was proposed that B-cells in the human blood are a mixture of clones equipped with different sets and ratios of glycosyltransferases. It was also proposed that each glycoform of IgG might have a different function. This hypothesis was realized by the comparative studies of the function of IgG samples before and after removal of galactose residues, fucose residue, or sialic acid residues.

Keywords: Abbreviations; ADCC; antibody dependent cell-mediated cytotoxicity; CHO; Chinese hamster ovary; Fuc; l; -fucose; Gal; galactose; GlcNAc; N; -acetylglucosamine; IgG; immunoglobulin G; IVIG; intravenous γ-globulin; Man; mannose; Neu5Ac; N; -acetylneuraminic acid; NMR; nuclear magnetic resonance; SNA; Sambucus nigra; agglutininImmunoglobulin G; N-linked sugar chain; Bence Jones glycoprotein; Fab; Fc; Fc-receptor

Core 2 GlcNAc modification and megalin ligand-binding activity by Heng Zhang; Shigemi Yoshioka; Masao Miyazaki; Reiji Kannagi; Akemi Suzuki (pp. 479-485).

Megalin, a receptor-like transporter glycoprotein, is expressed on kidney proximal tubular cells and reabsorbs small-molecular-weight proteins from the glomerular filtrate. Here, we report that mouse megalins differently modified with core 2 β6GlcNAc transferase had different kinetic properties to a fluorescence-labeled ligand, retinol-binding protein (RBP). BALB/c mice, a wild-type strain in terms of the expression of kidney-specific core 2 β6GlcNAc transferase, express megalin carrying the core 2 extended Le^x epitope, while DBA/2 mice, a mutant-strain of the core 2 β6GlcNAc transferase, express megalin lacking the epitope. We purified these two types of megalin using lentil lectin chromatography and measured the ligand-binding activities of the megalins using Cy5-labeled RBP by applying gel permeation chromatography (GPC) and fluorescence correlation spectroscopy (FCS). The analysis by GPC indicated that the apparent V_max of the interaction between Cy5-labeled RBP and the megalins of BALB/c and DBA/2 mice was 60 μM and 30 μM, respectively, and the apparent K_m was 11 μM and 17 μM, respectively. Scatchard analysis demonstrated the presence of two binding sites. Linear regression analysis resulted in a two-binding-site model characterized by a high-affinity site ( K_dBALB=12.0 μM; K_dDBA=20.9 μM) and a low-affinity site ( K_dBALB=36.2 μM; K_dDBA=58.8 μM). FCS analysis exhibited quite different K_m and V_max values from those obtained by GPC, but similar K_m values for the two types of megalin, and a lower V_max value for DBA/2 megalin than BALB/c megalin. These results suggest that the core 2 GlcNAc extended glycan chains on megalin can change the ligand-binding affinity and capacity.

Keywords: Abbreviations; RBP; retinol-binding protein; GPC; gel permeation chromatography; FCS; fluorescence correlation spectroscopy; DOC; deoxycholate; KDN; deaminoneuraminic acid; DBP; vitamin D-binding proteinGlycoconjugate; Megalin; Glycoprotein; Core 2 β6GlcNAc transferase; Cy5-labeled retinol-binding protein

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1 by Stacy M. Alvares; Clarence A. Dunn; Tod A. Brown; Elizabeth E. Wayner; William G. Carter (pp. 486-496).

Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and Detergent-Resistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell–cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals-possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell–cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell–cell contacts contributes to changes in cell behavior.

Keywords: Abbreviations; CDCP1; CUB Domain Containing Protein 1; ECM; extracellular matrix; MβCD; methyl-β-cyclodextrin; TEM; Tetraspanin Enriched Microdomain; DRM; detergent resistant microdomainGp140/CUB Domain Containing Protein 1; Detergent-resistant microdomain; Src family kinase; Tetraspanin; Adhesion

A new approach for drug discovery from glycobiology and phage-displayed peptide library technology by Takao Taki; Dai Ishikawa; Koichi Ogino; Michinori Tanaka; Naoto Oku; Tomohiro Asai; Iuliana Popa; Jacques Portoukalian (pp. 497-503).

Peptides which mimic functional activities of glycosphingolipids were prepared by a technology of phage-displayed peptide library using monoclonal antibodies against glycosphingolipids. These peptides were named glyco-replica peptides. Peptides prepared with anti-GD1α antibody by this technology were found to contain WHW as common motif, and they showed suppressive activity not only on adhesion between hepatic sinusoidal endothelial cells and lymphosarcoma RAW117-H10 cells, but also on metastasis of the tumor cell to the liver and lung. The WHW motif seems to be important to mimic the functional activity of the ganglioside GD1α. Next, we prepared GD3-replica peptides using a monoclonal antibody against GD3 (4F6). A peptide, GD3-P4 with highest affinity to 4F6 was used to immunize mice to examine if the mice show their immune response to raise antibodies against GD3. We confirmed the immune response and succeeded in the production of a monoclonal antibody (3D2) against GD3. The monoclonal antibody 3D2 showed specific binding to GD3 on a thin-layer chromatography plate and also melanoma tissues. Interestingly, the amino acid sequence of the CDR regions of light and heavy chains showed high similarity with those of the original GD3 monoclonal antibody (4F6) used for the preparation of GD3-replica peptide. The technology of the phage-displayed peptide library was applied to in vivo bio-panning study using an angiogenesis experimental model. The obtained peptides were found to show strong binding property to the neo-vasculature system and to be quite useful to carry an anti-tumor drug to the tumor tissue. Based on these experimental results, we discuss about some applications of this method to drug discovery.

Keywords: Phage displayed peptide library; Glyco-replica peptide; Glycolipid; Ganglioside; Angiogenesis; Melanoma; Drug discovery; Glycobiology; Metastasis; Molecular mimicry

Over-expression of mammalian sialidase NEU3 reduces Newcastle disease virus entry and propagation in COS7 cells by Luigi Anastasia; Javier Holguera; Anna Bianchi; Francesca D'Avila; Nadia Papini; Cristina Tringali; Eugenio Monti; Enrique Villar; Bruno Venerando; Isabel Muñoz-Barroso; Guido Tettamanti (pp. 504-512).

The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell–cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [³H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 °C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.

Keywords: Abbreviations; DMEM; Dulbecco's Modified Eagle's Medium; F; fusion protein; F0; precursor of F protein; DRMs; Detergent resistant microdomains (detergent resistant membranes); FBS; fetal bovine serum; GFP; green fluorescent protein; HN; hemagglutinin-neuraminidase glycoprotein; HPTLC; high performance thin-layer chromatography; MmNEU3; membrane-associated sialidase from mice (; Mus musculus; ); MOI; multiplicity of infection; NDV; Newcastle Disease Virus; Neu5Ac; N; -acetylneuraminic acid; PBS; phosphate buffered saline; pfu; plaque forming units; R18; octadecylrhodamine B chloride. The ganglioside nomenclature of Svennerholm (1963) is usedCOS7 cell; NDV; Sialidase NEU3; Lipid raft; GD1a ganglioside

Focal adhesion kinase as well as p130Cas and paxillin is crucially involved in the enhanced malignant properties under expression of ganglioside GD3 in melanoma cells by Kazunori Hamamura; Momoko Tsuji; Yuki Ohkawa; Hideyuki Nakashima; Sayaka Miyazaki; Takeshi Urano; Noriyuki Yamamoto; Minoru Ueda; Koichi Furukawa; Keiko Furukawa (pp. 513-519).

Mass spectrometry analysis of immunoprecipitates from serum-treated GD3-expressing melanoma cells with PY20 (anti-phosphotyrosine antibody) revealed that focal adhesion kinase (FAK) is more strongly activated in GD3-expressing cells than in GD3-negative cells. Involvent of FAK in the increased proliferation and invasion in GD3-expressing melanomas was demonstrated by siRNA-mediated knockdown. Also, it was shown that FAK is located up-stream of p130Cas and paxillin in the enhanced signaling pathway. GD3 expression enhanced the association of FAK with p130Cas after treatment with fetal calf serum. Thus, focal adhesion kinase as well as p130Cas and paxillin should be a crucial molecule undergoing stronger tyrosine phosphorylation in GD3-expressing melanoma cells. Molecules linking GD3 and FAK such as integrins in the enhanced signaling pathway remain to be investigated.

Keywords: Keyword; Focal adhesion kinase; Ganglioside; GD3; Melanoma; Proliferation; Invasion; Tyrosine phosphorylation

N-glycan of ErbB family plays a crucial role in dimer formation and tumor promotion by Motoko Takahashi; Shunichi Yokoe; Michio Asahi; Seung Ho Lee; Wei Li; Daisuke Osumi; Eiji Miyoshi; Naoyuki Taniguchi (pp. 520-524).

More and more evidence indicates that N-glycan regulates signal transduction by modulating receptor functions. Previous studies suggested that glycosylation of EGFR is involved in dimerization and endocytosis. We further determined the role of N-glycosylation of ErbB family. A series of human ErbB3 mutants that lack each of the 10 N-glycosylation sites were prepared and transfected to Flp-In-CHO cells for stable expression. A crosslinking study showed that Asn 418 to Gln mutant (N418Q) of ErbB3 underwent autodimerization without its ligand, heregulin, and the heterodimer formation with ErbB2 was also increased. The N418Q mutant of ErbB3 co-expressed with ErbB2 promoted downstream signaling, anchorage-independent cell growth and the tumor growth in athymic mice. These findings suggest that the specific N-glycan in domain III of ErbB family plays an essential role in regulating receptor dimerization and transforming activity. We assume that the N-glycans affect the conformation of ErbB family, which is crucial for their activity. Together with findings from other laboratories, it is suggested that N-glycosylation controls ErbB signaling by various mechanisms.

Keywords: Abbreviations; BS; ³; bis-(sulfosuccinimidyl)-suberate; CHO; Chinese hamster ovary; EGFR; epidermal growth factor receptor; ERK; extracellular signal-regulated kinase; GlcNAc; N; -acetylglucosamine; HRG; heregulin; PI3K; phosphoinositide 3-kinaseErbB; EGFR; N; -glycan; Glycosylation; Dimerization; Oligomerization

Current relevance of incomplete synthesis and neo-synthesis for cancer-associated alteration of carbohydrate determinants—Hakomori's concepts revisited by Reiji Kannagi; Jun Yin; Keiko Miyazaki; Mineko Izawa (pp. 525-531).

Incomplete synthesis and neo-synthesis are two major concepts for cancer-associated alterations of cell surface carbohydrate determinants, formulated by Hakomori and collaborators almost 25 years ago. These concepts are still as relevant and useful as ever for cancer-associated alteration of carbohydrate determinants. Incomplete synthesis of carbohydrate determinants occurs through the epigenetic silencing of glycogenes through DNA methylation and/or histone modification in the early stage cancers. The natural selection of more malignant cancer cells occurs through acquisition of hypoxia resistance by constitutively activated hypoxia inducible factors (HIFs) in the advanced stages of cancers. HIFs induce transcription of several important glycogenes, and lead to neo-synthesis of carbohydrate determinants. For instance, expression of sialyl Lewis A/X is induced by epigenetic silencing of glycogenes in the early stages, and is further accelerated in the advanced stages by hypoxia-induced transcription of several glycogenes. Expression of GM2 ganglioside is induced in cancers by altered glycosyltransferase activities, and its N-glycolyl sialic acid content increases by hypoxia-induced transcription of a sialic acid transporter gene. N-glycolyl GM2 thus reflects two cancer-associated genetic abnormalities in a single determinant, and has high cancer specificity. Every carbohydrate determinant is synthesized through multiple steps, each of which is affected by cancer-associated genetic abnormality. Superiority of carbohydrate determinants as cancer-specific molecules over protein determinants is demonstrated in that a single carbohydrate determinant can reflect multiple cancer-associated genetic abnormalities.

Keywords: Abbreviations; 5-aza-C; 5-aza-cytidine; COX2; cyclo-oxygenase-2; HIF; hypoxia inducible factor; ITIM; immunoreceptor tyrosine-based inhibition motif; Siglec; sialic acid binding immunoglobulin (Ig)-like lectins; VEGF; vascular endothelial growth factorEpigenetic silencing; DNA methylation; Tumor hypoxia; Selectin; Siglec; Hypoxia-inducible factor

Roles of plasma membrane-associated sialidase NEU3 in human cancers by Taeko Miyagi; Tadashi Wada; Kazunori Yamaguchi (pp. 532-537).

Altered sialylation of glycosphingolipids is observed in cancer as a ubiquitous phenotype, leading to the appearance of tumor-associated antigens, aberrant adhesion and disturbance of transmembrane signaling. To understand the pathological significance of aberrant alterations of gangliosides in cancer, our studies have been focused on sialidase, which is responsible for the removal of sialic acids from glycoproteins and glycolipids. Among human sialidases so far identified, sialidase NEU3 is a key enzyme for ganglioside degradation because of its uniqueness both in its localization in the plasma membrane and in specifically hydrolyzing gangliosides. NEU3 is markedly up-regulated in many types of cancers including colon and renal carcinomas and suppresses apoptosis of cancer cells. The present paper briefly summarizes our recent results on the sialidase alterations and their significance in cancer. NEU3 is indeed closely related to malignancy and thus may be a potential target for cancer diagnosis and therapy.

Keywords: Sialidase; Gangliosides; Cancer; Plasma membrane; Apoptosis; Transmembrane signaling

Anti-GM1 antibodies as a model of the immune response to self-glycans by Gustavo A. Nores; Ricardo D. Lardone; Romina Comín; María E. Alaniz; Ana L. Moyano; Fernando J. Irazoqui (pp. 538-545).

Glycans are a class of molecules with high structural variability, frequently found in the plasma membrane facing the extracellular space. Because of these characteristics, glycans are often considered as recognition molecules involved in cell social functions, and as targets of pathogenic factors. Induction of anti-glycan antibodies is one of the early events in immunological defense against bacteria that colonize the body. Because of this natural infection, antibodies recognizing a variety of bacterial glycans are found in sera of adult humans and animals. The immune response to glycans is restricted by self-tolerance, and no antibodies to self-glycans should exist in normal subjects. However, antibodies recognizing structures closely related to self-glycans do exist, and can lead to production of harmful anti-self antibodies. Normal human sera contain low-affinity anti-GM1 IgM-antibodies. Similar antibodies with higher affinity or different isotype are found in some neuropathy patients. Two hypotheses have been developed to explain the origin of disease-associated anti-GM1 antibodies. According to the “molecular mimicry” hypothesis, similarity between GM1 and Campylobacter jejuni lipopolysaccharide carrying a GM1-like glycan is the cause of Guillain–Barré syndrome associated with anti-GM1 IgG-antibodies. According to the “binding site drift” hypothesis, IgM-antibodies associated with disease originate through changes in the binding site of normally occurring anti-GM1 antibodies. We now present an “integrated” hypothesis, combining the “mimicry” and “drift” concepts, which satisfactorily explains most of the published data on anti-GM1 antibodies.

Keywords: Anti-glycan antibodies; Anti-GM1 antibodies; Neuropathy; Binding site drift; Molecular mimicry; Integrated hypothesis

Mucin-type O-glycosylation and its potential use in drug and vaccine development by Mads Agervig Tarp; Henrik Clausen (pp. 546-563).

Mucin-type O-glycans are found on mucins as well as many other glycoproteins. The initiation step in synthesis is catalyzed by a large family of polypeptide GalNAc-transferases attaching the first carbohydrate residue, GalNAc, to selected serine and threonine residues in proteins. During the last decade an increasing number of GalNAc-transferase isoforms have been cloned and their substrate-specificities partly characterized. These differences in substrate specificities have been exploited for in vitro site-directed O-glycosylation. In GlycoPEGylation™, polyehylene glycol (PEG) is transferred to recombinant therapeutics to specific acceptor sites directed by GalNAc-transferases. GalNAc-transferases have also been used to control density of glycosylation in the development of glycopeptide-based cancer vaccines. The membrane-associated mucin-1 (MUC1) has long been considered a target for immunotherapeutic and immunodiagnostic measures, since it is highly overexpressed and aberrantly O-glycosylated in most adenocarcinomas, including breast, ovarian, and pancreatic cancers. By using vaccines mimicking the glycosylation pattern of cancer-cells, it is possible to overcome tolerance in transgenic animals expressing the human MUC1 protein as a self-antigen providing important clues for an improved MUC1 vaccine design. The present review will highlight some of the potential applications of site-directed O-glycosylation.

Keywords: Mucin-type; O; -glycosylation; Glycosyltransferases; Mucins; Site-directed; O; -glycosylation; GalNAc-transferases; Cancer vaccines

Changes in adhesive and migratory characteristics of hepatocellular carcinoma (HCC) cells induced by expression of α3β1 integrin by Hiromi Mizuno; Masaharu Ogura; Yuta Saito; Wakana Sekine; Rikio Sano; Toshie Gotou; Teruaki Oku; Saotomo Itoh; Kouji Katabami; Tsutomu Tsuji (pp. 564-570).

The invasive and metastatic potentials of hepatocellular carcinoma are positively correlated with the expression level of α3β1 integrin, a high-affinity adhesion receptor for laminin isoforms including laminin-5. In this study, we investigated changes in the adhesive and invasive behaviors of human HCC HepG2 cells after transfection with cDNA for α3 integrin in order to elucidate the direct involvement of this integrin in these cellular processes. We introduced cDNA for splice variants of α3 integrin (α3A and α3B) into the cells, and selected two transfectant clones (HepG2-3A and HepG2-3B), which express the α3A and α3B integrins, respectively. Both transfectant cells adhered almost equally to laminin-5-coated plates in an α3 integrin-dependent manner, indicating that transfected α3Aβ1 and α3Bβ1 integrins were functionally active in these cells. The migratory and invasive potentials of the transfectant cells were assessed by scratch wound assay and in vitro chemoinvasion assay. The results demonstrated that the migration of HepG2-3A and HepG2-3B cells but not of mock transfectant (HepG2-M) cells was stimulated on the plates coated with laminin-5. Furthermore, HepG2-3A and HepG2-3B cells were found to be more invasive into laminin-5-containing matrices than were HepG2-M cells. These results strongly suggest that enhanced expression of α3β1 integrin on HCC cells is directly involved in their malignant phenotypes such as invasion and metastasis.

Keywords: Abbreviations; TGF; transforming growth factor; HCC; hepatocellular carcinoma; ECM; extracellular matrix; RT-PCR; reverse transcription-polymerase chain reactionα3 integrin; Hepatocellular carcinoma; Cell adhesion; Invasion; Laminin

Sphingolipid therapy in myocardial ischemia–reperfusion injury by Susheel Gundewar; David J. Lefer (pp. 571-576).

Sphingolipids are known to play a significant physiological role in cell growth, cell differentiation, and critical signal transduction pathways. Recent studies have demonstrated a significant role of sphingolipids and their metabolites in the pathogenesis of myocardial ischemia–reperfusion injury. Our laboratory has investigated the cytoprotective effects of N, N, N-trimethylsphingosine chloride (TMS), a stable N-methylated synthetic sphingolipid analogue on myocardial and hepatic ischemia–reperfusion injury in clinically relevant in vivo murine models of ischemia–reperfusion injury. TMS administered intravenously at the onset of ischemia reduced myocardial infarct size in the wild-type and obese (ob/ob) mice. Following myocardial I/R, there was an improvement in cardiac function in the wild-type mice. Additionally, TMS also decreased serum liver enzymes following hepatic I/R in wild-type mice. The cytoprotective effects did not extend to the ob/ob mice following hepatic I/R or to the db/db mice following both myocardial and hepatic I/R. Our data suggest that although TMS is cytoprotective following I/R in normal animals, the cytoprotective actions of TMS are largely attenuated in obese and diabetic animals which may be due to altered signaling mechanisms in these animal models. Here we review the therapeutic role of TMS and other sphingolipids in the pathogenesis of myocardial ischemia–reperfusion injury and their possible mechanisms of cardioprotection.

Keywords: Myocardial infarction; N; ,; N; ,; N; -trimethylsphingosine; Diabetes; Obesity; db/db; ob/ob

Interleukin-4 induces specific pp-GalNAc-T expression and alterations in mucin O-glycosylation in colonic epithelial cells by Akira Kanoh; Hideyuki Takeuchi; Kentaro Kato; Michihiko Waki; Katsuaki Usami; Tatsuro Irimura (pp. 577-584).

Mucus hypersecretion occurs as a consequence of the Th2 immune response in epithelia, yet it was not previously known whether the degree of O-glycosylation was modulated under such conditions. A colonic carcinoma cell line LS174T was used to assess the effect of interleukin (IL)-4 on the mRNA levels of eight pp-GalNAc-Ts. A three- to four-fold increase in pp-GalNAc-T1, T4, and T7 levels was observed. Lysates of untreated or IL-4-treated cells were examined for their ability to transfer GalNAc residues onto a peptide corresponding to the tandem repeat portion of human MUC2. The number of incorporated GalNAc residues was greater after incubation with lysates of IL-4-treated cells than with lysates of untreated cells. Mucin-like large glycoproteins secreted by IL-4-treated cells had higher binding capacity to PNA and VVA-B₄ than those secreted by untreated cells. The results indicated that IL-4-treated LS174T cells are able to produce mucins with a higher degree of O-glycosylation than untreated counterparts.

Keywords: Abbreviations; HPLC; high performance liquid chromatography; pp-GalNAc-T; UDP-; N; -acetyl-; d; -galactosamine: polypeptide UDP-; N; -acetylgalactosaminyltransferase; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresisIL-4; pp-GalNAc-T; Mucin; O; -glycosylation; Lectin

Regulation of tumor phenotypes by caveolin-1 and sphingolipid-controlled membrane signaling complexes by Alessandro Prinetti; Simona Prioni; Nicoletta Loberto; Massimo Aureli; Vanna Chigorno; Sandro Sonnino (pp. 585-596).

Aberrant (glyco)sphingolipid expression deeply affects several properties of tumor cells that are involved in tumor progression and metastasis formation: cell adhesion (to the extracellular matrix or to the endothelium of blood vessels), motility, recognition and invasion of host tissues. In particular, (glyco)sphingolipids might contribute to the modulation of integrin-dependent interactions of tumor cells (determining their adhesion, motility and invasiveness) with the extracellular matrix as well as with host cells present in the stromal compartment of the tumor. A model based on solid experimental evidence has been proposed: (glyco)sphingolipids at the cell surface interact with plasma membrane receptors (e.g., integrin receptors and growth factor receptors) and adapter molecules (including tetraspanins) forming signaling complexes that are able to influence the activity of signal transduction molecules oriented at the cytosolic surface of the plasma membrane (mainly the Src kinases pathway members). The function of these signaling complexes appears to be strictly dependent on their (glyco)sphingolipid composition, and likely on specific sphingolipid–protein interactions. From this point of view, particularly intriguing is the connection between (glyco)sphingolipids and caveolin-1, a membrane protein that plays multiple roles as a suppressor of tumor growth and metastasis in ovarian, breast and colon human carcinomas.

Keywords: Abbreviation; GSL; glycosphingolipid(s)Sphingolipid; Caveolin-1; GM3 ganglioside; Glycosynapse; Lipid raft; Tumor cell motility

Lysophospholipid signaling: Beyond the EDGs by William J. Valentine; Yuko Fujiwara; Ryoko Tsukahara; Gabor Tigyi (pp. 597-605).

As our understanding of the myriads of biological effects caused by lysophospholipids expands, we become witnesses to another miracle of nature that has endowed the simplest lysophospholipids with functions seemingly ubiquitous to every mammalian cell. A decade after the discovery of the EDG family lysophospholipid receptors, the field has gained unimaginable impetus explaining the biological effects of sphingosine-1-phosphate and lysophosphatidic acid (LPA). The discovery of LPA receptors in the purinergic G-protein-coupled receptor (GPCR) gene cluster refined this picture and added complexity to our concepts of lysophospholipid cell signaling. The intracellular lysophospholipid targets – identified and not yet identified – make us realize the dual mediator and second messenger roles of lysophospholipids. In this paper we provide new data obtained concerning LPA-elicited responses using cell lines naturally lacking or intentionally knocked out of many of the known LPA GPCR, widely used by investigators in the field as cells with LPA receptor “null background.” Our observations raise caution about the lack of LPA responsiveness in these cells and underline the unprecedented complexity and redundancy of lysophospholipid-evoked cellular responses.

Keywords: Lysophospholid; Sphingosine-1-phosphate; Lysophosphatidic acid; Receptor; Mitogen activated protein kinase; LPA; Endothelial differentiation gene

Plasma sphingosine 1-phosphate metabolism and analysis by Yutaka Yatomi (pp. 606-611).

The importance of sphingosine 1-phosphate (Sph-1-P) as an intercellular sphingolipid mediator has been established in various systems, and this is especially true in the areas of vascular biology and immunology. Blood platelets store Sph-1-P abundantly and release this bioactive lysophospholipid extracellularly upon stimulation, while vascular endothelial cells and smooth muscle cells respond dramatically to this platelet-derived bioactive lipid. Most of the responses elicited by extracellular Sph-1-P are believed to be mediated by G protein-coupled cell surface receptors, i.e., S1Ps. It is likely that regulation of Sph-1-P biological activity could be important for therapeutics, including but not limited to control of vascular disorders. Furthermore, elucidation of the mechanisms by which the levels of Sph-1-P in the blood are regulated seems important. Accordingly, the application of Sph-1-P analysis to laboratory medicine may be an important task in clinical medicine. In this review, Sph-1-P-related metabolism in the plasma will be summarized. Briefly, the levels and bioactivities of plasma Sph-1-P in vivo may be regulated by various factors, including Sph-1-P release from platelets (and red blood cells, based upon the recent reports), Sph-1-P distribution between albumin and lipoproteins, and S1P expression and lipid phosphate phosphatase activity on the cell surface. Then, application of Sph-1-P analysis to laboratory medicine will be discussed.

Keywords: Sphingosine 1-phosphate; Vascular biology; Platelet; Red blood cell; Laboratory medicine

Lysenin: A sphingomyelin specific pore-forming toxin by Hidehiko Shogomori; Toshihide Kobayashi (pp. 612-618).

Sphingomyelin is a major sphingolipid in mammalian cells. Recent results indicate that sphingomyelin is a reservoir of lipid second messengers, ceramide and sphingosine-1-phosphate. Sphingomyelin is also a major component of sphingolipid and cholesterol-rich membrane domains (lipid rafts). Lysenin is a pore-forming toxin that specifically binds sphingomyelin. The binding of lysenin to sphingomyelin is dependent on the membrane distribution of the lipid, i.e. the toxin selectively binds sphingomyelin clusters. Development of a non-toxic lysenin mutant revealed the spatial and functional heterogeneity of sphingolipid-rich membrane domains.

Keywords: Lipid domain; Lipid rafts; Cholesterol; Glycolipids

920 MHz ultra-high field NMR approaches to structural glycobiology by Koichi Kato; Hiroaki Sasakawa; Yukiko Kamiya; Maho Utsumi; Michiko Nakano; Noriko Takahashi; Yoshiki Yamaguchi (pp. 619-625).

Although NMR spectroscopy has great potential to provide us with detailed structural information on oligosaccharides and glycoconjugates, the carbohydrate NMR analyses have been hampered by the severe spectral overlapping and the insufficiency of the conformational restraints. Recently, ultra-high field NMR spectrometers have become available for applications to structural analyses of biological macromolecules. Here we demonstrate that ultra-high fields offer not only increases in sensitivity and chemical shift dispersion but also potential benefits for providing unique information on chemical exchange and relaxation, by displaying NMR spectral data of oligosaccharide, glycoprotein, and glycolipid systems recorded at a 21.6 T magnetic field (corresponding to 920 MHz¹H observation frequency). The ultra-high field NMR spectroscopy combined with sugar library and stable-isotope labeling approaches will open new horizons in structural glycobiology.

Keywords: Abbreviations; Aβ; amyloid β; protein; 3D; three-dimensional; CD; α-cyclodextrin; CSA; chemical shift anisotropy; HSQC; heteronuclear single-quantum correlation; Ig; immunoglobulin; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; PA; pyridylaminated; RDC; residual dipolar coupling; TROSY; transverse relaxation-optimized spectroscopyUltra-high field NMR spectroscopy; Structural glycobiology; Stable-isotope labeling; Oligosaccharide; Glycoprotein; Glycolipid; Ganglioside GM1; Amyloid β

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