The Lectins of Sophora japonica: I. Purification Properties and N-Terminal Amino Acid Sequences of Two Lectins from Leaves

Two lectins, Leaf Lectin I and Leaf Lectin II (LLI and LLII) were purified from the leaves of Sophora japonica. Like the Sophora seed lectin, LLI and LLII are tetrameric glycoproteins containing a single subunit with respect to size. The subunits of LLI (32 kilodaltons) and LLII (34 kilodaltons) are slightly larger than those of the seed lectin (29.5 kilodaltons). The three Sophora lectins display indistinguishable specificities, amino acid compositions, specific hemagglutinin activities, and extinction coefficients. Although very closely related to the seed lectin, the leaf and seed lectins are not immunologically identical and they differ in subunit molecular weights, carbohydrate content, and in the pH sensitivity of their hemagglutinin activities. N-terminal amino acid sequence analysis shows that although they are homologous proteins, the three Sophora lectins are products of distinct genes.     

Isolation and properties of N-acetyllactosamine-specific lectins from nine erythrina species

Lectins from seeds of nine species of Erythrina have been purified by affinity chromatography on columns of lactose coupled to Sepharose and their properties compared with those of the lectin from Erythrina cristagalli. All lectins are glycoproteins of M, ca 60 000 composed of two identical or nearly identical subunits. They contain between 3–10% carbohydrates comprised of N-acetylglucosamine, mannose, fucose and xylose. The amino acid composition of all Erythrina lectins is very similar. The N-terminal amino acid is valine, with the exception of the lectin from E. flabelliformis in which it is alanine. To the extent tested, identities or near identities have been found in the N-terminal sequences (up to 15 residues in some cases) of the lectins. Hapten inhibition experiments of agglutination have shown that the lectins are specific for N-acetyllactosamine, this disaccharide being 10–30 times more inhibitory than D-galactose and 10–20 times more than N-acetyl-D-galactosamine. All lectins agglutinate human erythrocytes equally well, irrespective of blood type, at minimal concentrations of 5–20 μg/ml. Six of the lectins are also very effective in agglutinating rabbit erythrocytes and are mitogenic for human peripheral blood lymphocytes, whereas three of them are considerably weaker hemagglutinins for rabbit erythrocytes, and two of these are also very weak mitogens. Our results, while demonstrating striking similarities in the molecular properties and sugar specificity of all Erythrina lectins studied, suggest the existence of differences at or close to the carbohydrate-binding site.     

A comparative study on lectins from four Erythrina species

A comparison of some physicochemical and structural properties of the lectins from the seeds of different species of the genus Erythrina is presented. The amino acid compositions of E. indica, E. arborescens, E. lithosperma and E. suberosa lectins are closely similar and resemble those of E. cristagalli [Eur. J. Biochem. (1982) 123, 247–2521 and E. corallodendron [Can. J. Biochem. (1981) 59, 315–320]. They are rich in acidic and hydroxy amino acids and poor in sulphur containing amino acids. All contain valine as the only N-terminal amino acid. They are glycoproteins containing high mannose type complex oligosaccharide chains. In addition to mannose they contain arabinose, xylose, fucose, glucose and galactose. Glucosamine is the amino sugar present. E. indica, E. arborescens and E. lithosperma lectins bind to Concanavalin A-Sepharose but not to lentil and pea lectin-Sepharose indicating the presence of terminal non-reducing α-d-mannose and/or internal 2-O-α-linked mannose residues in these lectins, and the absence of α(1 → 6) linkedl-fucose residues in the core regions of the oligosaccharide moieties. These lectins are metalloproteins containing about 2 g atoms of Mn²⁺ and 3 g atoms of Ca²⁺. The M_rs of E. arborescens, E. lithosperma and E. suberosa lectins are 58 000, 57 000 and 59 000, respectively. Each lectin consists of two noncovalently bound subunits which are of identical or very similar M_rs. The UV spectra of E. indica, E. arborescens and E. lithosperma lectins have similar features and in the presence of inhibitory sugars the absorbance at 278–281 nm and 290 nm increases in all cases indicating involvement of tryptophan residue(s) in sugar binding. The properties of Erythrina lectins show striking similarities and indicate close phylogenic relationships among these lectins.     

Erythro- and lymphoagglutinins of Phaseolus acutifolius

A potent lymphoagglutinin which had low affinity for red cells or fetuin and another lectin which reacted strongly with red cells and fetuin but was a poor agglutinin for lymphocytes were isolated from seeds of Phaseolus acutifolius. A number of other lectin components with intermediate activity towards these cells was also isolated. All the lectins had very similar amino acid and carbohydrate composition, sedimentation patterns, partial specific volume and molecular weight values of about 116 600 and were thus smaller than the related Phaseolus vulgaris lectins (M_r = 119 000). The lectins contained four subunits with only minor size and charge differences between the lympho- and erythroagglutinating subunits and their electrophoretic mobility in SDS gel electrophoresis was anomalously high. The existence of lympho- and erythroagglutinating subunits in two members of the genus Phaseolus supports their close morphological similarity.     

Structural characterization of a galectin isolated from the marine sponge Chondrilla caribensis with leishmanicidal potential

BackgroundSolving primary structure of lectins leads to an understanding of the physiological roles within an organism and its biotechnological potential. Only eight sponge lectins have had their primary structure fully determined.MethodsThe primary structure of CCL, Chondrilla caribensis lectin, was determined by tandem mass spectrometry. The three-dimensional structure was predicted and the protein-carbohydrate interaction analysed by molecular docking. Furthermore, the anti-leishmanial activity was observed by assays with Leishmania infantum.ResultsThe amino acid sequence consists of 142 amino acids with a calculated molecular mass of 15,443 Da. The lectin has a galectin-like domain architecture. As observed in other sponge galectins, the signature sequence of a highly conserved domain was also identified in CCL with some modifications. CCL exhibits a typical galectin structure consisting of a β-sandwich. Molecular docking showed that the amino acids interacting with CCL ligands at the monosaccharide binding site are mostly the same as those conserved in this family of lectins. Through its interaction with L. infantum glycans, CCL was able to inhibit the development of this parasite. CCL also induced apoptosis after eliciting ROS production and altering the membrane integrity of Leishmania infantum promastigote.ConclusionsCCL joins the restricted group of sponge lectins with determined primary structure and very high biotechnological potential owing to its promising results against pathogens that cause Leishmaniasis.General significanceAs the determination of primary structure is important for biological studies, now CCL can become a sponge galectin with an exciting future in the field of human health.     

Complexity of lectins from the hard roe of perch (Perca fluviatilis L.)

Fish eggs are a rich source of lectins, the sugar-binding (glyco)proteins. In this paper we aim to further characterise perch roe lectins using several protein characterisation techniques including affinity chromatography and protein sequencing. Perch roe lectins are comprised of two subunits, subunit A and subunit B which have molecular weights of 12,400 and 12,000, respectively. These subunits form multiple aggregates A_nB_n in which the two subunits are present in differing ratios and, also as an `homogeneous' aggregates of one of the subunits A_n or B_n. Lectins A_n (designated A thereafter) and lectin B_n (designated B thereafter) formed by one type of subunit only (subunit A or B) were isolated in a pure state. Lectin B could also be isolated by spontaneous precipitation occurring during incubation of the perch roe extract at 4°C. Lectin B has a higher affinity for d-glucose than lectin A, whereas both lectins (A and B) have a similar affinity for l-fucose. The N-terminal region of subunit B showed the following amino acid sequence: EPAXPPWGTQFG-, whereas the N-terminus of subunit A was blocked and therefore could not be directly sequenced. Differences between subunits A and B were also found in amino acid composition. This unusual complexity and variability of perch roe lectins is likely to have physiological significance which, as yet, remains to be determined.     

Purification and properties of d-galactose-binding lectins from some erythrina species: Comparison of properties of lectins from E. indica,E. arborescens,E. suberosa, and E. lithosperma

The lectins of the seeds of four species of the genus Erythrina, namely E. indica, E. arborescens, E. lithosperma, and E. suberosa were isolated by affinity chromatography on acid-treated ECD-Sepharose 6B. The lectins were found homogeneous in polyacrylamide gel electrophoresis and immunochemical tests. In SDS-gel electrophoresis, E. indica and E. lithosperma lectins each gave two bands with subunit molecular weights of 30,000 and 33,000 in the case of the former and 26,000 and 28,000 in the case of the latter. E. arborescens and E. suberosa gave single bands corresponding to polypetide chain molecular weight of 28,000. The lectins were found to be glycoproteins with their neutral sugar contents ranging from 4–9%. In carbohydrate specificity all the lectins were d-galactose specific. Their close similarity was also demonstrated by their homologous cross-reaction against the antiserum to E. indica lectin. In hemagglutinating activity toward human erythrocytes, E. indica and E. suberosa lectins showed higher activity toward the O group and E. arborescens toward the B group. The results show the similarity of the lectins derived from different species of the same genus in respect of immunochemical properties and carbohydrate specificity. In studies on E. indica lectin, the protein was found homogeneous by electrophoretic, immunochemical, and sedimentation experiments. Its molecular weight of 68,000 determined from sedimentation and diffusion data indicated that the molecule was a dimer of two noncovalently bound unequal subunits whose SDS-gel electrophoretic molecular weights are noted above. The lectin was devoid of cysteine and methionine and contained valine as its N-terminal amino acid. It had 9% neutral sugars and 1.5% glucosamine. Equilibrium dialysis studies with lactose showed that the values of the association constant K at different temperatures were of similar orders of magnitude to other lectins and the dimeric molecule possessed two noninteracting binding sites.     

ELISA measurement of immunochemical cross-reactions among Lathyrus lectins as an assessment of their phylogenetical relationship

The immunochemical cross-reactions among Lathyrus and other Vicieae lectins have been quantified by using an ELISA (enzyme-linked immunosorbent assay) technique, in order to assess their phylogenetical relationship. The data are consistent with those arising from the comparison of both the amino acid compositions of the lectins and the amino acid sequences of their light and heavy subunits. They confirm that two-chain lectins from the tribe of Vicieae are phylogenetically closely related.     

The nature of lectins fromDolichos lablab

The lectins from the seedsof Dolichos lablab var.lignosus (field bean) andDolichos lablab var.typicus (lablab bean) have been isolated in a homogeneous form by affinity chromatography on D-mannose linked Sepharose. Both the lectins are glycoproteins and have a molecular weight of 60,000 andS _20,w value of 5.2 and seem to be made up of 4 similar subunits (apparent molecular weight 15,000). The carbohydrate content ofthe lectins is mostly fucose (2–5 mol per mol of protein), mannose (5–8 mol per mol of protein) and N-acetyl glucosamine (1–2 mol per mol of protein). The amino acid composition of both the lectins was similar and methionine and half cystine could not be detected, Both the lectins have similar tryptic peptide map. Alanine and serine were the only N and C-terminal amino acids for both lectins. The lectins were found to contain low amounts of bound metals such as manganese, magnesium and calcium. The near ultra-violet circular dichroism spectra of the lectins are similar to that of Sainfoin. Circular dichroism data indicate that tyrosine and tryptophan residues are involved in sugar binding. The lectins are nonspecific for human blood groups and they agglutinate a variety of other erythrocytes. Among a number of sugars, D-glucose and D-mannose inhibited the haemag-glutinating activity ofthe lectins. The lectins were antigenically similar     

Computational analyses of protein coded by rice (Oryza sativa japonica) cDNA (GI: 32984786) indicate lectin like Ca2+ binding properties for Eicosapenta Peptide Repeats (EPRs)

Eicosapenta peptide repeats (EPRs) occur exclusively in flowering plant genomes and exhibit very high amino acid residue conservation across occurrence. DNA and amino acid sequence searches yielded no indications about the function due to absence of similarity to known sequences. Tertiary structure of an EPR protein coded by rice (Oryza sativa japonica) cDNA (GI: 32984786) was determined based on ab initio methodology in order to draw clues on functional significance of EPRs. The resultant structure comprised of seven α-helices and thirteen anti-parallel β-sheets. Surface-mapping of conserved residues onto the structure deduced that (i) regions equivalent to β α4- the primary function of EPR protein could be Ca²⁺ binding, and (iii) the putative EPR Ca²⁺ binding domain is structurally similar to calcium-binding domains of plant lectins. Additionally, the phylogenetic analysis showed an evolving taxa-specific distribution of EPR proteins observed in some GNA-like lectins.     

Lectins from latices of Euphorbia and Elaeophorbia species

Crude latex sera from 17 members of the genus Euphorbia and from Elaeophorbia drupifera (Euphorbiaceae) contained a wide range of agglutinating abilities. Homogeneous lectins were isolated from latices of Euphorbia coerulescens, E. hermentiana, E. lactea, E. lactea cristata, E. lathyris, E. trigona and Elaeophorbia drupifera. The M,s of the lectins ranged from 60 to 67 000, and the unit weights from 27 to 38 000. pI measurements showed that each latex contained from five to 13 isolectins. The amino acid compositions of the seven lectins were determined: those from E. hermentiana, E. lactea, E. lactea cristata, E. trigona and Elaeophorbia drupifera are related.     

The complete amino acid sequences of the β1- and β2-subunits of the isolectins LoL1 and LoL11 from seeds of Lathyrus ochrus (L.) DC

The complete amino acid sequences of the β₁- and β₂-subunits of the isolectins (LoL1 and LoL11) from seeds of Lathyrus ochrus were determined by analysis of peptides derived from the proteins by digestion with trypsin, chymotrypsin, pepsin and the S. aureus V₈ protease, as well as fragments produced by cleavage with iodosobenzoic acid. Both β-subunits consisted of singlepolypeptide chains of 181 amino acids, which differed from one another in only 3 positions. The homology of the Lathyrus ochrus isolectins with the other two-chain lectins of the tribe Vicieae, and the single-chain lectins of other tribes of the Leguminosae is discussed.     

Related glycoprotein lectins from root stocks of wild cucumbers

The lectin from root stocks of the Californian wild cucumber (Marah macrocarpus) has a molecular structure similar to that of the root stock lectin from white bryony (Bryonia dioica). These two Cucurbitaceae lectins (M, around 60-65 000) are composed of two different subunits which are held together by disulphide bridges. They resemble each other in amino acid and carbohydrate composition and are serologically related. It appears that, during the evolution of the Cucurbitaceae family, the genes encoding root stock lectins of at least some perennial species have been conserved fairly well.     

Structure Predictions of Two Bauhinia variegata Lectins Reveal Patterns of C-Terminal Properties in Single Chain Legume Lectins

Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and –II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.     

Isolation and characterization of a lectin from the seeds of Erythrina Edulis

A galactose-specific lectin was isolated from the seeds of Erythrina edulis. The protein was purified by affinity chromatography of the globulin fraction on an allyl-galactoside polyacrylamide gel. The hemagglutination properties, amino acid composition, A₂₈₀, MW of the protein and of its subunits, carbohydrate content, electrophoretic pattern and isoelectric point were determined. Comparison of its properties with those of other Erythrina lectins shows that the protein is a distinct member of this group of lectins.     

The Purification, Properties, and Localization of an Abundant Legume Seed Lectin Cross-Reactive Material from Spartium junceum

The seeds of Spartium junceum contained a large quantity of lectin-like protein that did not appear to be either a hemagglutinin or active lectin. The cross-reactive material (CRM), like most legume seed lectins, was a tetrameric glycoprotein of about 130,000 M_r. The singlesized subunits of about 33,000 M_r were not covalently associated. The amino acid composition was typical of legume lectins and was rich in hydroxy-amino acids and poor in sulfur-containing amino acids. The Spartium CRM contained about 3.5% covalently associated carbohydrate, most likely of the high-mannose type, since the CRM was precipitated by concanavalin A. The CRM was localized by electron-microscopic immunocytochemistry and found to be exclusively in protein-filled vacuoles (protein bodies). Because this protein was so similar immunologically, structurally, and in its physiology, to classic legume seed lectins, it is most likely a lectin homolog. Similar seed lectin CRMs appear to be both common and widespread in the Leguminosae.     

Characterization of a lectin from the seeds of Erythrina vespertilio

A lectin was isolated from the seeds of Erythrina vespertilio by affinity chromatography on lactose-Sepharose 6B. The lectin has an M, of 59 000 and consists of two non-covalently associated subunits (M, ∼ 30 000). The lectin is devoid of cysteine but has six methionine residues/mol and a neutral sugar content of 9.7% The carbohydrate composition was mannose, N-acetylglucosamine, fucose, xylose and galactose in amounts of 15.0, 4.0, 1.0, 5.0 and 25 mol/59 000 g, respectively. Alkaline gel electrophoresis and isoelectric focusing showed that the affinity purified lectin consists of a family ofisolectins. Valine was the only N-terminal amino acid found and the N-terminal sequence was homologous with that found for other legume lectins. The lectin was inhibited by galactosyl containing carbohydrates; p-nitrophenyl-β-galactoside was the best inhibitor and the lectin showed a slight preference for β-galactosides. Comparison of its properties with those of other Erythrina lectins shows that most of the lectins of this genus are closely related.     

The Lectins of Sophora japonica: II. Purification, Properties, and N-Terminal Amino Acid Sequences of Five Lectins from Bark

Five N-acetyl-galactosamine-specific lectins were isolated from the bark of the legume tree Sophora japonica. These lectins are immunologically and structurally very similar, but not identical, to the Sophora seed and leaf lectins. The carbohydrate specificities and hemagglutinin activities of these lectins are indistinguishable at pH 8.5 but their activities differ markedly at pH values below 8. All five lectins are tetrameric glycoproteins made up of different combinations of subunits of about 30,000, 30,100, 33,000 M_r containing 3% to 5% covalently attached sugar. These lectins are the overwhelmingly dominant proteins in bark, but they do not appear to be present in other tissues. Amino terminal sequence analysis indicates that at least two distinct lectin genes are expressed in bark.