Intercellular adhesion in the cellular slime mold Polysphondylium pallidum inhibited by interaction of asialofetuin or specific univalent antibody with endogenous cell surface lectin.

Previous work has shown that, as amoebae of the cellular slime mold Polysphondylium pallidum become aggregation competent, they accumulate on their cell surface a carbohydrate-binding protein (lectin) named pallidin. These amoebae also possess cell surface receptors, presumed to contain complex oligosaccharides with a high affinity for the endogenous lectin. If lectin-receptor interactions mediate cell-cell contact, then appropriate concentrations of pallidin inhibitors should block cell cohesion. Two potent macromolecular antagonists of the lectin were employed: the desialylated form of the glycoprotein fetuin and the univalent antibody (Fab) prepared against pallidin. We studied the effects of these inhibitors on rotation-mediated aggregation of P. pallidum amoebae under a variety of assay conditions. Amoebae exposed to hypertonic conditions or to antimetabolites (“Permissive conditions”) were selectively blocked from associating by microgram quantities of the lectin inhibitors, whereas cells in isotonic buffer (“nonpermissive condition”) were only slightly affected. A comparison of the morphology of agglutinates formed under the various conditions allows several explanations for the different susceptibilities to inhibition by antipallidin reagents. Although not conclusive, the work supports a model of cell adhesion in this simple eukaryotic system based at least in part on specific interactions between carbohydrate-binding proteins and receptors on adjoining cells.     

Identification and purification of an endogenous receptor for the lectin pallidin from Polysphondylium pallidum

We report the identification and purification of an endogenous carbohydrate-containing receptor of pallidin, the cell surface lectin implicated in mediating cell-cell adhesion in the cellular slime mold Polysphondylium pallidum. The receptor is identified in an aqueous extract of crude P. pallidum membranes as a potent inhibitor of the hemagglutination activity of pallidin. The inhibitor is purified to apparent homogeneity by affinity precipitation with pallidin followed by fractionation of the solubilized precipitate on Sepharose 4B. The hemagglutination inhibitor (HAI) is metabolically radiolabeled, indicating that it is a biosynthetic product of the amoebae and not an ingested food substance. The HAI is released into the extracellular medium by living, differentiated amoebae. This release is markedly facilitated by the addition of D-galactose, a specific saccharide that binds to pallidin. Hence, the HAI appears to have an in situ association with pallidin at the cell surface. Exogenously added HAI promotes the agglutination of differentiated amoebae in a gyrated suspension at very low concentrations. The results are consistent with a model of cell-cell adhesion in which the HAI is a multivalent, extracellular aggregation factor that is recognized by pallidin molecules on adjacent cells. The HAI would then be analogues to the aggregation factors identified in marine sponges.     

An endogenous lectin and its glycoprotein ligands are triggering basal and axon-induced Schwann cell proliferation

The proliferation of Schwann cells (the myelinating cells ofthe peripheral nervous system) is stimulated by the contactwith axonal membranes. It is suggested that the endogenous carbohydrate-bindingprotein (lectin) cerebellar soluble lectin (CSL) bound to ligandsat the surface of axonal preparations is mitogenic for Schwanncells. Both autocrine and axon-stimulated Schwann cell proliferationsseem to be dependent on the presence of CSL and its ligandsat the Schwann cell surface, as suggested by the effects ofN-glycosylation inhibitors and anti-CSL Fab fragments. Thesedata suggest that CSL regulates Schwann cell proliferation byclustering of a few glycoprotein ligands at the cell surface,consequently modulating phosphorylations. adhesion CSL N-glycan MAG signal     

Contact sites in aggregating cells of Polysphondylium pallidum

Aggregating cells of the cellular slime mold Polysphondylium pallidum are completely dissociated by univalent antibody fragments (Fab) directed against membrane antigens. The blocking effect on cell adhesion is species specific: Fab against P. pallidum has little effect on cells of Dictyostelium discoideum, and vice versa. Suspended cells of these species agglutinate together, but within the agglutinates they sort out into separate areas.Absorption of the Fab with growth phase cells removes only part of its blocking activity. This indicates the expression of a new class of target sites of adhesion blocking Fab during cell differentiation from the growth phase to the aggregation competent stage. Another class of target sites is already present on the surface of growth phase cells. In both developmental stages cell adhesion is largely resistant to EDTA.The major target sites of adhesion blocking Fab appear to differ from carbohydrate-binding proteins known as pallidin. Removal of the adhesion blocking activity by absorption of Fab with intact cells does not deplete for anti-pallidin Fab. Cell adhesion is only weakly affected by Fab specific for pallidin I and II.     

Introduction to the Symposium and Studies on the Surfaces of Separated and Synchronized Tumor and Embryonic Cell Populations

Tumor and embryonic cell surfaces are examined in this symposiumwith respect to their roles in cell-cell interactions and inearly development and malignancy. Three sets of studies havebeen recently performed in my laboratory to help elucidate thenature of tumor and embryonic cell surfaces and the means bywhich these cells adhere to each other. We separated an in vivo129/J ascites mouse teratoma into specific subpopuladons ofcells by velocity sedimentation in shallow density gradients.The teratoma consistently separated into two major populations:"large" and "small" cells. Only the large cells displayed "malignant-like"surface characteristics in terms of their agglutinability withcarbohydrate binding lectins. The teratoma cells were also synchronizedin culture with thymidine plus colcemid. In these synchronizedcultures, cellular adhesiveness and glutamine synthetase specificactivity displayed oscillatory patterns with peaks of glutaminesynthetase specific activity occurring just prior to peaks ofadhesivenesss. Also, both glutamine synthetase specific activityand cellular adhesiveness were enhanced by two compounds: actinomycinD and hydrocortisone. Based upon previous work that implicatesL-glutamine in intercellular adhesion, it is not unreasonableto speculate that glutamine synthetase specific activity andcellular adhesiveness may be causally related. The problem ofaltered tumor cell adhesiveness is important because it seems,in part, to be responsible for tumor spread. Finally, the seaurchin embryo system was utilized to identify specific cellsurface carbohydrates that may be involved in intercellularadhesion. In 15 separate experiments with each sugar and with15 different saccharides, D-galactose and N-acetyl-D-galactosaminewere the best inhibitors of rotation-medicated reaggregationof 24-hr sea urchin embryo cells dissociated by removal of divalentcations. ß-galactosidase also inhibited reaggregationof these cells. These results implicate galactopyranosyl-likeresidues in the adhesion of 24-hr sea urchin embryo cells witheach other.     

Differential expression of an endogenous mannose-binding protein R1 during muscle development and regeneration delineating its role in myoblast fusion

The role of the endogenous brain carbohydrate-binding proteinR1 in muscle cell development and regeneration was analysedboth in vivo and in vitro. In vivo, R1 was developmentally regulated,with an embryonic 65 000 subunit and a neonatal 67 000 subunit,being replaced progressively by a 135 000 adult form. LectinR1 was intracellularly localized at birth and in the prenatalperiod. During development and at the time of myoblast fusion,the antigen was progressively found at the surface, where itremained at low levels in the adult. In vitro, in pure myoblastcultures, only the embryonic form was present. The ultrastructuralstudies indicated that the lectin could participate in the membranefusion process during myoblast fusion. The specific role inmyoblast fusion, derived from the ultrastructural localizationof R1, was evidenced by a strong inhibitory effect of anti-R1 Fab fragments (10–100 µg/ml), relative to controlFab fragments. In vivo, the embryonic subunit pattern and subcellulardistribution of R1 reappeared in muscle cells after lesion ofthe adult muscle. This suggested that, as observed in vitro,R1 participated in vivo in the phenomenon of myoblast fusion.Similar modifications in subunit expression were observed inmuscles after denemation (the embryonic form of lectin R1 reappearingafter lesion), suggesting that R1 could be involved in the processof neuromuscular junction formation. Thus, it is proposed thatthe carbohydrate-binding protein R1 is an important recognitionmolecule for the formation of myotubes. Its potential involvementin a recognition process between axons and muscle cells duringneuromuscular junction formation is discussed. culture development fusion N-glycan glycoprotein lectin mannose myoblast     

Elucidation of the mechanism enhancing the avidity of lectin with oligosaccharides on the solid phase surface

The mechanism underlying molecular recognition of lectins waselucidated by a novel solid phase binding assay system basedon surface plasmon resonance. When the apparent affinities ofinteractions between chitooligosaccharides and wheat germ agglutininwere compared between lectin-immobilized and oligosaccharide-immobilizedassay systems, the affinity constants (Ka) calculated for theformer system were in good agreement with the previously reportedvalues measured in solution. On the other hand, in the lattersystem, the calculated Ka could be more than 10,000 times higherthan the values in solution at lower lee tin concentrations.To elucidate the reason for this, we systematically investigatedthe effects of the oligosaccha-ride immobilized density andthe lectin valence on the apparent affinity in the oligosaccharide-immobilizedassay system. Both the apparent association (k_ass) and dissociationrate constants (k_diss) showed a tendency to decrease as theoligosaccharide density increased. This effect was most remarkablefor the interaction possessing an extremely fast intrinsic K_ass.Oligomerization of lectin enhanced the avidity due to a significantreduction in k_diss. These phenomena could be explained by consideringthe nonhomogeneous conditions under which binding occurred.The reaction in a nonhomogeneous state is limited by the masstransport effect, and the effect of rebinding becomes so largethat it cannot be disregarded. These findings are the firstto demonstrate the importance of the mass transport effect inmodulating the affinity of lectin for oligosaccharides on asolid phase surface. avidity clustering effect lectin mass transport surface plasmon resonance     

Wild-type and cultured Ehrlich ascites tumour cells differ in tumorigenicity, lectin binding patterns and binding to basement membranes

Three different variants of the Ehrlich ascites tumour (EAT)cell were derived and the lectin surface reactivities, as wellas the malignant characteristics of each variant, were studied.Wild-type cells (EAT-wt) were selected for growth on basementmembranes and tissue culture plastic to give EAT-c cells. TheEAT-c were passaged in mice by i.p. injection, giving rise toa third variant (EAT-c/m). Each of these three cell variantswas characterized for: (i) specific lectin agglutinability patterns;(ii) the ability to produce ascites tumours in mice; (iii) theability to produce solid tumours; and (iv) the attachment toand growth on basement membranes and purified extracellularmatrix molecules. Analysis of the total protein and carbohydratecontent of each cell line showed that there was an increasein the glycosylation of the EAT-c cells compared to EAT-wt cells.After repeated passage of the EAT-c/m cells in mice, the glycosylationlevel of the EAT-c/m cells returned to that of the EAT-wt cellline. In addition, the EAT-c cells displayed an increase inthe number of terminal non-reducing sugars which could indicateeither an increased degree of branching or the presence of additionalN- and/or O-linked oligosaccharide chains of the cellular glycoproteins.This phenotype was retained by the EAT-c/m cells which had beenpassaged repeatedly in mice. The most significant increase wasin the content of sialic acid-containing glycoproteins foundin the EAT-c cells. The sialic acid-binding lectin Maackia amurensisleukoagglutinin (MAL) agglutinated all three EAT cell variants,while the sialic acid-binding Sambucus nigra (elderberry bark)lectin (SNA) agglutinated only the EAT-c and early-passage EAT-c/mcells. These findings indicate the presence of 2,3-linked sialicacid on all three variants, but only the cultured cells andearly-passage EAT-c/m cells possess the Neu5Ac2,6 linkage. TheEAT-c cells attached avidly to wells coated with either lamininor fibronectin, as well as an extracellular matrix producedby cultured bovine endothelial cells, but the EAT-wt and EAT-c/mcells did not. Paradoxically, the EAT-c cells were incapableof producing solid tumours when injected into a basement membrane-richskeletal muscle bed, whereas the EAT-wt and EAT-c/m cells producedrapidly growing tumours when injected into the same environment.Lectin agglutination patterns established that ascitic tumourcells within the peritoneal cavity were derived from injectedEAT-c cells. Ehrlich cell laminin lectin sialic acid tumour     

Cloning and expression of a Xenopus laevis oocyte lectin and characterization of its mRNA levels during early development

cDNA clones encoding a soluble, calcium-dependent, melibiose-bindinglectin from Xenopus laevis oacytes have been isolated, characterized,and expressed in bacteria This lectin has been shown by othersto be localized in oocyte cortical granules where it ultimatelyis released and participates in the formation of the fertilizationenvelope. A lectin with similar specificity has been purifiedby others from blastula and immunolocalized to specific locationsin developing embryos, which suggests it may also function afterfertilization in regulating cell adhesion and migration. Wehave used melibiose affinity chromatography to isolate the oocytelectin (monomer molecular masses of about 45 and 43 kDa) andshown that after exhaustive treatment with N-glycanase, onlyone major protein band at 35 kDa was observed, suggesting thata single polypeptide with variable N-Linked glycosylation isexpressed in the oocyte. After obtaining internal peptide sequences,a PCR-based cloning approach allowed the isolation of full lengthcDNAs from an ovary     

Neuronal-Glial Interactions in Retina Development

The development of embryonic retinoblasts into phenotypicallymature Müller glial cells has been shown to be dependenton close juxtapositional relationships between heterotypicellsof the retina. In this report, I review experiments in whichwe have attempted to examine the role of actual cell contactin the regulation of biochemical differentiation of retinalglial cells. Probes which bind to cell surface components includingantibodies to the retina cell membrane and plant lectins weretested for their ability to interfere with normal histogenesisand glial maturation in a reaggregation-basedin vitro developmentassay. Data are discussed which show that antibodies to thecell surface and the succinylated derivative of the plant lectinconcanavalin A can markedly impair both histogenesis and glialmaturation potential if introduced into cultures of reaggregatingdissociated embryonic retina cells. Preliminary analyses ofmembrane components which react with the lectin have been performed.The results suggest that certain specific membrane glycopeptidesare expressed by dissociated retina cells in an age-dependentmanner. Also, the results show that decline in the ability ofthe embryonic cells to elaborate these surface components correlateswith the capacity of the cells toreform developmentally regulatoryneuronal-glial communication "linkages"     

Pallidin. Purification and characterization of a carbohydrate-binding protein from Polysphondylium pallidum implicated in intercellular adhesion.

A carbohydrate-binding protein from Polysphondylium pallidum, a species of cellular slime mold, was purified to homogeneity by adsorption to formalinized erythrocytes and elution with D-galactose. The protein, for which we propose the name PALLIDIN, is assayed by its activity as an agglutinin of erythrocytes. It was previously shown to have different carbohydrate-binding specificities than discoidin, a carbohydrate-binding protein from Dictyostelium discoideum, another species of slime mold. Evidence has been presented previously that each of these proteins is detectable on the cell surface. In the present report we show that the physico-chemical properties of pallidin are different from discoidin. Pallidin has a subunit molecular weight of 24 800 +/- 1100 determined by polyacrylamide electrophoresis in the presence of dodecyl sulfate and 2-mercaptoethanol, compared to 26 100 +/- 1000 for discoidin. The weight-average molecular weight of pallidin is 250 000 +/- 50 000 determined by equilibrium sedimentation in the presence of D-galactose compared to 100 000 +/- 2000 for discoidin. In equilibrium sedimentation studies, pallidin exhibited some heterogeneity at equilibrium while discoidin was homogeneous. The amino acid composition of pallidin is generally similar but clearly different from the composition of discoidin. The isoelectric point of pallidin is 7.0 compared to 6.1 for discoidin. Like discoidin, pallidin contains no detectable hexosamine or neutral sugar. These results establish that agglutinins from two species of cellular slime molds are distinct. The different properties of the cell-surface agglutinins, pallidin and discoidin, are consistent with their suggested role in species-specific cellular recognition and adhesion in the species of slime mold from which they are derived.     

Characterization of Cell Surface Lectin-binding Patterns of Human Airway Epithelium

Glycosylated structures on the cell surface have a role in cell adhesion, migration, and proliferation. Repair of the airway epithelium after injury requires each of these processes, but the normal cell surface glycosylation of non-mucin producing airway epithelial cells is unknown. We examined cell surface glycosylation in human airway epithelial cells in tissue sections and in human airway epithelial cell lines in culture. Thirty-eight lectin probes were used to determine specific carbohydrate residues by lectin-histochemistry. Galactose or galactosamine-specific lectins labeled basal epithelial cells, lectins specific for several different carbohydrate structures bound columnar epithelial cells, and fucose-specific lectins labeled all airway epithelial cells. The epithelial cell lines 1HAEo– and 16HBE14o– bound lectins that were specific to basal epithelial cells. Flow cytometry of these cell lines with selected lectins demonstrated that lectin binding was to cell surface carbohydrates, and revealed possible hidden tissue antigens on dispersed cultured cells. We demonstrate specific lectin-binding patterns on the surface of normal human airway epithelial cells. The expression of specific carbohydrate residues may be useful to type epithelial cells and as a tool to examine cell events involved in epithelial repair.     

Encystment of Pythium aphanidermatum Zoospores is Induced by Root Mucilage Polysaccharides, Pectin and a Monoclonal Antibody to a Surface Antigen

The infection of roots by the pathogenic Oomycete Pythium aphanidermatuminvolves interactions between the fungal zoospores and rootsurface mucilage polysaccharides. After initial recognitionat the root surface the zoospores are triggered to encyst duringwhich adhesive glycoproteins are secreted followed by a fibrillarcyst wall. In this paper a simple in vitro assay has been usedto assess the ability of a variety of macromolecules to inducezoospore encystment. Mucilage polysaccharides of the cress rootsurface trigger encystment. Whole mucilage was fractionatedby gel filtration and a fraction low in uronic acid, containing5% fucose, was shown to be more effective in triggering encystmentthan a uronic acid-rich fraction. Encystment can also be inducedby commercial pectin. The lectin Con A, and PA1, one of a rangeof monoclonal antibodies specific for zoospore surface antigens,also triggered encystment. In Western blotting experiments PA1recognizes protein epitopes of a 75 kDa surface antigen. Theresults suggest that at least one mechanism of zoospore triggeringmay involve a specific zoospore surface receptor. Key words: Pythium aphanidermatum, recognition, encystment, zoospore, mucilage, root, monoclonal antibodies, polysaccharides     

The Bark Lectin of Robinia pseudoacacia: Purification and Partial Characterization

A lectin was purified from the bark of Robinia pseudoacaciaby sequential ion-exchange chromatography on DEAE-Sepharoseand CM-Toyopearl. The purified lectin was estimated to havea molecular weight of 106 kDa and to be a homotetramer of subunitswith a molecular weight of 29 kDa. Antibodies raised againstthe bark lectin cross-reacted with a 29-kDa polypeptide duringWestern blot analysis, showing that the antibodies are specificfor the bark lectin. The antibodies against the lectin fromRobinia bark cross-reacted with polypeptides in extracts ofthe seeds and bark of Sophora japonica, indicating that thelectin from Robinia bark is immunologically related to the lectinsof Sophora. However, the antibodies did not cross-react withproteins from Robinia seeds and leaves. The first twenty aminoacid residues from the N-terminus of the lectin from Robiniabark were determined and compared with those of the Sophoralectins. (Received July 13, 1991; Accepted December 12, 1991)     

Comparative cross-linking activities of lactose-specific plant and animal lectins and a natural lactose-binding immunoglobulin G fraction from human serum with asialofetuin

Plant and animal lectins bind and cross-link certain multiantennaryoligosaccharides, glycopeptides, and glycoproteins. This canlead to the formation of homogeneous cross-linked complexes,which may differ in their stoichiometry depending on the natureof the sugar receptor involved. As a precisely defined ligand,we have employed bovine asialofetuin (ASF), a glycoprotein thatpossesses three asparagine-linked triantennary complex carbohydratechains with terminal LacNAc residues. In the present study,we have compared the carbohydrate cross-linking properties oftwo Lac-specific plant lectins, an animal lectin and a naturallyoccurring Lac-binding polyclonal iminunoglobulin G subfractionfrom human serum with the ligand. Quantitative precipitationstudies of the Lac-specific plant lectins, Viscum album agglutininand Ricinus communis agglutinin, and the Lac-specific 16 kDadimenc galectin from chicken liver demonstrate that these lectinsform specific, stoichiometric cross-linked complexes with ASF.At low concentrations of ASF, 1:9 ASF/lectin (monomer) complexesformed with both plant lectins and the chicken lectin. Withincreasing concentrations of ASF, 1:3 ASF/lectin (monomer) complexesformed with the lectins irrespective of their source or size.The naturally occurring polyclonal antibodies, however, revealeda different cross-linking behavior. They show the formationof 1:3 ASF/antibody (per Fab moiety) cross-linked complexesat all concentrations of ASF. These studies demonstrate thatLac-specific plant and animal lectins as well as the Lac-bindingimmunoglobulin subfraction form specific stoichiometric cross-linkedcomplexes with ASF. These results are discussed in terms ofthe structure-function properties of multivalent lectins andantibodies. asialofetuin Lac-specific lectins immunoglobulin subfraction     

Pallidin is a component of a multi-protein complex involved in the biogenesis of lysosome-related organelles

The Hermansky–Pudlak syndrome defines a group of genetic disorders characterized by defective lysosome-related organelles such as melanosomes and platelet dense bodies. Hermansky–Pudlak syndrome can be caused by mutations of at least four genes in humans and 15 genes in mice. One of these genes is mutated in the pallid mouse strain and encodes a novel protein named pallidin (L. Huang, Y. M. Kuo and J. Gitschier, Nat Genet 1999; 23: 329–332). Pallidin has no homology to any other known protein and no recognizable functional motifs. We have conducted a biochemical characterization of human pallidin using a newly developed polyclonal antibody. We show that pallidin is a ubiquitously expressed ∼ 25 kDa protein found both in the cytosol and peripherally associated to membranes. Sedimentation velocity analyses show that native pallidin has a sedimentation coefficient of ∼ 5.1 S, much larger than expected from the molecular mass of the pallidin polypeptide. In line with this observation, cosedimentation and coprecipitation analyses reveal that pallidin is part of a hetero-oligomeric complex. One of the subunits of this complex is the product of another Hermansky–Pudlak syndrome gene, muted. Fibroblasts derived from the muted mouse strain exhibit reduced levels of pallidin, suggesting that the absence of the muted protein destabilizes pallidin. These observations indicate that pallidin is a subunit of a novel multi-protein complex involved in the biogenesis of lysosome-related organelles.     

Molecular properties of the partially SDS-resistant lectin from the albumen gland of Helix pomatia and demonstration of lectin-related molecules on the surface of H. pomatia haemocytes

Lectins (agglutinins) are components of the immunobiologicalrecognition system of vertebrates and invertebrates. The presentstudy focused on the molecular properties of the agglutininfrom the albumen gland of Helix pomatia (HPA) and on the occurrenceof lectin-related molecules on the surface of H. pomatia haemocytes.According to the current model (Hammarström et al., 1972,Scandinavian Journal of Immunology, 1: 259–301), the hexamericHPA of about 79 kDa is composed of three non-covalently associateddimers (26 kDa), each consisting of two disulphide-bridged 13kDa monomers. However, on native-gradient polyacrylamide gelelectrophoresis (PAGE), we obtained high molecular weight bandsrepresenting lectin polymers. The stepwise dissociation of thesewas achieved by incubation with SDS at temperatures from 20to 40°C (1 h) and at 100°C (10 min). The results obtainedon SDS–PAGE included the occurrence of partially SDS-resistanthexamers of about 66 kDa, of two dimer bands of 22 and 19 kDa,and of two minor heteromonomer fractions. Complete dissociationinto heteromonomers of 13 and 11 kDa was achieved by boilingthe lectin (10 min) with SDS under reducing conditions. Fornative lectin molecules, both monomers occurred as disulphide-linkedhomodimers. Monomers or dimers electroeluted from an SDS–gel,reassociated to SDS-resistant oligomers upon re-electrophoresis.Finally, molecules antigenetically related to the lectin wereextracted from the membrane of H. pomatia haemocytes. Anti-HPAantibodies recognized peptides with an apparent molecular weightof about 30 and 56 kDa, which were shown to represent cell-surfacemolecules. (Received 4 March 2008; accepted 9 September 2008)     

Mannose Binding Lectins of Vicia tetrasperma Seed and Their Immunological Relationship to Other Legume Lectins of Similar Specificity

Mannose specific lectins of Vicia tetrasperma were purifiedby affinity chromatography with Sephadex G-100, and ion exchangechromatography. Chromatofocusing using PBE-94 gel was successfullyemployed to separate the major isolectins, lectin I and II.Both lectins had the same molecular weight of 78,000 and weretetramers composed of a uniform subunit with a molecular weightof 18,700. Amino acid compositions of these lectins were quitesimilar to each other, rich in aspartic acid (and/or asparagine)and hydroxyl amino acids, and lacking methionine and cysteine.Agar gel double diffusion using anti V. tetrasperma lectin antiserumrevealed that lectins from V. cracca, Pisum sativum, and Lensculinaris, all of which have mannose binding properties, wereantigenically identical. The antiserum reacted with the analogouslectins from V.faba, V. hirsuta, and V. angustifolia, but formationof a spur in the diffusion assay showed that they were slightlydifferent from V. tetrasperma lectin. (Received December 24, 1985; Accepted March 12, 1986)     

Lectin-induced apoptosis of tumour cells

The mechanisms of cytotoxic activity of Griffonia simplicifolia1-B₄ (GS1B₄) and wheat germ agglutinin (WGA) lectins againstvarious murine tumour cell lines were studied. Tumour cellsthat lack lectin-binding carbohydrates were resistant to lysisby these lectins. However, YAC-1 cells that expressed GS1B⁴lectin-binding sites showed low sensitivity to lysis. To furtheranalyse the relative importance of cell surface carbohydratesin lectin cytotoxicity, BL6–8 melanoma cells, which donot express the     

Peptides inhibit selectin-mediated cell adhesion in vitro, and neutrophil influx into inflammatory sites in vivo

The selectins are cell adhesion molecules whose carbohydrate-bindingdomain (C-type lectin) is thought to be involved in leukocyteadhesion to activated vascular endothelium in the inflammatoryprocess. A series of peptides, based on a conserved region (⁴⁸YYWIGIRK⁵⁵-NH₂)of the lectin domain of E-, L- and P-selectins, were analysedfor their ability to block selectin-mediated cell adhesion invitro, and neutrophil infiltration into sites of inflammationin vivo. The peptides inhibited the adhesion of myeloid cellsto recombinant forms of E- and P-selectin. The adhesion of myeloidcells to human endothelial cells, stimulated to express E-selectin,was also inhibited by the peptides. Finally, the peptides blockedthe adhesion of lymphocytes, expressing L-selectin, to highendothelial venules in lymph nodes which contain the ligandfor L-selectin. A clear structure-activity relationship wasestablished when peptides of different amino acid chain lengthswere tested in these assays. Peptides lacking tyrosine residues(e.g. WIGIR-NH₂) at their amino terminus were poor inhibitorsof selectin-mediated cell adhesion in vitro. The peptides thatwere found to be inhibitors of cell adhesion in vitro were alsofound to inhibit (up to 70%) neutrophil infiltration into sitesof inflammation in a thioglycollate-induced peritonitis mousemodel system. They also significantly reduced (>50%) themigration of neutrophils into cytokine-treated skin. These resultsstrongly suggest that compounds based on these tyrosine-containing,selectin-derived peptides could be used as anti-inflammatorytherapeutic agents. inflammation neutrophils peptides selectins