Homophilic Binding Properties of Galectin-3: Involvement of the Carbohydrate Recognition Domain

Abstract: Galectin-3, an animal lectin specific for β-galactosides, is composed of three different domains. The N-terminal half of the molecule (N domain) consists of a short N-terminal segment followed by glycine-, proline-, and tyrosine-rich tandem repeats. The C-terminal domain (C domain) harbors the carbohydrate recognition domain homologous to other members of the galectin family of lectins. Galectin-3 aggregates in solution, and participation of the N domain of the molecule in this process has already been demonstrated. Using a solid-phase radioligand binding assay, which allows the direct analysis of galectin-3 self-association, here we provide evidence that the carbohydrate recognition domain of the lectin is involved in carbohydrate-dependent homophilic interactions: (a) Radiolabeled galectin-3 binds to immobilized galectin-3, and the addition of unlabeled galectin-3 in solution increases the rate of binding of radiolabeled lectin; (b) binding of radiolabeled galectin-3 to immobilized galectin-3 is inhibited by the C domain; (c) binding of radiolabeled galectin-3 to immobilized galectin-3 or the C domain is inhibited by lactose but not by sucrose; and (d) the radiolabeled C domain does not bind to immobilized C domain. Taken together, these data suggest that in addition to the N domain, the homophilic interactions of galectin-3 are mediated by the C domain.     

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.     

Glycoconjugates with Neu5Ac(α2,6)Gal(β1,4)GlcNAc sequences: a selective lectin-histochemical property of Kurloff cells in guinea pig thymus

Summary The Kurloff cell (KC), a natural killer lymphocyte, contains a large (10-m diameter) periodic acid-Schiff (PAS)-positive lysosome-like inclusion body called the Kurloff body (KB), which exhibits strong acid phosphatase activity. The presence of Sambucus nigra agglutinin (SNA)-reactive Neu5Ac(2,6)-D-Gal/Gal-NAc(1,4)GlcNAc oligosaccharide sequences and the absence of the corresponding Neu5Ac(2,3) Maackia amurensis agglutinin (MAA)-reactive sequence in the major 35-kDa N-glycosylproteins of the complex or hybrid type extracted from purified KC were established by Western-lectin-blotting of cytosolic extracts from purified KC. Moreover, these SNA-reactive sequences, or at least part of them, were shown to be borne by sialidase-sensitive KC acid isophosphatases. Thymic sections rich in KC, from estrogenized guinea pigs were examined by affino-histochemistry with these sialic acid-reactive lectins. The SNA-reactivity of thymic sections was quasiexclusively confined to KC clusters, whereas the whole thymic section was negative for MAA. KC were not SNA-reactive following preincubation and incubation with 200 mM lactose. When submitted to enzymatic or mild chemical desialylation processes, the SNA-reactivity of the KC clusters was enhanced. The SNA-reactivity of KC clusters was completely abolished following prolonged chemical desialylation, whereas the PAS-positivity of KB remained unchanged. Even after a prolonged sialidase treatment, this SNA-reactivity was only reduced. Moreover, after both these desialylation processes, KC developed a heavier Ricinus communis agglutinin-reactivity, thus confirming the presence of penultimate Gal residues in their abundant SNA-reactive oligosaccharide sequences Neu5Ac(2,6)Gal(1,4)GlcNAc. Such a selective lectin histochemical property provides a marker for detecting KC.     

Plasma membrane alterations of maturing goat (Capra indicus) spermatozoa: Lectin-binding and freeze-fracture study

Summary A qualitative and quantitative analysis of lectin-binding sites has been undertaken on spermatozoa recovered from different regions of the epididymis of the goat (Capra indicus) using fluorescein isothiocyanate-linked lectins (Bauhinia purpurea BPA, Concanavalin A Con A, Dolichos biflorus DBA, Maclura pomifera MPA, Arachis hypogaea or peanut agglutinin PNA, Glycine max or soyabean agglutinin SBA, Ulex europaeus UEA, and Triticum vulgaris or wheat-germ agglutinin WGA), in conjunction with scanning and transmission electron microscopy, and freeze-fracture techniques. Flow cytometric analysis has also been used to quantitize binding affinity. Spermatozoa from caput to cauda epididymidis show no significant variation in lectinbinding ability, but the samples removed from the corpus epididymidis contain a greater number of binding sites. The passage of spermatozoa through the epididymidis is accompanied by a redistribution of the plasma membrane lectin-receptors covering the sperm head and tail. Receptors for BPA, DBA, PNA and SBA are specifically restricted to the anterior region of the acrosome in caudal spermatozoa. Freeze-fracture replicas, examined to study changes in organisation of intramembranous particles of the plasma membrane during sperm maturation, reveal distinct changes in their distribution in the acrosome, post-acrosome and spermatozoon tail, especially in the corpus and cauda epididymidis.     

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.     

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.     

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     

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.