Purification and molecular cloning of a new galactose-specific lectin from <Emphasis Type="Italic">Bauhinia variegata</Emphasis> seeds

A new galactose-specific lectin was purified from seeds of a Caesalpinoideae plant, Bauhinia variegata, by affinity chromatography on lactose-agarose. Protein extracts haemagglutinated rabbit and human erythrocytes (native and treated with proteolytic enzymes), showing preference for rabbit blood treated with papain and trypsin. Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives, especially lactose. SDS-PAGE showed that the lectin, named BVL, has a pattern similar to other lectins isolated from the same genus, Bauhinia purpurea agglutinin (BPA). The molecular mass of BVL subunit is 32 871 Da, determined by MALDI-TOF spectrometry. DNA extracted from B. variegata young leaves and primers designed according to the B. purpurea lectin were used to generate specific fragments which were cloned and sequenced, revealing two distinct isoforms. The bvl gene sequence comprised an open reading frame of 876 base pairs which encodes a protein of 291 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to have 263 amino acid residues and 28 963 Da in size.     

A lectin from Bryonia dioica root stocks

An N-acetylgalactosamine-specific lectin has been isolated from root stocks of Bryonia dioica by affinity chromatography on fetuin-agarose. It is a dimeric protein composed of two different subunits of relative molecular masses 32,000 and 30,000, held together by intermolecular disulphide bonds. Although most abundant in root stocks, the lectin occurs in all vegetative parts of the plant but not in seeds. Bryony lectin differs from other Cucurbitaceae lectins and from all known N-acetylgalactosamine-specific lectins.Abbreviations BDA Bryonia dioica agglutinin - M_r relative molecular mass - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Sensitive and specific detection of Xanthomonas axonopodis pv. citri by PCR using pathovar specific primers based on hrpW gene sequences

A sensitive and specific assay was developed to detect citrus bacterial canker caused by Xanthomonas axonopodis pv. citri, in leaves and fruits of citrus. Primers XACF and XACR from hrpW homologous to pectate lyase, modifying the structure of pectin in plants, were used to amplify a 561 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally or artificially infected leaves of citrus. The PCR product was only produced from X. axonopodis pv. citri among 26 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference microbes.     

Cloning and sequence analysis of theMaackia amurensis haemagglutinin cDNA

Maackia amurensis haemagglutinin (MAH) is a leguminous lectin which preferentially binds to a cluster of sialylatedO-linked carbohydrate chains (Konami Y, Yamamoto K, Osawa T, Irimura T (1994)FEBS Lett 342:334–38). In the present study a 950 bp cDNA clone encoding MAH was isolated from a cDNA library constructed from germinatedMaackia amurensis seeds. From the nucleotide sequence, MAH was predicted to consist of 285 amino acid residues containing a signal peptide of 29 amino acids. The results also confirmed our previous findings from the amino acid sequence analysis, which indicated that two highly conserved amino acid residues in all other well-known leguminous lectins were replaced in MAH. These residues were lysine-105 and aspartic acid-135. The corresponding amino acid residues in other leguminous lectins were glycine and asparagine, respectively. These differences were due to the presence of nucleotides AAA and GAT in place of AAT/C and GGA/T.Abbreviations MAH Maackia amurensis haemagglutinin.     

Primary structure characterization of Bothrops jararacussu snake venom lectin

The complete amino acid sequence of the lectin from Bothrops jararacussu snake venom (BJcuL) is reported. The sequence was determined by Edman degradation and amino acid analysis of the S-carboxymethylated BJcuL derivative (RC-BJcuL) and from its peptides originated from enzymatic digestion. The sequence of amino acid residues showed that this lectin displays the invariant amino acid residues characterized in C-type lectins. Amino acids analysis revealed a high content of acidic amino acids and leucine. These findings suggest that BJcuL, like other snake venom lectins, possesses structural similarities to the carbohydrate recognition domain (CRD) of calcium-dependent animal lectins belonging to the C-type -galactoside binding lectin family.     

Biochemical characterization of a lectin from Delonix regia seeds

A lectin from Delonix regia (DRL) seeds was purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on diethylaminoethyl-Sepharose and reverse-phase high-performance liquid chromatography on a C18 column. Hemagglutinating activity was monitored using rat erythrocytes. DRL showed no specificity for human erythrocytes of ABO blood groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single protein in the presence of 0.1 M of dithiothreitol (DTT) and in nonreducing conditions. Native-PAGE showed that DRL is a monomer with a molecular mass of about 12 kDa, as determined by denaturing gel electrophoresis and gel filtration chromatography. An amino acid composition revealed the absence of cysteine residues, the presence of 1 mol methionine/mol protein and a high proportion of acidic amino acids and glycine. The N-terminal sequence of DRL was determined by Edman degradation, and up to 16 amino acid residues showed more than 90% homology with other lectins from the Leguminosae family. The optimal pH range for lectin activity was between pH 8.0 and 9.0, and the lectin was active up to 60°C. The lectin required Mn²⁺ for hemagglutinating activity and remained active after reduction with 0.1 M of DTT, but lost activity in the presence of 8 M of urea. Sodium metaperiodate had no effect on the activity of DRL.     

Molecular cloning and characterization of a mannose-binding lectin gene from <Emphasis Type="Italic">Pinellia cordata</Emphasis>

Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein was successfully expressed in Escherichia coli with the molecular weight expected.     

Isolation and Biochemical Characterization of a Galactoside Binding Lectin from <Emphasis Type="Italic">Bauhinia variegata</Emphasis> Candida (BvcL) Seeds

A new lectin (BvcL) from seeds of a primitive Brazilian Caesalpinoideae, the Bauhinia variegata candida was purified and biochemical characterized. BvcL was isolated by gel filtration chromatography on Sephadex G75 and affinity chromatography on immobilized d-lactose column. SDS-PAGE showed that BvcL under non-reducing condition presents two bands of 68 and 32 kDa and a single band of 32 kDa in reducing condition. However, only one band was seen in native PAGE. The hemagglutination activity of BvcL was not specific for any human blood group trypsin-treated erythrocytes. Carbohydrate inhibition analysis indicated that BvcL is inhibited by lactose, galactose, galactosamine and other galactoside derivates. Amino acid analysis revealed a large content of Ser, Gly, Thr, Asp and Glu and low concentrations of Met, Cys and His. Intrinsic fluorescence of BvcL was not significantly affected by sugar binding galactose; and aromatic-region CD is unusually high for plant lectins. The N-terminal amino acid sequence of 17 residues showed 90% sequential homology to galactose-specific legume lectins of the subfamily Caesalpinoideae.     

Differences in properties of peanut seed lectin and purified galactose- and mannose-binding lectins from nodules of peanut

Two lectins were purified by affinity chromatography from mature peanut (Arachis hypogaea L.) nodules, and compared with the previously characterised seed lectin of this plant. One of the nodule lectins was similar to the seed lectin in its molecular weight and amino-acid composition and ability to bind derivatives of galactose. However, unlike the seed lectin, this nodule lectin appeared to be a glycoprotein and the two lectins were only partially identical in their reaction with antibodies prepared against the seed lectin. The other nodule lectin also appeared to be a glycoprotein but bound mannose/glucose-like sugar derivatives, and differed from the seed lectin in molecular weight, antigenic properties and amino-acid composition.Abbreviations Gal galactose - Gle glucose - GNL galactose-binding nodule lectin - Fru fructose - MNL mannosebinding nodule lectin - M _r rerative molecular mass - PBS phosphate-buffered saline - PSL peanut seed lectin - SDS sodium dodecyl sulphate - Sorb sorbitol     

Genomic Cloning and Characterization of a Novel Lectin Gene from <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis>

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.     

Isolation and characterization of a new mannose-binding lectin gene from<Emphasis Type="Italic">Taxus media</Emphasis>

In this paper, we report the cloning and characterization of the first mannose-binding lectin gene from a gymnosperm plant species,Taxus media. The full-length cDNA ofT. media agglutinin (TMA) consisted of 676 bp and contained a 432 bp open reading frame (ORF) encoding a 144 amino acid protein. Comparative analysis showed that TMA had high homology with many previously reported plant mannose-binding lectins and thattma encoded a precursor lectin with a 26-aa signal peptide. Molecular modelling revealed that TMA was a new mannosebinding lectin with three typical mannose-binding boxes like lectins from species of angiosperms. Tissue expression pattern analyses revealed thattma is expressed in a tissue-specific manner in leaves and stems, but not in fruits and roots. Phylogenetic tree analyses showed that TMA belonged to the structurally and evolutionarily closely related monocot mannose-binding lectin superfamily. This study provides useful information to understand the molecular evolution of plant lectins.     

New affinity procedure for the isolation and further characterization of the blood group B specific lectin from the red marine alga Ptilota plumosa

The red marine alga Ptilota plumosa has been shownto contain an anti-human blood group B lectin. We report here a new isolationprocedure by affinity chromatography on Sephadex G-200 and characterisation ofthe isolated lectin. The M _r , determined by gelfiltration, was 52,500. SDS-PAGE revealed a single protein band withM _r 17,440, indicating the native lectin was atrimer of subunits with the same M_r, as reported for the lectinsfromtwo other Ptilota species, P.filicinaand P. serrata. Analysis of amino acid composition showedslightly more basic than acidic amino acids. This was in contrast to theP. filicina and P. serrata lectinspreviously found to contain a higher proportion of acidic than basic aminoacids. Haemagglutination inhibition tests showed the P.plumosa lectin was inhibited by galactose, glucose and theirderivatives with p-nitrophenyl--D-galactoside moststrongly inhibitory. All glycoproteins tested failed to inhibit the lectin. Theamino acid composition, human blood group-B specificity and lack of inhibitionby glycoproteins indicate the lectin from P. plumosapossesses unique characteristics among marine algal lectins.     

Purification and characterization of two major lectins fromVigna mungo (blackgram)

Blackgram (Vigna mungo L. Hepper)seeds contain two galactose-specific lectins, BGL-I and BGL-II. BGL-I was partially purified into two monomeric lectins which were designated as BGL-I-1 (94 kDa) and BGL-I-2 (89 kDa). BGL-II is a monomeric lectin of 83 kDA. The purified lectins were associated with galactosidase activities. BGL-I-1 and BGL-II were copurified with α-galactosidase activity while BGL-I-2 was largely associated with β-galactosidase activity. These lectins agglutinate trypsin treated rabbit erythrocytes, but not the human erythrocytes of A, B or O groups. They were stable between pH 3·5 and 7·5 for their agglutination. The lectins did not show any metalion requirement. They were inactivated at 50°C. The lectin activity was inhibited by D-galactose (0·1 mM). The Scatchard plots of galactose binding to these lectins are nonlinear and biphasic curves indicative of multiple binding sites. The data show that the monomeric lectins have both lectin and galactosidase activities suggestive of a bifunctional protein.     

The seed lectins of black locust (robinia pseudoacacia) are encoded by two genes which differ from the bark lectin genes

Two lectins were isolated from Robinia pseudoacacia (black locust) seeds using affinity chromatography on fetuin-agarose, and ion exchange chromatography on a Neobar CS column. The first lectin, R. pseudoacacia seed agglutinin I, referred to as RPsAI, is a homotetramer of four 34 kDa subunits whereas the second lectin, referred to as RPsAII, is composed of four 29 kDa polypeptides. cDNA clones encoding the polypeptides of RPsAI and RPsAII were isolated and their sequences were determined. Both polypeptides are translated from mRNAs of ca. 1.2 kb encoding a precursor carrying a signal peptide. Alignment of the deduced amino acid sequences of the different clones indicates that the 34 and 29 kDa seed lectin polypeptides show 95% sequence identity. In spite of this striking homology, the 29 kDa polypeptide has only one putative glycosylation site whereas the 34 kDa subunit has four of these sites. Carbohydrate analysis revealed that the 34 kDa possesses three carbohydrate chains whereas the 29 kDa polypeptide is only partially glycosylated at one site. A comparison of the deduced amino acid sequences of the two seed and three bark lectin polypeptides demonstrated unambiguously that they are encoded by different genes. This implies that five different genes are involved in the control of the expression of the lectins in black locust.Abbreviations LECRPAs cDNA clone encoding Robinia pseudoacacia seed lectin - LoLI Lathyrus ochrus isolectin I - PsA Pisum sativum agglutinin - RPbAI Robinia pseudoacacia bark agglutinin I - RPbAII Robinia pseudoacacia bark agglutinin II - RPsAI Robinia pseudoacacia seed agglutinin I - RPsAII Robinia pseudoacacia seed agglutinin II     

Structural and functional characterization of basic PLA2 isolated from Crotalus durissus terrificus venom

The venom of Crotalus durissus terrificus was fractionated by reverse-phase HPLC to obtain crotapotins (F5 and F7) and PLA₂ (F15, F16, and F17) of high purity. The phospholipases A₂ (PLA₂s) and crotapotins showed antimicrobial activity against Xanthomonas axonopodis pv. passiflorae, although the unseparated crotoxin did not. The F17 of the PLA₂ also revealed significant anticoagulant activity, althrough for this to occur the presence of Glu 53 and Trp 61 is important. The F17 of the PLA₂ showed allosteric behavior in the presence of a synthetic substrate. The amino acid sequence of this PLA₂ isoform, determined by automatic sequencing, was HLLQFNKMLKFETRKNAVPFYAFGCYCGWGGQRRPKDATDRCCFVHDCCYEKVTKCNTKWDFYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPDSRCREPSETC. Analysis showed that the sequence of this PLA₂ isoform differed slightly from the amino acid sequence of the basic crotoxin subunit reported in the literature. The homology with other crotalid PLA₂ cited in the literature varied from 60% to 90%. The pL was estimated to be 8.15, and the calculated molecular weight was 14664.14 as determined by Tricine SDS-PAGE, two-dimensional electrophoresis, and MALDI-TOFF. These results also suggested that the enzymatic activity plays an important role in the bactericidal effect of the F17 PLA₂ as well as that of anticoagulation, although other regions of the molecule may also be involved in this biological activity.     

Structural analysis of ConBr reveals molecular correlation between the carbohydrate recognition domain and endothelial NO synthase activation

Diocleinae lectins are highly homologous in their primary structure which features metal binding sites and a carbohydrate recognition domain (CRD). Differences in the biological activity of legume lectins have been widely investigated using hemagglutination inhibition assays, isothermal titration microcalorimetry and co-crystallization with mono- and oligosaccharides. Here we report a new lectin crystal structure (ConBr) extracted from seeds of Canavalia brasiliensis, predict dimannoside binding by docking, identify the α-aminobutyric acid (Abu) binding pocket and compare the CRD of ConBr to that of homologous lectins. Based on the hypothesis that the carbohydrate affinity of lectins depends on CRD configuration, the relationship between tridimensional structure and endothelial NO synthase activation was used to clarify differences in biological activity. Our study established a correlation between the position of CRD amino acid side chains and the stimulation of NO release from endothelium.     

Lectins from seeds of Crotalaria pallida (smooth rattlebox)

A lectin from the seeds of Crotalaria pallida (CPL), with an apparent molecular mass of 30 kDa, determined by SDS-polyacrylamide gel electrophoresis, showed human type A and B erythrocytes agglutination activity, which is inhibited by raffinose and galactose. The lectin requirement for divalent cation was demonstrated with EDTA/EGTA blocking hemagglutination activity. Although the N-terminal amino acid sequence of CPL is identical to another lectin from Crotalaria striata, which is taxonomically synonymous to Crotalaria pallida, these lectins differ in amino acid composition and hemagglutination properties.     

Isolation of a chitin-binding lectin, with insecticidal activity in chemically-defined synthetic diets, from two wild brassica species with resistance to cabbage aphidBrevicoryne brassicae

A lectin, with a chitin-binding domain and chitinase activity, is present in significant quantities in the wild brassica speciesB. fruticulosa andB. spinescens but at low levels in cultivated cabbage cv. Offenham Compacta. The lectin, purified >1000 fold after binding to chitin, migrated on SDS-PAGE gels as a single band with a M _r of 14.500. The amino acid composition of the lectin fromB. spinescens indicated high concentrations of asparagine/aspartic acid, glycine, leucine and serine in common with other chitin-binding lectins with insecticidal and antifungal activities. Brassica lectin and the closely related agglutinin from wheatgerm and nettle show significant insecticidal activity when presented toBrevicoryne brassicae in chemically-defined synthetic diets.     

Structure-function relationships amongLathyrus lectins

The structure-function relationship of eight structurally very similarLathyrus lectins, which are two-chain lectins from theVicieae tribe, has been studied. Hemagglutination activity, inhibition of hemagglutination, glycoprotein reactivity and mitogenic activity were determined for each of the lectins. Despite the close structural resemblance, marked differences in the carbohydrate binding activity of theLathyrus lectins were recorded. These functional differences were related to small conformational changes due to minor differences in amino acid sequence of eachLathyrus lectin.     

Cloning and characterization of the lectin cDNA clones from onion,shallot and leek

Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells.cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level.Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5–13 kDa) after post-translational modifications.Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.