Catfish (Clarias batrachus) serum lectin recognizes polyvalent Tn [alpha-D-GalpNAc1-Ser/Thr], Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc1-Ser/Thr], and II [beta-D-Galp(1-->4)-beta-D-GlcpNAc1-] mammalian glycotopes

A new calcium dependent GalNAc/Gal specific lectin was isolated from the serum of Indian catfish, Clarias batrachus and designated as C. batrachus lectin (CBL). It is a disulfide-linked homodecameric lectin of 74.65kDa subunits and the oligomeric form is essential for its activity. Binding specificity of CBL was investigated by enzyme-linked lectin-sorbent assay using a series of simple sugars, polysaccharides, and glycoproteins. GalNAc was more potent inhibitor than Gal; and alpha glycosides of both were more inhibitory than their beta counterparts. CBL showed maximum affinity for human tumor-associated Tn-antigens (GalNAcalpha1-Ser/Thr) at the molecular level and was 3.5 times higher than GalNAc. CBL interacted strongly with polyvalent Tn and Talpha (Galbeta1,3GalNAcalpha1-) as well as multivalent-II (Galbeta1,4GlcNAcbeta1-) antigens containing glycoproteins and intensity of inhibition was 10(3)-10(5) times more than monovalent ones. The overall specificity of CBL lies in the order of polyvalent Tn, Talpha and II>monovalent TnMe-alphaGalNAc>monovalent Talpha> Me-betaGalNAc>Me-alphaGal>monovalent T>GalNAc>monovalent F>monovalent II>Me-betaGal>Gal.     

Effect of a fungal lectin from Xerocomus chrysenteron (XCL) on the biological parameters of aphids

Aphids are important pests of crop plants in Europe. Increasing resistance of aphids to insecticides and their side effects on the environment and non target organism's including human's stimulated research on alterative methods of aphid control, including the use of entomotoxic proteins. Lectins are carbohydrate binding proteins that are widely distributed in nature; they have been isolated from microorganisms, fungi, plants and animals. Several of these proteins were tested for their potential biocide effect on plenty of pests. A fungal lectin, namely Xerocomus Chrysenteron lectin (XCL) was previously purified and was shown to be toxic for several pests including aphids. XCL was clearly the most toxic lectin against M. persicae. In this work, bioassays using artificial diets incorporating a broad range of XCL concentrations (from 10 microg x ml(-1) to 5000 microg x ml(-1)) were developed to assess the negative effects of XCL on the biological parameters (development duration, weight and fecundity) of M. persicae a polyphagous aphid found on more than 400 host plant species and transmitting more than 100 viral diseases. A significant mortality of aphids was observed, corresponding to the LC50 and LC90 of 0, 46 and 6, 02 mg/ml respectively after 24hrs. Significant differences of M. persicae weight, development duration and fecundity (P < 0.05) was observed between the tested XCL concentrations. Conavalia ensifomris lectin (ConA) was included as lectin reference on the bioassay experiments and was shown to be less toxic and induced lower negative changes in M. persicae biological parameters when compared with XCL.     

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.     

Interaction of rice (Oryza sativa) lectin with N-acetylglucosaminides. Fluorescence studies.

The interaction of lectin isolated from rice (Oryza sativa) embryos with N-acetylglucosaminides was studied by equilibrium dialysis and fluorescence. Equilibrium dialysis with 4-methylumbelliferyl-(GlcNac)2 showed that rice lectin (Mr 38000) contains four equivalent saccharide-binding sites. Addition of the N-acetylglucosaminides GlcNac, (GlcNac)2 and (GlcNac)3 enhanced the intrinsic fluorescence of rice lectin and this was accompanied by a 10nm blue-shift of its maximum fluorescence with (GlcNac)2 and (GlcNac)3. These changes in intensity allowed determination of the association constants, which increased with the number of saccharide units: at 20 degrees C, Ka = (1.3 +/- 0.1) X 10(3), (5.1 +/- 0.4) X 10(4) and (2.6 +/- 0.1) X 10(5) M-1 for GlcNac, (GlcNac)2 and (GlcNac)3 respectively. The binding enthalpy, delta H0, for the three glucosaminides were very low and ranged from -12.1 to -20.6 kJ X mol-1. The results are compared with those obtained with wheat-germ agglutinin, another GlcNac-specific gramineaous lectin.     

Resistance to green leafhopper (Nephotettix virescens) and brown planthopper (Nilaparvata lugens) in transgenic rice expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA)

Transgenic rice plants expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA) were screened for resistance to green leafhopper (Nephotettix virescens; GLH), a major homopteran pest of rice. Survival was reduced by 29% and 53% (P<0.05) respectively, on plants where GNA expression was tissue-specific (phloem and epidermal layer) or constitutive. Similar levels of resistance in GNA-expressing transgenic rice were previously reported for rice brown planthopper (Nilaparvata lugens; BPH). GNA binding to glycoproteins in gut tissues showed that BPH contained more "receptors" than GLH, and that the binding affinity was stronger, particularly in the midgut. Subsequent toxicity of GNA is thus unlikely to be directly related to the amount of lectin bound. GNA was not detected in the honeydew of either insect species when they were fed on GNA-expressing plants, in contrast to results from artificial diet studies. This result suggests that GNA is not being delivered to the insect efficiently. When offered a free choice vs control plants, BPH nymphs tended to avoid plants expressing GNA; avoidance was less pronounced and took longer to develop on plants where GNA expression was tissue-specific, In contrast to BPH, GLH nymphs were attracted to plants expressing GNA, whether constitutively or in a tissue-specific manner.     

Recognition profile of Bauhinia purpurea agglutinin (BPA)

Bauhinia purpurea agglutinin (BPA) is a Galbeta1-3GalNAc (T) specific leguminous lectin that has been widely used in multifarious cytochemical and immunological studies of cells and tissues under pathological or malignant conditions. Despite these diverse applications, knowledge of its carbohydrate specificity was mainly limited to molecular or submolecular T disaccharides. Thus, the requirement of high density polyvalent or multi-antennary carbohydrate structural units for BPA binding and an updated affinity profile were further evaluated by enzyme-linked lectinosorbent (ELLSA) and inhibition assays. Among the glycoproteins (gps) tested and expressed as 50% nanogram inhibition, the high density polyvalent GalNAcalpha1-Ser/Thr (Tn) and Galbeta1-3/4GlcNAc (I/II) glycotopes present on macromolecules generated a great enhancement of binding affinity for BPA as compared to their monomers. The most potent inhibitors were a Tn-containing gp (asialo OSM) and a I/II containing gp (human blood group precursor gp), which were up to 1.7 x 10(4) and 2.3 x 10(3) times more potent than monovalent Gal and GalNAc, respectively. However, multi-antennary glycopeptides, such as tri-antennary Galbeta1-4GlcNAc, which was slightly more active than II or Gal, gave only a minor contribution. Regarding the carbohydrate structural units studied by the inhibition assay, blood group GalNAcbeta1-3/4Gal (P/S) active glycotopes were active ligands. The overall binding profile of BPA was: high density polyvalent T/Tn and II clusters > Tn-glycopeptides (M.W. <3.0 x 10(3))/Talpha monomer > monovalent P/S > Tn monomer and GalNAc > tri-antennary II > Gal > Man and Glc (inactive). These findings give evidence for the binding of this lectin to dense cell surface T, Tn and I/II glycoconjugates and should facilitate future usage of this lectin in biotechnological and medical applications.     

Properties of the lectin from the hog peanut (Amphicarpaea bracteata)

An N-acetyl-D-galactosamine-specific lectin has been isolated from the two seed forms of the hog peanut (Amphicarpaea bracteata) using an affinity support containing the synthetic type A blood group trisaccharide alpha-D-GalNAc-(1,3)-[alpha-L-Fuc-(1,2)]-beta-D-Gal (Synsorb A). The affinity-purified lectin appears to be identical in both seed types. Gel filtration on Sephadex G-200 gives a single symmetrical peak corresponding to Mr 135,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows four subunit forms, each of which contains carbohydrate. Limited amino terminal sequencing indicates heterogeneity in two of the first 10 residues. The lectin contains no cysteine. There are four equivalent, noninteracting GalNAc binding sites per 135,000-Da molecule, having an association constant for methyl N-acetyl-alpha-D-galactosaminide of 4.0 X 10(4) M-1. Precipitin and hapten inhibition studies show the lectin to be specific for terminal, nonreducing D-GalNAc units, with a preference for the alpha-anomer and enhanced specificity for the disaccharide, GalNAc alpha 1,3GalNAc. There is also a single adenine binding site per Mr 135,000 lectin molecule with an association constant of 1.3 X 10(6) M-1.     

Phytohemagglutinin (PHA)-resistant Chinese hamster ovary cell mutants acquire sensitivity to the lectin after fusion with liposomes containing PHA receptor glycoproteins

Phytohemagglutinin receptor glycoproteins have been inserted into phospholipid vesicles and these have been fused with phytohemagglutinin-resistant chinese hamster ovary cells. Our results show that the fused cells acquire "neoreceptors" for the lectin phytohemagglutinin. Fluorescence activated cell sorting analyses show that approximately 40% of the cells fused with the receptor-containing vesicles. Studies with 125I-labelled lectin showed that fused cells bound three times more ligand than untreated mutant cells. Furthermore, lectin receptors were functionally inserted in the mutant cell plasma membrane. Fused cells cultured in the presence of lectin (200 micrograms ml-1) lost rapidly (8 hours or less) their ability to incorporate [3H] thymidine. Whereas mutant cells cultured for 16 hours in the presence of 50-400 micrograms ml-1 of lectin remained viable, fused cells showed a 45% decrease in 3H-labelled nucleotide incorporation. The method described here should be of general applicability for the study of lectin-dependent cytotoxicity in chinese hamster ovary cell lines.     

Renal alterations promoted by the lectins from Canavalia ensiformis (ConA) and Dioclea guianensis (DguiL) seeds

The lectin from the seeds of Canavalia ensiformis (ConA) and Dioclea guianensis (DguiL) was tested upon its renal effects using the isolated perfusion rat kidney method. Both lectins (10 microg/ml) affected perfusion pressure and renal vascular resistance, but DguiL showed a much greater action than ConA. However, ConA, but not DguiL, affected potassium tubular transport.     

Binding of Porphyrins by the Tumor-Specific Lectin, Jacalin [Jack Fruit (Artocarpus integrifolia) Agglutinin]

Jacalin (Artocarpus integrifolia agglutinin) specifically recognizes thetumor-associated T-antigenic disaccharide structure,Gal13GalNAc. Porphyrins and their derivatives are currently used asphotosensitizers in photodynamic therapy to treat malignant tumors. In thisstudy, the interaction of several free base porphyrins and their metalderivatives with jacalin is investigated by absorption and fluorescencespectroscopy. Each lectin subunit was found to bind one porphyrin moleculeand the association constants were estimated to be in the range of2.4×10³M^–1 to 1.3×10⁵M^–1 at room temperaturefor the interaction of different porphyrins with jacalin. These values arein the same range as those obtained for the interaction of monosaccharidesto jacalin. Both free lectin and lectin saturated with the specificsaccharide were found to bind different porphyrins with comparable bindingstrength indicating that porphyrin binding takes place at a site differentfrom the sugar binding site. Further, both anionic and cationic porphyrinswere found to interact with the lectin with comparable affinity, clearlyindicating that the charge on the porphyrin does not play any role in thebinding process and that most likely the interaction is mediated byhydrophobic forces. These results suggest that jacalin and other lectins maypotentially be useful for targeted delivery of porphyrins to tumor tissuesin photodynamic therapy.     

Isolation and characterization of a second lectin (SNA-II) present in elderberry (Sambucus nigra L.) bark

A second lectin (SNA-II) has been isolated from elderberry (Sambucus nigra L.) bark by affinity chromatography on immobilized asialo-glycophorin. This lectin is a blood group nonspecific glycoprotein containing 7.8% carbohydrate and which is rich in asparagine/aspartic acid, glutamine/glutamic acid, glycine, valine, and leucine. Gel filtration on Superose 12 gave a single symmetrical peak corresponding to Mr, 51,000; SDS-acrylamide electrophoresis gave a single polypeptide, Mr, 30,000. Hence SNA-II appears to be a homodimer. The lectin is a Gal/GalNAc-specific lectin which is precipitated by glycoproteins containing GalNAc-terminated oligosaccharide chains (e.g., asialo-ovine submaxillary and hog gastric mucins), and by glycoproteins and polysaccharides having multiple terminal nonreducing D-galactosyl groups as occur in asialoglycophorin, asialo-laminin and Type 14 pneumococcal polysaccharide. The carbohydrate binding specificity of SNA-II was studied by sugar hapten inhibition of the asialo-glycophorin precipitation reaction. The lectin's binding site appears to be most complementary to Gal-NAc linked alpha to the C-2, C-3, or C-6 hydroxyl group of galactose. These disaccharide units are approximately 100 times more potent than melibiose, 60 times more potent than N-acetyllactosamine, and 30 times more potent than lactose. Interestingly, the blood group A-active trisaccharide containing an L-fucosyl group linked alpha 1-2 to galactose was 10-fold poorer as an inhibitor than the parent oligosaccharide (GalNAc alpha 1-3Gal), suggesting steric hindrance to binding by the alpha-L-fucosyl group; this explains the failure of the lectin to exhibit blood group A specificity.     

A single step purification of a sialic acid binding lectin (AchatininH) from Achatina fulica snail

A sialic acid binding lectin, Achatinin_H, was purified in single step from the hemolymph of the land snail, Achatina fulica, by the affinity chromatography on sheep submaxillary mucin coupled to Sepharose 4B. The yield of the lectin was found to be 3 mg from 100 ml of hemolymph. The homogeneity of the lectin was established by alkaline gel electrophoresis, immunodiffusion, immunoelectrophoresis and analytical isoelectrophoresis. The molecular weight of the native protein was 242000, having identical subunits of Mr 15000. The lectin agglutinated rabbit erythrocytes in the presence of Ca^2–. The inhibition study clearly suggests that the binding site of the lectin recognizes sialic acid as the immunodominant sugar. This was further confirmed by the observation that there was a marked decrease of agglutinating activity of the lectin with neuraminidase treated rabbit erythrocytes and asialofetuin was unable to inhibit the activity of Achatinin_H. Among the inhibitors used the glycoconjugate containing 2-6 linkages of N-acetylneuraminic acid with subterminal galactopyranose or 2-acetamido-2-deoxy-galactopyranose residue was found to be better inhibitor than that containing 23 linkages of N-acetyl neuraminic acid. Besides that sialoglycoprotein containing both N and O type of glycosidic linkages plays an important role in binding with the lectin. Fetuin was found to be the best inhibitor.     

Concanavalin A inhibits development of tomato moth (Lacanobia oleracea) and peach-potato aphid (Myzus persicae) when expressed in transgenic potato plants

The effects of concanavalin A (ConA), a glucose/mannose-specific lectin from jackbean (Canavalia ensiformis), on insect crop pests from two different orders, Lepidoptera and Homoptera, were investigated. When fed to larvae of tomato moth (Lacanobia oleracea) at a range of concentrations (0.02–2.0% of total protein) in artificial diet, ConA decreased survival, with up to 90% mortality observed at the highest dose level, and retarded development, but had only a small effect on larval weight. When fed to peach-potato aphids (Myzus persicae) at a range of concentrations (1–9μM) in liquid artificial diet, ConA reduced aphid size by up to 30%, retarded development to maturity, and reduced fecundity (production of offspring) by >35%, but had little effect on survival. With both insects, there was a poor correlation between lectin dose and the quantitative effect. Constitutive expression of ConA in transgenic potatoes driven by the CaMV 35S promoter resulted in the protein accumulating to levels lower than predicted, possibly due to potato not being able to adequately reproduce the post-translational processing of this lectin which occurs in jackbean. However, the expressed lectin was functionally active as a haemagglutinin. Bioassay of L. oleracea larvae on ConA-expressing potato plants showed that the lectin retarded larval development, and decreased larval weights by >45%, but had no significant effect on survival. It also decreased consumption of plant tissue by the larvae. In agreement with the diet bioassay results, ConA-expressing potatoes decreased the fecundity of M. persicae by up to 45%. ConA thus has potential as a protective agent against insect pests in transgenic crops. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fine sugar specificity of the mistletoe (Viscum album) lectin I

The behaviour ofN-acetyllactosamine-type oligosaccharides and glycopeptides on a column of mistletoe lectin I (MLI) immobilized on Sepharose 4B was examined. The immobilized lectin does not show any affinity for asialo-N-glycosylpeptides and related oligosaccharides, which possess one to four unmaskedN-acetyllactosamine sequences. However, substitution of at least one of theN-acetyllactosamine sequences by sialic acid residues, either at O-3 or O-6 of galactose, slightly enhances the affinity of the lectin. Such sialylatedN-glycosylpeptides or oligosaccharides are eluted from the lectin column by the starting buffer as retarded fractions. Surprisingly, the affinity of the immobilized MLI is higher for P1 antigen-containing glycopeptide isolated from turtle-dove ovomucoid and for glycopeptides from bovine thyroglobulin containing terminal non-reducing Gal1–3Gal sequences. These structures are strongly bound on the lectin column and their elution is obtained with 0.15M galactose in the starting buffer.In memory of Hartmut Franz.     

The inactivation by concanavalin A of the ecto-ATPase and ectoacetylcholinesterase of intact skeletal muscles

The (Ca2+ or Mg2+)-activated ectophosphohydrolase of intact frog muscle liberates, in situ, about 37 mumol inorganic phosphate/g muscle in 20 min at 20 degrees C with 10 mM ATP. Pretreatment with concanavalin A (ConA) at 4 degrees C for 18 h caused ectoenzyme inactivation which plateaued at 35-40% of the control rate. The inhibition was concentration dependent, being maximal at about 500 micrograms ConA/mL Ringer's solution. The lectin mediated its effect via the membrane glycoproteins since the inhibition was specifically prevented by alpha-methyl D-mannopyranoside. As the temperature increased from 10 to 40 degrees C, the ectoenzyme activity of untreated muscles increased linearly between 10 and 35 degrees C, with a "break point" and a clear change in slope at 35 degrees C. When treated with ConA the activity increased linearly from 10 to 40 degrees C, eliminating the transition temperature. The findings suggested that a phase transition toward fluidity in the lipid bilayer may have occurred at 35 degrees C and that this was abolished by the lectin binding. Hence we perturbed the surface membrane phospholipids of muscle pretreated with the lectin. Phospholipase C increased the activation by the lectin; phospholipase D had no effect, but phospholipase A2 completely prevented it. The lectin may require the more fluid fatty acyl chains of membrane lipids to achieve inhibition of this ecto-ATPase. Ectoacetylcholinesterase, in situ, and its inactivation by ConA were measured directly on whole, intact skeletal muscles.     

The isolation and some properties of a lectin (Haemagglutinin) from Cucurbita phloem exudate

The occurrence of high haemagglutinating (lectin) activity in phloem exudate from three cucurbit species is reported. The protein responsible for this lectin activity in Cucurbita maxima Duch. has been isolated by cation exchange chromatography on Sepharose and identified by gel electrophoresis. The lectin showed agglutinating activity at concentrations as low as 0.1 g/ml. No sugar, including those transported in the phloem of these species, interacted with agglutination. The lectin could not be extracted from cucurbit seed, but appeared in 5-day old seedlings. The possible role of a lectin in the sieve element is discussed.     

Physicochemical Properties and Oxidative Inactivation of Soluble Lectin from Water Buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) Brain

Lectins are carbohydrate-binding proteins present in a wide variety of plants and animals, which serve various important physiological functions. A soluble β-galactoside binding lectin has been isolated and purified to homogeneity from buffalo brain using ammonium sulphate precipitation (40–70%) and gel permeation chromatography on Sephadex G_50–80 column. The molecular weight of buffalo brain lectin (BBL) as determined by SDS-PAGE under reducing and non-reducing conditions was 14.2 kDa, however, with gel filtration it was 28.5 kDa, revealing the dimeric form of protein. The neutral sugar content of the soluble lectin was estimated to be 3.3%. The BBL showed highest affinity for lactose and other sugar moieties in glycosidic form, suggesting it to be a β-galactoside binding lectin. The association constant for lactose binding as evidenced by Scatchard analysis was 6.6 × 10³ M⁻¹ showing two carbohydrate binding sites per lectin molecule. A total inhibition of lectin activity was observed by denaturants like guanidine HCl, thiourea and urea at 6 M concentration. The treatment of BBL with oxidizing agent destroyed its agglutination activity, abolished its fluorescence, and shifted its UV absorption maxima from 282 to 250 nm. The effect of H₂O₂ was greatly prevented by lactose indicating that BBL is more stable in the presence of its specific ligand. The purified lectin was investigated for its brain cell aggregation properties by testing its ability to agglutinate cells isolated from buffalo and goat brains. Rate of aggregation of buffalo brain cells by purified protein was more than the goat brain cells. The data from above study suggests that the isolated lectin may belong to the galectin-1 family but is glycosylated unlike those purified till date.     

Analysis of saccharide binding to Artocarpus integrifolia lectin reveals specific recognition of T-antigen (beta-D-Gal(1----3)D-GalNAc)

The binding of Artocarpus integrifolia lectin to N-dansylgalactosamine (where dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) leads to a 100% increase in dansyl fluorescence with a concomitant blue shift in the emission maximum by 10 nm. This binding is carbohydrate-specific and has an association constant of 1.74 X 10(4) M-1 at 20 degrees C. The lectin has two binding sites for N-dansylgalactosamine. The values of -delta H and -delta S for the binding of N-dansylgalactosamine are in the range of values reported for several lectin-monosaccharide interactions, indicating an absence of nonpolar interaction of the dansyl moiety of the sugar with the combining region of the protein. Dissociation of the bound N-dansylgalactosamine from its complex with the lectin and the consequent change in its fluorescence on addition of nonfluorescent sugars allowed evaluation of the association constant for competing ligands. The thermodynamic parameters for the binding of monosaccharides suggest that the OH groups at C-2, C-3, C-4, and C-6 in the D-galactose configuration are important loci for interaction with the lectin. The acetamido group at C-2 of 2-acetamido-2-deoxygalactopyranose and a methoxyl group at C-1 of methyl-alpha-D-galactopyranoside are presumably also involved in binding through nonpolar and van der Waals' interactions. The T-antigenic disaccharide Gal beta 1----3GalNAc binds very strongly to the lectin when compared with methyl-beta-D-galactopyranoside, the beta(1----3)-linked disaccharides such as Gal beta 1----3GlcNAc, and the beta(1----4)-linked disaccharides, N-acetyllactosamine and lactose. The major stabilizing force for the avid binding of T-antigenic disaccharide appears to be a favorable enthalpic contribution. The combining site of the lectin is, therefore, extended. These data taken together suggest that the Artocarpus lectin is specific toward the Thomsen-Friedenreich (T) antigen. There are subtle differences in the overall topography of its combining site when compared with that of peanut (Arachis hypogaea) agglutinin. The results of stopped flow spectrometry for the binding of N-dansylgalactosamine tot he Artocarpus lectin are consistent with a simple single-step bimolecular association and unimolecular dissociation rate processes. The value of K+1 and K-1 at 21 degrees C are 8.1 X 10(5) M-1 s-1 and 50 s-1, respectively. The activation parameters indicate an enthalpy-controlled association process.     

Molecular cloning,expression, and cytokinin (6-benzylaminopurine) antagonist activity of peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis>) lectin SL-I

Isolation and purification of a α-methyl-mannoside specific lectin (SL-I) of peanut was reported earlier [Singh and Das (1994) Glycoconj J 11:282–285]. Native SL-I is a glycoprotein having ∼31 kDa subunit molecular mass and forms dimer. The gene encoding this lectin is identified from a 6-day old peanut root cDNA library by anti-SL-I antibody and N-terminal amino acid sequence homology to the native lectin. Nucleotide sequence derived amino acid sequence of the re-SL-I shows amino acid sequence homology with the N-terminal and tryptic digests’ amino acid sequence of the native SL-I (nSL-I). Presence of a putative glycosylation (QNPS) site and a hydrophobic adenine-binding (VLVSYDANS) site is also identified in SL-I. Homology modeling of the lectin suggests it to be an archetype of legume lectins. It is expressed as a ~30 kDa apoprotein in E. coli and has the carbohydrate specificity and secondary structure identical to its natural counterpart. The lectin SL-I inhibits cytokinin 6-benzylaminopurine (BA)-induced “delayed leaf senescence” and “cotyledon expansion”. Equilibrium dialysis revealed a single high-affinity binding site for adenine (7.6 × 10⁻⁶ M) and BA (1.09 × 10⁻⁵ M) in the SL-I dimer and thus suggesting that the cytokinin antagonist effect of SL-I is mediated by the direct interaction of SL-I with BA.The nucleotide sequence data reported here are available in the DDBJ/EMBL/GenBank databases under the Accession No. AJ585523     

On the specificity of the D-galactose-binding lectin (PA-I) of Pseudomonas aeruginosa and its strong binding to hydrophobic derivatives of D-galactose and thiogalactose.

The D-galactose-binding lectin (PA-I) from the bacterium Pseudomonas aeruginosa, isolated by affinity chromatography on Sepharose, was examined for its relative affinities for simple sugars and their derivatives using equilibrium dialysis and hemagglutination inhibition tests. The lectin, which was found to bind 0.68 mol of D-galactose per subunit of 12.8 kDa, exhibited an association constant (Ka) of 3.4 x 10(4) M-1 for D-galactose and higher affinities for hydrophobic and thio derivatives of D-galactose (with highest affinity for the hydrophobic thio derivatives). alpha-Methyl-galactoside was a stronger inhibitor than the beta-methyl derivative and alpha-lactose was a weak inhibitor but the hydrophobic phenylated derivatives of the beta-configuration of D-galactose were more potent inhibitors than the respective alpha-galactosides.