Poster Sessions CP06: Cell Surface,Cell Membrane. Glucuronyl glycoconjugates replace sulfoglucuronyl glycoconjugates in HNK‐1 knockout mice

Sulfoglucuronyl carbohydrate (SGC), reactive with HNK‐1 antibody, is expressed in several glycolipids, glycoproteins and proteoglycans of the nervous system. The interaction of SGC with SGC‐binding protein, SBP‐1 has been implicated in cell‐cell recognition, neurite outgrowth and neuronal migration during development. In sulfotransferase (ST) null mutant mice, which lack SGC, synaptic transmission in pyramidal cells of the hippocampus was increased and long‐term potentiation was reduced. However, ST null mice are viable, fertile and have wild type anatomy of all major brain areas and many non‐neural organs. Failure to observe severe phenotype in the ST null mice prompted us to determine the compensatory molecular replacement of SGC by analyzing the carbohydrate of glycolipids and glycoprotefins of the mutant nervous system. In the ST null mice, SGC containing molecules were absent and they were replaced by the precursor glucuronyl carbohydrate (GC) containing molecules. Other relevant glycolipids and proteins were not affected. The GC molecules in the mutant were localized at the same anatomical sites as the SGC molecules in the wild type. In vitro binding studies showed that similar to sulfoglucuronyl glycolipids, glucuronyl glycolipids interacted with SBP‐1, but with a lower binding capacity. In vitro studies with explant cultures of cerebellum indicated that neurite outgrowth and cell migration were not significantly affected, possibly due to interaction of SBP‐1 with the GC molecules. The results indicated that in vivo SBP‐1–GC interaction was sufficient enough for normal neurite outgrowth and cell migration in the mutant and thus having a minimal abnormal phenotype.     

A novel approach for the topographical localization of glycolipids on the cell surface

In this study we have developed a prototype system for distinguishing between the topographical distribution of glycolipids versus glycoproteins on the ultrastructural level. Direct modification of membrane-based sialic acids with biotin groups labels both glycolipids and glycoproteins. In this case, subsequent ultrastructural localization of biotinylated sites would not discern between these two classes of glycoconjugate in an unambiguous manner. When biotinylated cells are fixed prior to interaction with ferritin-conjugated avidin, the mean distance of marker molecules from the membrane bilayer is 8.0 nm. In contrast, if the cells are allowed to cap through the action of ferritin-avidin conjugates on unfixed cells, the average distance (13.0 nm) of the marker molecules appears even more distant from the membrane on the capped portion of the cell (uropod), whereas those on the head region are positioned in close proximity to the bilayer (3.7 nm). In order to exclusively label cell surface glycolipids on the ultrastructural level, bovine brain gangliosides were biotinylated in vitro and the haptenized gangliosides were incorporated into intact cells. In this case, marker molecules denoting the incorporated gangliosides were found in relatively close juxtaposition to the membrane surface, in a manner strikingly similar to the labeling pattern of the head region on capped cells. These results support the concept that, in the native state, the carbohydrate portion of glycolipids is positioned closer to the membrane bilayer than that of glycoproteins.     

Incorporation of lone pairs into the electrostatic term in the empirical intra- and intermolecular energy calculations

The method hitherto used for estimating the electrostatic term in empirical intramolecular calculations of stable conformations of biologically important molecules and macromolecules and intermolecular calculations of molecular associations or packing energy in molecular crystals had been analyzed. It has been shown that the contribution of atomic hybridization moments is omitted in the calculation of electrostatic interactions from net atomic charges localized on nuclei which have been determined by standard quantum-chemical methods. This contribution plays an important part in determining electrostatic interactions, mainly in molecules containing atoms with lone pairs. Simultaneously, a modified method for calculating the electrostatic term comprising the interaction of the lone pairs, which are represented by atomic hybridization moments, has been proposed. The relationship between the atomic hybridization moment and the bond angle has been expressed for some typical configurations occurring in biologically important molecules. Finally, this new approach is illustrated by results of the conformational analysis of some model compounds for biomolecules and compared with the approach used so far for the estimation of the electrostatic interaction in empirical methods of calculation of the intra- and intermolecular energy.     

New EPR Method for Cellular Surface Characterization

An electron paramagnetic resonance (EPR)-based membrane surface characterization method is presented to detect the properties of the carbohydrate-rich part of membrane surfaces as well as carbohydrate interaction with other membrane constituents and water-soluble molecules. The proposed method relies on the spin-labeling and spectral decomposition based on spectral simulation and optimization with EPRSIM software. In order to increase the sensitivity of characterization to the carbohydrate-rich part of the membrane surface, the sucrose-contrasting approach is introduced. With this method, which was established on model membranes with glycolipids and tested on erythrocyte membrane, we were able to characterize the surface and lipid bilayer lateral heterogeneity. Additionally, some properties of the interaction between glycocalyx and lipid bilayer as well as between glycocalyx and sucrose molecules were determined. The experiments also provided some information about the anchoring and aggregation of the glycosylated molecules. According to the results, some functions of the glycosylated surface are discussed.     

HNK-1 sulfotransferase null mice express glucuronyl glycoconjugates and show normal cerebellar granule neuron migration in vivo and in vitro

Sulfoglucuronyl carbohydrate (SGC), reactive with antibody against human natural killer cell antigen, is expressed in several glycolipids, glycoproteins and proteoglycans of the nervous system and has been implicated in cell-cell recognition, neurite outgrowth and neuronal migration during development, through its interaction with SGC-binding protein (SBP) 1. However, sulfotransferase (ST) null mutant mice, which lack SGC, were shown to have normal development with usual gross anatomy of the nervous system and other organs. Failure to observe a severe phenotype in the ST null mice prompted us to determine the compensatory molecular replacement of SGC by analyzing the carbohydrate of glycolipids and glycoproteins of the ST mutant nervous system. In the ST null mice, SGC-containing molecules were absent; instead the precursor glucuronyl carbohydrate (GC)-containing molecules accumulated. Other relevant glycolipids and proteins were not affected. The GC molecules in the mutant were localized at the same anatomical sites in the nervous system as the SGC molecules in the wild type. In vitro binding studies showed that, similar to sulfoglucuronyl glycolipids, glucuronyl glycolipids interacted with SBP-1, but with a lower binding capacity. In vitro studies with explant cultures of cerebellum indicated that neurite outgrowth and cell migration were not significantly affected in the mutant, possibly owing to interaction of SBP-1 with GC molecules. The results suggested that in vivo SBP-1-GC interaction was sufficient to allow normal neurite outgrowth and cell migration in the mutant, giving rise to a wild-type phenotype. However, the role of other compensatory molecules involved in these processes cannot be completely ruled out.     

HNK-1 carbohydrate synthesis in retinoic acid-induced P19 cells

Sulfoglucuronyl carbohydrate (SGC), reactive with HNK-1 antibody, is expressed in several glycolipids, glycoproteins and proteoglycans of the nervous system. The interaction of SGC with SGC-binding protein, SBP-1 has been implicated in cell-cell recognition, neurite outgrowth and neuronal migration during development. In sulfotransferase (ST) null mutant mice, which lack SGC, synaptic transmission in pyramidal cells of the hippocampus was increased and long-term potentiation was reduced. However, ST null mice are viable, fertile and have wild type anatomy of all major brain areas and many non-neural organs. Failure to observe severe phenotype in the ST null mice prompted us to determine the compensatory molecular replacement of SGC by analyzing the carbohydrate of glycolipids and glycoprotefins of the mutant nervous system. In the ST null mice, SGC containing molecules were absent and they were replaced by the precursor glucuronyl carbohydrate (GC) containing molecules. Other relevant glycolipids and proteins were not affected. The GC molecules in the mutant were localized at the same anatomical sites as the SGC molecules in the wild type. In vitro binding studies showed that similar to sulfoglucuronyl glycolipids, glucuronyl glycolipids interacted with SBP-1, but with a lower binding capacity. In vitro studies with explant cultures of cerebellum indicated that neurite outgrowth and cell migration were not significantly affected, possibly due to interaction of SBP-1 with the GC molecules. The results indicated that in vivo SBP-1–GC interaction was sufficient enough for normal neurite outgrowth and cell migration in the mutant and thus having a minimal abnormal phenotype.     

Recognition at cell surfaces: phytohaemagglutinin-lymphocyte interaction.

Many aspects of cell behaviour are regulated by the interaction of extracellular ligands with specific receptors exposed on the cell surface. The receptors correspond to membrane proteins and expecially glycoproteins. A key event in regulation is the transmission across the surface membrane of the information resulting from receptor-ligand interaction. The activation of lymphocytes by Phaseolus vulgaris phytohaemagglutinin (PHA) provides a convenient experimental model for the study of the molecular basis of receptor-ligand interaction and the molecular consequences of interaction. The receptor mediating lymphocyte activation by PHA is probably a unique glycoprotein which is present to the extent of about 3 X 10(4) molecules/cell. The PHA-receptor complex solubilized in 1% sodium deoxycholate has a molecular size of about 3 X 10(5). The primary event in the activation process is probably an increase in the permeability of the surface membrane to Ca2+. This may be achieved by PHA cross-linking ('patching') the receptors to form a polar channel that permits an influx of Ca2+.     

Functional expression of a myo-inositol/H+ symporter from Leishmania donovani.

The vast majority of surface molecules in such kinetoplastid protozoa as members of the genus Leishmania contain inositol and are either glycosyl inositol phospholipids or glycoproteins that are tethered to the external surface of the plasma membrane by glycosylphosphatidylinositol anchors. We have shown that the biosynthetic precursor for these abundant glycolipids, myo-inositol, is translocated across the parasite plasma membrane by a specific transporter that is structurally related to mammalian facilitative glucose transporters. This myo-inositol transporter has been expressed and characterized in Xenopus laevis oocytes. Two-electrode voltage clamp experiments demonstrate that this protein is a sodium-independent electrogenic symporter that appears to utilize a proton gradient to concentrate myo-inositol within the cell. Immunolocalization experiments with a transporter-specific polyclonal antibody reveal the presence of this protein in the parasite plasma membrane.     

Interaction of negatively charged liposomes with nuclear membranes: adsorption, lipid mixing and lysis of the vesicles

Fluorescence energy transfer studies reveal that negatively charged lipid vesicles interact with nuclei from mouse liver cells. This interaction was observed with charged lipid vesicles composed of PA or PS but not with the uncharged PC or PE:PC vesicles. The vesicles were prepared by bath sonication and contained either a fluorescent marker in the lipid bilayer or in the vesicular interior. The negatively charged vesicles showed an adsorption to the nuclear membrane visible by fluorescence microscopy. The results obtained by resonance energy transfer experiments are interpreted in terms of a mixing of the lipids from the vesicles with the nuclear membrane. Encapsulation studies documented a staining of the nuclei only if the dye molecules of high or low molecular weight were encapsulated inside negatively charged vesicles. As consequence of the vesicle-nuclei interaction morphological changes on the nuclear surface became visible.     

Shapes of bilayer vesicles with membrane embedded molecules

The interdependence of the lateral distribution of molecules which are embedded in a membrane (such as integral membrane proteins) and the shape of a cell with no internal structure (such as phospholipid vesicles or mammalian erythrocytes) has been studied. The coupling of the lateral distribution of the molecules and the cell shape is introduced by considering that the energy of the membrane embedded molecule at a given site of the membrane depends on the curvature of the membrane at that site. Direct interactions between embedded molecules are not considered. A simple expression for the interaction of the membrane embedded molecule with the local membrane curvature is proposed. Starting from this interaction, the consistently related expressions for the free energy and for the distribution function of the embedded molecules are derived. The equilibrium cell shape and the corresponding lateral distribution of the membrane embedded molecules are determined by minimization of the membrane free energy which includes the free energy of the membrane embedded molecules and the membrane elastic energy. The resulting inhomogeneous distribution of the membrane embedded molecules affects the cell shape in a nontrivial manner. In particular, it is shown that the shape corresponding to the absolute energy minimum at given cell volume and membrane area may be elliptically non-axisymmetric, in contrast to the case of a laterally homogeneous membrane where it is axisymmetric.     

Specific and non specific interactions involving Le X determinant quantified by lipid vesicle micromanipulation

Carbohydrate-carbohydrate recognition is emerging today as an important type of interaction in cell adhesion. One Ca(2+)mediated homotypic interaction between two Lewis( X ) determinants (Le( X )) has been proposed to drive cell adhesion in murine embryogenesis. Here, the adhesion energies of lipid vesicles functionalized with glycolipids bearing monomeric or dimeric Le( X ) determinants were measured in NaCl or CaCl(2) media with the micropipette aspiration technique. These experiments on Le( X ) with an environment akin to that provided by biological membrane confirmed the existence of this specific calcium dependent interaction of monomeric Le( X ). In contrast, dimeric Le( X ) produced a repulsive contribution. By using a simple model involving the various contributions to the adhesion free energy, specific and non specific interactions could be separated and quantified. The involvement of calcium ions has been discussed in the monomeric and dimeric Le( X ) lipids.     

Contribution of interfacial tension changes during cellular interaction to the energy balance

The energetics of cell-cell and cell-substrate interactions has been analyzed in terms of the lyophobic colloid stability theory adapted to biological conditions. Some important differences that exist between lyophobic particles and living cells are recognized and taken into account. The protein-aceous coat exterior to the lipid cell membrane (glycocalyx) is treated as a very thick Stern layer which has a constant electric capacitance. The cell itself is viewed as a fluid droplet due to the semi-fluid state of the cell membrane, and its outer boundary is assumed to have a constant electric charge density. When particles with constant surface charge density interact, their surface potential increases. Then the potential at the lipid-protein interface will also increase, hence the interfacial tension should decrease. The magnitude of the interfacial tension change at the lipid-protein interface occurring during the interaction of cells has been calculated for various thicknesses of the glycocalyx. This term, obtained for cells with a relatively thin proteinaceous coat, was found to dominate the energy balance, making the total energy of interaction negative.     

Mean field analysis of FKBP12 complexes with FK506 and rapamycin: Implications for a role of crystallographic water molecules in molecular recognition and specificity

Mean field analysis of FKBP12 complexes with FK506 and rapamycin has been performed by using structures obtained from molecular docking simulations on a simple, yet robust molecular recognition energy landscape. When crystallographic water molecules are included in the simulations as an extension of the FKBP12 protein surface, there is an appreciable stability gap between the energy of the native FKBP12–FK506 complex and energies of conformations with the “native-like” binding mode. By contrast, the energy spectrum of the FKBP12–rapamycin complex is dense regardless of the presence of the water molecules. The stability gap in the FKBP12–FK506 system is determined by two critical water molecules from the effector region that participate in a network of specific hydrogen bond interactions. This interaction pattern protects the integrity and precision of the composite ligand-protein effector surface in the binary FKBP12–FK506 complex and is preserved in the crystal structure of the FKBP12–FK506–calcineurin ternary complex. These features of the binding energy landscapes provide useful insights into specific and nonspecific aspects of FK506 and rapamycin recognition. Proteins 28:313–324, 1997. © 1997 Wiley-Liss, Inc.     

Generation of Stable Lipid Raft Microdomains in the Enterocyte Brush Border by Selective Endocytic Removal of Non-Raft Membrane

The small intestinal brush border has an unusually high proportion of glycolipids which promote the formation of lipid raft microdomains, stabilized by various cross-linking lectins. This unique membrane organization acts to provide physical and chemical stability to the membrane that faces multiple deleterious agents present in the gut lumen, such as bile salts, digestive enzymes of the pancreas, and a plethora of pathogens. In the present work, we studied the constitutive endocytosis from the brush border of cultured jejunal explants of the pig, and the results indicate that this process functions to enrich the contents of lipid raft components in the brush border. The lipophilic fluorescent marker FM, taken up into early endosomes in the terminal web region (TWEEs), was absent from detergent resistant membranes (DRMs), implying an association with non-raft membrane. Furthermore, neither major lipid raft-associated brush border enzymes nor glycolipids were detected by immunofluorescence microscopy in subapical punctae resembling TWEEs. Finally, two model raft lipids, BODIPY-lactosylceramide and BODIPY-GM₁, were not endocytosed except when cholera toxin subunit B (CTB) was present. In conclusion, we propose that constitutive, selective endocytic removal of non-raft membrane acts as a sorting mechanism to enrich the brush border contents of lipid raft components, such as glycolipids and the major digestive enzymes. This sorting may be energetically driven by changes in membrane curvature when molecules move from a microvillar surface to an endocytic invagination.     

Overview of the diversity of erythrocyte blood group antigens

During the differentiation and maturation of erythrocytes, the surface molecules of erythrocytes are gradually expressed and stabilized. These molecules are to be antigenic in addition to their functions of maintaining cell membrane structural stability, material transport and exchange of cells and signal transmission between cells. The antigenic molecules on the erythrocyte surface are called erythrocyte blood group antigens. The blood group antigens and their corresponding blood group antibodies in vivo are important indicators for clinical blood transfusion and organ transplantation, and also form the basis for research on blood group related diseases. Three hundred and sixty-eight erythrocyte blood group antigens have been confirmed so far, which are classified into 39 blood group systems, 5 blood group collections and 2 blood group series. Based on the diversity of blood group antigens and their composition of glycolipids, glycoproteins and other molecules, this study mainly reviews the classification, molecular structure, antibody response and gene regulation of blood group antigens, and explains the main reasons for the diversity of blood group antigens.     

Tetraspanins in cellular immunity

Tetraspanins are a superfamily of integral membrane proteins involved in the organization of microdomains that consist of both cell membrane proteins and cytoplasmic signalling molecules. These microdomains are important in regulating molecular recognition at the cell surface and subsequent signal transduction processes central to the generation of an efficient immune response. Tetraspanins, both immune-cell-specific, such as CD37, and ubiquitously expressed, such as CD81, have been shown to be imp-ortant in both innate and adaptive cellular immunity. This is via their molecular interaction with important immune cell-surface molecules such as antigen-presenting MHC proteins, T-cell co-receptors CD4 and CD8, as well as cytoplasmic molecules such as Lck and PKC (protein kinase C). Moreover, the generation of tetraspanin-deficient mice has enabled the study of these proteins in immunity. A variety of tetraspanins have a role in the regulation of pattern recognition, antigen presentation and T-cell proliferation. Recent studies have also begun to elucidate roles for tetraspanins in macrophages, NK cells (natural killer cells) and granulocytes.     

Desialylation of surface receptors as a new dimension in cell signaling

Terminal sialic acid residues are found in abundance in glycan chains of glycoproteins and glycolipids on the surface of all live cells forming an outer layer of the cell originally known as glycocalyx. Their presence affects the molecular properties and structure of glycoconjugates, modifying their function and interactions with other molecules. Consequently, the sialylation state of glycoproteins and glycolipids has been recognized as a critical factor modulating molecular recognitions inside the cell, between the cells, between the cells and the extracellular matrix, and between the cells and certain exogenous pathogens. Until recently sialyltransferases that catalyze transfer of sialic acid residues to the glycan chains in the process of their biosynthesis were thought to be mainly responsible for the creation and maintenance of a temporal and spatial diversity of sialylated moieties. However, the growing evidence suggests that in mammalian cells, at least equally important roles belong to sialidases/neuraminidases, which are located on the cell surface and in intracellular compartments, and may either initiate the catabolism of sialoglycoconjugates or just cleave their sialic acid residues, and thereby contribute to temporal changes in their structure and functions. The current review summarizes emerging data demonstrating that mammalian neuraminidase 1, well known for its lysosomal catabolic function, is also targeted to the cell surface and assumes the previously unrecognized role as a structural and functional modulator of cellular receptors.     

Where catabolism meets signalling: neuraminidase 1 as a modulator of cell receptors

Terminal sialic acid residues are found in abundance in glycan chains of glycoproteins and glycolipids on the surface of all live cells forming an outer layer of the cell originally known as glycocalyx. Their presence affects the molecular properties and structure of glycoconjugates, modifying their function and interactions with other molecules. Consequently, the sialylation state of glycoproteins and glycolipids has been recognized as a critical factor modulating molecular recognitions inside the cell, between the cells, between the cells and the extracellular matrix, and between the cells and certain exogenous pathogens. Sialyltransferases that attach sialic acid residues to the glycan chains in the process of their initial synthesis were thought to be mainly responsible for the creation and maintenance of a temporal and spatial diversity of sialylated moieties. However, the growing evidence also suggests that in mammalian cells, at least equally important roles belong to sialidases/neuraminidases, which are located on the cell surface and in intracellular compartments, and may either initiate the catabolism of sialoglycoconjugates or just cleave their sialic acid residues, and thereby contribute to temporal changes in their structure and functions. The current review summarizes emerging data demonstrating that neuraminidase 1 (NEU1), well known for its lysosomal catabolic function, can be also targeted to the cell surface and assume the previously unrecognized role as a structural and functional modulator of cellular receptors.     

Acidic pH modulates the interaction between human granulocyte-macrophage colony-stimulating factor and glycosaminoglycans.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls growth and differentiation of hematopoietic cells. Previous reports have indicated that the mitogenic activity of GM-CSF may be modulated by the glycosidic moiety of proteoglycans associated with the membrane of stromal cells. In this work, we have performed in vitro studies of the interaction between GM-CSF and glycosaminoglycans. The addition of heparin promoted a marked blue shift in the fluorescence emission spectrum of GM-CSF as well as a 30-fold increase in the intensity of light scattering, which indicates formation of large molecular weight complexes between the two molecules. Interestingly, heparin-induced changes in the spectral properties of GM-CSF were only observed at acidic pH. The dependence on acidic pH, together with a strict dependence on glycosaminoglycan sulfation and the fact that high ionic strength destabilized the interaction, indicates that the association between GM-CSF and glycosaminoglycans is mediated by electrostatic interactions. These interactions probably involve sulfate groups in the glycosaminoglycans and positively charged histidine residues in GM-CSF. We propose that negatively charged glycolipids present on the plasma membrane of the hematopoietic and/or the stromal cell could promote an acidic microenvironment capable of triggering interaction between GM-CSF and membrane-bound proteoglycans in vivo.     

Direct molecular interaction of caveolin-3 with KCa1.1 channel in living HEK293 cell expression system

Caveolin family is supposed to be essential molecules for the formation of not only caveola structure on cell membrane but also functional molecular complexes in them with direct and/or indirect interaction with other membrane and/or submembrane associated proteins. The direct coupling of caveolin-1 (cav1) with large conductance Ca²⁺-activated K⁺ channel, KCa1.1 has been established in several types of cells and in expression system as well. The possible interaction of caveolin-3 (cav3), which shows expression in some differential tissues from cav1, with KCa1.1 remains to be determined. In the present study, the density of KCa1.1 current expressed in HEK293 cells was significantly reduced by the co-expression of cav3, as well as cav1. The co-localization and direct interaction of GFP- or CFP-labeled cav3 (GFP/CFP-cav3) with YFP- or mCherry-labeled KCa1.1 (KCa1.1-YFP/mCherry) were clearly demonstrated by single molecular image analyses using total internal reflection fluorescence (TIRF) microscopy and fluorescence resonance energy transfer (FRET) analyses with acceptor photobleaching method. The deletion of suggested cav1-binding motif in C terminus region of KCa1.1 (KCa1.1ΔCB-YFP) resulted in the marked decrease in cell surface expression, co-localization and FRET efficiency with CFP-cav3 and CFP-cav1. The FLAG-KCa1.1 co-immunoprecipitation with GFP-cav3 or GFP-cav1 also supported their direct molecular interaction. These results strongly suggest that cav3 possesses direct interaction with KCa1.1, presumably at the same domain for cav1 binding. This interaction regulates KCa1.1 expression to cell surface and the formation of functional molecular complex in caveolae in living cells.