Conformational stability of legume lectins reflect their different modes of quaternary association: solvent denaturation studies on concanavalin A and winged bean acidic agglutinin.

Thermodynamic parameters associated with the unfolding of the legume lectin, WBA II, were determined by isothermal denaturation. The analysis of isothermal denaturation data provided values for conformational stability and heat capacity for WBA II unfolding. To explore the role of intersubunit contact in stability, we carried out similar studies under identical conditions on Concanavalin A, a legume lectin of nearly similar size, buried hydrophobic surface area and tertiary structure to that of WBA II but with a different oligomerization pattern. Both proteins showed a reversible two-state unfolding with guanidine hydrochloride. As expected, the change in heat capacity upon unfolding was similar for both proteins at 3.5 and 3.7 kcal mol(-1) K(-1) for Concanavalin A and WBA II, respectively. Although the deltaG(H20) at the maximum stability of both proteins is around 16 kcal/mol, Concanavalin A exhibits greater stability at higher temperatures. The T(g) obtained for Concanavalin A and WBA II were 21 degrees C apart at 87.2 and 66.6 degrees C, respectively. The higher conformational stability at higher temperatures and the T(g) of Concanavalin A as compared to that of WBA II are largely due to substantial differences in the degree of subunit contact in these dimeric proteins. Ionic interactions and hydrogen bonding between the monomers of the two proteins also seem to play a significant role in the observed stability differences between these two proteins.

Legume lectin family, the 'natural mutants of the quaternary state', provide insights into the relationship between protein stability and oligomerization.

Legume lectins family of proteins, despite having the same 'jelly roll' tertiary structural fold at monomeric level, exhibit considerable variation in their quaternary structure arising out of small changes in their sequence. Nevertheless, their folding behavior and stability correlates very well with their patterns of assembly into dimers and tetramers. A conservation of their fold during evolution, its wide distribution in many protein families together with the availability of structural information on them make them interesting as proteins to explore the effect of inter- versus intra-subunit interactions in the stability of multimeric proteins. Additionally, as 'natural mutants' of quaternary association, proteins of legume lectin family provide interesting paradigms for studies addressing the effect of subunit oligomerization on the stability, folding and function as well as the evolution of multimeric structures.

Expression of winged bean basic agglutinin in Spodoptera frugiperda insect cell expression system.

In this paper we report the successful expression of the winged bean basic agglutinin (WBA I) in insect cells infected with a recombinant baculovirus carrying the WBA I gene and its characterization in terms of its carbohydrate binding properties. The expressed protein appears to have a lower molecular weight than the native counterpart which is consistent with the lack of glycosylation of the former. Moreover, the expressed protein maintains its dimeric nature. Hence, a role for glycosylation in modulation of dimerization of WBA I is ruled out unlike Erythrina corallodendron (EcorL). Despite this the protein is active, with its sugar specificity unaltered.

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.

Carbohydrate specificity and salt-bridge mediated conformational change in acidic winged bean agglutinin.

Structures of two crystal forms of the dimeric acidic winged bean agglutinin (WBAII) complexed with methyl-alpha-D-galactose have been determined at 3.0 A and 3.3 A resolution. The subunit structure and dimerisation of the lectin are similar to those of the basic lectin from winged bean (WBAI) and the lectin from Erythrina corallodendron (EcorL). The conformation of a loop and its orientation with respect to the rest of the molecule in WBAII are, however, different from those in all the other legume lectins of known structure. This difference appears to have been caused by the formation of two strategically placed salt bridges in the former. Modelling based on the crystal structures provides a rationale for the specificity of the lectin, which is very different from that of WBAI, for the H-antigenic determinant responsible for O blood group reactivity. It also leads to a qualitative explanation for the thermodynamic data on sugar-binding to the lectin, with special emphasis on the role of a tyrosyl residue in the variable loop in the sugar-binding region in generating the carbohydrate specificity of WBAII.

Expression, purification and characterization of peanut (Arachis hypogaea) agglutinin (PNA) from baculovirus infected insect cells.

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Galbeta1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 x 10(6) cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) cross-reacted with re-PNA. The subunit molecular weight (30 kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.

A predominantly hydrophobic recognition of H-antigenic sugars by winged bean acidic lectin: a thermodynamic study.

The thermodynamics of binding of winged bean (Psophocarpus tetragonolobus) acidic agglutinin to the H-antigenic oligosaccharide (Fucalpha1-2Galbeta1-4GlcNAc-oMe) and its deoxy and methoxy congeners were determined by isothermal titration calorimetry. We report a relatively hydrophobically driven binding of winged bean acidic agglutinin to the congeners of the above sugar. This conclusion is arrived, from the binding parameters of the fucosyl congeners, the nature of the enthalpy-entropy compensation plots and the temperature dependence of binding enthalpies of some of the congeners. Thus, the binding site of winged bean acidic agglutinin must be quite extended to accommodate the trisaccharide, with non-polar loci that recognize the fucosyl moiety of the H-antigenic determinant.

Structure of basic winged-bean lectin and a comparison with its saccharide-bound form.

The crystal structure of the saccharide-free form of the basic form of winged-bean agglutinin (WBAI) has been solved by the molecular-replacement method and refined at 2.3 A resolution. The final R factor is 19.7% for all data in the resolution range 8.0-2.3 A. The asymmetric unit contains two half-dimers, each located on a crystallographic twofold axis. The structure of the saccharide-free form is compared with that of the complex of WBAI with methyl-alpha-D-galactoside. The complex is composed of two dimers in the asymmetric unit. The intersubunit interactions in the dimer are nearly identical in the two structures. The binding site of the saccharide-free structure contains three ordered water molecules at positions similar to those of the hydroxyl groups of the carbohydrate which are hydrogen bonded to the protein. Superposition of the saccharide-binding sites of the two structures shows that the major changes involve expulsion of these ordered water molecules and a shift of about 0.6 A of the main-chain atoms of the variable loop.

Crystallization and preliminary crystallographic analysis of winged bean acidic lectin.

The acidic lectin (WBAII) from the winged bean (Psophocarpus tetragonolobus) binds to the H-antigenic determinant on human erythrocytes and to the T-antigenic disaccharide Gal-beta1,3-GalNAc. Two crystal forms of WBAII were obtained in the presence of methyl-alpha-D-galactose. Form I belongs to space group R3 with unit-cell dimensions a = b = 182.11, c = 44.99 A and has one dimer in the asymmetric unit. Form II belongs to space group C2 with unit-cell dimensions a = 135.36, b = 127.25, c = 139.98 A, beta = 95. 9 degrees and has four dimers in the asymmetric unit. Intensity data were collected to 3.0 A and to 3.5 A from crystals of form I and II, respectively. The structures were solved by the molecular-replacement method using the coordinates of the basic form of winged bean lectin.

Molten globule-like state of peanut lectin monomer retains its carbohydrate specificity. Implications in protein folding and legume lectin oligomerization.

A central question in biological chemistry is the minimal structural requirement of a protein that would determine its specificity and activity, the underlying basis being the importance of the entire structural element of a protein with regards to its activity vis à vis the overall integrity and stability of the protein. Although there are many reports on the characterization of protein folding/unfolding intermediates, with considerable secondary structural elements but substantial loss of tertiary structure, none of them have been reported to show any activity toward their respective ligands. This may be a result of the conditions under which such intermediates have been isolated or due to the importance of specific structural elements for the activity. In this paper we report such an intermediate in the unfolding of peanut agglutinin that seems to retain, to a considerable degree, its carbohydrate binding specificity and activity. This result has significant implications on the molten globule state during the folding pathway(s) of proteins in general and the quaternary association in legume lectins in particular, where precise subunit topology is required for their biologic activities.

Thermodynamic characterization of the conformational stability of the homodimeric protein, pea lectin.

The conformational stability of the homodimeric pea lectin was determined by both isothermal urea-induced and thermal denaturation in the absence and presence of urea. The denaturation profiles were analyzed to obtain the thermodynamic parameters associated with the unfolding of the protein. The data not only conform to the simple A2 if 2U model of unfolding but also are well described by the linear extrapolation model for the nature of denaturant-protein interactions. In addition, both the conformational stability (DeltaGs) and the DeltaCp for the protein unfolding is quite high, at about 18.79 kcal/mol and 5.32 kcal/(mol K), respectively, which may be a reflection of the relatively larger size of the dimeric molecule (Mr 49 000) and, perhaps, a consequent larger buried hydrophobic core in the folded protein. The simple two-state (A2 if 2U) nature of the unfolding process, with the absence of any monomeric intermediate, suggests that the quaternary interactions alone may contribute significantly to the conformational stability of the oligomer-a point that may be general to many oligomeric proteins.

Molecular basis of recognition by Gal/GalNAc specific legume lectins: influence of Glu 129 on the specificity of peanut agglutinin (PNA) towards C2-substituents of galactose.

The ability to discriminate between galactose and N- acetylgalactosamine, observed in some lectins, is crucial for their biological activity as well as their usefulness as tools in biology and medicine. However, the molecular basis of differential binding of lectins to these two sugars is poorly understood. Peanut agglutinin (PNA) is one of the few galactose-specific legume lectins which does not bind N- acetylgalactosamine at all and is, therefore, ideal for the study of the basis of specificity towards C-2 substituted derivatives of galactopyranosides. Examination of the three-dimensional structure of PNA in complex with lactose revealed the presence of both a longer loop and bulkier residues in the region surrounding the C-2 hydroxyl of the galactopyranoside ring, which can sterically prevent the accommodation of a bulky substituent in this position. One such residue, is a glutamic acid at position 129 which protrudes into the binding site and perhaps directly obstructs any substitution at the C-2 position. Two mutants in bacterially expressed PNA were therefore constructed. These were E129D and E129A, in which Glu129 was replaced by Asp and Ala, respectively. The specificity of the mutants for galactose, galactosamine, and N- acetylgalactosamine was examined through observing the inhibition of hemagglutination and binding of the lectin to immobilized asialofetuin. The results showed that the affinity of E129A and E129D for C-2-substituted derivatives of the galactose varies. The mutant E129D showed significant binding towards N- acetylgalactosamine, suggesting that the residue Glu 129 is crucial in imparting exclusive galactose-specificity upon PNA. This study not only attempts to provide an explanation for the inability of PNA to accommodate C-2-substituted derivatives at its primary subsite, but also seeks to present a basis for engineering lectins with altered specificities.

Differential scanning calorimetric studies of the glycoprotein, winged bean acidic lectin, isolated from the seeds of Psophocarpus tetrogonolobus.

Differential scanning calorimetry of solutions of WBAII and in presence of sugar ligands shows that WBAII dimer dissociates to its constituent monomeric subunits at the denaturation temperature. The thermal denaturation of WBAII consists of the unfolding of two independent domains of WBAII similar to that of basic winged bean lectin and ECorL and in contrast to concanavalin A (conA), pea and lentil lectin, which unfold as single entities. Apparently, the glycosylation reduces the structural integrity of WBAII as compared to conA, pea and lentil lectin. The increase in the denaturation temperature of the sugar-lectin complexes yields binding constants close to the binding constants extrapolated from the ITC results and confirms the mechanism proposed for its thermal unfolding.

Cloning and sequencing of winged bean (Psophocarpus tetragonolobus) basic agglutinin (WBA I): presence of second glycosylation site and its implications in quaternary structure.

We report cloning of the DNA encoding winged bean basic agglutinin (WBA I). Using oligonucleotide primers corresponding to N- and C-termini of the mature lectin, the complete coding sequence for WBA I could be amplified from genomic DNA. DNA sequence determination by the chain termination method revealed the absence of any intervening sequences in the gene. The DNA deduced amino acid sequence of WBA I displayed some differences with its primary structure established previously by chemical means. Comparison of the sequence of WBA I with that of other legume lectins highlighted several interesting features, including the existence of the largest specificity determining loop which might account for its oligosaccharide-binding specificity and the presence of an additional N-glycosylation site. These data also throw some light on the relationship between the primary structure of the protein and its probable mode of dimerization.