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.     

Purification and Characterization of Griffonia simplicifolia Leaf Lectins

Leaves from mature Griffonia simplicifolia plants were examined for the presence of leaf lectins possessing sugar binding specificities similar to the four known seed lectins (GS-I, GS-II, GS-III, GS-IV). Three (GS-I, -II, -IV) of the four known G. simplicifolia seed lectins were present in the leaves. Leaf G. simplicifolia lectins I and IV were similar to the respective seed lectins. Leaf GS-II, however, was composed of two types of subunits (M_r = 33,000 and 19,000), whereas the seed lectin consists of only one type of subunit (M_r 32,500). Seed and leaf GS-II lectins also had different isoelectric points. All leaf and seed lectins were similar with respect to their hemagglutination and glycoconjugate precipitation properties and all subunits contained covalently bound carbohydrate. Leaf GS-IV appeared slightly under-glycosylated compared to seed GS-IV.

The fate of GS-I and GS-II seed lectins in aging cotyledons was investigated. GS-I isolectins usually contain isolectin subtypes associated with each main isolectin. Upon inbibition and germination, these GS-I isolectin subtypes disappeared. Over time, GS-II lectin did not change its disc gel electrophoretic properties.

     

Purification and characterization of arcelin seed protein from common bean

Arcelin, a seed protein originally discovered in wild bean accessions, was purified, characterized, and compared to phaseolin, the major seed protein of common bean, and to phytohemagglutinin (PHA), the major bean seed lectin. Arcelin and PHA has several characteristics in common. Both were glycoproteins having similar subunit M_r, deglycosylated M_r, and amino acid compositions. The two proteins were related antigenically and they had the same developmental timing of accumulation. Arcelin also had some hemagglutinating activity, a characteristic associated with lectins. However, several features distinguished arcelin from PHA. Arcelin had a more basic isoelectric point than PHA, greater numbers of basic amino acid residues, additional cysteine residues, and one methionine residue, which PHA lacks. Native PHA protein is a tetramer of subunits, and although a small component of native arcelin protein was also tetrameric, most of the arcelin preparation was dimeric. The hemagglutinating activity of arcelin was specific only for some pronase-treated erythrocytes. It did not agglutinate native erythrocytes, nor did it bind to thyroglobulin or fetuin affinity resins as did PHA. Although arcelin has lectin-like properties, we believe the distinctions between arcelin and PHA warrant the designation of arcelin as a unique bean seed protein.     

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.     

Subcellular Localizations of Two Dolichos biflorus Lectins

The subcellular localizations of the Dolichos biflorus seed lectin and the structurally related lectin (cross-reactive material [CRM]) from the stems and leaves of this plant were determined by immunofluorescence, immunocytochemistry, and cell fractionation procedures. Subcellular fractionation of the cotyledons using a nonaqueous procedure to minimize disruption of the protein bodies showed that the majority of the seed lectin was associated with the protein body fraction and some lectin was also present in the starch granules. Immunofluorescence and immunocytochemistry at the light microscopic level showed that the seed lectin was mainly localized at the peripheries of these organelles. Lectin was also found in the cytoplasm of the cells, although the amount appeared to be dependent upon the degree of protein body disruption.

Immunofluorescence and immunocytochemistry studies of the stem and leaf lectin (CRM) indicated that a significant portion of this lectin may be associated with the cell walls, although lectin was also seen in the cytoplasm of plasmolyzed cells. Extraction and cell fractionation studies showed that a large portion of the CRM is readily solubilized and most of the remainder is pelleted at 1000g. The CRM can be extracted from these pellets by treatment with cellulase and pectinase; other reagents such as NaCl, detergents, and EDTA could also release significant amounts of CRM. These studies suggest that the CRM is noncovalently bound to the cell walls. A comparison of the distribution of exogenously supplied [¹²⁵I]CRM with the endogenous CRM during extraction and cell fractionation indicates that soluble CRM is not adsorbed to the 1000g pellet during fractionation.

The different subcellular distributions of these two structurally related lectins suggest that different tissues of the same plant may utilize lectins for different functions.

     

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.     

Characterization and comparison of arcelin seed protein variants from common bean

Four variants of arcelin, an insecticidal seed storage protein of bean, Phaseolus vulgaris L., were investigated. Each variant (arcelin-1, -2, -3, and -4) was purified, and solubilities and M_rs were determined. For arcelins-1, -2, and -4, the isoelectric points, hemagglutinating activities, immunological cross-reactivities, and N-terminal amino acid sequences were determined. On the basis of native and denatured M_rs, the variants were classified as being composed of dimer protein (arcelin-2), tetramer protein (arcelins-3 and -4), or both dimer and tetramer proteins (arcelin-1). Although the dimer proteins (arcelins-1d and -2) could be distinguished by M_rs and isoelectric points, they were identical for their first 37 N-terminal amino acids and had similar immunological cross-reactions, and bean lines containing these variants had a DNA restriction fragment in common. The tetramer proteins arcelin-1t and arcelin-4 also could be distinguished from each other based on M_rs and isoelectric points; however, they had similar immunological cross-reactions and they were 77 to 93% identical for N-terminal amino acid composition. The similarities among arcelin variants, phytohemagglutinin, and a bean α-amylase inhibitor suggest that they are all encoded by related members of a lectin gene family.     

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     

Purification and properties of a lectin from lonchocarpus capassa (apple-leaf) seed

A lectin has been purified from L. capassa seed by ammonium sulphate fractionation and affinity chromatography on a column of D-galactose-derivatized Sepharose. The lectin is a glycoprotein which contains 3.8% neutral carbohydrates comprised of mannose, N-acetylglucosamine, xylose and fucose. The subunit M, of the lectin is 29 000, it has only alanine as N-terminal amino acid and contains 240 amino acids with a high content of acidic and hydroxy amino acids, single residues of methionine and histidine and the absence ofcystine. The lectin of L. capassa seed is a metalloprotein in that it contains 0.8 mol Ca²⁺ and 0.4 mol Mn²⁺ per mol. It agglutinates untreated human A, O and B type erythrocytes and rabbit erythrocytes. N-Acetyl-D-galactosamine was the best inhibitor. D-Galactose and various carbohydrates containing this sugar inhibit the hemagglutinating activity of the lectin. The lectin is also inhibited by D-glucose. The amino-terminal sequence of the lectin from L. capassa seed shows a significant degree of homology with many lectins from leguminous plants and is related to concanavalin A by a circularly permuted sequence homology.     

Isolation and properties of a lectin from sainfoin (Onobrychis viciifolia, Scop.)

A glycoprotein capable of binding simple carbohydrates and causing hemagglutination has been isolated from seeds of the legume plant sainfoin (Onobrychis viciifolia, Scop. var Eski). The phytolectin was prepared by affinity chromatography of pH 7.0 sodium phosphate extracts on columns of Sepharose-4B containing covalently attached D-mannose. Molecular weight determinations showed the lectin to be a dimer consisting of 26 000 dalton, non-covalently associated monomers. Amino acid analyses indicated high amounts of aspartate, glutamate, threonine and serine which accounted for 41% of all amino acids. One residue of cysteine was present and methionine was totally absent. The lectin contained 2.6% (w/w) neutral carbohydrate and two residues of N-acetylglucosamine/monomer. Carbohydrate-binding specificity was directed toward D-mannose and D-glucose and their alpha-glycosidic derivatives. The purified protein agglutinated cat erythrocytes at 5 micrograms/ml. Antiserum to seed lectin showed a single common immunoprecipitation line in Ouchterlony double diffusion against both the seed and root antigen. Lectin isolated from sainfoin seedling roots showed molecular weight, amino acid and carbohydrate values similar to that of the seed lectin.     

Protein subunits and amino acid composition of wild lentil

Three wild species of lentil, Lens orientalis, L. ervoides and L. nigricans were investigated for protein subunits of the albumin protein fraction (APF), globulin protein fraction (GPF) and for protein and free amino acid composition. The APF and GPF formed 12.7–16.8 % and 34.7–49.0 %, respectively, of the meal nitrogen. SDS-PAGE showed APF to contain 15 to 20 major and a similar number of minor protein subunits ranging in M_r at least from 14 400 to 94 000. The GPF was also heterogenous and contained some subunits having M_r similar to APF subunits but none < 15 000. The three wild lentil species were distinguishable by their protein subunit composition. The protein amino acid composition of the wild species was identical and similar to that of the cultivated lentil. The wild species, like the cultivated species (L. culinaris), contained major amounts of free arginine, glutamic and aspartic acids, serine and a number of unidentified amino acids. L. orientalis, L. nigricans and the cultivated lentil contained two acidic and two basic unidentified amino acids. However, L. ervoides was distinctly different in that it contained only the two acidic plus one neutral unidentified amino acid, but none of the two basic unidentified amino acids.     

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.     

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.     

Isolation and properties of a lectin from the roots of Pisum sativum (Garden pea)

Abstract The roots of pea (Pisum sativum L. ev. Feltham First) seedlings contained haemagglutinating activity and a protein which reacted with antibodies directed against pea seed lectin. This protein was shown to be present on the surface of root hairs and in the root cortical cells by immunofluorescence. Lectin (haemagglutinin) was purified from pea seedling roots by both immunoaffinity chromatography and affinity chromatography on Sephadex G-100. The pea root lectin was similar to the seed lectin when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and was antigenically identical: however, the isoelectric focussing band patterns of the proteins differed. The sugar specificity of the root lectin differed from that of the seed lectin, and the haemagglutinating activity of the root lectin was less than the seed lectin. These results are discussed with reference to the hypothesis that lectins mediate in the symbiotic association of legume and Rhizobium through their carbohydrate-binding properties.     

Identification and characterization of an immunologically conserved adenovirus early region 11,000 Mr protein and its association with the nuclear matrix

Antisera from some hamsters bearing adenovirus-induced tumors contain antibodies to an 11,000 M_r adenovirus-induced protein. In adenovirus-infected HeLa cells, this early viral protein was specifically associated with the nuclear matrix fraction. After two-dimensional gel electrophoresis, two forms of the 11,000 M_r protein at pI 5.6 and pI 5.4 were found. Only the pI 5.4 form of this protein was associated with the nuclear matrix fraction. Adenoviruses from groups A, B, C, D and E all produced an early viral protein (10,000 to 12,000 M_r) that reacted with group C antibody to the 11,000 M_r protein. To date, this is the only known early viral protein that is immunologically conserved in all of the human adenovirus groups.The positions of two methionine and seven leucine residues were determined by sequencing the first 35 amino acids from the N terminus of the adenovirus serotype 2 group C 11,000 M_r protein. The positions of these amino acid residues were compared to the adenovirus serotype 2 nucleotide sequence, which uniquely localized the structural gene of the 11,000 M_r protein to region E4, subregion 3 in type 2 adenovirus. A frameshift mutant, which contained a deletion of one base-pair in the structural gene of the 11,000 M_r protein, was isolated and mapped by marker rescue and nucleotide sequence analysis. This mutant failed to produce immunologically detectable 11,000 M_r protein. The mutant had a viable phenotype, producing normal levels of infectious virus in both HeLa cells and WI38 cells in culture. These experiments identify the first adenovirus early region 4 protein detected in virus-infected cells.     

Isolation and Partial Characterization of a New Lectin from Seeds of the Greater Celandine (Chelidonium majus)

Seeds of the greater celandine (Chelidonium majus L.) contain a lectin which could be isolated using a combination of affinity chromatography on chitin and ion exchange chromatography on sulphopropyl-Sephadex. The purified lectin was partially characterized with respect to its biochemical and physicochemical properties. It is a small dimeric protein composed of two different subunits of M_r 9,500 and 11,500, respectively. Its amino acid composition is typified by high contents of glycine and cysteine. No covalently bound carbohydrate could be detected. Hapten inhibition experiments indicated that the lectin exhibits specificity towards oligomers of N-acetylglucosamine, the potency of inhibition increasing with chain length up to four residues. The greater celandine lectin is the first lectin to be isolated from a species belonging to the plant family Papaveraceae (poppy family). Although it represents a new type of plant lectin, resemblances to phytohemaglutinins from diverse taxonomic origin are obvious.     

Isolation and partial characterization of a lectin from ground elder (Aegopodium podagraria) rhizomes

A lectin has been isolated from rhizomes of ground elder (Aegopodium podagraria) using a combination of affinity chromatography on erythrocyte membrane proteins immobilized on cross-linked agarose and hydroxyapatite, and ion-exchange chromatography. The molecular structure of the lectin was determined by gelfiltration, sucrose density-gradient centrifugation and gel electrophoresis under denaturing conditions. It has an unusually high M_r (about 480000) and is most probably an octamer composed of two distinct types of subunits with slightly different M_r (about 60000). Hapten inhibition assays indicated that the Aegopodium lectin is preferentially inhibited by N-acetylgalactosamine. Nevertheless, it does not agglutinate preferentially blood-group-A erythrocytes. The ground-elder lectin is a typical non-seed lectin, which occurs virtually exclusively in the underground rhizomes. In this organ it is an abundant protein as it represents up to 5% of the total protein content. The lectin content of the rhizome tissue varies strongly according to its particular location along the organ. In addition, the lectin content changes dramatically as a function of the seasons. The ground-elder lectin differs from all other plant lectins by its unusually high molecular weight. In addition, it is the first lectin to be isolated from a species of the family Apiaceae.Abbreviations APA Aegopodium podagraria agglutinin - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Blood group B-specific lectin of Plecoglossus altivelis (Ayu fish) eggs

A lectin that agglutinates human blood group B erythrocytes but not blood group A and O erythrocytes was isolated from eggs of Ayu sweet fish (Plecoglossus altivelis). The lectin also agglutinates Ehrlich ascites carcinoma cells but not rat ascites hepatoma AH109 or rat sarcoma 150 cells tested. The lectin agglutination was most effectively inhibited by monosaccharides with the first type of configuration, i.e., L-rhamnose, L-mannose and L-lyxose at a concentration of 0.03 mM. The lectin agglutination was moderately inhibited by monosaccharides with the second type of configuration, i.e., D-galactose, D-fucose and D-galacturonic acid at a concentration of 0.4 mM. However, the agglutination was not inhibited by various other monosaccharides and oligosaccharides that have other types of configuration. The basis for an apparent B-specific hemagglutination may be due to the steric similarity of the C₂ and C₄ of the galactosyl series, the B-specific determinant, and the L-rhamnosyl series, which are the best inhibitors of the lectin activity. The lectin was affinity purified on an L-rhamnosyl-Sepharose column and was characterized as a homogeneous low molecular weight protein (M_r 14 000) with an abundance of hydrophobic amino acids and dicarboxylic amino acid.     

Purification,Chemical, and Immunochemical Properties of a New Lectin from Mimosoideae (Parkia Discolor)

ABSTRACT

A glucose/mannose-binding lectin was isolated from seeds of Parkia discolor (Mimosoideae) using affinity chromatography on Sephadex G-100 gel. The protein presented a unique component in SDS-PAGE corresponding to a molecular mass of 58,000 Da, which is very similar to that of a closely related lectin from Parkia platycephala. Among the simple sugars tested, mannose was the best inhibitor, but biantennary glycans, containing the trimannoside core, present in N-glycoproteins, also seem to be powerful inhibitors of the haemagglutinating activity induced by the purified lectin. The protein was characterised by high content of glycine and proline and absence of cysteine. Rabbit antibodies, anti-P. platycephala seed lectin, recognised the P.discolor lectin. However, no cross-reaction was observed when a set of other legume lectins from sub-family Papilionoideae and others from families Moraceae and Euphorbiaceae were assayed with the Parkia lectins. This suggests that Parkia lectins comprise a new group of legume lectins exhibiting distinct characteristics.