Alpha-galactoside-binding isolectins from wild jack fruit seed (Artocarpus hirsuta): purification and properties

Five isolectins with marked specificity for alpha-linked galactose were purified from the wild jack (Artocarpus hirsuta) seeds by affinity chromatography on cross-linked guar gum. They were composed of a glycosylated subunit A (Mr = 16 kDa) and a nonglycosylated subunit B (Mr = 11 kDa) in noncovalent association. The isolectins which eluted as a single peak of Mr 45 kDa on gel filtration in Biogel P-100 and in a TSK G-3000 SW high pressure column, were resolved into five peaks on electrophoresis at pH 4.5. Sodium dodecyl sulphate polyacrylamide gel electrophoreogram of the major isolectin band suggested that the isolectins may be the five possible tetrameric combinations of A and B subunits. The combined isolectins bound only two molecules of 4-methyl umbelliferyl alpha-D-galactoside with a binding constant of 4.75 x 10(4) M-1. The pH optimum of sugar binding was 7.0. The isolectins specifically bound to human IgG and IgA but not to IgM.     

Purification and characterization of a lectin from the mushroom, Flammulina veltipes

A lectin was purified to homogeneity from the mushroom, Flammulina veltipes, by zinc acetate treatment and CM-cellulose column chromatography. Its molecular weight was estimated to be 20,000 by gel filtration and polyacrylamide gel electrophoresis. The lectin does not contain carbohydrate, half-cystine, methionine, or histidine. On gel filtration sith Sepharose 6B in the presence of 6M guanidine-HCl, the purified lectin dissociated into two nonidentical subunits, FVA-L (molecular weight, 12,000) and FVA-S (8,000). The hemagglutinating activity was retained only in the FVA-L subunit. The lectin is mitogenic with respect to mouse spleen lymphocytes.     

Physical-chemical characterization and carbohydrate-binding activity of the A and B subunits of the Bandeiraea simplificolia I isolectins.

Bandeiraea simplicifolia I plant seed isolectins comprise a family of tetrameric alpha-D-galactopyranosyl-binding glycoproteins composed of various combinations of teo different kinds of subunits designated A and B. Subtypes of the A (Aa, Ab, Ac, Ad, and Ae) and B (Ba, Bb, Bc, Bd, and Be) subunits were demotypes varies from seed to seed (e.g., some seeds contain only B subunits, others only A subunits), subtypes Ac and Bc predominate in a natural mixture of the isolectins. Two-dimensional agar gel diffusion studies indicate that, in addition to common structural features, each subunit contains its own distinct antigenic determinants. Although the A and B subunits have closely similar amino acid compositions, they differ markedly in one respect: the B subunit has one methionine residue whereas the A subunit contains no methionine. The neutral carbohydrate content of both subunits is identical. The ability of biopolymers and synthetic glycoproteins to precipitate A4 and B4, as well as the capacity of sugars and oligosaccharides to inhibit precipitate formation, was examined. On the basis of these studies, it is suggested that hydrogen bonding occurs between the hydrogen atoms of the C-3 and C-4 hydroxyl groups of alpha-D-GalNAcp and alpha-D-galp units and the A and B subunits, respectively.     

Purification and characterization of a lectin (plant hemagglutinin) with N blood group specificity from Vicia graminea seeds.

A lectin with N blood group specificity was isolated from Vicia graminea seeds. This lectin was purified from a crude extract by precipitation with ammonium sulfate, DEAE-cellulose chromatography and Sephadex G-150 gel filtration. Purification steps were followed by increase of specific activity. Its homogeneity was demonstrated by polyacrylamide gel electrophoresis, immunoelectrophoresis, electrofocusing and ultracentrifugation. This lectin is an acid glycoprotein with 7.3% carbohydrate, a high percentage of serine and contains no sialic acid. The native lectin has a molecular weight about 100 000 and dissociates into four subunits of 25 000 as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preliminary hemagglutination inhibition has shown that the lectin was not inhibited by any of the monosaccharides contained in N blood group substances; however it was inhibited by the erythrocyte membrane major glycoprotein and the tryptic fragments obtained from erythrocytes.     

粘虫幼虫血淋巴中的凝集素

粘虫Mythlmna separata Walker幼虫血淋巴中含有凝集某些脊椎动物红细胞的凝集素,凝集活性可被乳糖、岩藻糖或神经氨酸抑制.用CNBr-sepharose 4B 进行亲和层析从血淋巴中分离的凝集素成分比较复杂,聚丙烯酰胺凝胶电泳显示三条区带,SDS聚丙烯酰胺凝胶电泳出现6个亚基,亚基分子量分别为71000、65000、56000、35000、33000及31000道尔顿.     

Further characterization of a lectin and its in vivo receptor from Geodia cydonium

From the results of two-dimensional isoelectric focusing, SDS-gel electrophoresis and from immunochemical data it became evident that lectin I and lectin II (corresponding to fractions Geodia I and Geodia II isolated on immobilized lactose) from the sponge Geodia cydonium are apparently identical mixtures of several isolectins, the pI values of their subunits ranging, in contrast to our previous report, from 4.8–7.5. The hypothetical concept of sugar-mediated, specific lectin-lectin interactions (self-recognition) could not be verified by binding of FITC-labelled isolectins (Geodia I) to the lectin subunits, which had been purified by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The concept should also be dismissed on the basis of carbohydrate analyses revealing in contradiction with previous results the exclusive presence of alkali-labile bound tetraglucose on the purified isolectins (1 mol/mol lectin protein). The combining site of the isolectins was shown by a quantitative microprecipitation inhibition assay to be most complementar to oligosaccharides of the β-galactoside series and to interact specifically with particular structural elements of the subterminal sugar(s). Carbohydrates of the anti aggregation receptor, which are assumed to represent the functional ligand of the Geodia-isolectins in vivo, could be demonstrated to have a high affinity for the lectin combining site, exceeding that of the best disaccharide inhibitor, lactose, by five orders of magnitude. A preliminary chemical characterization of the receptor carbohydrate revealed that D-galactose and D-glucose (each approx. 200 mol/mol receptor) are organized in an oligosaccharide, which could be cleaved from the protein by trifluoroacetolysis.     

Isolation and characterization of a group of isolectins with galactose/N-acetylgalactosamine specificity from hemolymph of the giant silk moth Hyalophora cecropia

A series of isolectins from Hyalophora cecropia was purified by affinity chromatography on d-GalNAc-Sepharose. The yield of the lectin was 0.4 mg/ml of hemolymph and the concentration was about the same in larvae and pupae. The molecular weight of the lectin was around 160,000 and the sizes of the subunits were 41 and 38 kD for the A and the B chains, respectively. The isolectins are believed to be tetramers with varying proportions between the two subunits. Inhibition studies indicate that the A chain has a binding site with specificity for d-Gal/d-GalNAc while the B chain has specificity for an unknown structure on rabbit erythrocytes not necessarily of carbohydrate nature. A d-Gal/d-GalNAc specific lectin was found also in Anthereae pernyi but the yield was only 0.03 mg/ml of hemolymph.     

A structural comparison of the A and B subunits of Griffonia simplicifolia I isolectins

A structural comparison between the A and B subunits of the five tetrameric Griffonia simplicifolia I isolectins (A4, A3B, A2B2, AB3, B4) was undertaken to determine the extent of homology between the subunits. The first 25 N-terminal amino acids of both A and B subunits were determined following the enzymatic removal of N-terminal pyroglutamate blocking groups with pyroglutamate aminopeptidase. Although 21 amino acids were common to both subunits, there were four unique amino acids in the N-terminal sequence of A and B. Residues 8, 9, 17, and 19 were asparagine, leucine, lysine, and asparagine in subunit A and threonine, phenylalanine, glutamic acid, and serine in subunit B. The last six C-terminal amino acids, released by digestion with carboxypeptidase Y, were the same for both subunits: Arg-(Phe, Val)-Leu-Thr-Ser-COOH. Subunit B, which contains one methionyl residue, was cleaved by cyanogen bromide into two fragments, a large (Mr = 31,000) and a small (Mr = 2700) polypeptide. Failure of the small fragment to undergo manual Edman degradation indicated an N-terminal blocking group, presumably pyroglutamate. Both subunits were digested with trypsin and the tryptic peptides were analyzed using reverse-phase HPLC. Tryptic glycopeptides were identified by labeling the carbohydrate moiety of the A and B subunit using sodium [3H] borohydride. Cysteine-containing tryptic peptides were similarly identified by using [1-14C]iodoacetamide. Approximately 30% of the tryptic peptides were common to both subunits. Thus, although the N- and C-terminal regions of A and B are similar, the subunits each possess unique sequences.     

Isolectins from Dictyostelium purpureum. Purification and characterization of seven functionally distinct forms

Purpurin, the lectin from Dictyostelium purpureum, has been resolved into seven tetrameric isolectins by polyacrylamide gel electrophoresis at pH 8.9. The isolectins are assembled from four distinct subunits resolved by electrophoresis in sodium dodecyl sulfate and by tryptic peptide mapping. Two of the subunits combine randomly with each other to form mixed tetramers (I4, I3II1, I2II2, I1II3, II4) in binomial proportions. The other two subunits (III and IV) form only homotetramers. The isolectins can be functionally discriminated and separated on the basis of their relative affinities for columns derivatized with complementary saccharides. On the basis of relative sensitivity to hapten inhibitors of hemagglutination, isolectins III4 and IV4 are distinct from each other and from isolectins composed of subunits I and II. However, isolectins of I and II are not distinguishable on the basis of hemagglutination inhibition. None of the subunits are glycosylated, and all form tetramers with molecular weights of approximately 88,000. The existence of multiple functionally distinct forms suggests that lectin function in cellular slime molds may be more complex than presently envisioned.     

Purification and characterization of N-acetyl-D-galactosamine-binding lectin from Falcata japonica

An N-acetyl-D-galactosamine-binding lectin from Falcata japonica seeds was purified by affinity column chromatography of N-acetyl-D-galactosamine coupled to epoxy-activated Sepharose 6B. A 1000-fold purification of lectin was obtained from the crude extracts. The purified lectin agglutinated blood group A red cells, but neither blood group B nor O red cells. Polyacrylamide gel electrophoresis of the lectin showed one diffuse band. Molecular weights of 125,000 and 117,000 were estimated by gel filtration and ultracentrifugal analysis, respectively. SDS-polyacrylamide gel electrophoresis of the lectin also showed a single band which has a molecular weight of 34,000. Therefore, the lectin molecule was estimated to be a tetramer composed of four identical non-covalently bound subunits. F. japonica lectin was a glycoprotein containing 5% total carbohydrate, and the amino acid composition was characterized by a high content of aspartic acid, serine and glycine, a low content of methionine and the absence of cysteine.     

Purification and characterization of a human lectin specific for penultimate galactose residues

A novel lectin has been found in human plasma. The lectin was purified by affinity chromatography using an adsorbent in which 2-O-alpha-D-glucopyranosyl-O-beta-D-galactopyranosylhydroxylysine (Glc-Gal-Hyl) was coupled to Sepharose. The molecular weight of the lectin was determined by gradient gel electrophoresis to be approximately 240,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulphate, the subunit had the molecular weight of 29,500. Composition analysis has shown the lectin is a glycoprotein in which 12% of the molecule consists of carbohydrate. Native human, horse, calf, sheep, rabbit, and rat erythrocytes were agglutinated by the lectin in the presence of calcium. Glc-Gal-Hyl, N-acetylated Glc-Gal-Hyl, and stachyose inhibited the hemagglutination, whereas monosaccharides, maltose, cellobiose, lactose, raffinose, galactosylhydroxylysine, and N-acetylated galactosylhydroxylysine were not inhibitory. The lectin is strongly inhibited by the desialylated bovine erythrocyte glycoprotein, which contains galactose beta 1-3galactose beta-sequence at the nonreducing termini of the sugar chains, whereas disialylated orosomucoid did not inhibit the lectin. These results indicate that the lectin recognizes the penultimate galactose residue in a hapten molecule in contrast to usual galactose-binding proteins or galactose-specific lectins, which recognize exposed, terminal galactose residues of sugar chains.     

Studies on hemagglutinins (lectins) from plasma of murrel fish, family Channidae

Hemagglutinating activity can be identified in the plasma of different species of murrel fish. This activity may be divided into four types according to their agglutinability towards erythrocytes from different sources. Type I plasma agglutinates human blood group A erythrocytes, type II can agglutinate neuraminidase treated human A B O erythrocytes, type III shows no agglutinating activity towards human erythrocytes, while type IV agglutinates human erythrocytes non-specifically. All of them bind to DEAE-cellulose but elute out by different salt concentrations. Type IV plasma is found to be a combination of three separate hemagglutinins, which are separable by sequential binding to human A B O erythrocytes. Blood group A specific lectin activity is purified from this plasma using formalinised A group erythrocytes. The apparent homogeneity of this purified lectin is established by polyacrylamide gel electrophoresis, isoelectric focusing and immunodiffusion. This agglutinin is antigenically identical with that isolated from type I plasma by affinity chromatography on N-acetyl-D-galactosamine coupled to epoxy-activated cellulose column. Their molecular weights are also found to be identical (Mr 140,000) in polyacrylamide gel electrophoresis, having two identical subunits. Forssman glycolipid (0.03 mM) was found to be the most potent inhibitor of agglutination, although Gal beta 1-3 GalNAc (0.09 mM) is also a good inhibitor. Exhaustive dialysis of the purified lectin (hemagglutinin) against EDTA denatures it irreversibly by dissociating it to its subunit structure. Thus human A group agglutinating activity isolated from type I and type IV plasma are identical.     

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。     

Purification and characterization of a lectin from the beetle, Allomyrina dichotoma

A lectin was purified from the hemolymph of Allomyrina dichotoma larvae by affinity chromatography on acid-treated Sepharose 4B. The purified lectin showed two protein bands on polyacrylamide gel electrophoresis. These two lectin bands (allo A-I and -II) were separated by DEAE-Cellulofine column chromatography. By gel filtration on Sephadex G-100, the molecular weights of allo A-I and -II were estimated to be 65,000 and 66,500, respectively. On the other hand, by SDS-polyacrylamide gel electrophoresis after cross-linking of subunits with glutaraldehyde, they are estimated to be 38,000 and 39,000, respectively. On SDS-polyacrylamide gel electrophoresis, it was proved that both allo A-I and -II lectin consisted of two subunits, respectively. The molecular weights were 17,500 and 20,000 for allo A-I, and 19,000 and 20,000 for allo A-II. The isoelectric points of allo A-I and -II were estimated to be 6.4 and 5.9, respectively. On double immunodiffusion, allo A-I and -II gave single precipitin lines, which fused completely with each other, against the antibody to crude allo A. The hemagglutinating activity of allo A-I and -II was inhibited only by beta-linked D-galactose such as lactose and lactulose.     

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.     

Glycolipid-lectin interactions: detection by direct binding of 125I-lectins to thin layer chromatograms

Glycolipids that bind 125I-labeled lectins are detected by autoradiography after thin layer chromatography of glycolipid standards or crude lipid extracts. Soybean agglutinin, Bandeiraea simplicifolia I isolectins A4 and B4, and Helix pomatia lectin are used to detect corresponding cell surface, glycolipid receptors in human and bovine erythrocytes. When lipid extracts from A and AB erythrocyte stroma are analyzed with Helix pomatia lectin, a polymorphic expression of blood group A glycolipid determinants is detected. The Bandeiraea simplicifolia isolectins react weakly with human erythrocyte glycolipids but bind at least 4 glycolipids in bovine stroma extracts. Soybean agglutinin reacts with glycolipids in all erythrocytes analyzed. This technique extends lectin specificity studies from inhibition analyses in aqueous systems using available, known structures to identification of specific, lectin-binding glycolipids in crude lipid extracts of cell membranes.     

Isolation and characterization of a lectin from edible mushroom, Volvariella volvacea

A lectin was purified from edible mushroom, Volvariella volvacea by extraction with 5% cold acetic acid in the presence of 0.1% 2-mercaptoethanol, followed by ammonium sulfate fractionation, and DEAE-C-52 and CM-C-52 column chromatographies. The molecular weight was measured to be 26,000, and the lectin consisted of two non-identical subunits as demonstrated by gel filtration and polyacrylamide gel electrophoresis. The lectin does not contain half-cystine, methionine, or histidine. The LD50 of the lectin is 17.5 mg per kg body weight of mice. The lectin has a moderate inhibitory effect on the growth of tumor cells.     

Purification and characterization of two types of Cytisus sessilifolius anti-H(O) lectins by affinity chromatography.

Two anti-H(O) lectins were separated from extracts of Cytisus sessilifolius seeds by successive affinity chromatographies on columns of di-N-acetylchitobiose- and galactose-Sepharose 4B. One was found to be inhibited most by di-N-acetylchitotriose or tri-N-acetylchitotriose [Cytisus-type anti-H(O) lectin designated as Cytisus sessilifolius lectin I (CSA-I)] and the other anti-H(O) lectin was inhibited by galactose or lactose and designated as Cytisus sessilifolius lectin II (CSA-II). These two anti-H(O) lectins were further purified by gel filtration on TSK-Gel G3000SW. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular masses of the purified lectins I and II were found to be 95,000 and 68,000 Da, respectively, by gel filtration on TSK-Gel G3000SW. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol, both lectins gave a single component of molecular masses of 27,000 +/- 2,000 and 34,000 +/- 2,000 Da, respectively, suggesting that the lectins I and II were composed of four and two apparently identical subunits, respectively. Lectins I and II contain 38% and 13% carbohydrate, respectively, and only very small amounts of cysteine and methionine, but they are rich in aspartic acid, serine and glycine. The N-terminal amino-acid sequences of these two lectins were determined and compared with those of several lectins already published.     

Purification and characterization of a new type lactose-binding Ulex europaeus lectin by affinity chromatography

A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.     

Characterization of the carbohydrate moiety of Clerodendron trichotomum lectins. Its structure and reactivity toward plant lectins

Lectins were isolated from fruits and leaves of Clerodendron trichotomum by affinity chromatography on lactamyl-Sepharose. The purified lectins (C. trichotomum agglutinin: CTA) were homogeneous on SDS/polyacrylamide gel electrophoresis, and the carbohydrate moiety was characterized by physicochemical and immunochemical methods. The asparagine-linked oligosaccharides were released by treatment with N-oligosaccharide glycopeptidase (almond, EC 3.5.1.52) of peptic glycopeptides obtained from fruit CTA, and separated by gel filtration and thin-layer chromatography. The structure of the predominant oligosaccharide was determined as Xyl beta 1----2 (Man alpha 1----6)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc by high-performance liquid chromatography, sugar analysis and 1H-NMR spectroscopy. The reactivity of the carbohydrate moiety of CTA toward various lectins was studied. Fruit and leaf CTAs were applied to polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets and detected with horseradish-peroxidase-conjugated lectins. Concanavalin A, lentil lectin, pea lectin, Vicia faba lectin and Ulex europeus agglutinin I, but not wheat germ lectin, bound to fruit CTA. The results indicate new binding properties of these plant lectins: a beta-xylosyl residue substituted at C-2 of the beta-mannosyl residue of N-linked oligosaccharide does not affect the binding with mannose-specific lectins, lentil, pea and Vicia faba lectins can bind to N-linked oligosaccharides containing an alpha-L-fucosyl residue attached to C-3 of the asparagine-linked N-acetyl-D-glucosamine residue, and Ulex europeus agglutinin I can bind to the (alpha 1----3)-linked fucose residue of the N-linked oligosaccharide.