Five alpha-D-galactopyranosyl-binding isolectins from Bandeiraea simplicifolia seeds.

The alpha-D-galactopyranosyl-binding lectin previously purified from Bandeiraea simplicifolia seeds (Hayes, C.H., and Goldstein, I.J. (1974) J. Biol. Chem. 249, 1904) is shown to consist of five isolectins separable on polyacrylamide gel electrophoresis at pH 9.5. The isolectins are tetrameric structures composed of various combinations of two different glycoprotein subunits designated A and B. The A and B subunits appear to be immunochemically indistinguishable against rabbit antisera prepared from the isolectin mixture. The A subunit contains no methionine, whereas the B subunit contains 1 residue. The subunits migrate differently on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, although each subunit contains 1 residue of cysteine, they react differently toward 5,5'-dithiobis(2-nitrobenzoic acid). The carbohydrate binding specificity of the two subunits differs significantly: the A subunit exhibits a primary specificity for alpha-D-GalNAcp but also reacts with alpha-D-Galp units, whereas the B subunit shows a sharp specificity toward alpha-D-Galp residues. The differences in carbohydrate binding specificity were exploited in separating the isolectins. B. simplicifolia I isolectins (A4) and (A3B) were purified on a Bio-Gel melibionate column, and (A2B2), (AB3), and (B4) were separated on a column of insolubilized blood group A substance.     

A simple procedure for the isolation of l-fucose-binding lectins from Ulex europaeus and Lotus tetragonolobus

l-Fucose-binding lectins from Ulex europaeus and Lotus tetragonolobus were isolated by affinity chromatography on columns of l-fucose-Sepharose 6B. l-Fucose was coupled to Sepharose 6B after divinyl sulfone-activation of the gel to give an affinity adsorbent capable of binding more than 1.2 mg of Ulex lectin/ml of gel, which could then be eluted with 0.1M or 0.05M l-fucose. Analysis of the isolated lectins by hemagglutination assay, by gel filtration, and by polyacrylamide discelectrophoresis revealed the presence of isolectins, or aggregated species, or both. The apparent mol. wt. of the major lectin fraction from Lotus was 35 000 when determined on Sephadex G-200 or Ultrogel AcA 34. In contrast, the apparent mol. wt. of the major lectin fraction from Ulex was 68 000 when chromatographed on Sephadex G-200 and 45 000 when chromatographed on Ultrogel AcA 34. The yields of lectins were 4.5 mg/100 g of Ulex seeds and 394 mg/100 g of Lotus seeds.     

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.     

Lectins from<Emphasis Type="Italic">Griffonia simplicifolia</Emphasis> seeds

The physical-chemical and carbohydrate binding specificity ofGriffonia simplicifolia I (GS I) isolectins, one of the 4 lectins isolated fromGriffonia simplicifolia seeds, are described.Association constants for the binding of methyl α- and β-D-galactopyranoside and methyl 2-acetamido-2-deoxy-α-D-galactopyranoside to the A₄, A₂ B₂ and B₄ isolectins are reported.Precipitation reactions of theGriffonia simplicifolia isolectins with guaran and type B blood group substance are described.The hypothesis that subunit B is a precursor of subunit A, a process involving proteolytic cleavage of the B subunit, was tested by conducting structural studies on the 2 subunits. The results indicated that the A and B subunits are probably products of 2 separate but closely related, possibly contiguous genes.     

Distribution of glucose/mannose-specific isolectins in pea (Pisum sativum L.) seedlings

We report on the distribution and initial characterization of glucose/mannose-specific isolectins of 4- and 7-d-old pea (Pisum sativum L.) seedlings grown with or without nitrate supply. Particular attention was payed to root lectin, which probably functions as a determinant of host-plant specificity during the infection of pea roots by Rhizobium leguminosarum bv. viciae. A pair of seedling cotyledons yielded 545±49 g of affinity-purified lectin, approx. 25% more lectin than did dry seeds. Shoots and roots of 4-d-old seedlings contained 100-fold less lectin than cotyledons, whereas only traces of lectin could be found in shoots and roots from 7-d-old seedlings. Polypeptides with a subunit structure similar to the precursor of the pea seed lectin could be demonstrated in cotyledons, shoots and roots. Chromatofocusing and isoelectric focusing showed that seed and non-seed isolectin differ in composition. An isolectin with an isoelectric point at pH 7.2 appeared to be a typical pea seed isolectin, whereas an isolectin focusing at pH 6.1 was the major non-seed lectin. The latter isolectin was also found in root cell-wall extracts, detached root hairs and root-surface washings. All non-seed isolectins were cross-reactive with rabbit antiserum raised against the seed isolectin with an isolectric point at pH 6.1. A protein similar to this acidic glucose/mannose-specific seed isolectin possibly represents the major lectin to be encountered by Rhizobium leguminosarum bv. viciae in the pea rhizosphere and at the root surface. Growth of pea seedlings in a nitrate-rich medium neither affected the distribution of isolectins nor their hemagglutination activity; however, the yield of affinity-purified root lectin was significantly reduced whereas shoot lectin yield slightly increased. Agglutination-inhibition tests demonstrated an overall similar sugar-binding specificity for pea seed and non-seed lectin. However root lectin from seedlings grown with or without nitrate supplement, and shoot lectin from nitrate-supplied seedlings showed a slightly different spectrum of sugar binding. The absorption spectra obtained by circular dichroism of seed and root lectin in the presence of a hapten also differed. These data indicate that nutritional conditions may affect the sugar-binding activity of non-seed isolectin, and that despite their similarities, seed and non-seed isolectins have different properties that may reflect tissue-specialization.Abbreviations IEF isoelectric focusing - MW molecular weight - pI isoelectric point - Psl1, Psl2 and Psl3 pea isolectins - SDSPAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis The authors wish to thank Professors L. Kanarek and M. van Poucke for helpful discussions.     

A single step purification of a sialic acid binding lectin (AchatininH) from Achatina fulica snail

A sialic acid binding lectin, Achatinin_H, was purified in single step from the hemolymph of the land snail, Achatina fulica, by the affinity chromatography on sheep submaxillary mucin coupled to Sepharose 4B. The yield of the lectin was found to be 3 mg from 100 ml of hemolymph. The homogeneity of the lectin was established by alkaline gel electrophoresis, immunodiffusion, immunoelectrophoresis and analytical isoelectrophoresis. The molecular weight of the native protein was 242000, having identical subunits of Mr 15000. The lectin agglutinated rabbit erythrocytes in the presence of Ca^2–. The inhibition study clearly suggests that the binding site of the lectin recognizes sialic acid as the immunodominant sugar. This was further confirmed by the observation that there was a marked decrease of agglutinating activity of the lectin with neuraminidase treated rabbit erythrocytes and asialofetuin was unable to inhibit the activity of Achatinin_H. Among the inhibitors used the glycoconjugate containing 2-6 linkages of N-acetylneuraminic acid with subterminal galactopyranose or 2-acetamido-2-deoxy-galactopyranose residue was found to be better inhibitor than that containing 23 linkages of N-acetyl neuraminic acid. Besides that sialoglycoprotein containing both N and O type of glycosidic linkages plays an important role in binding with the lectin. Fetuin was found to be the best inhibitor.     

Distribution of Lectins in the Jumbo Virginia and Spanish Varieties of the Peanut, Arachis hypogaea L

Peanut lectin was purified from seed meal of the Spanish and Jumbo Virginia varieties of peanut (Arachis hypogaea L.) by affinity chromatography on lactose coupled to Sepharose 4B. Polyacrylamide gel isoelectric focusing resolved the lectin preparation from Jumbo Virginia seeds into seven isolectins (pI 5.7, 5.9, 6.0, 6.2, 6.3, 6.5, and 6.7). Seed meal from the Spanish variety contained six isolectins which were indistinguishable from the pI 5.7, 5.9, 6.2, 6.3, 6.5, and 6.7 isolectins from Jumbo Virginia. Quantitative, lactose-specific hemagglutination was used to examine the lectins in tissues of both peanut varieties. In young (3- to 9-day-old) seedlings of each variety, more than 90% of the total amount of lectins detected in the plants was in the cotyledons. Most of the remainder was in hypocotyls, stems, and leaves; young roots contained no more than 4 micrograms of lectin per plant. Lectins were present in all nonroot tissues of 21- to 30-day-old seedlings, except 27-day-old Spanish hypocotyls. As cotyledons of each variety senesced, several of the more basic isolectins decreased to undetectable levels, but the acidic isolectins remained until at least 15 days after planting. Some of the seed isolectins and several apparently new lactose-binding lectins were also identified in affinity-purified extracts of 5-day-old roots and hypocotyls. Rabbit antibodies raised against the Jumbo Virginia seed isolectin preparation reacted with seed, cotyledon, and hypocotyl lectin preparations from both varieties. Analysis of seed lectin preparations from seven varieties of A. hypogaea and of a related species (A. villosulicarpa) indicated that isolectin composition in Arachis may be a characteristic of both the species and the subspecies (botanical type) to which the variety belongs.     

Identification of an Arylphorin-type Storage Protein in the Sweet Potato Hornworm, Agrius convolvuli

A major hemolymph protein (M_r 480,000) in the larvae of the sweet potato hornworm, Agrius convolvuli, was purified and characterized. This protein was isolated with a high yield from the hemolymph of day 3 fifth final instar larvae by ammonium sulfate precipitation and Phenyl-Sepharose and Q-Sepharose column chromatographies. The protein has two subunits, an M_r 84,000 subunit (α) and an M_r 80,000 subunit (β), and the native protein was composed of a heterohexamer (α₃β₃). The two subunits have similar amino acid compositions, with high contents of aromatic amino acids (about 15% phenylalanine plus tyrosine) and low levels of methionine. The N-terminal amino acid sequences of both subunits showed high homologies with insect arylphorin-type storage proteins. The protein concentration in the hemolymph increased steeply from day 3 final instar larva and reached a maximum level of 42 mg/ml in females and 41 mg/ml in males among wandering larvae. The concentration in the hemolymph declined once during the larval–pupal transformation but remained high during the early–mid pupal period and almost disappeared after adult emergence. These quantitative changes were the same for males and females. Based on these characteristics, we identified the hemolymph protein as an arylphorin-type storage protein.     

Characterization of isolectins inTetracarpidium conophorum seeds (Nigerian walnut)

A lectin preparation obtained fromTetracarpidium conophorum (Nigerian walnut) by affinity chromatography of seed extracts on lactose-agarose has been shown to contain two components by gel filtration on Sephadex G150. The larger componentTetracarpidium conophorum agglutinin I (TCAI) is a disulphide-bonded 70 kDa homodimer whereas the second component TCAII is a 34 kDa monomeric protein. Amino terminal aminoacid sequencing shows identity in TCAI and TCAII for the first fifteen residues after which the sequences diverge. TheN-terminal sequences of TCAI and TCAII show identity with sequences in the B-chains of ricin andRicinus communis agglutinin I (RCAI) in eleven of the initial fifteen residues. Thereafter TCAI appears to be homologous to the ricin B chain whereas TCAII is more homologous with the B chain of RCAI. A limited screening of the carbohydrate-binding specificity of TCAII by affinity chromatography of defined oligosaccharides on TCAII Sepharose columns shows that the binding specificity reported earlier for affinity purifiedTetracarpidium conophorum isolectins (Sato S, Animashaun T, Hughes RC (1991)J Biol Chem 266:11485–94) reflects the binding properties of TCAII which is the major isolectin in unfractionated lectin preparations.     

Isolation, purification, and some properties of a lectin and abrin from Abrus precatorius linn.

An alternative and simple procedure has been described for the simultaneous separation of the lectin and abrin from the seeds of Abrus precatorius and their purification free from each other. Both the lectin and abrin have been crystallized, the latter as salt-free crystals. One variety of abrin which was nonhemagglutinating and did not bind d-galactose was obtained. The lectin found homogeneous by immunodiffusion, immunoelectrophoresis and sedimentation had a molecular weight of 132,000 which underwent pH-dependent reversible association-dissociation at pH 7 and 2, dissociating into non-covalently bound subunits of approximately 64,000 molecular weight. The protein was stable in the pH range 2–10. The abrin molecule did not undergo any change at low pH values. The C- and NH₂-terminal groups of the lectin were found to be Ala-Leu (or Leu-Ala) and valine, respectively. Crystalline lectin showed the presence of three isolectins in isoelectric focusing.     

The specificity of frutalin lectin using biomembrane models

Frutalin is a homotetrameric α-d-galactose (d-Gal)-binding lectin that activates natural killer cells in vitro and promotes leukocyte migration in vivo. Because lectins are potent lymphocyte stimulators, understanding the interactions that occur between them and cell surfaces can help to the action mechanisms involved in this process. In this paper, we present a detailed investigation of the interactions of frutalin with phospho- and glycolipids using Langmuir monolayers as biomembrane models. The results confirm the specificity of frutalin for d-Gal attached to a biomembrane. Adsorption of frutalin was more efficient for the galactose polar head lipids, in contrast to the one for sulfated galactose, in which a lag time is observed, indicating a rearrangement of the monolayer to incorporate the protein. Regarding ganglioside GM1 monolayers, lower quantities of the protein were adsorbed, probably due to the farther apart position of d-galactose from the interface. Binary mixtures containing galactocerebroside revealed small domains formed at high lipid packing in the presence of frutalin, suggesting that lectin induces the clusterization and the forming of domains in vitro, which may be a form of receptor internalization. This is the first experimental evidence of such lectin effect, and it may be useful to understand the mechanism of action of lectins at the molecular level.     

Studies on the interactions between glycosylated β-peptides and the lectin Vicia villosa by saturation transfer difference NMR spectroscopy

Saturation transfer difference (STD) NMR spectroscopy was used to study the interaction of the lectin Vicia villosa (VVLB₄) with α-d-GalNAc glycosylated β³-peptides. The data were compared to those obtained with the monosaccharides d-Gal, d-GalNAc, and d-Glc as well as with those obtained with the Tn antigen α-glycopeptide (d-GalNAc-α-O-Ser/Thr), molecule naturally recognized by V. villosa. Evidence that the lectin also recognizes glycosylated β³-peptides and has close contact with both the sugar and amino acid moieties was obtained.     

Isolation and Characterization of a Sialic Acid-specific Lectin from Hemolymph of the Southeast Asian Horseshoe Crab Tachypleus gigas

A lectin was isolated from hemolymph of the Southeast Asian horseshoe crab Tachypleus gigas by using glycophorin HA affinity chromatography and Sephacryl S-300 gel filtration. The purified lectin had a molecular mass of approximately 396 kDa and was composed of 13 identical subunits with molecular masses of 31 kDa. The serological specificity of the purified lectin was specifically inhibited by sialic acids sialoglycoproteins, but not by neutral sugars, hexosamines, N-acetylhexosamines, or asialoglycoproteins. Although the N-terminal amino acid sequence of the lectin from T. gigas was identical to that from American horseshoe crab (liphemin) by the same purification method and cross reacted with the anti-liphemin serum, the calcium concentration of hemagglutinating activity of the purified lectin showed a smaller optimal concentration than that of liphemin.     

Musca domestica hemolymph ferritin

We describe a method for the purification of ferritin from Musca domestica larval hemolymph. Musca ferritin occurs in hemolymph predominantly as a native protein with molecular weight equal to 550,000 and subunits of 26,000. The average iron content of purified ferritin was determined to be 3,000 ± 600 iron atoms per molecule. The iron contents of ferritin was heterogeneous; both fully iron loaded molecules and apoferritin are probably present in the Musca hemolymph. The anti-ferritin serum raised in rabbit was able to recognize native ferritin but was not reactive with the protein subunits isolated by SDS-PAGE. The ferritin concentration in hemolymph attains a maximum of 0.28 mg/ml in the wandering stage larvae, decreasing to 0.13 mg/ml at the middle of pupal stadium. The ferritin contents of midgut and fat bodies were also determined. Fat body ferritin content is greatly reduced when the feeding larva passes into wandering stage. © 1996 Wiley-Liss, Inc.     

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.     

Molecular and kinetic properties of purified γ-glutamyl transpeptidase from yeast (Saccharomyces cerevisiae)

The enzyme γ-glutamyl transpeptidase was purified from the yeast Saccharomyces cerevisiae by a procedure involving protamine sulfate treatment, chromatography on DEAE-Sephadex A 50, salt fractionation, successive chromatography on Sephadex G 150 and lentil lectin sepharose 6B. The procedure achieves 25 % yield and 4200-fold purification. The final preparation is a glycoprotein (M, 90 000) containing 31.4 % carbohydrates and composed of two non-identical subunits (M, 64 000 and 23 000). The specificity patterns of the yeast enzyme are rather similar to those of mammalian and higher plant transpeptidases. The enzyme mechanism might be of the double displacement (ping-pong) type.     

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.     

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.     

A genetic basis for the origin of six different isolectins in hexaploid wheat

Wheat (Triticum aestivum) germ agglutinin represents a complex mixture of multiple isolectin forms. Upon ion exchange chromatography at pH 3.8, three isolectins can be separated, each of which is composed of two identical subunits. At pH 5.0, however, three additional isolectins can be distinguished, which are built up of two different subunits (heteromeric lectins). Evidence is presented that these heterodimers are normal constituents of the wheat embryo cells. Analyses of the isolectin patterns in extracts from Triticum monococcum, Triticum turgidum dicoccum and Triticum aestivum, provide evidence that each genome, either in simple or complex (polyploid) genomes, directs the synthesis of a single lectin subunit species. In addition, a comparison of the isolectin pattern in these wheat species of increasing ploidy level, made it possible to determine unequivocally the genome by which the individual lectin subunits in polyploid species are coded for. The possible use of lectins in studies on the origin of individual genoms in polyploid species is discussed.Abbreviations CL cereal lectin - PBS phosphate buffered saline - SP Sephadex sulfopropyl Sephadex - WGA wheat germ agglutinin     

Multiple molecular forms of peanut lectin: Classification of isolectins and isolectin distribution among genotypes of the genus Arachis

Peanut lectin (or an immunologically indistinguishable material) is present in seeds of 4556 genotypes of the peanut, Arachis hypogaea, and in 65 genotypes of related species of Arachis. Seeds of one line of A. villosa and three lines of unclassified Arachis spp. are devoid of the lectin. Peanut lectin from 116 A. hypogaea genotypes is resolved by isoelectric focusing into three related isolectin profiles, which are designated the V, S, and V₂ types. Each is composed of from six to eight separate isolectins. Peanut lectin from A. monticola, A. pusilla, and one genotype of Arachis spp. is of the V type; isolectin profiles from other wild Arachis genotypes are variable, but comprise several distinct groups. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolves peanut lectin preparations from 37 genotypes into the lectin subunit of M_r 30,000 and a second polypeptide of M_r 60,000. Lectin preparations from five genotypes lack the M_r 60,000 polypeptide band and have a subunit that migrates slightly faster (and therefore probably is of lower molecular weight) than the subunits of all other tested lines. Peanut lectin preparations from 62 lines have specific hemagglutinating activities ranging from 1024 to 4196 with desialyzed human Type O erythrocytes. The lectin from one genotype exhibits substantially less hemagglutinating activity and is hemolytic.