Specific binding of peanut agglutinin to GM1-doped solid supported lipid bilayers investigated by shear wave resonator measurements

This study deals with the specific interaction between the lectin peanut agglutinin (PNA) from Arachis hypogaea and the ganglioside G_M1 which was incorporated in a solid supported lipid bilayer immobilized on a gold electrode placed on top of an AT-cut quartz crystal. Bilayer formation was reached by self-assembly processes. The first monolayer consists of octanethiol attached to the gold surface via chemisorption and the second monolayer was immobilized by vesicle fusion on the preformed hydrophobic surface. We managed to keep unspecific binding to a minimum by using a phospholipid matrix consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Lectin binding to ganglioside G_M1 containing membranes was determined by a decrease of the resonant frequency of the quartz crystal. The minimum amount of receptor within the membrane which is necessary to obtain a complete protein monolayer was found to be less than 2 mol%. The adsorption isotherm of PNA to G_M1 was recorded and analyzed to be of Langmuir type, exhibiting a binding constant of PNA to the ganglioside of 8.3 ⋅ 10⁵ M^–1. The good agreement of the calculated Langmuir adsorption isotherm with the obtained experimental data implies that protein multilayers are not formed and that interactions between the adsorbents can be neglected. Furthermore, the association constants of two different saccharides, β-Galp-(1 → 3)-GalNAc exhibiting a strong binding to PNA in solution, and β-D-galactose with a much lower affinity were estimated by determining the equilibrium concentration of PNA attached to the surface. Moreover we were able to remove the attached lectin monolayer by digestion of the protein with pronase causing an increase in the resonant frequency which almost reversed the frequency shift to lower frequencies during adsorption. An even more complex system was built up by the use of digoxigenin-labeled PNA which also binds to the solid supported membrane containing the receptor G_M1. The immobilized lectin was recognized by anti-digoxigenin-F_ab-fragments, which is measurable by a further decrease of the resonant frequency. For all binding processes we found larger frequency shifts for a complete protein monolayer than predicted by Sauerbrey's equation, clearly showing that in addition to mass loading viscoelastic changes occur at the lipid-protein interface. Received: 22 July 1996 / Accepted: 12 September 1996     

A constraint scaffold enhances affinity of a bivalent N-acetylglucosamine ligand against wheat germ agglutinin

Bivalent glycoconjugates have a minimal valence with avidity potential on protein-carbohydrate interactions as well as simplicity of chemical structures enabling simple synthesis with low cost. Understanding the way to maximize the affinities of bivalent glycoconjugates is important for the development of cost-effective tools for therapeutic and diagnostic research. However, there has been little discussion about the effects of constraints imposed from ligand scaffolds on the binding abilities. We synthesized three kinds of biantennary N-acetylglucosamine glycosides with different scaffolds using isobutenyl bis(propargyl)ether as a common scaffold precursor. Decoration of the scaffold branches with GlcNAc moieties through copper-catalyzed azide-alkyne cycloaddition and grafting of the alkenyl focal point to another bivalent biotin dendron through thiol-ene and nucleophilic substitution reactions were successfully carried out in an orthogonal manner. The association constants of the ligands against wheat germ agglutinin were determined by a fluorometric titration assay. A bivalent biotin counterpart provided higher affinity than an isobutyl scaffold, whereas an isobutenyl scaffold yielded more enhancement than a bivalent biotin counterpart. The present work suggested that the constraint and steric bulk of ligand scaffolds are possible factors for improving binding properties of glycoconjugates against lectins or proteins.     

Schistosoma mansoni: Tegumental damage as a consequence of lectin binding

Adult worms of Schistosoma mansoni exhibit gross tegumental damage following incubation in concanavalin A or Ricinus communis agglutinin. However, incubation with wheat germ agglutinin induces only minimal surface damage, while soybean agglutinin has no damaging effect upon the worms. Damage induced by Ricinus communis agglutinin or concanavalin A may be prevented by the addition of the appropriate competing sugar. In contrast, incubation of 3-hr artificially transformed schistosomula in concanavalin A and other lectins does not produce any disruption of the tegument. These results indicate that the surface membrane of the adult schistosome is readily disrupted by ligand binding and appears to be particularly sensitive and fragile. The membrane of the schistosomulum, however, is more resistant to the effects of lectin binding. Adult worms incubated in culture medium alone (ELAC or RPMI 1640) show background changes which seem to be related to the tonicity of the medium. Such results advocate that preliminary assessment of schistosome integrity be carried out prior to any experimental procedures which preclude the addition of serum to the basic incubation medium. Schistosomula do not exhibit comparable sensitivity.     

Cell surface changes of the presumptive ectoderm following neural-inducing treatment by concanavalin A

Summary Scanning electron microscopic studies revealed that Concanavalin A (ConA) induces characteristic changes of the cell surface and the cell architecture of the presumptive ectoderm associated with differentiation into neural tissues. In Con A-treated cells, the filopodia with which cells were connected to each other disappeared from the interior (blastocoelic) surface and the cellular adhesivity decreased significantly. Thereafter, the cells underwent from those of the control explants. After cultivation for 60 h, a certain pattern of cell arrangement, which resembled the architecture of neural tissues, was observed among randomly arranged cells in the explants treated with Con A. The morphological changes specifically observed in Con A-treated explants were different from those found in explants treated with succinyl Con A (S-Con A) orDolichos biflorus agglutinin (DBA), which is unable to induce formation of the neural tissues. The molecular organization of the plasma membrane appears to be important in the mechanism of neural induction.     

Lectins and also bacteria modify the glycosylation of gut surface receptors in the rat

Oral exposure to lectins or the presence or absence of bacteria in the rat small intestine were shown by histological methods using anti-lectin antibodies or digoxigenin-labelled lectins to have major effects on the state of glycosylation of lumenal membranes and cytoplasmic glycoconjugates of epithelial cells. Taken together with the dramatic effects of exposure to lectins on gut function, metabolism and bacterial ecology, this can be used as a basis for new perspectives of biomedical manipulations to improve health. Abbreviations: DIG, digoxigenin-labelled; POD, peroxidase-labelled; Spf, specific pathogen-free; TBS, Saline (0.9% w/v NaCl solution) buffered with 0.05m Tris-glycine, pH 7.8; PAP, antibody-peroxidase-antiperoxidase. For lectins see Table 1.     

Endogenous Lectins as Cell Surface Transducers

Interactions between cells or between cell and substratum involve specificreceptors and their ligands. Among the various cell surface receptorsidentified during the last decades, the carbohydrate-binding proteins,e.g., lectins are of peculiar interest because glycolipids, glycoproteinsand proteoglycans have been shown to interact with lectins on the surfaceof animal cells. Animal lectins are recognized as molecules playingimportant roles in a variety of biological processes through binding toglycoconjugates and lectin-like receptors such as selectins, sialoadhesins(CD22, CD33), natural killer receptors (NKR-P1, CD69 and CD94/NKG2),hyaluronate receptors (CD44, RHAMM, ICAM-1), B-cell associated antigen(CD23, CD72), 2 leukocyte integrin (CD11b/CD18) or the well-knownreceptors for mannose, mannose-6-phosphate or asialoglycoprotein havebeen suggested to be able to mediate the transfer of information fromthe outside to the inside of the cell. This review focuses on the mostrecent advances in our understanding of the molecular basis ofoutside-in signaling mediated by lectins. Lectin-likereceptors are involved in signal transduction in a great variety of ways;at the molecular level, they mimic in most of the cases the function ofgrowth factor receptor either coupled to tyrosine kinase activity or toheterotrimeric G protein. They lead to a multiplicity of cellular eventsfollowing their activation depending on factors such as cellular type,species and/or tissue. Nevertheless the potential of surface lectins astransducers is emphasized by the observation that in a few cases lectin-likereceptors induce either novel signal transduction mechanism or newintracellular events with regards to what it has been observed as aconsequence of growth factor receptor activation. This observation bringsthe idea that lectins may offer, as cell surface transducers, an alternativeor additional signaling potential to cell.     

Differential affinities of <Emphasis Type="Italic">Erythrina cristagalli</Emphasis> lectin (ECL) toward monosaccharides and polyvalent mammalian structural units

Previous studies on the carbohydrate specificities of Erythrina cristagalli lectin (ECL) were mainly limited to analyzing the binding of oligo-antennary Galβ1→4GlcNAc (II). In this report, a wider range of recognition factors of ECL toward known mammalian ligands and glycans were examined by enzyme-linked lectinosorbent and inhibition assays, using natural polyvalent glycotopes, and a glycan array assay. From the results, it is shown that GalNAc was an active ligand, but its polyvalent structural units, in contrast to those of Gal, were poor inhibitors. Among soluble natural glycans tested for 50% molecular mass inhibition, Streptococcus pneumoniae type 14 capsular polysaccharide of polyvalent II was the most potent inhibitor; it was 2.1 × 10⁴, 3.9 × 10³ and 2.4 × 10³ more active than Gal, tri-antennary II and monomeric II, respectively. Most type II-containing glycoproteins were also potent inhibitors, indicating that special polyvalent II and Galβ1-related structures play critically important roles in lectin binding. Mapping all information available, it can be concluded that: [a] Galβ1→4GlcNAc (II) and some Galβ1-related oligosaccharides, rather than GalNAc-related oligosaccharides, are the core structures for lectin binding; [b] their polyvalent II forms within macromolecules are a potent recognition force for ECL, while II monomer and oligo-antennary II forms play only a limited role in binding; [c] the shape of the lectin binding domains may correspond to a cavity type with Galβ1→4GlcNAc as the core binding site with additional one to four sugars subsites, and is most complementary to a linear trisaccharide, Galβ1→4GlcNAcβ1→6Gal. These analyses should facilitate the understanding of the binding function of ECL. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inducible lectins with galectin properties and human IL1α epitopes opsonize yeast during the inflammatory response of the ascidian <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

Studies on inducible ascidian lectins may shed light on the evolutionary emergence of cytokine functions. Here, we show that the levels of opsonins, with IL1α-epitopes, increase in Ciona intestinalis hemolymph as a response to an inflammatory stimulus and, in particular, to intratunic injection of lipopolysaccharide (LPS). The inflammatory agent promptly (within 4 h) enhances Ca²⁺-independent serum hemagglutinating and opsonizing activities, which are both inhibited by D-galactose and D-galactosides (α-lactose, N-acetyl-D-lactosamine, thio-digalactoside), suggesting that anti-rabbit erythrocyte lectins with galectin properties are involved as opsonins. Inducible galectin molecules contain interleukin-1α (IL1α) epitopes, and their activities are specifically inhibited by anti-human recombinant IL1α antibody. Analysis by SDS-polyacrylamide gel electrophoresis has revealed that the density of the bands of several serum proteins increases within 4 h after LPS injection, correlated with the enhanced serum activity. Moreover, Western blot patterns demonstrate that several serum proteins (59, 37, 30, 23, 15 kDa) cross-react with the antibody as early as 4 h post-injection. Although we have not been able to establish whether, in adition to galectins, various types of D-galactose-specific lectins are contained in the serum, we show, for the first time in invertebrates, that galectin molecules with opsonic properties can be enhanced in response to a non-specific inflammatory stimulus, and that their release can be further stimulated by LPS. Finally, we reveal that multiple galectins share human IL1α epitopes, probably because of steric configuration and the oligomerization process. This work was supported by a research grant from the Italian Ministry of Education (PRIN 2004 4057000 to Nicolò Parrinello), co-funded by the University of Palermo.     

Spondias purpurea Exudate polysaccharide as affinity matrix for the isolation of a galactose-binding-lectin

Spondias purpurea L., popularly known as ciriguela, is native and widespread tree from Mexico through Northern Peru and Brazil, particularly in semi-arid zones. This tree exudes a water soluble polysaccharide, constituted of a (1→3) linked galactan backbone substituted at C6 with d-galactose, d-xylose, l-arabinose, l-rhamnose and glucuronic acid units. Brazilian polysaccharide differs from Venezuelan on the amount of acid and arabinose as well as the presence of fucose and glucose as minor sugar. The d-galactose substitution (1→6) confers to the polysaccharide the peculiar capacity of binding -d-galactose specific lectins after cross-linking with epichlorohydrin. The gel obtained was able to specifically retain d-galactose-binding-lectins, among with those from Artocarpus incisa, Artocarpus integrifolia, Erythrina velutina and Ricinus communis. On the other hand, no glucose-binding-lectins were retained.