Defining the carbohydrate specificities of Erythrina corallodendron lectin (ECorL) as polyvalent Galbeta1-4GlcNAc (II) > monomeric II > monomeric Gal and GalNAc.

BACKGROUND: Erythrina corallodendron lectin (ECorL) is one of the potent applied lectins. In previous studies, the carbohydrate specificities of this lectin were limited to monosaccharides, simple oligosaccharides and several clusters. However, the polyvalent factor has not been investigated. METHODS: The binding properties at the combining sites of ECorL were characterized by sensitive enzyme-linked lectinosorbent (ELLSA) and inhibition assays, using our collection of ligands and polyvalent natural glycans with known glycotopes. RESULTS: Results of both binding and inhibition assays revealed a very high affinity between ECorL and Galbeta1-4GlcNAc (II)-containing glycoproteins. Among soluble natural glycans tested for inhibition, the high-density polyvalent II glycotopes, such as Streptococcus pneumoniae type 14 capsular polysaccharide which is composed of repeating poly-II residues, resulted in 2.4 x 10(4), 1.4 x 10(3) and 8.6 x 10(2)-fold higher affinities to ECorL than the monomeric Gal, linear II and tri-antennary II, respectively, at the non-reducing end in N-linked glycopeptides (Tri-II). The ECorL-glycan interaction was also strongly inhibited by most of the other high-density II-containing glycoproteins. Although GalNAc was as potent an inhibitor as Gal, its polyvalent structural units were poor inhibitors. CONCLUSIONS: [1] Galbeta1-4GlcNAc (II) and other Galbeta1 -related oligosaccharides are essential for binding. [2] Their polyvalent form in glycoproteins is the most important binding factor for ECorL, while II monomer and oligo-antennary II forms play only a limited role in binding. [3] Although GalNAc is more active than Gal for ECorL, its reactivity is not changed by polyvalent effects. This lectin may be used as a tool to study glycobiology in basic and medical sciences.

Differential affinities of Erythrina cristagalli 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 Galbeta1-->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 x 10(4), 3.9 x 10(3) and 2.4 x 10(3) 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 Galbeta1-related structures play critically important roles in lectin binding. Mapping all information available, it can be concluded that: [a] Galbeta1-->4GlcNAc (II) and some Galbeta1-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 Galbeta1-->4GlcNAc as the core binding site with additional one to four sugars subsites, and is most complementary to a linear trisaccharide, Galbeta1-->4GlcNAcbeta1-->6Gal. These analyses should facilitate the understanding of the binding function of ECL.

Lectinochemical studies on the binding properties of a toxic lectin (ricin) isolated from the seeds of Ricinus communis.

BACKGROUND: Ricin (RCA2 or RCA60) is a highly toxic heterodimeric protein found in the seeds of the castor plant Ricinus communis. It is a potential biohazard. In the present study, the fine specificity of ricin was defined. METHODS: The combining site of ricin was characterized by quantitative precipitin (QPA) and precipitin inhibition assays (QPIA). RESULTS: Of 31 glycoproteins and pneumococcus type XIV capsular polysaccharide tested, only twelve of them precipitated over 50% of the toxin N added, reflecting poor precipitability of the lectin with the compounds tested. This can be explained by only a single chain (B chain of the molecules) participating in binding. The blood group active glycoproteins after mild acid hydrolysis or Smith degradation, as well as sialic-acid containing glycoproteins after removal of sialic acid, in general, had substantially increased activity. Of the monosaccharides tested for inhibition of precipitation of ricin, p-nitrophenyl betaGal was the best; this compound was 1.3-fold better than its alpha-anomer. While methyl betaGal was twice as active as its alpha anomer, Gal and blood group B active disaccharides (Galalpha1-3Gal) were 2.5 times more active than GalNAc. Among the oligosaccharides tested, Galbeta1-3GalNAc (T) Gal beta1-3/4GlcNAc (I/II), Galbeta1-4Glc (L) and human blood group I Ma trisaccharide (Galbeta1-4GlcNAcbeta1-6Gal) were about equally active and the best inhibitors. They were about 2.0 and 2.4 more active than Galalpha1-4Gal (E) sequence and B determinant, respectively. CONCLUSION: From the present results, it is concluded that: (a) this toxin has a broad range of affinity for the beta-anomer of Gal; (b) its combining site is probably of a shallow groove type and as large as a trisaccharide; (c) Galbeta--is the major combining site of the lectin; and (d) hydrophobic interaction gives a significant contribution for binding. This information should facilitate future usage of this lectin in glycobiological research and medical applications.

Fine specificity of domain-I of recombinant tandem-repeat-type galectin-4 from rat gastrointestinal tract (G4-N).

Galectins, a family of beta-galactoside-specific endogenous lectins, are involved in regulating diverse activities such as proliferation/apoptosis, cell-cell (matrix) interaction and cell migration. It is presently unclear to what extent the carbohydrate fine specificities of the combining sites of mammalian galectins overlap. To address this issue, we performed an analysis of the carbohydrate-recognition domain (CRD-I) near the N-terminus of recombinant rat galectin-4 (G4-N) by the biotin/avidin-mediated microtitre plate lectin-binding assay with natural glycoproteins (gps)/polysaccharide and by the inhibition of galectin-glycan interactions with a panel of glycosubstances. Among the 35 glycans tested for lectin binding, G4-N reacted best with human blood group ABH precursor gps, and asialo porcine salivary gps, which contain high densities of the blood group Ii determinants Galbeta1-3GalNAc (the mucin-type sugar sequence on the human erythrocyte membrane) and/or GalNAcalpha1-Ser/Thr ( Tn ), whereas this lectin domain reacted weakly or not at all with most sialylated gps. Among the oligosaccharides tested by the inhibition assay, Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc was the best. It was 666.7 and 33.3 times more potent than Gal and Galbeta1-3GlcNAc, respectively. G4-N has a preference for the beta-anomer of Gal at the non-reducing ends of oligosaccharides with a Galbeta1-3 linkage, over Galbeta1-4 and Galbeta1-6. The fraction of Tn glycopeptide from asialo ovine submandibular glycoprotein was 8.3 times more active than Galbeta1-3GlcNAc. The overall carbohydrate specificity of G4-N can be defined as Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (lacto- N -tetraose)>Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc (lacto- N -neo-tetraose) and Tn clusters>Galbeta1-4Glc and GalNAcbeta1-3Gal>Galbeta1-3GalNAc>Galbeta1-3GlcNAc>Galbeta1-4GlcNAc>GalNAc>Gal. The definition of this binding profile provides the basis to detect differential binding properties relative to the other galectins with ensuing implications for functional analysis.