Molecular basis of the differences in binding properties of the highly related C-type lectins DC-SIGN and L-SIGN to Lewis X trisaccharide and Schistosoma mansoni egg antigens

The dendritic cell-specific C-type lectin DC-SIGN functions as a pathogen receptor that recognizes Schistosoma mansoni egg antigens through its major glycan epitope Galbeta1,4(Fucalpha1,3)GlcNAc (Lex). Here we report that L-SIGN, a highly related homologue of DC-SIGN found on liver sinusoidal endothelial cells, binds to S. mansoni egg antigens but not to the Lex epitope. L-SIGN does bind the Lewis antigens Lea, Leb, and Ley, similar as DC-SIGN. A specific mutation in the carbohydrate recognition domain of DC-SIGN (V351G) abrogates binding to all Lewis antigens. In L-SIGN Ser363 is present at the corresponding position of Val351 in DC-SIGN. Replacement of this Ser into Val resulted in a "gain of function" L-SIGN mutant that binds to Lex, and shows increased binding to the other Lewis antigens. These data indicate that Val351 is important for the fucose specificity of DC-SIGN. Molecular modeling and docking of the different Lewis antigens in the carbohydrate recognition domains of L-SIGN, DC-SIGN, and their mutant forms, demonstrate that Val351 in DC-SIGN creates a hydrophobic pocket that strongly interacts with the Fucalpha1,3/4-GlcNAc moiety of the Lewis antigens. The equivalent amino acid residue Ser363 in L-SIGN creates a hydrophilic pocket that prevents interaction with Fucalpha1,3-GlcNAc in Lex but supports interactions with the Fucalpha1,4-GlcNAc moiety in Lea and Leb antigens. These data demonstrate for the first time that DC-SIGN and L-SIGN differ in their carbohydrate binding profiles and will contribute to our understanding of the functional roles of these C-type lectin receptors, both in recognition of pathogen and self-glycan antigens.     

The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x

Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S. mansoni. We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN). Monoclonal antibodies against the carbohydrate antigens Lewisx (Lex) and GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDNF) inhibit binding of DC-SIGN to SEAs, suggesting that these glycan antigens may be critically involved in binding. In a solid-phase adhesion assay, DC-SIGN-Fc binds polyvalent neoglycoconjugates that contain the Lex antigen, whereas no binding was observed to Galbeta1-4GlcNAc, and binding to neoglycoconjugates containing only alpha-fucose or oligosaccharides with a terminal alpha1-2-linked fucose is low. These data indicate that binding of DC-SIGN to Lex antigen is fucose-dependent and that adjacent monosaccharides and/or the anomeric linkage of the fucose are important for binding activity. Previous studies have shown that DC-SIGN binds HIV gp120 that contains high-mannose-type N-glycans. Site-directed mutagenesis within the carbohydrate recognition domain (CRD) of DC-SIGN demonstrates that amino acids E324 and E347 are involved in binding to HIV gp120, Lex, and SEAs. By contrast, mutation of amino acid Val351 abrogates binding to SEAs and Lex but not HIV gp120. These data suggest that DC-SIGN recognizes these ligands through different (but overlapping) regions within its CRD. Our data imply that DC-SIGN not only is a pathogen receptor for HIV gp120 but may also function in pathogen recognition by interaction with the carbohydrate antigens Lex and possibly LDNF, which are found on important human pathogens, such as schistosomes and the bacterium Helicobacter pylori.     

Specific interaction between Lex and Lex determinants. A possible basis for cell recognition in preimplantation embryos and in embryonal carcinoma cells

The Lex determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc-beta 1----R) has been implicated as having a role in mediating compaction of the mouse embryo at the morula stage (Fenderson, B., Zehavi, U., and Hakomori, S. (1984) J. Exp. Med. 160, 1591-1596). Here, we present evidence suggesting a role for Lex in F9 embryonal carcinoma cell adhesion and a mechanism for Lex recognition based on carbohydrate-carbohydrate interaction. Homotypic aggregation of F9 cells was inhibited by lacto-N-fucopentaose III, and F9 cells showed a preferential interaction with Lex liposomes. The following observations suggest that the structure capable of recognizing Lex per se on F9 cells is Lex: (i) Cell surface-labeled components solubilized in octylglucoside, affinity-bound on an Lex-octyl-Sepharose column, contained glycoproteins reactive with anti-Lex antibody. (ii) Liposomes containing Lex showed significant interaction with Lex glycolipid, but not other glycolipids, coated on a plastic surface. (iii) Liposomes containing Lex glycolipid were found to self-aggregate, whereas liposomes containing paragloboside (nLc4) or sialylparagloboside (IV3NeuAcnLc4) did not. (iv) The diffusibility of 3H-labeled lacto-N-fucopentaitol III (but not I or II), incubated with Lex liposome, from the lower to the upper Boyden chamber through a semipermeable membrane was inhibited. In all these experiments (i-iv), the interaction of Lex to Lex (or Lex to lacto-N-fucopentaose III) was clearly observed only in the presence of Ca2+ and Mg2+ and was enhanced by the presence of Mn2+. These interactions were inhibited by EDTA. The results suggest the novel hypothesis that carbohydrate-carbohydrate interactions may play an important role in controlling cell recognition during F9 cell aggregation and during embryonic development.     

A carbohydrate domain common to both sialyl Le(a) and sialyl Le(X) is recognized by the endothelial cell leukocyte adhesion molecule ELAM-1.

The specificity of endothelial cell leukocyte adhesion molecule-1, ELAM-1, for binding to a panel of carbohydrate structures was determined by a sensitive cell binding assay with immobilized synthetic glycoconjugates. ELAM-1 cDNA transfectants were found to bind Sialyl Lea (sialylated lacto-N-fucopentaose II) or sialylated Lewis a antigen (NeuAc alpha 2-3Gal beta 1-3(Fuc alpha 1-4)GlcNAc), as well as or slightly better than Sialyl Lex (sialylated lacto-N-fucopentaose III) or sialylated Lewis X antigen (NeuAc alpha 2-3 Gal beta 1-4(Fuc alpha 1-3)GlcNAc). A monoclonal antibody, HECA-452, which has been identified recently as recognizing ELAM-1 ligands in addition to those containing Sialyl Lex, was also found to bind both Sialyl Lex and Sialyl Lea. Hard sphere exo-anomeric (HSEA) calculations were performed on these two hexasaccharides. The conformations indicate that Sialyl Lea and Sialyl Lex show a high degree of similarity in both the nonreducing and reducing termini. As Lea and Lex show much weaker reactivity, the determinants recognized by ELAM-1 and HECA-452 probably involve neuraminic acid and fucose residues which on one face of both Sialyl Lex and Sialyl Lea can be similarly positioned. The finding that Sialyl Lea is a potent ligand for ELAM-1 is important, as circulating Sialyl Lea and Sialyl Lex containing mucins which are elevated in the serum of many cancer patients may block leukocyte interactions with ELAM-1 and may contribute to the pathological immunodepression observed in these patients.     

Immobilized glycoconjugates for cell recognition studies

Specific cell-cell recognition and adhesion may involve cell surface glycoconjugates on one cell binding the complementary carbohydrate receptors on an apposing cell surface. Such interactions have been modeled by immobilizing simple synthetic glycosides, glycoproteins, glycosaminoglycans, and glycolipids on otherwise inert plastic surfaces and incubating them with intact cells. Using this approach, the ability of several cell types to recognize specific carbohydrates has been demonstrated. This carbohydrate-directed cell adhesion may depend on cell surface carbohydrate receptors which mediate both the initial specific adhesion and complex postrecognition cellular responses. While the relationship of the cell adhesion demonstrated here to cell-cell recognition in vivo has yet to be determined, this well-controlled biochemical approach may reveal new information on the way in which cells analyze and respond to their immediate external environment.     

Carbohydrate-carbohydrate interaction as a major force initiating cell-cell recognition

Sponges were the earliest multicellular organisms to evolve through the development of cell recognition and adhesion processes mediated by cell surface proteoglycans. Information on sponges has an extra added value because, as a group, they are the oldest Metazoans alive and contribute more to our understanding of life on earth than knowledge of other animal groups. Although the proteoglycans are emerging as key players in various physiological and pathophysiological cellular events, little is known about the carbohydrate moiety of the proteoglycan molecule. Until recently there was no evidence provided for the existence of specific and biologically significant carbohydrate-carbohydrate interaction. We show here that the interaction between single oligosaccharides of surface proteoglycans is relatively strong (in the 200-300 piconewtons range) and in the same range as other relevant biological interactions, like those between antibodies and antigens. This carbohydrate-carbohydrate recognition is highly species-specific and perfectly mimics specific cell-cell recognition. Both the strength and the species-specificity of the carbohydrate-carbohydrate interaction are guaranteed by polyvalency, by compositional and architectural differences between carbohydrates, and by the arrangement of the carbohydrate chain in a three-dimensional context. Ca(2+)-ions are essential and probably provide coordinating forces. Our findings confirm the existence and character of species-specific carbohydrate-carbohydrate recognition fundamental to cell recognition and adhesion events.     

Immunity to schistosomiasis: glycans are potential antigenic targets for immune intervention

The major humoral immune responses in animals infected with Schistosoma mansoni are directed toward carbohydrate antigens. Among these antigens are complex-type N-glycans expressing LDN [GalNAcbeta1-4GlcNAc-R], LDNF [GalNAcbeta1-4(Fucalpha1-3)GlcNAc-R], and polymeric Lewis x (Lex) [Galbeta1-4(Fucalpha1-3)GlcNAc]n-R epitopes. We have now evaluated the potential of the three glycan antigens as targets for immune-mediated intervention of infections and serodiagnosis. A variety of approaches were employed, including ELISA, Western blot, immunohistology, and in vitro complement lysis assays, to determine the immunogenicity of the glycans in infected humans, their localization on the parasites and their efficacy as targets for parasite lysis. Our results show that S. mansoni-infected patients, with either intestinal or hepatosplenic disease, generate predominantly IgM, but also IgG and IgA, antibodies to LDN, LDNF, and Lex. However, immune responses to Lex are generally lower than responses to LDN and LDNF and less specific to schistosome infections. Western blot analysis with monoclonal antibodies (mAb) to LDN, LDNF, and Lex determinants show that the glycan antigens occur on multiple glycoproteins from cercariae, 3-h, 48-h, and lung stage schistosomula, as well as adults and eggs. Immunohistological studies demonstrate that LDN, LDNF, and Lex are expressed on the parasite surface at all stages of development in the vertebrate host. Importantly, a mAb to LDN in the presence of complement efficiently kills schistosomula in vitro, as demonstrated by flow-cytometric assays that quantify cytolysis by propidium iodide uptake into damaged parasites. These findings raise the possibility that LDN and LDNF may be targets for vaccination and/or serodiagnosis of chronic schistosomiasis in humans.     

Electrochemiluminescent biosensing of carbohydrate-functionalized CdS nanocomposites for in situ label-free analysis of cell surface carbohydrate

A facile electrochemiluminescent (ECL) strategy for in situ label-free monitoring of carbohydrate expression on living cells was designed by integrating the specific recognition of lectin to carbohydrate with a carbohydrate-functionalized CdS nanocomposite. The mercaptopropionic acid-capped CdS quantum dots were firstly immobilized on carbon nanotubes modified electrode and then functionalized with carbohydrate using mannan as a model on the surface. The carbohydrate-functionalized CdS nanocomposite showed high ECL sensitivity and good stability, and could be used for competitive recognition to concanavalin A with the target cells in solution, which led to a change of ECL intensity due to the resistance of concanavalin A. The change depended on both the cell number and the expression level of cell surface carbohydrate. A wide linear response to cells ranging from 2×10(3) to 1×10(7) cells mL(-1) with a detection limit of 1.2×10(3) cells mL(-1) was obtained. The proposed biosensor could be used to in situ evaluate cell surface glycan, and the average number of mannose moieties on single living BGC cell was detected to be 8.7×10(7). This sensitive strategy was further used for facile monitoring of dynamic carbohydrate expression on living cells in response to drugs. The proposed method could be further expanded to high-throughput detection with the addition of more specific glycan-lectin pairs to the repertoire.     

Glycoproteins and lectins in cell adhesion and cell recognition processes

Summary The discovery of endogenous lectins having specific and high affinity for the carbohydrate portions of glycoproteins has opened up new directions in the field of cell adhesion and cell recognition. Two endogenous lectins, termed as CSL and R1, initially isolated from the rat cerebellum and having a wide distribution in mammalian tissues, have been shown to participate in essential mechanisms of cell adhesion. The membrane-bound lectin R1 seems to be involved in transient recognition between neuronal cells, followed by elimination of the glycoprotein ligands at the surface of the recognized cell. In contrast, CSL is a molecule involved in adhesion between various normal or transformed cells since it participates in the formation of tight junctions. The glycoprotein ligands recognized with higher affinity by these two lectins seem to possess a special structure which defines a sub-class of oncofetal HNK-1 glycans. The over-expression of the glycoprotein ligands of these lectins in most transformed cells provides new tools for understanding the underlying mechanism of malignant transformation as well as the generation of signals through cell adhesion.     

Glycopeptides inhibit allospecific cytotoxic T lymphocyte recognition in an MHC-specific manner

Attempts to block specific T cell recognition with soluble extracts have been uniformly unsuccessful. However, we found that glycopeptides prepared from three MHC-different tumor cell lines were able to inhibit binding of allospecific cytotoxic T lymphocytes (CTL) to an appropriate tumor cell or Con A blast target cell. Inhibition was only observed when the MHC of the cell providing the glycopeptide was the same as the MHC of the target cell being recognized. This result was obtained by using both fully allogeneic CTL and CTL generated between B10 congenic mice differing only at the MHC. This suggests that the inhibition depends on the MHC expressed by the target cell. Because we extensively pronase digested cell glycoproteins and then enriched for glycopeptides containing small amounts of peptide, we attribute the inhibition to the carbohydrate portion of the glycopeptide. Our observations suggest that CTL may, in part, recognize carbohydrate molecules on the target cell surface whose specific structure(s) is influenced or regulated by genes in or near the MHC. They also suggest that the T cell receptor complex has some lectin-like properties.     

Glycolipid-mediated cell-cell recognition in inflammation and nerve regeneration

Cell surface complex carbohydrates have emerged as key recognition molecules, mediating physiological interactions between cells. Typically, glycans on one cell surface are engaged by complementary carbohydrate binding proteins (lectins) on an apposing cell, initiating appropriate cellular responses. Although many cell surface lectins have been identified in vertebrates, only a few of their endogenous carbohydrate ligands have been established. Each major class of cell surface glycans-glycoproteins, glycolipids, and proteoglycans-has been implicated as physiologically relevant lectin ligands. The current minireview focuses on findings that implicate glycosphingolipids as especially important molecules in cell-cell recognition in two different systems: the recognition of human leukocytes by E-selectin on the vascular endothelium during inflammation and the recognition of nerve cell axons by myelin-associated glycoprotein in myelin-axon stabilization and the regulation of axon regeneration.     

Effect of lectins on endocytosis and secretion of lysosomal enzymes by cultured fibroblasts.

1. Pretreatment of cultured human skin fibroblasts with convanavalin A and wheat germ agglutinin inhibited endocytosis of alpha-N-acetylglucosaminidase and increased extracellular accumulation of beta-N-acetylglucosaminidase. 2. These effects were dose-dependent, reversible and could be prevented by haptenic carbohydrates, such as methyl alpha-D-mannoside or N-acetylglucosamine. 3. Pretreatment of fibroblasts with di- and monovalent succinylated concanavalin A inhibited alpha-N-acetylglucosaminidase endocytosis, but had no effect on extracellular beta-N-acetylglucosaminidase accumulation. 4. Concanavalin A-alpha-N-acetylglucosaminidase complexes become internalized via the recognition of the lectin. Complex formation prevents recognition of the phosphorylated carbohydrate on lysosomal enzymes that interacts with cell surface receptors specific for lysosomal enzymes. The inhibitory effect of all lectins tested on lysosomal enzyme endocytosis suggests that the cell surface receptors for lysosomal enzymes interact either directly with lectins or are closely linked to lectin receptors. The effect of polyvalent lectins on extracellular lysosomal enzyme accumulation is ascribed to their alteration of membrane fluidity.     

Isolation of cell surface antigen mutants of Myxococcus xanthus by use of monoclonal antibodies

Monoclonal antibodies (MAbs) with affinities for molecules on the cell surface of the procaryote Myxococcus xanthus were used in a screening strategy for the isolation of mutants lacking particular cell surface molecules. From a large library of independent mutants created by Tn5 transposon mutagenesis, mutants were isolated which lacked reactivities with MAb 1604 (a MAb specific for a cell surface protein) and MAbs 2600, 1733, 1514, 1412, and 783 (MAbs specific for carbohydrate epitopes on the O antigen of lipopolysaccharide [LPS]). The defect in antibody recognition was shown by genetic crosses and DNA hybridization experiments to be caused by the Tn5 transposon acting as a mutation at a single locus. Quantitative enzyme-linked immunosorbent assays showed that particular mutant strains had no detectable affinity for the specific MAb probe. LPS mutants were resistant to myxophage Mx8, and this provided a selection method for isolating a large number of new LPS mutants. A class of Mx8-resistant mutants lacked reactivity with MAb 1514 and therefore was defective in the O antigen of LPS. A class of Mx1-resistant mutants lacked reactivity with MAb 2254, a MAb specific for a carbohydrate epitope on the core of LPS. A comparison of MAb binding to different mutant strains revealed a principle for mapping epitopes and showed that MAbs 1514 and 2254 recognize side-chain carbohydrates rather than backbone carbohydrates within the LPS molecule.     

Perspectives for establishment and reorganization of carbohydrate-directed CD antibodies. Report of the carbohydrate section

Complex glycosylated glycoproteins, glycolipids and proteoglycans are expressed on the cell surface and are also found as constituents of the extracellular matrix (ECM). Interactions of the carbohydrate moiety of these macromolecules with specific receptors (lectins) are involved in many functions of immune cells such as cell-cell or cell-ECM adhesion, recognition, and neutralization of pathogens and regulation of apoptosis. For studies on live cells mAbs recognizing distinct oligosaccharide structures are useful tools because in contrast to other analytical methods of carbohydrate biochemistry they are able to react with glycans in the complex sterical context of the cell surface. In general expression patterns of carbohydrate mAbs depend on (i) the number and type of carriers to which the glycans are linked (glycoproteins, glycolipids), (ii) the steric situation on the cell surface, and (iii) modifications of the basic glycotope (different branching, chain length, masking by sialylation, sulphation or fucosylation).     

Immunohistochemical demonstration of glycoconjugates bearing the type 2 chain-backbone structure in human fetal, normal and neoplastic gastrointestinal tract.

Immunohistochemical distributions of carbohydrate antigens based on the type 2 chain in normal as well as fetal and neoplastic tissues of human gastrointestinal tract were investigated with a monoclonal antibody (MAb) H11 (specific for type 2 chain) alone and in combination with the two MAbs MSG15 (for alpha 2----6 sialylated type 2 chain) and IB9 (for the alpha 2----6 sialylated type 2 chain and glycoproteins having NeuAc alpha 2----6Gal-NAc), and 188C1 (for short- and long-chain Lex antigens) and FH2 (for the long-chain Lex antigen). In the pyloric mucosa of secretors, the type 2 chain is oncodevelopmentally expressed, but in non-secretors it is detected in surface mucous cells of normal gastric mucosa. The alpha 2----6 sialylation, which is confined to endocrine cells of normal pyloric mucosa, occurs in fetal and carcinoma tissues. Irrespective of the secretor status, the short- and the long-chain Lex antigens can be detected in mature and immature glandular mucous cells of normal gastric mucosa, respectively; both antigens are also expressed in fetal and carcinoma tissues. In the colon, the type 2 chain and its alpha 2----6 sialylated counterpart are expressed in an oncodevelopmental manner. The short- and the long-chain Lex antigens are significantly enhanced in colonic carcinoma. The glycoproteins with NeuAc alpha 2----6GalNAc residues appear in gastric and colonic carcinoma as well as intestinalized gastric mucosa and transitional mucosa. Thus, some of these antigens were distinctively expressed in certain epithelial cells lining the normal gastrointestinal tract depending on maturation and patients' secretor status, and some were oncodevelopmental or carcinoma-associated antigens of the human gastrointestinal tract.     

Gangliosides support neural retina cell adhesion

Cell surface carbohydrates and complementary carbohydrate receptors may mediate cell-cell recognition during neuronal development. To model such interactions, we developed a technique to test the ability of cell surface lipids (particularly glycosphingolipids) to mediate specific cell recognition and adhesion (Blackburn, C.C., and Schnaar, R.L. (1983) J. Biol. Chem. 258, 1180-1188). When cells were incubated on plastic microwells adsorbed with various glycolipids, carbohydrate-specific cell adhesion was readily detected. We report here the use of this method to study adhesion of embryonic chick neural retina cells to purified cell surface lipids. Rapid and specific cell adhesion was observed when the neural retina cells were incubated on surfaces adsorbed with gangliosides (an important class of neuronal cell surface glycoconjugates) but not on surfaces adsorbed with various neutral glycosphingolipids, phospholipids, or sulfatide. This suggests that the observed cell adhesion was specific for the carbohydrate moiety of the adsorbed ganglioside and was not due to nonspecific ionic or hydrophobic interactions. Although the surface density of adsorbed lipid required to support cell adhesion was the same for all gangliosides examined, the extent of adhesion varied when different purified gangliosides were used. Ganglioside-specific adhesion was not dependent on the presence of calcium (at 37 degrees C) and was attenuated by pretreatment of the cells with trypsin. The extent of ganglioside-directed neural retinal cell adhesion varied with embryonic age. These results imply that gangliosides may play a role in cell-cell recognition in the developing nervous system.     

Soluble fibronectin interaction with cell surface and extracellular matrix is mediated by carbohydrate-to-carbohydrate interaction

Cell adhesion and spreading on solid phase fibronectin (FN), coated on plate or presented in extracellular matrix, are mediated by integrin receptors alpha5beta1, alpha4beta1, etc., although binding of "soluble-form FN" to cell surface varies extensively depending on glycosylation status of FN per se. Deposition or incorporation at the cell surface or pericellular matrix of soluble-form FN from body fluids or synthesized de novo takes place through a yet-unknown (perhaps integrin-independent) mechanism. Here we present evidence that the mechanism involves carbohydrate-to-carbohydrate interaction. Binding or incorporation of soluble-form placental or hepatoma FN to cell surface or pericellular matrix is highly dependent on the specific glycosylation status of FN per se and combination with glycosylation status of the cell surface, and is greatly promoted by a certain type of coexisting (shedded) glycosphingolipid. A few lines of study indicate that the process is mediated by interaction of FN carbohydrate with cell surface carbohydrate. The great enhancement of the binding process by glycosphingolipid is based on dual interaction of glycosphingolipid carbohydrate with FN carbohydrate and with cell surface carbohydrate. Here we present an example of promotion of binding of soluble-form FN from placenta or from hepatoma cells, having a specific carbohydrate epitope termed "disialyl-I," to K562 or VA13 cell surface in the presence of glycosphingolipid Gg3, which interacts specifically with disialyl-I.     

Binding and cross-linking properties of galectins

The galectins are a family of animal lectins that possess similar carbohydrate binding specificities and conserved consensus sequences. The biological properties of mammalian galectins include the regulation of inflammation, cell adhesion, cell proliferation and cell death. Evidence suggests that the biological activities of the galectins are related to their multivalent binding properties since most galectins possess two carbohydrate recognition domains and are therefore bivalent. For example, galectin-1, which is dimeric, binds and cross-links specific glycoprotein counter-receptors on the surface of human T-cells leading to apoptosis [J. Immunol. 163 (1999) 3801]. Different galectin-1 counter-receptors associated with specific phosphatase or kinase activities formed separate clusters on the surface of the cells as a result of the lectin binding to the carbohydrate chains of the respective glycoproteins. Importantly, monovalent galectin-1 is inactive in this system. This indicates that the separation and organization of signaling molecules that result from galectin-1 binding is involved in the apoptotic signal. The separation of specific glycoprotein receptors induced by galectin-1 binding was modeled on the basis of molecular and structural studies of the binding of lectins to multivalent carbohydrates resulting in the formation of specific two- and three-dimensional cross-linked lattices [Biochemistry 36 (1997) 15073]. In this article, the binding and cross-linking properties of galectin-1 and other lectins are reviewed as a model for the biological signal transduction properties of the galectin family of animal lectins.     

Site localization of membrane-bound proteins on whole cell level using atomic force microscopy

This study presents molecular recognition method, which is based on specific force measurements between modified AFM (atomic force microscopy) tip and mammalian cell. The presented method allows recognition of specific cell surface proteins and receptor sites by nanometer accuracy level. Here we demonstrate specific recognition of membrane-bound Osteopontin (OPN) sites on preosteogenic cell membrane. By merging specific force detection map of the proteins and topography image of the cell, we create a new image (recognition image), which demonstrates the exact locations of the proteins relative to the cell membrane. The recognition results indicate the strong affinity between the modified tip and the target molecules, therefore, it enables the use of an AFM as a remarkable nanoscale tracking tool on the whole cell level.     

Lysis of target cells infected with vesicular stomatitis virus (VSV) in the presence of tunicamycin by anti-VSV cytotoxic T lymphocytes

We have analyzed the requirement for the expression of the major surface glycoprotein (G protein) of vesicular stomatitis virus (VSV) on target cells for recognition and lysis by anti-VSV cytotoxic T lymphocytes (CTL). In addition, we have attempted to determine if the carbohydrate moieties on the G protein are required for recognition and lysis by anti-VSV CTL. When VSV (Orsay) is grown at 30 degrees C in the presence of tunicamycin (TM), glycosylation of G protein is inhibited; however, nonglycosylated G protein is found on the surface of the cell and active virus particles are produced. In contrast, VSV (Orsay) grown at 39 degrees C in the presence of TM produces low titers of virus and the presence of G protein on the surface of cells is not detectable. The susceptibility of these target cells to lysis by anti-VSV CTL was analyzed. The results suggest that expression of the G protein is required for target cell lysis by anti-VSV CTL. However, the presence of the carbohydrate moieties on the G protein are nt an absolute requirement for recognition by anti-VSV CTL. VSV-infected target cells incubated in the presence of TM were lysed by anti-VSV CTL up to 50 to 80% of the infected target cell control. This result suggests either that some clones of anti-VSV CTL recognize carbohydrate moieties or that carbohydrate moieties play some as yet undefined nonantigenic role in the recognition of the target antigen by the CTL receptor.