Erythrina Corallodendron Lectin Research Articles

Role of glycosylation in structure and stability of Erythrina corallodendron lectin (EcorL): A molecular dynamics study

The effect of glycosylation on structure and stability of glycoproteins has been a topic of considerable interest. In this work, we have investigated the solution conformation of the oligosaccharide and its effect on the structure and stability of the glycoprotein by carrying out a series of long Molecular dynamics (MD) simulations on glycosylated Erythrina corallodendron lectin (EcorL) and nonglycosylated recombinant Erythrina corallodendron lectin (rEcorL). Our results indicate that, despite the similarity in overall three dimensional structures, glycosylated EcorL has lesser nonpolar solvent accessible surface area compared to nonglycosylated EcorL. This might explain the experimental observation of higher thermodynamic stability for glycosylated EcorL compared to nonglycosylated EcorL. Analysis of the simulation results indicates that, dynamic view of interactions between protein residues and oligosaccharide is entirely different from the static picture seen in the crystal structure. The oligosaccharide moiety had dynamically stable interactions with Lys 55 and Tyr 53, both of which are separated in sequence from the site of glycosylation, Asn 17. It is possible that glycosylation helps in forming long-range contacts between amino acids, which are separated in sequence and thus provides a folding nucleus. Thus our simulations not only reveal the conformations sampled by the oligosaccharide, but also provide novel insights into possible molecular mechanisms by which glycosylation can help in folding of the glycoprotein by formation of folding nucleus involving specific contacts with the oligosaccharide moiety.

Modification of the sugar specificity of a plant lectin: structural studies on a point mutant ofErythrina corallodendronlectin

A mutant of Erythrina corallodendron lectin was generated with the aim of enhancing its affinity for N-acetylgalactosamine. A tyrosine residue close to the binding site of the lectin was mutated to a glycine in order to facilitate stronger interactions between the acetamido group of the sugar and the lectin which were prevented by the side chain of the tyrosine in the wild-type lectin. The crystal structures of this Y106G mutant lectin in complex with galactose and N-acetylgalactosamine have been determined. A structural rationale has been provided for the differences in the relative binding affinities of the wild-type and mutant lectins towards the two sugars based on the structures. A hydrogen bond between the O6 atom of the sugars and the variable loop of the carbohydrate-binding site of the lectin is lost in the mutant complexes owing to a conformational change in the loop. This loss is compensated by an additional hydrogen bond that is formed between the acetamido group of the sugar and the mutant lectin in the complex with N-acetylgalactosamine, resulting in a higher affinity of the mutant lectin for N-acetylgalactosamine compared with that for galactose, in contrast to the almost equal affinity of the wild-type lectin for the two sugars. The structure of a complex of the mutant with a citrate ion bound at the carbohydrate-binding site that was obtained while attempting to crystallize the complexes with sugars is also presented.

Effect of glycosylation on the structure of Erythrina corallodendron lectin.

The three-dimensional structure of the recombinant form of Erythrina corallodendron lectin, complexed with lactose, has been elucidated by X-ray crystallography at 2.55 A resolution. Comparison of this non-glycosylated structure with that of the native glycosylated lectin reveals that the tertiary and quaternary structures are identical in the two forms, with local changes observed at one of the glycosylation sites (Asn17). These changes take place in such a way that hydrogen bonds with the neighboring protein molecules in rECorL compensate those made by the glycan with the protein in ECorL. Contrary to an earlier report, this study demonstrates that the glycan attached to the lectin does not influence the oligomeric state of the lectin. Identical interactions between the lectin and the non-covalently bound lactose in the two forms indicate, in line with earlier reports, that glycosylation does not affect the carbohydrate specificity of the lectin. The present study, the first of its kind involving a glycosylated protein with a well-defined glycan and the corresponding deglycosylated form, provides insights into the structural aspects of protein glycosylation.

Probing genetic variation and glycoform distribution in lectins of the Erythrina genus by mass spectrometry

Six leguminous lectins from the seeds of plants of the Erythrina genus, namely E. caffra (ECafL), E. cristagalli (ECL), E. flabelliformis (EFL), E. lysistemon (ELysL), E. rubrinerva (ERL), and E. vespertilio (EVL), were examined to establish their sequence homology and to determine the structure and sites of attachment of their glycans. Tryptic digests of these lectins were analyzed by capillary electrophoresis coupled to electrospray mass spectrometry (CE-ESMS). Assignments were made by comparing the molecular masses of the observed tryptic peptides with those of Erythrina corallodendron lectin (ECorL), the sequence of which had been established previously. Glycan structure and genetic variations in the amino acid sequence were probed by tandem mass spectrometry. Small differences were found between the sequences of the various lectins examined and all of them exhibited C-terminal processing resulting in proteins with a C-terminal Asn residue. The major glycan of these glycoproteins was shown to be the heptasaccharide Man 3XylFucGlcNAc 2, consistent with previous investigations on ECL and ECorL. A minor glycan heterogeneity was observed for most lectins examined except for that of ECafL and ECorL where an extra hexose residue was observed on the reducing GlcNAc residue of the heptasaccharide.

Role of N-linked glycan in the unfolding pathway of Erythrina corallodendron lectin.

Erythrina corallodendron lectin (ECorL) exhibits an exquisitely structured oligosaccharide chain. Interestingly, the bacterially expressed, nonglycosylated counterpart, rECorL, possesses an essentially identical carbohydrate specificity and agglutinating activity as the glycosylated lectin, thus suggesting that the overall structure of the two are identical. This paper reports the unfolding behavior of E. corallodendron lectin in its glycosylated (EcorL) and nonglycosylated (rECorL) forms. ECorL shows a two-state unfolding pattern during isothermal melts and differential scanning calorimetry (DSC). The T(g) of ECorL as obtained from isothermal melts is 74 degrees C at pH 7.4. The T(p) obtained from DSC studies is between 74.8 to 68.1 degrees C in the pH range of 5.26-7.77. The recombinant lectin (rECorL), which is devoid of carbohydrate, shows, in contrast to the glycosylated protein, a non-two-state unfolding profile as measured by both probes mentioned, but the number of intermediates during unfolding could not be ascertained. Simulated annealing on ECorL, with the sugars removed, reveals that the protein Calpha backbones overlap, indicating that the overall structure, including the mode of dimerization, of rECorL is insignificantly altered as compared to ECorL. The alterations in the folding behavior of rECorL as compared to that observed in ECorL may be due to the fact that, unlike most other glycoproteins, one of the glycans in ECorL is unusually structured and forms many hydrogen bonds with the protein. It therefore appears that while the covalently linked sugar does not contribute appreciably to the final folded structure of ECorL, it does alter its folding process in a significant manner.

Isolation of the gene and large-scale expression and purification of recombinant Erythrina cristagalli lectin

Using polymerase chain reaction, the coding sequence for Erythrina cristagalli lectin (ECL) has been cloned and expressed in Escherichia coli. The amplified DNA sequence of ECL is highly homologous to that previously reported for Erythrina corallodendron lectin (ECorL), confirming the absence of introns in the ECL gene. The polypeptide sequences of ECL and ECorL have been compared and five amino acids have been identified that differentiate the two proteins. Recombinant E. cristagalli lectin ( recECL) was expressed in E. coli from a genomic clone encoding the mature E. cristagalli lectin gene. Constitutive expression localised recombinant protein in inclusion bodies, which were solubilised, and recECL, subsequently refolded and purified by lactose affinity chromatography. Significant advantages were observed for purification from inclusion bodies rather than from a clone optimised to express soluble protein. A large-scale purification scheme has been developed that can prepare functional recECL from inclusion bodies with a yield of 870 mg/L culture. By the range of characterisation methods employed in this study, it has been demonstrated that recECL is functionally equivalent to native ECL obtained from the E. cristagalli plant. In addition, characterisation of the binding of radiolabelled recECL to cultured dorsal root ganglia demonstrated that recECL binds to a single pool of receptors.

Isolation, purification, and physicochemical characterization of a d-galactose-binding lectin from seeds of Erythrina speciosa

A lectin was isolated from the saline extract of Erythrina speciosa seeds by affinity chromatography on lactose–Sepharose. The lectin content was about 265 mg/100 g dry flour. E. speciosa seed lectin (EspecL) agglutinated all human RBC types, showing no human blood group specificity; however a slight preference toward the O blood group was evident. The lectin also agglutinated rabbit, sheep, and mouse blood cells and showed no effect on horse erythrocytes. Lactose was the most potent inhibitor of EspecL hemagglutinating activity (minimal inhibitory concentration (MIC)=0.25 mM) followed by N-acetyllactosamine, MIC=0.5 mM, and then p-nitrophenyl α-galactopyranoside, MIC=2 mM. The lectin was a glycoprotein with a neutral carbohydrate content of 5.5% and had two p I values of 5.8 and 6.1 and E 1% 1 cm of 14.5. The native molecular mass of the lectin detected by hydrodynamic light scattering was 58 kDa and when examined by mass spectroscopy and SDS–PAGE it was found to be composed of two identical subunits of molecular mass of 27.6 kDa. The amino acid composition of the lectin revealed that it was rich in acidic and hydroxyl amino acids, contained a lesser amount of methionine, and totally lacked cysteine. The N-terminal of the lectin shared major similarities with other reported Erythrina lectins. The lectin was a metaloprotein that needed both Ca 2+ and Mn 2+ ions for its activity. Removal of these metals by EDTA rendered the lectin inactive whereas their addition restored the activity. EspecL was acidic pH sensitive and totally lost its activity when incubated with all pH values between pH 3 and pH 6. Above pH 6 and to pH 9.6 there was no effect on the lectin activity. At 65 °C for more than 90 min the lectin was fairly stable; however, when heated at 70 °C for 10 min it lost more than 80% of its original activity and was totally inactivated at 80 °C for less than 10 min. Fluorescence studies of EspecL indicated that tryptophan residues were present in a highly hydrophobic environment, and binding of lactose to EspecL neither quenched tryptophan fluorescence nor altered λ max position. Treating purified EspecL with NBS an affinity-modifying reagent specific for tryptophan totally inactivated the lectin with total modification of three tryptophan residues. Of these residues only the third modified residue seemed to play a crucial role in the lectin activity. Addition of lactose to the assay medium did not provide protection against NBS modification which indicated that tryptophan might not be directly involved in the binding of haptenic sugar d-galactose. Modification of tyrosine with N-acetylimidazole led to a 50% drop in EspecL activity with concomitant acetylation of six tyrosine residues. The secondary structure of EspecL as studied by circular dichroism was found to be a typical β-pleated-sheet structure which is comparable to the CD structure of Erythrina corallodendron lectin. Binding of lactose did not alter the EspecL secondary structure as revealed by CD examination.

How proteins bind carbohydrates: lessons from legume lectins.

The pioneering studies of Irvin Liener on soybean agglutinin (SBA) in the early 1950s served as the starting point of our involvement in lectin research during the past four decades. Initially we characterized SBA extensively as a glycoprotein and showed that its covalently linked glycan is an oligomannoside commonly present in animal glycoproteins. We have also introduced the use of the lectin to the study of normal and malignant cells and to the purging of bone marrow for transplantation. Our recent work focuses on the combining site of Erythrina corallodendron lectin, closely related to SBA. In this legume lectin, as in essentially all other members of the same protein family, irrespective of their sugar specificity, interactions with a constellation of three invariant residues (aspartic acid, asparagine, and an aromatic residue) are essential for ligand binding. Lectins from other families, whether of plants or animals, also combine with carbohydrates by H-bonds and hydrophobic interactions, but the amino acids involved may differ even if the specificity of the lectins is the same. Therefore, nature finds diverse solutions for the design of binding sites for structurally similar ligands, such as mono- or oligosaccharides. This diversity strongly suggests that lectins are products of convergent evolution.

High-resolution Crystal Structures of Erythrina cristagalli Lectin in Complex with Lactose and 2′-α- l-Fucosyllactose and Correlation with Thermodynamic Binding Data

The primary sequence of Erythrina cristagalli lectin (ECL) was mapped by mass spectrometry, and the crystal structures of the lectin in complex with lactose and 2′-α- l-fucosyllactose were determined at 1.6 Å and 1.7 Å resolution, respectively. The two complexes were compared with the crystal structure of the closely related Erythrina corallodendron lectin (ECorL) in complex with lactose, with the crystal structure of the Ulex europaeus lectin II in complex with 2′-α- l-fucosyllactose, and with two modeled complexes of ECorL with 2′-α- l-fucosyl- N-acetyllactosamine. The molecular models are very similar to the crystal structure of ECL in complex with 2′-α- l-fucosyllactose with respect to the overall mode of binding, with the l-fucose fitting snugly into the cavity surrounded by Tyr106, Tyr108, Trp135 and Pro134 adjoining the primary combining site of the lectin. Marked differences were however noted between the models and the experimental structure in the network of hydrogen bonds and hydrophobic interactions holding the l-fucose in the combining site of the lectin, pointing to limitations of the modeling approach. In addition to the structural characterization of the ECL complexes, an effort was undertaken to correlate the structural data with thermodynamic data obtained from microcalorimetry, revealing the importance of the water network in the lectin combining site for carbohydrate binding.

Chemical characteristics of dimer interfaces in the legume lectin family.

The Erythrina corallodendron lectin (EcorL) crystallizes in monoclinic and hexagonal crystal forms. Comparison of the newly determined hexagonal form (PDB code 1fyu) with the monoclinic form shows that the dimeric structure of EcorL reflects the inherent biological structure of the protein and is not an artifact of the crystal packing. To further understand the factors determining the dimerization modes of legume lectins, EcorL, concanavalin A (ConA), and Griffonia simplicifolia (GS4) were taken as representatives of the three unique dimers found in the family. Six virtual homodimers were generated. The hydropathy, amino acid composition, and solvation energy were calculated for all nine homodimers. Each of the three native dimers has a distinct chemical composition. EcorL has a dominant hydrophobic component, and ConA has a strong polar component, but in GS4 the three components contribute equally to the interface. This distribution pattern at the interface is unique to the native dimers and distinct from the partition observed in the virtual dimers. Amino acid composition of other members of the family that dimerize like EcorL or ConA maintain the same pattern of amino acids distribution observed in EcorL and ConA. However, lectins that dimerize like GS4 do not show a particularly distinct distribution. In all cases, the calculated solvation energy of the native dimer was lower than that of the virtual dimers, suggesting that the observed mode of dimerization is the most stable organization for the given sequence and tertiary structure. The dimerization type cannot be predicted by sequence analysis.

The epitope of the H‐type 2 trisaccharide recognized by Erythrina corallodendron lectin. Evidence for both attractive polar and strong hydrophobic interactions for complex formation involving a lectin

Erythrina corallodendron lectin (ECorL), originally regarded as specific for N-acetyllactosamine (LacNAc), shows preferential binding of the II-type 2 human blood group determinant (alpha -L-Fuc-(1-2)-beta -D-Gal-(1-4)-beta -D-GlcNAc-OH). In the present study, various deoxy- and mono-O-methyl derivatives of beta -H-type 2 methyl glycoside and beta -LacNAc methyl glycoside were employed in an enzyme-linked lectin assay to further examine the specificity requirements of ECorL and the contribution of the fucose moiety to the binding of the trisaccharide to the lectin. Based on the relative affinities of the ligands, the X-ray structure of the ECorL/ LacNAc complex, and the site-directed mutagenesis studies of the lectin, the epitope of the II-type 2 trisaccharide recognized by ECorL has been delineated. The fucose moiety is shown to provide a strong hydrophobic interaction at its 2-OH, most likely with Trp 135 of the lectin combining site, in addition to the polar interactions of the key hydroxyl groups at positions 3 and 4 of the galactose and the stacking of the latter residue on Phe131. Comparison of the epitopes of H-type-2-OMe recognized by three other legume lectins, from Galactia tenuiflora, Psophocarpus tetragonolobus, and Ulex europaeus, shows that all four are different, in spite of the high similarity between their tertiary structures. Our findings provide a striking illustration of how a single oligosaccharide can carry different messages for communication with different recognition molecules.

A Highly Practical Synthesis of Cyclodextrin-Based Glycoclusters Having Enhanced Affinity with Lectins

A simple and highly practical method for the synthesis of cyclodextrin-scaffolded glycoclusters recognized specifically by lectins is described. Nucleophilic displacement of iodide from heptakis 6-deoxy-6-iodo-β-cyclodextrin by unprotected sodium thiolates derived from 3-(3-thioacetyl propionamido)propyl glycosides proceeded smoothly in mild condition and gave novel per-glycosylated cyclodextrins (glycocyclodextrins, glycoCDs) having d-galactose, N-acetyl- d-glucosamine, lactose or N-acetyllactosamine residues in high yields (78–88%). It was demonstrated that all these per-glycosylated β-cyclodextrins showed amplified inhibitory effects on the erythrocytes agglutination induced by wheat germ ( Triticum vulgaris) agglutinin (WGA) or Erythrina corallodendron lectin (ECorL).

Structure/thermodynamics relationships of lectin-saccharide complexes: the Erythrina corallodendron case.

Molecular dynamics (MD) simulations of Erythrina corallodendron lectin binding to a monosaccharide, alpha-galactose, and a disaccharide, N-acetyl lactosamine, have been performed in order to investigate the relationship between structure and thermodynamics. A simulated annealing protocol has been used to generate ensembles of structures for the two complexes, from which both qualitative and quantitative information on binding dynamics have been extracted. The ensembled averaged lectin-saccharide interaction enthalpy is equivalent for both sugars, whereas the calculation based on the X-ray structures does show a difference. Within large statistical errors, the calculated 'binding enthalpy' is also the same for the two systems. These errors arise largely from terms involving solvent and are a typical limitation of current MD simulations. Significant qualitative differences in binding between the two complexes are, however, observed over the ensembles. These could be important for unraveling the structure/thermodynamic relationship. Stated simply, there are a greater number of binding options available to the disaccharide compared to the monosaccharide. The implications of alternative binding states on thermodynamic parameters and the 'breaking of enthalpy-entropy compensation' are discussed. The role of solvent in lectin-saccharide complex formation is suggested to be significant.

The primary structure of the acidic lectin from winged bean ( Psophocarpus tetragonolobus): insights in carbohydrate recognition, adenine binding and quaternary association

The amino acid sequence of the winged bean acidic lectin (WBA II) was determined by chemical means and by recombinant techniques. From the N- and C-terminal sequence, obtained chemically, primers were designed for PCR amplification of the genomic DNA. The PCR product was cloned and sequenced to get the complete primary structure of WBA II. Peptide fragments for sequencing were also obtained by tryptic cleavages of the native lectin. The WBA II sequence showed a high degree of homology with that of WBA I and Erythrina corallodendron lectin (ECorL), especially in the regions involved in subunit association, where there is a very high conservation of residues. This perhaps implies the importance of this particular region in subunit interactions in this lectin. In addition, many of the residues, involved in carbohydrate binding in legume lectins, appear to be conserved in WBA II. The distinct differences in anomeric specificity observed amongst WBA I, WBA II, ECorL and peanut agglutinin (PNA) may be explained by subtle differences in sequence/structure of their D-loops. WBA II binds adenine quite strongly; a putative adenine binding sequence has been identified.

Structures of the Erythrina corallodendron lectin and of its complexes with mono- and disaccharides

The structures of the Erythrina corallodendron lectin (EcorL) and of its complexes with galactose, N-acetylgalactosamine, lactose and N-acetyllactosamine were determined at a resolution of 1.9 to 1.95 Å. The final R-values of the five models are in the range 0.169 to 0.181. The unusual, non-canonical, dimer interface of EcorL is made of β-strands from the two monomers, which face one another in a “hand-shake” mode. The galactose molecule in the primary binding site is bound in an identical way in all four complexes. Features of the electrostatic potential of the galactose molecule match those of the potential in the combining site, thus probably pointing to the contribution of the electrostatic energy to determining the orientation of the ligand. No conformational change occurs in the protein upon binding the ligand. Subtle variations in the binding mode of the second monosaccharide (glucose in the complex with lactose and N-acetylglucosamine in the complex with N-acetyllactosamine) were observed. The mobility of Gln219 is lower in the complexes with the disaccharides than in the complexes with the monosaccharides, indicating further recruitment of this residue to ligand binding through more extensive hydrogen bonding in the former complexes. Water molecules that have been located in the combining sites of the five structures undergo rearrangement in response to binding of the different ligands. The new structural information is in qualitative agreement with thermodynamic data on the binding to EcorL.

Carbohydrate specificity and quaternary association in basic winged bean lectin: X-ray analysis of the lectin at 2.5 Å resolution

The structure of basic Winged Bean Agglutinin (WBAI) with two dimeric molecules complexed with methyl-α- d-galactopyranoside in the asymmetric unit, has been determined by the molecular replacement method and refined with 2.5 Å X-ray intensity data. The polypeptide chain of each monomer has the characteristic legume lectin tertiary fold. The structure clearly defines the lectin-carbohydrate interactions. It reveals how the unusually long variable loop in the binding region endows the lectin with its characteristic sugar specificity. The lectin forms non-canonical dimers of the type found in Erythrina corallodendron lectin (EcorL) even though glycosylation, unlike in EcorL, does not prevent the formation of canonical dimers. The structure thus further demonstrates that the mode of dimerisation of legume lectins is not necessarily determined by the covalently bound carbohydrate but is governed by features intrinsic to the protein. The present analysis and our earlier work on peanut lectin (PNA), show that legume lectins are a family of proteins in which small alterations in essentially the same tertiary structure lead to wide variations in quaternary association. A relationship among the non-canonical modes of dimeric association in legume lectins is presented.

Structural features of the combining site region of Erythrina corallodendron lectin: role of tryptophan 135.

The role of Trp 135 and Tyr 108 in the combining site of Erythrina corallodendron lectin (ECorL) was investigated by physicochemical characterization of mutants obtained by site-directed mutagenesis, hemagglutination-inhibition studies, and molecular modeling, including dynamics simulations. The findings demonstrate that Trp 135 in ECorL: (1) is required for the tight binding of Ca2+ and Mn2+ to the lectin because mutation of this residue into alanine results in loss of these ions upon dialysis and concomitant reversible inactivation of the mutant; (2) contributes to the high affinity of methyl alpha-N-dansylgalactosaminide (MealphaGalNDns) to the lectin; and (3) is solely responsible for the fluorescence energy transfer between the aromatic residues of the lectin and the dansyl group in the ECorL-MealphaGalNDns complex. Docking of MealphaGalNDns into the combining site of the lectin reveals that the dansyl moiety is parallel with the indole of Trp 135, as required for efficient fluorescence energy transfer, in one of the two possible conformations that this ligand assumes in the bound state. In the W135A mutant, which still binds MealphaGalNDns strongly, the dansyl group may partially insert itself into the place formerly occupied by Trp 135, a process that from dynamics simulations does not appear to be energetically favored unless the loop containing this residue assumes an open conformation. However, a small fraction of the W135A molecules must be able to bind MealphaGalNDns in order to explain the relatively high affinity, as compared to galactose, still remaining for this ligand. A model for the molecular events leading to inactivation of the W135A mutant upon demetallization is also presented in which the cis-trans isomerization of the Ala 88-Asp 89 peptide bond, observed in high-temperature dynamics simulations, appears not to be a required step.

Involvement of laser photo-CIDNP (chemically induced dynamic nuclear polarization)-reactive amino acid side chains in ligand binding by galactoside-specific lectins in solution.

For proteins in solution the validity of certain crystallographic parameters can be ascertained by a combination of molecular-dynamics (MD) simulations and NMR spectroscopy. Using the laser photo-CIDNP (chemically induced dynamic nuclear polarization) technique as a measure for surface accessibility of histidine, tyrosine and tryptophan, the spectra of bovine galectin-1 and Erythrina corallodendron lectin (EcorL) are readily reconcilable with the crystallographic data for these two proteins. The results emphasise the role of Trp68/Trp69 for carbohydrate binding in bovine galectin-1/chicken galectins and of Trp194 in murine galectin-3. This feature derived from the crystal structure of bovine galectin-1 is maintained in solution for the prototype human homologue, two avian galectins and the chimera-type murine galectin-3, as the spectra corroborate the CIDNP-inferable spatial parameters of the four calculated models for binding-site architecture. In EcorL, Tyr106/Tyr108 are constituents of the extended combining pocket, which can be shielded in solution by ligand presence. Discrepancies between results from modelling and CIDNP measurements concern primarily the lack of reactivity of histidine residues for human and avian prototype galectins and of Tyr82/Tyr229 of the plant lectin. Site-directed mutagenesis of EcorL is assumed to provide information on the role of a certain residue for functional aspects. When single-site mutants of EcorL ([Ala106]EcorL, [Ala108]EcorL, [Ala229]EcorL) were subjected to molecular-dynamics (MD) simulations, the apparent surface accessibilities even of spatially separated amino acid side chains could non-uniformly be affected. This conclusion is supported by the assessment of the spectra for the mutant proteins. On the basis of these CIDNP-results modelling of the binding-site architecture of the lectin indicates the occurrence of notable alterations in the orientation of Tyr106/Tyr108 phenyl rings. The implied potential effect of single-site mutations on conformational features of a protein will deserve attention for the interpretation of studies comparing wild-type and mutant proteins.

Natural human antibodies to dietary lectins

Natural antibodies to self and non-self proteins, including dietary proteins, are a significant part of the immune repertoire of humans. Antibodies to three structurally related legume lectins ( Erythrina corallodendron lectin (ECorL), peanut agglutinin (PNA), and soybean agglutinin (SBA)) and to one cereal lectin (wheat germ agglutinin (WGA)) were purified by affinity chromatography from human sera and their binding specificity examined. The anti-SBA, anti-ECorL and anti-WGA antibodies exhibited high specificity, whereas the anti-PNA antibodies were polyreactive. Although the anti-WGA antibodies were highly specific for WGA, they also crossreacted slightly toward some other proteins. The anti-ECorL antibodies bound to native SBA, but the anti-SBA antibodies failed to bind to the native ECorL. Although the anti-SBA and anti-ECorL antibodies both exhibited specificity when interacting with native lectins, they bound to a wider range of denatured lectins, indicating a common or universal epitope which is recognized by many natural antibodies. Interestingly, the natural antibodies did not interfere with the agglutination properties of the lectins. These findings may provide a basis for studying the in vivo biological effects of anti-dietary protein antibodies, including those against carbohydrate-binding proteins.

Thermodynamics of Monosaccharide and Disaccharide Binding to Erythrina corallodendron Lectin

Isothermal titration calorimetry measurements of the binding of 2'-fucosyllactose, lactose, N-acetyllactosamine, galactopyranose, 2-acetamido-2-deoxygalactopyranoside, methyl alpha-N-dansylgalactosaminide (Me-alpha-DNS-GalN), methyl alpha-D-galactopyranoside, methyl beta-D-galactopyranoside, and fucose to Erythrina corallodendron lectin (ECorL), a dimer with one binding site per subunit, were performed at 283-286 and 297-299 K. The site binding enthalpies, DeltaHb, with the exception of Me-alpha-DNS-GalN, are the same at both temperatures and range from -47.1 +/- 1.0 kJ mol-1 for N-acetyllactosamine to -4.4 +/- 0.3 kJ mol-1 for fucose, and the site binding constants range from 3.82 +/- 0.9 x 10(5)M-1 for Me-alpha-DNS-GalN at 283.2 K to 0.46 +/- 0.05 x 10(3) M-1 for fucose at 297.2 K. The binding reactions are mainly enthalpically driven except for fucose and exhibit enthalpy-entropy compensation. The binding enthalpies of the disaccharides are about twice the binding enthalpies of the monosaccharides in contrast to concanavalin A where the binding enthalpies do not double for the disaccharides. Differential scanning calorimetry measurements show that denaturation of the ECorL dimer results in dissociation into its monomer subunits. The binding constants from the increase in denaturation temperature of ECorL in the presence of saccharides are in agreement with values from isothermal titration calorimetry results. The thermal denaturation of ECorL occurs around 333 K, well below the 344-360 K denaturation temperature of other legume lectins of similar size and tertiary structure, undoubtedly due to the difference in its quaternary structure relative to other legume lectins. This is also apparent from the independent unfolding of its two domains.