Structural and conformational features that affect the interaction of polylactosaminoglycans with immobilized wheat germ agglutinin

We examined the interaction between immobilized wheat germ agglutinin and the large, polylactosamine-containing glycans from human erythrocytes and human K-562 erythroleukemic cells. Three classes of interaction were identified. One class of glycan was merely retarded during chromatography. The other two classes were retained and could be distinguished by their ease of displacement with N-acetylglucosamine (GlcNAc); one was a moderate-affinity fraction displaced by 0.1 M GlcNAc and the other was a high-affinity fraction subsequently displaced by 1.0 M GlcNAc. A relatively small fraction of the K-562 polylactosamines were in the high-affinity class. We explored the role that fucose and sialic acid substitutions play in the strength of the lectin-glycan interaction. Although sialic acid is recognized by wheat germ agglutinin, sialylation was not required for the high-affinity interaction, and the presence of sialic acids actually prevented some glycans from binding with high affinity. In contrast, fucose is not part of the binding determinant, yet the removal of fucose resulted in decreased affinity. The possibility that some of these changes in affinity were the result of conformational changes was explored using matrices that had wheat germ agglutinin immobilized at different densities. At low wheat germ agglutinin densities, adult and fetal erythroglycans and K-562 glycophorin-like glycans were not retained by the matrix. As the density increased, the proportion of glycans that were retarded, and ultimately retained, increased. While these increases in the proportions retained occurred in parallel for the three different glycans, the apparent affinities of the glycan-lectin interactions differed. The glycophorin-like glycans were always readily displaced by 0.1 M GlcNAc, even at higher wheat germ agglutinin densities. In contrast, as the wheat germ agglutinin density increased, the proportion of erythroglycans that could be displaced by 0.1 M GlcNAc decreased; at 10 mg/ml immobilized wheat germ agglutinin, greater than 80% of the erythroglycans exhibited this tighter interaction. We suggest that this higher affinity interaction is the result of the large glycans spanning adjacent wheat germ agglutinin molecules, and is determined by the proximity of these molecules and the conformation of the glycans.     

Clearance of senescent erythrocytes: wheat germ agglutinin distribution on young and old human erythrocytes

To add an additional aspect to the process of recognition and removal of senescent human erythrocytes from the circulation, the binding of wheat germ agglutinin (WGA) to separated young, old and sialidase-treated human erythrocytes is evaluated with the immune-electron microscopical method. WGA/gold conjugate binding to old erythrocytes was lower (27%) than to young erythrocytes and even lower following treatment with sialidase (82%), exhibiting a clustered, non-continuous labeling pattern in all three erythrocyte populations, thus showing a possible redistribution of WGA binding sites. The decrease in bound WGA/gold particles correlates well with the previously reported decrease in surface sialic acid on old erythrocytes. The binding of WGA/gold are indicative of the changes occurring on erythrocyte membrane surfaces when interacting with different agglutinins.     

Anaplasma marginale, Eperythrozoon wenyoni: lectin reactions with bovine erythrocytes

Normal bovine erythrocytes were agglutinated with four of five lectins specific for different oligosaccharides. The order of reactivity was wheat germ greater than ricin greater than soybean greater than peanut. Concanavalin A did not agglutinate normal bovine erythrocytes. After neuraminidase treatment of normal bovine erythrocytes, each lectin agglutinated the cells with decreased concentrations of lectin, verifying that partial removal of sialic acid exposes more of each lectin's binding sites or alters the binding site such that fewer molecules of lectin are required to initiate agglutination. A change in agglutination of erythrocytes using soybean agglutinin and peanut agglutinin occurred when cells were obtained from cattle infected with Eperythrozoon wenyoni. The results suggested that an alteration in erythrocyte membranes occurred as a result of this infection as manifested by the increased recognition of both the soybean agglutinin and peanut agglutinin receptor carbohydrates. A similar effect was indicated with erythrocytes obtained during an acute Anaplasma marginale infection; however, an ensuing reticulocytosis masked the effect, requiring the use of fluoresceinated lectins to verify that increased binding of each lectin occurred with infected cells when compared to normal cells.     

A novel ligand from Plasmodium falciparum that binds to a sialic acid-containing receptor on the surface of human erythrocytes

Invasion of the merozoite form of Plasmodium falciparum into human erythrocytes involves multiple receptor-ligand interactions. The EBA175 protein of P. falciparum has been shown to be the ligand that binds to a sialic acid-dependent site on glycophorin A. We have identified a novel P. falciparum ligand, termed erythrocyte-binding antigen 140 (EBA140), that shares structural features and homology with EBA175. Subcellular localization of EBA140 suggests that it is located in the micronemes, the same localization as EBA175. EBA140 binds to a sialic acid-dependent receptor on the surface of human erythrocytes. Binding of EBA140 to this erythrocyte receptor is sensitive to neuraminidase and resistant to trypsin, proteinase K and pronase. The protease-resistant properties of the erythrocyte receptor suggests that it is not glycophorin A or C. Additionally, analysis of mutant erythrocytes from humans has shown that EBA140 does not bind glycophorin B. Interestingly, we have identified a parasite line that lacks the eba140 gene, suggesting that this protein is not essential for in vitro invasion. These results suggest that EBA140 may be involved in merozoite invasion using a sialic acid-dependent receptor on human erythrocytes.     

Effect of in vivo gamma-irradiation on the binding of wheat germ agglutinin on lymphocyte plasma membranes

Using quantitative fluorimetry with fluoresceinated wheat germ agglutinin, we have been able to investigate in vivo gamma radiation-induced damage at the outer membrane level of rat splenic lymphocytes, namely damage to the glucosidic moieties of membrane glycoproteins and glycolipids. This paper demonstrates that below an irradiation level of 1 gray (Gy), removal of sialic acid is the major feature leading to new exposed specific binding sites for wheat germ agglutinin, since this lectin is specific for sialic acid and N-acetyl-D-glucosamine. Our studies also suggest that above 1 Gy of irradiation more internal damage occurs, since we observed a striking decrease in wheat germ agglutinin binding sites.     

Glycoprotein characteristics of the sodium channel saxitoxin-binding component from mammalian sarcolemma

The saxitoxin-binding component of the excitable membrane sodium channel exhibits glycoprotein characteristics as evidenced by its specific interaction with various agarose-immobilized lectins. The detergent-solubilized saxitoxin-binding component interacts quantitatively with immobilized wheat germ agglutinin and concanavalin A and fractionally with immobilized Lens culinaris hemagglutinin and Ricinus communis agglutinin. These lectins preferentially bind $\text{N-}\text{acetylglucosamine}$ and sialic acid (wheat germ agglutinin), mannose (concanavalin A and Lens cunilaris and galactose (Ricinus communis). Removal of terminal sialic acid residues by neuraminidase markedly decreases binding to immobilized wheat germ agglutinin but uncovers sites capable of interacting with lectins specific for galactose and $\text{N-}\text{acetylgalactosamine}$. $\text{β-N-}\text{acetylglucosaminidase}$, an exoglycosidase has no effect on the binding of the channel protein to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component appears to be a glycoprotein containing terminal sialic acid residues and internal mannose, galactose, $\text{N-}\text{acetylglucosamine}$, and $\text{N-}\text{acetylgalactosamine}$ residues. The toxin binding site is spatially separated from the binding sites for the lectins studied. The effect of these sugar moieties must be considered when evaluating the biophysical parameters of the sodium channel.     

SabA is the H. pylori hemagglutinin and is polymorphic in binding to sialylated glycans

Adherence of Helicobacter pylori to inflamed gastric mucosa is dependent on the sialic acid-binding adhesin (SabA) and cognate sialylated/fucosylated glycans on the host cell surface. By in situ hybridization, H. pylori bacteria were observed in close association with erythrocytes in capillaries and post-capillary venules of the lamina propria of gastric mucosa in both infected humans and Rhesus monkeys. In vivo adherence of H. pylori to erythrocytes may require molecular mechanisms similar to the sialic acid-dependent in vitro agglutination of erythrocytes (i.e., sialic acid-dependent hemagglutination). In this context, the SabA adhesin was identified as the sialic acid-dependent hemagglutinin based on sialidase-sensitive hemagglutination, binding assays with sialylated glycoconjugates, and analysis of a series of isogenic sabA deletion mutants. The topographic presentation of binding sites for SabA on the erythrocyte membrane was mapped to gangliosides with extended core chains. However, receptor mapping revealed that the NeuAcalpha2-3Gal-disaccharide constitutes the minimal sialylated binding epitope required for SabA binding. Furthermore, clinical isolates demonstrated polymorphism in sialyl binding and complementation analysis of sabA mutants demonstrated that polymorphism in sialyl binding is an inherent property of the SabA protein itself. Gastric inflammation is associated with periodic changes in the composition of mucosal sialylation patterns. We suggest that dynamic adaptation in sialyl-binding properties during persistent infection specializes H. pylori both for individual variation in mucosal glycosylation and tropism for local areas of inflamed and/or dysplastic tissue.     

Specific erythrocyte binding capacity and biological activity of Plasmodium falciparum erythrocyte binding ligand 1 (EBL-1)-derived peptides

Erythrocyte binding ligand 1 (EBL-1) is a member of the ebl multigene family involved in Plasmodium falciparum invasion of erythrocytes. We found that five EBL-1 high-activity binding peptides (HABPs) bound specifically to erythrocytes: 29895 ((41)HKKKSGELNNNKSGILRSTY(60)), 29903 ((201)LYECGK-KIKEMKWICTDNQF(220)), 29923 ((601)CNAILGSYADIGDIVRGLDV(620)), 29924((621)WRDINTNKLSEK-FQKIFMGGY(640)), and 30018 ((2481)LEDIINLSKKKKKSINDTSFY(2500)). We also show that binding was saturable, not sialic acid-dependent, and that all peptides specifically bound to a 36-kDa protein on the erythrocyte membrane. The five HABPs inhibited in vitro merozoite invasion depending on the peptide concentration used, suggesting their possible role in the invasion process.     

The effect of oral contraceptives on rat platelet membrane glycoproteins

Female rats were administered oral contraceptives and the levels of sialic acid on platelet membrane and granule glycoproteins were compared to controls using a sialic acid assay and a fluorescein-conjugated wheat germ agglutinin binding assay and also by measuring the binding of ¹²⁵I-labelled wheat germ agglutinin to glycoprotein bands from platelets separated by polyacrylamide electrophoresis. The contraceptive-treated rats showed increased levels of glycoprotein sialylation which may partly explain the altered physiological function of the platelets.     

Reticulocyte-binding protein homologue 1 is required for sialic acid-dependent invasion into human erythrocytes by Plasmodium falciparum

The Apicomplexan parasite responsible for the most virulent form of malaria, Plasmodium falciparum, invades human erythrocytes through multiple ligand-receptor interactions. Some strains of P. falciparum are sensitive to neuraminidase treatment of the host erythrocyte and these parasites have been termed sialic acid-dependent as they utilize receptors containing sialic acid. In contrast, other strains can efficiently invade neuraminidase-treated erythrocytes and hence are sialic acid-independent. The molecular interactions that allow P. falciparum to differentially utilize receptors for merozoite invasion are not understood. The P. falciparum reticulocyte-binding protein homologue (PfRh or PfRBL) family have been implicated in the invasion process but their exact role is unknown. PfRh1, a member of this protein family, appears to be expressed in all parasite lines analysed but there are marked differences in the level of expression between different strains. We have used targeted gene disruption of the PfRh1 gene in P. falciparum to show that the encoded protein is required for sialic acid-dependent invasion of human erythrocytes. The DeltaPfRh1 parasites are able to invade normally; however, they utilize a pattern of ligand-receptor interactions that are more neuraminidase-resistant. Current data suggest a strategy based on the differential function of specific PfRh proteins has evolved to allow P. falciparum parasites to utilize alternative receptors on the erythrocyte surface for evasion of receptor polymorphisms and the host immune system.     

Relative susceptibilities to neuraminidase of the glycoproteins of the human erythrorcyte

Release of sialic acid from the glycoproteins of the normal human erythrocyte surface by neuraminidase was investigated. The glycoproteins of the membrane were separated by electrophoresis in sodium dodecylsulfate polyacrylamide gels. Sialic acid was determined in the sliced gel by a modification of the 2-thiobarbituric acid method, revealing three sialic acid-containing glycoproteins. Treatment of intact erythrocytes with neuraminidase to remove varying amounts of sialic acid indicates that all the glycoproteins are essentially equally accessible to the neuraminidase when 20%–60% of the sialic acid is removed. Similar but not quite identical results were obtained with isolated erythrocyte membranes.Treatment of intact cells with the lectins concanavalin A or phytohemagglutinin-P resulted in shielding of about 25% and 50%, respectively, of the sialic acid from neuraminidase. Concanavalin A blocked sialic acid release over long time periods and with high concentrations of neuraminidase. In contrast, the sialic acid shielding by phytohemagglutinin-P can be overcome by high concentrations of neuraminidase. Both lectins were found to shield the various glycoproteins selectively, with different patterns of shielding. Wheat germ agglutinin exhibited no detectable effect on the susceptibility of the erythrocyte sialic acid to neuraminidase.     

The Cell Adhesion Molecule “CAR” and Sialic Acid on Human Erythrocytes Influence Adenovirus In Vivo Biodistribution

Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad–erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models.     

Human erythrocyte band 3 functions as a receptor for the sialic acid-independent invasion of Plasmodium falciparum. Role of the RhopH3–MSP1 complex

Plasmodium falciparum takes advantage of two broadly defined alternate invasion pathways when infecting human erythrocytes: one that depends on and the other that is independent of host sialic acid residues on the erythrocyte surface. Within the sialic acid-dependent (SAD) and sialic acid-independent (SAID) invasion pathways, several alternate host receptors are used by P. falciparum based on its particular invasion phenotype. Earlier, we reported that two putative extracellular regions of human erythrocyte band 3 termed 5C and 6A function as host invasion receptor segments binding parasite proteins MSP1 and MSP9 via a SAID mechanism. In this study, we developed two mono-specific anti-peptide chicken IgY antibodies to demonstrate that the 5C and 6A regions of band 3 are exposed on the surface of human erythrocytes. These antibodies inhibited erythrocyte invasion by the P. falciparum 3D7 and 7G8 strains (SAID invasion phenotype), and the blocking effect was enhanced in sialic acid-depleted erythrocytes. In contrast, the IgY antibodies had only a marginal inhibitory effect on FCR3 and Dd2 strains (SAD invasion phenotype). A direct biochemical interaction between erythrocyte band 3 epitopes and parasite RhopH3, identified by the yeast two-hybrid screen, was established. RhopH3 formed a complex with MSP1₁₉ and the 5ABC region of band 3, and a recombinant segment of RhopH3 inhibited parasite invasion in human erythrocytes. Together, these findings provide evidence that erythrocyte band 3 functions as a major host invasion receptor in the SAID invasion pathway by assembling a multi-protein complex composed of parasite ligands RhopH3 and MSP1.     

Lectin-receptor interactions in liposomes.

The major sialoglycoprotein of mammalian erythrocytes has been incorporated into phosphatidylcholine membranes to generate a model system, glycoprotein-liposomes. Electron microscopic examination revealed these structures to be vesicles, approximately 300 A in diameter. An aqueous compartment inside the glycoprotein-liposomes has been identified by trapped volume studies with [14C]sucrose. These glycoprotein-liposomes were found to interact with the lectins, wheat germ agglutinin, and phytohemagglutinin, to form aggregates of mainly unfused vesicles. The aggregation process has been studied by electron microscopy, 90 degrees light scattering, and differential ultracentrifugation analysis. Hapten inhibitors of the lectins were found to inhibit the lectin-induced aggregation of the glycoprotein-liposomes. Binding of 125I-labeled wheat germ agglutinin to glycoprotein-liposomes was studied by differential ultracentrifugation. Hapten inhibitors of wheat germ agglutinin were also found to inhbit the binding of 125I-labled wheat germ agglutinin to the glycoprotein-liposomes. The characteristics of the lectin interactions with glycoprotein-liposomes appeared to be phenomenologically similar to lectin-cell interactions.     

Effects of phytohaemagglutinin, wheat-germ agglutinin, and concanavalin-A on the physical state of sialic acid and membrane proteins in human erythrocyte ghosts: a spin label study

Parallel experiments employing sialic acid- and protein specific spin labels have been performed to monitor the effects on the physical state of this carbohydrate and membrane proteins of human erythrocytes induced by the binding of three lectins, $\text{Phaseolus vulgaris}$ phytohaemagglutinin (PHA), wheat germ agglutinin (WGA), and Concanavalin-A (Con-A). PHA and WGA, both of which are known to bind at different sites on the principal sialoglycoprotein of human erythrocytes, glycophorin, had markedly different effects: compared to control values, PHA decreased the apparent correlation time of the sialic acid specific spin probe by 10% while this parameter was decreased by 33% by WGA. The protein specific spin label also monitored differential effects of these lectins: the relevant electron spin resonance parameter (the W/S ratio) was reduced 33% by PHA and increased by WGA over 17% from that of control values. Con-A, which is known to bind to the principal transmembrane protein, Band 3, had no effect on sialic acid or membrane proteins as assessed by the two spin labels employed. These results suggest that (1) the effects of binding of these different lectins, two of which bind to the same cell surface receptor, can be discriminated by use of spin labeling methods; (2) binding events occuring at the cell surface have distinct and pronounced effects on the physical state of proteins within the membrane; (3) the different results with PHA and WGA both of which bind to glycophorin are indicative of multiple and complex interactions of this glycoprotein with the membrane proteins in the erythrocyte; and (4) that the spin labelling technique has the potential to investigate the relationships between cell-surface binding events to membrane structural-functional interactions.     

Interaction of sulfated glycosaminoglycans with lectins

The sulfated glycosaminoglycans, such as keratan sulfate and chitin sulfate having 3-hydroxy free N-acetyl-beta-D-glucosaminyl residues as constituents, reacted with wheat germ agglutinin and Solanum tuberosum agglutinin by sugar-specific interaction. The glycosaminoglycans showed different inhibitory activities to the hemagglutination reaction of these lectins and keratan sulfate and its modified products formed insoluble complexes with both of the lectins at pH 7.0 in physiological saline solutions (0.15 M NaCl). S. tuberosum agglutinin was precipitated within a particularly narrow concentration range of keratan sulfate, and the formation of a soluble complex was observed by gel chromatography. These interactions were specifically inhibited by N,N'-diacetylchitobiose but not by 2 M NaCl. The specific interactions of the glycosaminoglycans with S. tuberosum agglutinin were confirmed by their ultraviolet difference spectra with two peaks at 285 and 298 nm attributable to the tryptophan residues in the binding site of the agglutinin. It was also found that S. tuberosum agglutinin and wheat germ agglutinin have different binding specificities. The presence of sulfate groups in either keratan sulfate or chitin sulfate did not interfere with their specific interactions with S. tuberosum agglutinin as strongly as with wheat germ agglutinin. The N-acetylneuraminic acid residues in keratan sulfate were found to be receptor sites for wheat germ agglutinin but not for S. tuberosum agglutinin.     

Effect of trypsinization on lectin binding to germ cells from ICR and T/t6 mice

Flow cytometry was used to quantify the binding of fluorescein isothiocyanate (FITC)-labeled lectins to testis cells from ICR and T/t6 mice before and after trypsin treatment. Soybean agglutinin, wheat germ agglutinin, and concanavalin A bound well to testis cells of both mouse strains. Limax flavus agglutinin (LFA) bound very slightly and Ulex europeas agglutinin (UEA) did not bind at all. Trypsinization increased binding of soybean agglutinin and decreased binding of wheat germ agglutinin in both mouse strains, providing evidence for masked carbohydrate-binding sites on the surface of germ cells. It did not affect binding of the other lectins. Trypsin treatment was an attempt to increase lectin binding, particularly the binding of LFA and UEA to the reported T/t-specific carbohydrates, sialic acid, and L-fucose, respectively. These studies indicate that the T/t6 locus alleles do not alter the surface carbohydrate content of testis cells sufficiently to be detected by lectin-binding differences.     

Proteolysis-associated deglycosylation of beta 1-adrenergic receptor in turkey erythrocytes and membranes

A protease that can be inhibited by glutathione, dithiothreitol, and o-phenanthroline but not by ethylenediaminetetraacetic acid converts the 50-kilodalton beta-adrenergic receptor in turkey erythrocyte membranes to a 40-kDa polypeptide which retains the specific ligand binding site. This conversion is attenuated in intact erythrocytes. The large 50-kDa peptide contains N-linked, complex carbohydrates and is retained on wheat germ agglutinin-Sepharose. The 40-kDa product of proteolysis does not bind to the wheat germ agglutinin and can thus be separated from the 50-kDa polypeptide by lectin chromatography. However, the large difference in molecular weights of the two receptor peptides cannot be accounted for solely by the different extent of glycosylation.     

Protein translocation across the endoplasmic reticulum. I. Detection in the microsomal membrane of a receptor for the signal recognition particle

Thin-section and critical-point-dried fracture-labeled preparations are used to determine the distribution and partition of glycophorin- associated wheat germ agglutinin (WGA) binding sites over protoplasmic and exoplasmic faces of freeze-fractured human erythrocyte membranes. Most wheat germ agglutinin binding sites are found over exoplasmic faces. Label is sparse over the protoplasmic faces. These results contrast with previous observations of the partition of band 3 component where biochemical analysis and fracture-label of concanavalin A (Con A) binding sites show preferential partition of this transmembrane protein with the protoplasmic face. Presence of characteristic proportions of WGA and Con A binding sites over each fracture face is interpreted to indicate the operation of a stochastic process during freeze-fracture. This process appears modulated by the relative expression of each transmembrane protein at either surface as well as by their association to components of the erythrocyte membrane skeleton.     

Effect of polyene antibiotics on the lectin-induced agglutination of transformed and untransformed cell lines.

Treatment of transformed Py3T3, SV101-3T3, and L1210 cells, as well as mitotic and Pronase-treated untransformed 3T3 cells, with the polyene antibiotics filipin, nystatin, and amphotericin B inhibited agglutination by wheat germ agglutinin. The effect of polyene antibiotic treatment was lectin and cell specific. Concanavalin A induced agglutination was not inhibited, wheat germ agglutination induced agglutination of untransformed 3T3 interphase cells was not influenced, and other aggregation phenomena, including those of erythrocytes with blood group specific antibodies or divalent cations, were unaffected by polyene treatments. This suggests that the formation of polyene-cholesterol complexes in transformed and erythrocyte cell membranes may specifically affect wheat germ agglutinin receptors and/or secondary events necessary for wheat germ agglutinin induced agglutination. Fluorescence studies of membrane filipin-cholesterol complexes showed that pretreating the cells with wheat germ agglutinin, but not concanavalin A, perturbed the fluorescence properties of filipin. Electron spin resonance studies with spin-labeled fatty acids revealed at best only a slight decrease in fatty acyl chain flexibility following filipin treatment. These studies indicate that there are not only quantitative differences between the agglutinability of transformed and untransformed cells with wheat germ agglutinin but that qualitative differences exist as well.