Structural analysis of toxic volkensin, a type 2 ribosome inactivating protein from Adenia volkensii Harm (kilyambiti plant): Molecular modeling and surface analysis by computational methods and limited proteolysis

Volkensin, isolated from Adenia volkensii, is one of the most toxic type 2 ribosome-inactivating protein (RIP), exerting its biological function by inhibiting protein synthesis. Despite the high sequence identity with type 2 RIPs, including ricin, volkensin shows interesting peculiar properties.In this work a computational model building of volkensin was performed. The volkensin electrostatic potential charge distribution, the hydrophobic profile and the surface topology analyses were also carried out to aid the understanding of structure–function relationships of this potent toxin. Volkensin surface topology was probed by applying a limited proteolysis approach with the aim to gain insights into volkensin conformational features.     

Cloning and expression of the B chain of volkensin, type 2 ribosome inactivating protein from Adenia volkensii harms: co-folding with the A chain for heterodimer reconstitution

Type 2 ribosome inactivating proteins (RIPs) include some potent plant toxins, among which ricin from Ricinus communis and abrin from Abrus precatorius seeds, have been known for more than a century. Two other type 2 RIPs belong to this class of proteins, both isolated from plants of the same family (Passifloraceae), modeccin and volkensin, from Adenia digitata and Adenia volkensii roots, respectively. Volkensin is probably the most potent plant toxin known, with an LD50 for rats of 50-60 ng/kg. Here we report the cloning, expression and renaturation of recombinant volkensin B chain. Furthermore, starting from separately expressed A and B chains, a co-association procedure was set-up, leading to in vitro heterodimeric volkensin reconstitution. The recombinant heterodimer was characterized by N-terminal sequence analysis and its hemagglutinating activity assessed. In parallel, we have explored the carbohydrate-binding properties of native volkensin with the aim to correlate toxin-specific properties (i.e., axonal transport along neurons) to lectin's sugar-binding preferences.     

Lectin-like binding of pertussis toxin to a 165-kilodalton Chinese hamster ovary cell glycoprotein

Chinese hamster ovary (CHO) cells cluster in the presence of pertussis toxin, a response that is correlated with the ADP-ribosylation of a Mr = 41,000 membrane protein by the toxin. A ricin-resistant line of CHO cells (CHO-15B) which specifically lacks the terminal NeuAc----Gal beta 4GlcNAc oligosaccharide sequence on glycoproteins did not cluster in response to pertussis toxin. These cells do contain the Mr = 41,000 protein substrate for the enzymatic activity of the toxin which suggests that pertussis toxin, like certain plant lectins, does not bind to or is not internalized by the CHO-15B cells. There was no evidence of pertussis toxin binding to gangliosides or neutral glycolipids isolated from CHO cells but the toxin bound to a Mr = 165,000 component in N-octyglucoside extracts of CHO cells that had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted to nitrocellulose. Plant lectins from Ricinus communis and Erythina cristagalli detected a similar size band in CHO cells and also did not react with CHO-15B cells. Unlike pertussis toxin, these plant lectins recognized two other major bands in CHO cell extracts and reacted best after sialidase treatment of nitrocellulose transfers containing CHO cell extracts. Conversely, sialidase treatment abolished binding a pertussis toxin and wheat germ agglutinin, a plant lectin that reacts with multivalent sialic acid residues on glycoproteins, to the Mr = 165,000 band. Purified B oligomer of pertussis toxin also uniquely detected a Mr = 165,000 component in CHO cell extracts while the A subunit of pertussis toxin was unreactive. These results indicate that pertussis toxin binds to a CHO cell glycoprotein with N-linked oligosaccharides and that sialic acid contributes to the complementary receptor site for the toxin. In addition, they suggest that a glycoprotein may serve as a cell surface receptor for pertussis toxin and that this interaction is mediated by a lectin-like binding site located on the B oligomer.     

A novel mannose-specific and sugar specifically aggregatable lectin from the bark of the Japanese pagoda tree (Sophora japonica)

A new D-mannose/D-glucose-specific lectin (B-SJA-II) was isolated from the bark of the Japanese pagoda tree, Sophora japonica. B-SJA-II was separated from a well known D-galactose/N-acetyl-D-galactosamine-specific lectin (B-SJA-I) by affinity chromatography on lactamyl-Sepharose, then purified by affinity chromatography on maltamyl-Sepharose. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, B-SJA-II gave four bands: subunit a-1 (Mr = 19,400), a-2 (Mr = 18,200), b-1 (Mr = 15,000), and b-2 (Mr = 13,200). Carbohydrate analysis and binding study with horseradish peroxidase-labeled lectins on the bands electroblotted onto polyvinylidene difluoride membrane showed that the three subunits other than b-2 have N-linked oligosaccharides typical of plant glycoproteins. The binding assay with horseradish peroxidase-glycoproteins revealed that all the subunits can bind sugar specifically with fetuin and asialofetuin. Furthermore, B-SJA-II aggregated to form precipitates in the absence of a specific sugar and became soluble upon addition of the specific sugar. The results indicate that each subunit has a sugar-binding site for the mannosyl core of N-linked oligosaccharide chains and recognizes each other sugar specifically to form aggregates. According to the N-terminal amino acid sequences obtained, the subunits are classified into two groups. The first group (a-1 and a-2) has an N-terminal sequence 50% identical with that of other S. japonica lectins (Hankins, C. N., Kindinger, J. I., and Shannon, L. M. (1988) Plant Physiol. 86, 67-70) and the amino acid sequence initiating at position 123 of concanavalin A (Cunningham, B. (1975) J. Biol. Chem. 250, 1503-1512), while the N-terminal sequence of the second group (b-1 and b-2) is homologous to that of concanavalin A, but completely different from that of the first group.     

Volkensin from Adenia volkensii Harms (kilyambiti plant), a type 2 ribosome-inactivating protein.

Volkensin, a type 2 ribosome-inactivating protein from the roots of Adenia volkensii Harms (kilyambiti plant) was characterized both at the protein and nucleotide level by direct amino acid sequencing and cloning of the gene encoding the protein. Gene sequence analysis revealed that volkensin is encoded by a 1569-bp ORF (523 amino acid residues) without introns, with an internal linker sequence of 45 bp. Differences in residues present at several sequence positions (reproduced after repeated protein sequence analyses), with respect to the gene sequence, suggest several isoforms for the volkensin A-chain. Based on the crystallographic coordinates of ricin, which shares a high sequence identity with volkensin, a molecular model of volkensin was obtained. The 3D model suggests that the amino acid residues of the active site of the ricin A-chain are conserved at identical spatial positions, including Ser203, a novel amino acid residue found to be conserved in all known ribosome-inactivating proteins. The sugar binding site 1 of the ricin B-chain is also conserved in the volkensin B-chain, whilst in binding site 2, His246 replaces Tyr248. Native volkensin contains two free cysteinyl residues out of 14 derived from the gene sequence, thus suggesting a further disulphide bridge in the B chain, in addition to the inter- and intrachain disulphide bond pattern common to other type 2 ribosome-inactivating proteins.     

野花生豆凝集素的纯化和性质

用猪胃粘蛋白-Sepharose 4B作亲和吸附剂,可从野花生豆(Crotalarta mucronata)的种子中分离纯化出对人类A型血专一凝集的凝集素。该凝集素可用pH30.,Gly-HCl-1mol/L NaCl溶液解吸附。纯化的凝集素在PAGE或SDS-PAGE中均显示单一蛋白带,表明凝集素分子内只有一种亚基。用SDS-PAGE测得其亚基分子量为49,000。氨基酸组成分析表明,该凝集素富含甘氨酸和谷氨酸,不合甲硫氮酸。纯化的野花生豆凝集素(简称CML)含有4.11％的中性糖。它对人A型血细胞有强烈凝集作用,对AB型血有弱凝集作用,但对B型和O型血均不凝集。其对A型血细胞的凝集作用可被N-乙酰半乳糖胺抑制,但对AB型血则无抑制作用。CML是一个促有絲分裂原,对人外周血中淋巴细胞有促有絲分裂作用。     

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (NADP): amino acid sequence of the subunits from isoenzyme I and structural relationship with isoenzyme II

The structural relationship between isoenzymes I and II of chloroplast glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NADP+ oxidoreductase (phosphorylating) EC 1.2.1.13) has been established at the protein level. The complete primary structure of subunits A and B of glyceraldehyde-3-phosphate dehydrogenase I from Spinacia oleracea has been determined by sequence analysis of the corresponding tryptic peptides, aligned by fragments derived from cyanogen bromide and Staphylococcus proteinase V8 digestions and by partially sequencing each intact subunit. Subunit A has an Mr of 36,225 and consists of 337 amino acid residues, whilst subunit B (Mr 39,355) consists of 368 residues. The amino acid sequence of subunit B, as determined through direct analysis of the protein, is identical to that recently deduced at cDNA level (Brinkmann et al. (1989) Plant Mol. Biol. 13, 81-94). The two subunits share a common portion of amino acid sequence which differs by 66 amino acid residues. Subunit B has an extra C-terminal sequence of 31 amino acid residues. Chloroplast glyceraldehyde-3-phosphate dehydrogenase II was partially characterized by sequencing the N-terminal portion of the intact protein and some of its tryptic peptides. The sequences of all the examined fragments fit precisely that of the corresponding regions of subunit A from glyceraldehyde-3-phosphate dehydrogenase I.     

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.     

A Novel Rhamnose-binding Lectin Family from Eggs of Steelhead Trout (Oncorhynchus mykiss) with Different Structures and Tissue Distribution

An L-rhamnose-binding isolectin named STL3 (subunit Mr, 21.5 k) was isolated from eggs of the steelhead trout (Oncorhynchus mykiss) in addition to STL1 (subunit Mr, 31.4 k) and STL2 (subunit Mr, 21.3 k) that had been already isolated. STLs were composed of non-covalently linked subunits. The primary structures of STL1 and STL3 were analyzed by the combined use of protein sequencing and cDNA sequencing. A cDNA encoding STL2, of which the protein sequence had been previously studied, was also analyzed. The STL1 subunit (289 amino acid residues) had different structural properties compared to those of the STL2 subunit (195 amino acid residues) and the STL3 subunit (195 amino acid residues); e.g., the number of repeated domain (three for STL1, and two for STL2 and STL3), although all of them were composed of tandemly repeated homologous domains (40 to 53% identities).

The lectin levels in various tissues and during the embryonic development showed that STL1 had different distribution and expression profiles from those of STL2 and STL3. Although STL1 could be detected in several tissues and serum of both male and female steelhead trout, STL2 and STL3 were only abundant in the ovary. STL2 and STL3 levels dramatically decreased just after hatching, however, the STL1 level increased temporarily. These results indicate that the multiple lectins from eggs of the steelhead trout form a novel rhamnose-binding lectin family with different structures and tissue distribution to share distinct functions in eggs.     

A novel rhamnose-binding lectin family from eggs of steelhead trout (Oncorhynchus mykiss) with different structures and tissue distribution

An L-rhamnose-binding isolectin named STL3 (subunit Mr, 21.5 k) was isolated from eggs of the steelhead trout (Oncorhynchus mykiss) in addition to STL1 (subunit Mr, 31.4 k) and STL2 (subunit Mr, 21.3 k) that had been already isolated. STLs were composed of noncovalently linked subunits. The primary structures of STL1 and STL3 were analyzed by the combined use of protein sequencing and cDNA sequencing. A cDNA encoding STL2, of which the protein sequence had been previously studied, was also analyzed. The STL1 subunit (289 amino acid residues) had different structural properties compared to those of the STL2 subunit (195 amino acid residues) and the STL3 subunit (195 amino acid residues); e.g., the number of repeated domain (three for STL1, and two for STL2 and STL3), although all of them were composed of tandemly repeated homologous domains (40 to 53% identities). The lectin levels in various tissues and during the embryonic development showed that STL1 had different distribution and expression profiles from those of STL2 and STL3. Although STL1 could be detected in several tissues and serum of both male and female steelhead trout, STL2 and STL3 were only abundant in the ovary. STL2 and STL3 levels dramatically decreased just after hatching, however, the STL1 level increased temporarily. These results indicate that the multiple lectins from eggs of the steelhead trout form a novel rhamnose-binding lectin family with different structures and tissue distribution to share distinct functions in eggs.     

A D-mannose-specific lectin from Gerardia savaglia that inhibits nucleocytoplasmic transport of mRNA

A new lectin has been isolated from the coral Gerardia savaglia by affinity chromatography, using locust gum as an absorbent, and D-mannose as eluant. Final purification was achieved by Bio-Gel P300 gel filtration. The agglutinin is a protein composed of two polypeptide chains with a Mr of 14800; the two subunits are not linked by disulfide bond(s). The isoelectric point is 4.8, the amino acid composition is rich in the acidic amino acids aspartic acid and glutamic acid. The absorption maximum for the protein was at 276 nm; with a molar absorption coefficient of 1.27 X 10(5) M-1 cm-1. The lectin precipitated erythrocytes from humans (A, B and O), sheep, rabbit and carp with a titer between 2(5) and 10(10); the affinity constant for lectin binding to sheep red blood cells was 2.8 X 10(8) M-1 and the number of binding sites, 3.2 X 10(5)/cell. Ca2+ ions are required for full activity; the pH optimum lies in the range between 6 and 11. Inhibition experiments revealed that the lectin is specific for D-mannose. The lectin is mitogenic only for those spleen lymphocytes from mice which had been activated by lipopolysaccharide. An interesting feature of this lectin is its ability to bind to glycoproteins present in nuclei from CV-1 monkey kidney cells. The fluorescein-isothiocyanate-labelled lectin reacted with six polypeptides in the nuclear envelope from rat liver (Mr 190,000, 115,000, 80,000, 62,000, 56,000 and 42,000) and with two polypeptides in the nuclear matrix or pore complex lamina fraction (Mr 190,000 and 62,000). The lectin inhibited the nuclear envelope mRNA translocation system in vitro. It is suggested that this effect is due to an interaction of the lectin with the nuclear glycoproteins gp190 and/or gp62.     

An anti-A1 lectin in the seeds of Amphicarpaea bracteata

An anti-A1 lectin has been isolated from the extract of Amphicarpaea bracteata seeds by affinity chromatography on Epoxy-activated Sepharose 6B coupled to N-acetyl-D-galactosamine. The yield of the purified lectin was 86 microgram/g of seeds. The purified lectin shows one main band on electrophoresis in sodium dodecyl sulfate-polyacrylamide. The amino acid and neutral sugar composition indicate that this lectin is an acidic glycoprotein with a neutral sugar content of approx. 2%. The composition of the lectin is different from that of the Dolichos biflorus lectin but the two lectins have some common characteristics. The most powerful inhibitors of the agglutination of A1 red blood cells by the A. bracteata lectin is N-acetyl-D-galactosamine. Much weaker inhibitors of the agglutination are alpha-lactose, D-fucose, and five other sugars.     

Structure-function relationship of islet-activating protein, pertussis toxin: biological activities of hybrid toxins reconstituted from native and methylated subunits

Islet-activating protein (IAP), pertussis toxin, is a hexameric protein composed of an A protomer and a B oligomer, the residual pentamer having such a subunit assembly that two different dimers, dimer 1 and dimer 2, are connected with each other by means of the smallest C subunit. Incubation of IAP with formaldehyde and pyridine-borane produced the modified toxin in which most of the free amino groups were dimethylated. The methylated and nonmethylated (native) IAP were disintegrated into their respective constituent components, which were then cross combined to reconstitute hybrid toxins with the original hexameric structure. The binding of the B oligomer to the mammalian cell surface via dimer 2 was, but the binding via dimer 1 was not, seriously impaired by methylation of amino groups in the protein. The binding of the B oligomer allowed the A protomer to enter cells and to catalyze ADP-ribosylation of a membrane Mr 41 000 protein. The diverse biological activities of IAP occurring by this mechanism were mimicked by not only methylated IAP but also all hybrid toxins, indicating that the free amino groups in the protein were not essential for the enzyme activity of the A protomer and that the A protomer was able to enter cells if the B oligomer bound to cells "monovalently" via dimer 1. An additional effect of the B oligomer binding, i.e., the direct stimulation, without the transport of the A protomer, of cells leading to mitosis in lymphocytes in vitro or increases in circulating lymphocytes in vivo, was not mimicked by hybrid toxins containing methylated dimer 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence of a full-length cDNA for rat lung beta-galactoside-binding protein: primary and secondary structure of the lectin

A full-length cDNA for rat lung beta-galactoside lectin (subunit Mr approximately 14,000, lectin 14K) was cloned and the nucleotide sequence determined. The deduced amino acid sequence agrees with the amino acid composition and direct amino acid sequence analysis of purified rat lung lectin peptides. We found that the amino-terminal alanine is blocked with an acetyl group. Comparison of the amino acid sequence with other proteins shows a high degree of homology only with other vertebrate lectin sequences, supporting the suggestion that these lectins may constitute a unique class of vertebrate proteins. The amino acid composition and sequence of lectin peptides, the sequence of lectin cDNA, and isoelectric focusing of purified lectin indicate that rat lung lectin 14K is composed predominantly of a single protein. In addition, rat uterus lectin 14K was found to be the same protein as that present in lung. We characterized the secondary and tertiary structure of rat lung lectin 14K by circular dichroism, by analytical ultracentrifugation, and by computer analysis of its primary structure. Results of these experiments suggest that lectin 14K is primarily a hydrophilic protein with an asymmetric, elongated structure consisting of approximately equal amounts of alpha helix, beta sheet, beta turn, and random coil. We found that Cys-2 and Cys-130 react most rapidly with iodoacetamide; one or both of these residues may be primarily responsible for the thiol requirement of lectin activity.     

Purification,some properties of a D-galactose-binding leaf lectin from Erythrina indica and further characterization of seed lectin

Lectin from a leaf of Erythrina indica was isolated by affinity chromatography on Lactamyl-Seralose 4B. Lectin gave a single band in polyacrylamide gel electrophoresis (PAGE). In SDS-gel electrophoresis under reducing and non-reducing conditions Erythrina indica leaf lectin (EiLL) split into two bands with subunit molecular weights of 30 and 33 kDa, whereas 58 kDa was obtained for the intact lectin by gel filtration on Sephadex G-100. EiLL agglutinated all human RBC types, with a slight preference for the O blood group. Lectin was found to be a glycoprotein with a neutral sugar content of 9.5%. The carbohydrate specificity of lectin was directed towards D-galactose and its derivatives with pronounced preference for lactose. EiLL had pH optima at pH 7.0; above and below this pH lectin lost sugar-binding capability rapidly. Lectin showed broad temperature optima from 25 to 50 degrees C; however, at 55 degrees C EiLL lost more than 90% of its activity and at 60 degrees C it was totally inactivated. The pI of EiLL was found to be 7.6. The amino acid analysis of EiLL indicated that the lectin was rich in acidic as well as hydrophobic amino acids and totally lacked cysteine and methionine. The N-terminal amino acids were Val-Glu-Thr-IIe-Ser-Phe-Ser-Phe-Ser-Glu-Phe-Glu-Ala-Gly-Asn-Asp-X-Leu-Thr-Gln-Glu-Gly-Ala-Ala-Leu-. Chemical modification studies of both EiLL and Erythrina indica seed lectin (EiSL) with phenylglyoxal, DEP and DTNB revealed an absence of arginine, histidine and cysteine, respectively, in or near the ligand-binding site of both lectins. Modification of tyrosine with NAI led to partial inactivation of EiLL and EiSL; however, total inactivation was observed upon NBS-modification of two tryptophan residues in EiSL. Despite the apparent importance of these tryptophan residues for lectin activity they did not seem to have a direct role in binding haptenic sugar as D-galactose did not protect lectin from inactivation by NBS.     

Purification and characterization of a mitogenic lectin and a lectin-binding protein from Vicia sativa

From the seeds of Vicia sativa, a novel mitogenic lectin was isolated. Purification was carried out by affinity chromatography on Sephadex G-100. The tetrameric lectin is a glycoprotein with a molecular weight of Mr 40 000; it consists of two large beta-subunits (Mr 14 000) and two small alpha-subunits (Mr 6000). The N-terminal sequence of both subunits and their amino acid compositions were determined. The lectin agglutinates human erythrocytes, preferring group B, and erythrocytes from rabbits and horses; no agglutination takes place with sheep erythrocytes. Agglutination is inhibited by mono-, di- and tri-saccharides with the configuration of glucose at the free 4-hydroxyl group. The lectin stimulates mitosis in lymphocytes of mice. From the seeds of the same plant, a protein was isolated which binds to the lectin described above. The lectin binder consists of subunits with a molecular weight of 53 500.     

The primary structure of coagulation factor IX/factor X-binding protein isolated from the venom of Trimeresurus flavoviridis. Homology with asialoglycoprotein receptors, proteoglycan core protein, tetranectin, and lymphocyte Fc epsilon receptor for immunoglobulin E

An anticoagulant protein, factor IX/factor X-binding protein (IX/X-bp), isolated from the venom of Trimeresurus flavoviridis, binds with factor IX and factor X in the presence of Ca2+ with a 1 to 1 stoichiometry (Atoda, H., and Morita, T. (1989) J. Biochem. (Tokyo) 106, 808-813). Analysis of S-pyridylethylated IX/X-bp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a 16.0-kDa band (designated the A chain) and a 15.5-kDa band (designated the B chain). These two chains were separated by reversed-phase high performance liquid chromatography, and their complete amino acid sequences were determined by sequencing of the peptides obtained after digestion with lysyl endopeptidase, chymotrypsin, and V8 protease from Staphylococcus aureus and after chemical cleavage with cyanogen bromide. The A chain had an amino-terminal sequence of Asp-Cys-Leu-Ser-Gly- and consisted of 129 residues with Mr 14,830. The B chain has an amino-terminal sequence of Asp-Cys-Pro-Ser-Asp- and consists of 123 residues of Mr 14,440. There was 47% identity between the A and the B chain. The sequence of IX/X-bp showed 25-37% identity with that of the C-type carbohydrate recognition domain-like structure of acorn barnacle lectin, human and rat asialoglycoprotein receptors, the human lymphocyte Fc epsilon receptor for immunoglobulin E, proteoglycan core protein, pancreatic stone protein, and tetranectin. The sequences of the first 18 amino acid residues of both the A and B chains were also, to a certain extent, homologous to the partial amino acid sequence of the b subunit of factor XIII, a member of the beta 2-glycoprotein I-like family. In this region, some similarity with the amino-terminal amino acid sequence of botrocetin was also observed.     

Lectin-associated proteins from the seeds of Leguminosae

The seeds of Pisum sativum (pea), Canavalia ensiformis, Vicia faba, Vicia sativa, and Ricinus communis were shown to contain proteins which are associated to the respective lectins (lectin binders). The lectin binders from Pisum sativum and Canavalia ensiformis were studied more closely. Both are single proteins not resembling the variety of membrane glycoproteins found in animals and plants which bind to lectins. The pea lectin binder is a tetrameric glycoprotein composed of identical subunits of the Mr 51 000. Its interaction with the lectin is abolished by acidic buffers or by glucose. The Concanavalin A binder, which does not contain sugar, is composed of one kind of subunit, Mr of 35 000. As in the case of the pea lectin binder, glucose and acid dissociate the lectin-lectin binder complex, but in contrast to the pea lectin binder low NaCl concentrations also cause this effect. During germination and growth, the Concanavalin A binder appears in the roots.     

The amino-acid sequence of multiple lectins of the acorn barnacle Megabalanus rosa and its homology with animal lectins

The amino-acid sequence of a lectin isolated from the coelomic fluid of the acorn barnacle Megabalanus rosa has been determined. The lectin (Mr 140,000) is a multimeric protein whose subunit consists of 173 amino acids and one carbohydrate chain attached to Asn-39. The amino-acid sequence was determined by the manual sequencing of peptides derived from the protein by digestion with Staphylococcus aureus V8 proteinase, lysine endopeptidase and chymotrypsin, as well as fragments produced by cleavage with cyanogen bromide. The amino-acid sequence of the lectin was compared with the sequence of one (Mr 64,000) of the multiple lectins of M. rosa. They are distinct molecules in spite of a significant homology in their amino-acid sequences. The amino-acid sequence includes some regions homologous to those in other invertebrate lectins, such as sea urchin and flesh fly lectins, and vertebrate lectins. This is the first report to show the amino-acid sequence of multiple lectins isolated from an invertebrate.     

Isolation and characterization of lectins from Vicia villosa. Two distinct carbohydrate binding activities are present in seed extracts

An uncharacterized lectin from Vicia villosa seeds has been reported to bind specifically to mouse cytotoxic T lymphocytes (Kimura, A., Wigzell, H., Holmquist, G., Ersson, B., and Carlsson, P., (1979) J. Exp. Med. 149, 473-484). We have found that V. villosa seeds contain at least three lectins which we have purified by affinity chromatography on a column of immobilized porcine blood group substances eluted with varying concentrations of N-acetylgalactosamine and by anion exchange chromatography. The three lectins are composed of two different subunits with Mr = 35,900 (subunit B) and 33,600 (subunit A), estimated from their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sedimentation equilibrium analysis suggests that the purified lectins are tetramers. They have been designated B4, A4, and A2B2 to indicate their apparent subunit compositions. The purified B4 and A4 lectins contain 6.7-9.8% carbohydrate by weight; in addition, both are rich in the acidic and hydroxylic amino acids and lack cysteine and methionine. The A4 lectin agglutinates A erythrocytes specifically and binds to A1 erythrocytes (273,000 sites/cell) with an association constant of 1.8 X 10(7) M-1. Although a blood group A agglutinating activity was recognized in the original preparation of V. villosa lectins, lectins with this activity were obtained in relatively small amounts from seed extracts. The predominant lectin in V. villosa seeds, B4, does not agglutinate A, B, or O erythrocytes.