Thermodynamic and kinetic analysis of carbohydrate binding to the basic lectin from winged bean (Psophocarpus tetragonolobus)

A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N-dansylgalactosamine and the lectin are consistent with a simple one-step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(-2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.     

Erythrocyte-binding studies on an acidic lectin from winged bean (Psophocarpus tetragonolobus).

An acidic lectin (WBA II) was isolated to homogeneity from the crude seed extract of the winged bean (Psophocarpus tetragonolobus) by affinity chromatography on lactosylaminoethyl-Bio-Gel. Binding of WBA II to human erythrocytes of type-A, -B and -O blood groups showed the presence of 10(5) receptors/cell, with high association constants (10(6)-10(8) M-1). Competitive binding studies with blood-group-specific lectins reveal that WBA II binds to H- and T-antigenic determinants on human erythrocytes. Affinity-chromatographic studies using A-, B-, H- and T-antigenic determinants coupled to an insoluble matrix confirm the specificity of WBA II towards H- and T-antigenic determinants. Inhibition of the binding of WBA II by various sugars show that N-acetylgalactosamine and T-antigenic disaccharide (Thomsen-Friedenreich antigen, Gal beta 1-3GalNAc) are the most potent mono- and di-saccharide inhibitors respectively. In addition, inhibition of the binding of WBA II to erythrocytes by dog intestine H-fucolipid prove that the lectin binds to H-antigenic determinant.     

Isolation,purification and some properties of a lectin from the winged bean (Psophocarpus tetragonolobus)

We have isolated by affinity chromatography a lectin from the seeds of the winged bean (Psophocrapus tetragonolobus) which agglutinated human (group A, B and O), sheep and rabbit, but not mouse erythrocytes. A molecular weight of 41,000 was obtained from gel filtration, and on sodium dodecyl sulphate polyacrylamide gel electrophoresis a single polypeptide chain of molecular weight 35,000 was seen both before and after reduction. Isoelectric focussing of the lectin on polyacrylamide gel gave a single band with a calculated isoelectric point of 4.0. The lectin was found to be rich in acidic amino acids; cysteine was not detected. Carbohydrate analysis revealed no covalently bound sugars.Abbreviations PBS phosphate-buffered saline - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - WBL winged bean lectin - HPLC high performance liquid chromatography     

Isolation of an N-acetyl-d-galactosamine-binding protein from winged bean (Psophocarpus tetragonolobus)

A lectin has been purified to homogeneity by affinity chromatography on a Sepharose-N-caproyl-d-galactosamine column from the local variety of winged bean (Psophocarpus tetragonolobus). The lectin agglutinated native erythrocytes of all blood groups. This hemagglutination was inhibited best by N-acetyl-d-galactosamine. A molecular weight of 41,000 was obtained for the lectin by gel filtration on Bio-Gel P-100. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis of the same lectin showed a single M_r 35,000 polypeptide.     

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.     

Localized agglutinin staining in muscle capillaries from normal and very old atrophic human muscle using winged bean (Psophocarpus tetragonolobus) lectin

 The winged bean (Psophocarpus tetragonolobus) agglutinin (total lectin) and its basic (WBA I) and acidic isoform (WBA II) were used to analyze capillaries in sections from human muscle. The microvessels were clearly labeled after incubation with the lectins in both normal muscle and in old muscles with age-related type II atrophy or muscle fiber grouping. Muscle fibers, nerves, and connective tissue remained unstained. The total lectin detected muscle capillaries from all blood group AB0 individuals. The isoform WBA I reacted only with blood vessels in blood group A and B individuals, while the blood vessels in blood group 0 individuals were demonstrated with WBA II. WBA I staining was inhibited by p-nitrophenyl α-galactopyranoside and N-acetylgalactosamine, whereas 2′-fucosyllactose and preincubation with an antibody against type-1 chain H abolished capillary staining with WBA II. The study demonstrates the usefulness of WBA as a marker of capillaries in human muscle. Accepted: 2 September 1996     

Characterization of the acidic lectins from winged bean seed (Psophocarpus tetragonolobus(L.)DC)

The seeds of winged bean, Psophocarpus tetragonolobus(L.)DC, contain two distinct groups of lectins characterized by different erythrocyte hemagglutinating specificities and isoelectric points. Three acidic lectins (I, II, and III) (pI approximately 5.5) were purified to apparent homogeneity by chromatography on Ultrogel AcA44 and SP-Sephadex C-25. These lectins are glycoproteins with relative molecular mass of 54,000. The total carbohydrate content of the acidic lectins was 7% and was comprised of mannose, N-acetylglucosamine, fucose, and xylose in amounts corresponding to 9.2, 4.8, 1.6, and 7.0 mol/54,000 g, respectively. Electrophoresis in dodecyl sulfate, in the presence and absence of 2-mercaptoethanol, gave a single subunit of apparent relative molecular mass 30-32,000, somewhat higher than expected from the native relative molecular mass. On isoelectric focusing in 8 M urea the subunits of the acidic lectins did not show any significant charge heterogeneity as found for the winged bean basic lectins. The acidic lectins have very similar amino acid compositions. They contain essentially no half-cystine, 1-2 methionine residues, and are rich in acidic and hydroxy amino acids. The amino-terminal sequences of lectins II and III were identical while the amino-terminal sequence of lectin I contained five differences in the first 25 residues; the acidic lectins showed extensive sequence homology with the winged bean basic lectins, the other one-chain subunit lectins and the beta subunit of the two-chain subunit legume lectins. The acidic lectins agglutinated trypsinized human (type A, B, AB, and O) erythrocytes but not trypsinized rabbit erythrocytes. They were inhibited by various D-galactose derivatives and D-galactose-containing disaccharides and trisaccharides. N-Acetylgalactosamine was the best inhibitor, and the specificity appears to be directed to beta-D-galactosides. However, compared with winged bean basic lectins and soybean lectin, the winged bean acidic lectins show a low affinity for the inhibitory sugars.     

Effect of raw winged bean [Psophocarpus tetragonolobus (L.) DC] tuber lectin on gastrointestinal tract of growing rats

The fraction containing high hemagglutinating activity was prepared from raw winged bean tubers and orally administered to growing rats. The food intake and body weights of these rats decreased as the level of lectin increased and significant lectin activity was detected in the faeces extracted from these rats which is anti-genically similar to the native lectin preparation. Microscopic examination has revealed morphological changes in the intestinal epithelial cells. The binding action of the lectin to the mucosal epithelia of the gastrointestinal tract is indicative of the deleterious effects caused by the winged bean tuber lectin.     

Conformation and stability of the chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L.) DC)

Spectrophotometric measurement was found to be a sensitive method for evaluating the stability of the chymotrypsin inhibitor from the winged bean. The thermal stability of this protein in aqueous solution was much greater at pH 3 than at pH 8 or pH 11. Evidence from u.v. absorption and from circular dichroism indicated that irreversible conformation changes occurred at higher temperature (greater than 70 degrees). Circular dichroism and optical rotatory dispersion studies at pH 8 show that the inhibitor is rich in beta-structure and virtually devoid of alpha-helix in aqueous solution. We conclude from experiments with denaturing solvents that the inhibitor is very stable and that high concentrations of denaturant are required before unfolding occurs. Chemical modification experiments with tetranitromethane were consistent with a tight stable structure; even in 6M guanidine hydrochloride only three of the five tyrosine residues in the inhibitor molecule were nitrated. However, tyrosine does not seem to be implicated at the reactive site of the inhibitor. Interaction of the inhibitor with alpha-chymotrypsin and chymotrypsin B was also followed by difference spectroscopy in the ultraviolet region. Difference spectra were detected that were characteristic of changes in the environment of both tyrosine and tryptophan chromophores. Comparison of the spectral data obtained for the interaction of the inhibitor with bovine alpha-chymotrypsin and with chymotrypsin B indicated that a tryptophan residue may be involved at the reactive site of the inhibitor. Spectral changes were also detected for the interaction between the chymotrypsin inhibitor and trypsin, although it is well established that the specificity of this inhibitor is restricted to the chymotrypsins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of the trypsin inhibitors from winged bean seed (Psophocarpus tetragonolobus (L) Dc.).

The trypsin inhibitors from winged bean seed were isolated by affinity chromatography on trypsin-Sepharose 4B and the components fractionated by chromatography on SP-Sephadex C-25 and Sephadex G-100. The major components, inhibitors 2 and 3 were found to be homogeneous proteins with molecular weights of about 20,000. The inhibitors stoichiometrically inhibited bovine trypsin in the molar ratio of 1 : 1 whereas the inhibition of bovine alpha-chymotrypsin was weak and non-stoichiometric. Amino acid analysis indicated that both the inhibitors contain four cysteine residues and are rich in aspartic acid, glutamic acid, glycine, valine and leucine; however, inhibitor 3 lacks histidine and methionine while inhibitor 2 contains one histidine and three methionines. A minor trypsin inhibitor fraction was also isolated which contained at least three proteins with a molecular weight of about 10,000 and a high content of half-cystine.     

Specificity and stability of the chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L) Dc)

The specificity of the winged bean chymotrypsin inhibitor is restricted to the chymotrypsins (EC 3.4.21.1 and EC 3.4.21.2). Trypsins (EC 3.4.21.4), elastase (EC 3.4.21.11), subtilisins (EC 3.4.21.14), proteinase K (EC 3.4.21.14) and Pronase (EC 3.4.24.4) are not inhibited. The inhibitor reacts with two molecules of chymotrypsin to form a stable complex (Mr approx. 70 0000) which was isolated by gel filtration on Sephadex G-100. When mixed with substrate, the interaction of the inhibitor with alpha-chymotrypsin is characterized by substrate-induced dissociation of the complex. In contrast, the interaction with chymotrypsin B is quantitative with no substrate-induced dissociation. The inhibitor reacts with alpha-chymotrypsin to form a 1 : 2 molar complex at all ratios of [I]/[E]; however, the interaction with chymotrypsin B is characterized by the formation of initially of a 1 : 1 molar complex at [I] greater than [E] followed by the formation of the 1 : 2 molar complex at [I] less than 2[E]; an intermediate species of Mr approx. 48 000 was demonstrated by gel filtration on Sephadex G-100. The inhibitor is stable over the pH range 2.0-11.5 and to heating up to 70 degrees C at pH 4.1 and 8.0, and up to 90 degrees C at pH 3.0. The inhibitor resists denaturation in 8.0 M urea at pH 8.0 and 4.0, is stable in 0.12 M beta-mercaptoethanol at pH 8.0; however, reduction in 8.0 M urea results in a loss of inhibitory activity. The inhibitor resists digestion with pepsin at pH 2.0, being only slowly degraded over a period of 7 days with an equimolar amount of pepsin.     

Purification and characterization of proteinase inhibitors from winged bean (Psophocarpus tetragonolobus (L.) DC.) seeds

Seven proteinase inhibitors were isolated from winged bean seeds by ion-exchange chromatographies. These inhibitors had molecular weights of around 20,000, included four half-cystine residues, and were Kunitz-type inhibitors. Two (WTI-2 and 3) inhibited bovine trypsin strongly and four (WCI-1, 2, 3, and 4) inhibited bovine alpha-chymotrypsin, but in different ways. One mole of WCI-2 or -3 could inhibit 2 mol of alpha-chymotrypsin. The remaining inhibitor (WTCI-1) could bind both bovine trypsin and alpha-chymotrypsin at the molar ratio of 1:1, but not simultaneously. All four chymotrypsin inhibitors cross-reacted with rabbit anti-WCI-3 serum, while the other inhibitors did not.     

Thermodynamic analysis of ligand binding to winged bean (Psophocarpus tetragonolobus) acidic agglutinin reveals its specificity for terminally monofucosylated H-reactive sugars

The sugar-specific binding of N-dansylgalactosamine to WBA II (n = 2; Ka = 5.6 x 10(3) M-1; delta H = -21 kJ.mol-1; delta S = -21.3 J.mol-1.K-1) was utilized in substitution titrations for evaluating the association constants for the interaction of sugars with the lectin. An axial hydroxyl at C-4 and equatorial hydroxyls at C-3 and C-6 as in D-galacto configuration are crucial for binding. Both axial and equatorial hydroxyls are tolerated at C-2. Conformationally akin disaccharides such as lactose, N-acetyllactosamine, Gal beta 1-3GlcNAc, and Gal beta 1-3GalNAc show similar affinities. 2'-Fucosyllactose and H-disaccharide display 146 and 13 times stronger affinity over lactose and galactose, yet fucose by itself is devoid of activity. An interesting feature, noted for the first time, in protein-sugar interactions is the positive entropy change for the binding of 2'-fucosyllactose, suggesting that nonpolar interactions play an important role in stabilization of the lectin-sugar complex. 3-Fucosyllactose, lactodifucotetraose, lacto-N-fucopentaose II and III are inactive, whereas lacto-N-fucopentaose I has 14-fold lower affinity as compared with 2'-fucosyllactose. Conformational analysis indicates that the substitution at subterminal glucose or GlcNAc by L-fucose in either alpha 1-3 or alpha 1-4 linkage leads to its projection so as to sterically hinder the access of 3'-fucosyllactose, lactodifucotetraose, and lacto-N-fucopentaose II and III to the binding site of winged bean agglutinin II. Similarly the projection of alpha 1-3 linked Gal/GalNAc also leads to steric hindrance and hence prevents the binding of blood group A and B reactive sugars. Considering its unique specificity winged bean agglutinin II should be useful in the isolation and characterization of terminally monofucosylated H-reactive oligosaccharides from those that are difucosylated or internally fucosylated.     

Amino acid sequences of two trypsin inhibitors from winged bean seeds (Psophocarpus tetragonolobus (L)DC.)

The trypsin inhibitor (WTI-1) purified from winged bean seeds is a Kunitz type protease inhibitor having a molecular weight of 19,200. WTI-1 inhibits bovine trypsin stoichiometrically, but not bovine alpha-chymotrypsin. The approximate Ki value for the trypsin-inhibitor complex is 2.5 X 10(-9) M. The complete amino acid sequence of WTI-1 was determined by conventional methods. Comparison of the sequence with that of soybean trypsin inhibitor (STI) indicated that the sequence of WTI-1 had 50% homology with that of STI. WTI-1 was separated into 2 homologous inhibitors, WTI-1A and WTI-1B, by isoelectric focusing. The isoelectric points of WTI-1A and WTI-1B were 8.5 and 9.4, respectively, and their sequences were presumed from their amino acid compositions.     

Direct somatic embryogenesis and plantlet regeneration from seedling leaves of winged bean,Psophocarpus tetragonolobus (L.) DC

Excised seedling leaf segments of winged bean [Psophocarpus tetragonolobus (L.) DC.] underwent direct somatic embryogenesis under appropriate incubation conditions. Initiation and development of the somatic embryos occurred using a two-step culture method. The culture procedure involved incubation for 28 days on MS basal medium supplemented with 0.1–0.5 mg/l NAA and 1.0–2.0 mg/l BA (induction medium) before transfer to MS medium supplemented with 0.1 mg/l IAA and 2.0 mg/l BA (embryo development medium). The initial exposure to low levels of NAA coincident with high levels of BA in the induction medium was essential for embryogenic induction. Maximum embryogenesis (43.3%) was obtained with 0.2 mg/l NAA and 2.0 mg/l BA, and at least 14 days on induction medium were required prior to transfer to the embryo development medium. The conversion frequency of cotyledonary embryos was 53.3% upon culture on MS medium containing 0.1 mg/l ABA for 7 days followed by transfer to MS medium supplemented with 0.1 mg/l IBA and 0.2 mg/l BA. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid     

Size and compositional heterogeneity of seeds in the winged bean pod (Psophocarpus tetragonolobus (L.)DC)

Differently positioned seeds in the mature pod of winged bean (Psophocarpus tetragonolobus (L.) DC) differ in mass and content of total protein and phosphorus and starch.     

Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [Psophocarpus tetragonolobus (L.) DC.]

Somatic embryos were obtained from callus cultures derived from leaf explants of the winged bean, Psophocarpus tetragonolobus (L.) DC. Initiation and development of the somatic embryos occurred with a two-step culture method. Callus cultures initiated on MS medium with NAA and BAP, upon transfer to a new medium with IAA and BAP, produced somatic embryos. Maximum embryogenesis of 60% was obtained on induction medium with 0.5 mg/l NAA plus 1.0 mg/l BAP followed by transfer to a secondary medium with 0.1 mg/l IAA and 2.0 mg/l BAP. Optimal embryo germination and plantlet development was achieved on MS medium with 0.2 mg/l BAP plus 0.1 mg/l IBA. The regenerated plants were successfully transferred to glasshouse conditions.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - BAP 6-benzylaminopurine - KN Kinetin