Separation and partial characterization of isolectins with different subunit compositions from Datura stramonium seeds

The lectin from Datura stramonium seeds was separated into three individual isolectins by hydrophobic-interaction chromatography on phenyl-Sepharose. Two of these isolectins are homodimers made up of two A- or two B-subunits, whereas the third is a heterodimer composed of one A- and one B-subunit. Analysis of the homodimeric AA- and BB-isolectins revealed that the A- and B-subunits have similar but not identical M_r values (32 000 and 28 000, respectively), amino acid and carbohydrate compositions. The A-subunit has a higher affinity for N-acetyl-D-glucosamine oligomers than the B-subunit, whereas the latter is more specific for the carbohydrate determinants of some animal glycoproteins such as fetuin, asialofetuin and ovomucoid.     

Isolectin composition of individual clones of Urtica dioica: Evidence for phenotypic differences

Analysis of the isolectin composition of 102 individual nettle ( Urtica dioica L.) clones by ion-exchange chromatography revealed the occurrence of at least 11 different isolectins, which all had the same molecular structure and exhibited identical carbohydrate-binding specificity and agglutination properties. All 11 isolectins, however, did not occur simultaneously; 34 combinations of either 1, 2, 3, 4 or 5 isolectins were found. Since the occurrence of multiple molecular forms of the nettle agglutinin cannot be explained by the (partial) autotetraploid character of stinging nettle it is postulated to rely on the expression of a family of closely related lectin genes.     

Lectin variability in Phaseolus coccineus

Lectin variability within Phaseolus coccineus is revealed by non-denatured electrophoretic patterns and immunological labelling of total seed protein extracts, showing that the different cultivars and wild varieties studied can be classified into three main categories according to the number of isolectins (three, two or one) present in each extract. Attempts in the purification of these isolectins were performed on three different affinity systems in which ligands were thyroglobulin (known to purify the P. vulgaris isolectins), pig red cell membrane ghosts (stroma) or antibodies against the P. vulgaris cv. Contender E₂L₂ isolectin. The P. coccineus isolectins exhibit varied affinities towards thyroglobulin and stroma, the cathodic and anodic (pH 4.5) isolectins being respectively retained by the two systems, whereas the antibody affinity system is the only one able to purify the totality of the isolectins present in an extract.     

Toxic isolectins from the mushroom Boletus venenatus

Ingestion of the toxic mushroom Boletus venenatus causes a severe gastrointestinal syndrome, such as nausea, repetitive vomiting, diarrhea, and stomachache. A family of isolectins (B. venenatus lectins, BVLs) was isolated as the toxic principles from the mushroom by successive 80% ammonium sulfate-precipitation, Super Q anion-exchange chromatography, and TSK-gel G3000SW gel filtration. Although BVLs showed a single band on SDS–PAGE, they were further divided into eight isolectins (BVL-1 to -8) by BioAssist Q anion-exchange chromatography. All the isolectins showed lectin activity and had very similar molecular weights as detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Among them, BVL-1 and -3 were further characterized with their complete amino acid sequences of 99 amino acids determined and found to be identical to each other. In the hemagglutination inhibition assay, both proteins failed to bind to any mono- or oligo-saccharides tested and showed the same sugar-binding specificity to glycoproteins. Among the glycoproteins examined, asialo-fetuin was the strongest inhibitor. The sugar-binding specificity of each isolectin was also analyzed by using frontal affinity chromatography and surface plasmon resonance analysis, indicating that they recognized N-linked sugar chains, especially Galβ1 → 4GlcNAcβ1 → 4Manβ1 → 4GlcNAcβ1 → 4GlcNAc (Type II) residues in N-linked sugar chains. BVLs ingestion resulted in fatal toxicity in mice upon intraperitoneal administration and caused diarrhea upon oral administration in rats.     

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.     

Comparison of the spectroscopic and saccharide binding properties of lentil and pea isolectins

The lentil isolectins, CMLcH A and CMLcH B, and pea isolectins, CMPSA A and CMPSA B, are compared in terms of their spectroscopic and saccharide binding properties. The paramagnetic contribution to the solvent proton magnetic relaxation dispersion profiles of solutions of the isolectins of each protein are found to be essentially identical. Electron paramagnetic resonance spectra suggest a high degree of octahedral symmetry at the Mn2+ site for both pairs of isolectins. The near-ultraviolet absorption spectra of CMLcH A and CMLcH B are identical, as are the spectra of CMPSA A and CMPSA B. Carbohydrate binding activities of the isolectins of each protein are compared using hemagglutination, precipitation, and precipitation-inhibition assays, and are found to be identical, although the activities of CMLcH and CMPSA differ somewhat. These results demonstrate that the spectroscopic and saccharide binding properties of the isolectins of CMLcH are essentially identical, as are those of the isolectins of CMPSA, and suggest that native mixtures of the isolectins may be treated as single proteins in further studies.     

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.     

Phytochemical,phylogenetic, and anti-inflammatory evaluation of 43 Urtica accessions (stinging nettle) based on UPLC–Q-TOF-MS metabolomic profiles

Several species of the genus Urtica (especially Urtica dioica, Urticaceae), are used medicinally to treat a variety of ailments. To better understand the chemical diversity of the genus and to compare different accessions and different taxa of Urtica, 63 leaf samples representing a broad geographical, taxonomical and morphological diversity were evaluated under controlled conditions. A molecular phylogeny for all taxa investigated was prepared to compare phytochemical similarity with phylogenetic relatedness. Metabolites were analyzed via UPLC–PDA–MS and multivariate data analyses. In total, 43 metabolites were identified, with phenolic compounds and hydroxy fatty acids as the dominant substance groups. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) provides a first structured chemotaxonomy of the genus. The molecular data present a highly resolved phylogeny with well-supported clades and subclades. U. dioica is retrieved as both para- and polyphyletic. European members of the U. dioica group and the North American subspecies share a rather similar metabolite profile and were largely retrieved as one, nearly exclusive cluster by metabolite data. This latter cluster also includes – remotely related – Urtica urens, which is pharmaceutically used in the same way as U. dioica. However, most highly supported phylogenetic clades were not retrieved in the metabolite cluster analyses. Overall, metabolite profiles indicate considerable phytochemical diversity in the genus, which largely falls into a group characterized by high contents of hydroxy fatty acids (e.g., most Andean-American taxa) and another group characterized by high contents of phenolic acids (especially the U. dioica-clade). Anti-inflammatory in vitro COX1 enzyme inhibition assays suggest that bioactivity may be predicted by gross metabolic profiling in Urtica.     

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     

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.     

Strict Binding Specificity of Small-Sized Lectins from the Red Alga Hypnea japonica for Core (α1-6) Fucosylated N-Glycans

Small-sized isolectins (9 KDa) from Hypnea japonica belong to a new lectin family. Here, we describe the carbohydrate-binding properties of the three isolectins (hypninA1, A2, and A3) and the amino acid sequence of hypninA3 (P85888). In frontal affinity chromatography with about 100 pyridylaminated oligosaccharides, the isolectins, which had no affinity for monosaccharides, commonly bound only core (α1-6) fucosylated N-glycans, and did not the other oligosaccharides examined, including (α1-2), (α1-3), and (α1-4) fucosylated glycans. The specific binding of hypninA3 with the fucosylated N-glycans (K _a; 0.52–7.58×10⁶ M^?1) was confirmed by surface plasmon resonance analyses on an immobilized glycoprotein with and without core (α1-6) fucose. Such specificity of hypninA is clearly distinct from those of other known fucose-binding lectins, making it a valuable tool for cancer diagnosis and quality control of medicinal antibodies. HypninA3 is a polypeptide composed of 90 amino acids containing four half-cystines.     

Asymmetrical conspecific seed-siring advantage between Silene latifolia and S. dioica

Background and Aims

Silene dioica and S. latifolia experience only limited introgression despite overlapping flowering phenologies, geographical distributions, and some pollinator sharing. Conspecific pollen precedence and other reproductive barriers operating between pollination and seed germination may limit hybridization. This study investigates whether barriers at this stage contribute to reproductive isolation between these species and, if so, which mechanisms are responsible.

Methods

Pollen-tube lengths for pollen of both species in styles of both species were compared. Additionally, both species were pollinated with majority S. latifolia and majority S. dioica pollen mixes; then seed set, seed germination rates and hybridity of the resulting seedlings were determined using species-specific molecular markers.

Key Results

The longest pollen tubes were significantly longer for conspecific than heterospecific pollen in both species, indicating conspecific pollen precedence. Seed set but not seed germination was lower for flowers pollinated with pure heterospecific versus pure conspecific pollen. Mixed-species pollinations resulted in disproportionately high representation of nonhybrid offspring for pollinations of S. latifolia but not S. dioica flowers.

Conclusions

The finding of conspecific pollen precedence for pollen-tube growth but not seed siring in S. dioica flowers may be explained by variation in pollen-tube growth rates, either at different locations in the style or between leading and trailing pollen tubes. Additionally, this study finds a barrier to hybridization operating between pollination and seed germination against S. dioica but not S. latifolia pollen. The results are consistent with the underlying cause of this barrier being attrition of S. dioica pollen tubes or reduced success of heterospecifically fertilized ovules, rather than time-variant mechanisms. Post-pollination, pre-germination barriers to hybridization thus play a partial role in limiting introgression between these species.     

Mannose Binding Lectins of Vicia tetrasperma Seed and Their Immunological Relationship to Other Legume Lectins of Similar Specificity

Mannose specific lectins of Vicia tetrasperma were purifiedby affinity chromatography with Sephadex G-100, and ion exchangechromatography. Chromatofocusing using PBE-94 gel was successfullyemployed to separate the major isolectins, lectin I and II.Both lectins had the same molecular weight of 78,000 and weretetramers composed of a uniform subunit with a molecular weightof 18,700. Amino acid compositions of these lectins were quitesimilar to each other, rich in aspartic acid (and/or asparagine)and hydroxyl amino acids, and lacking methionine and cysteine.Agar gel double diffusion using anti V. tetrasperma lectin antiserumrevealed that lectins from V. cracca, Pisum sativum, and Lensculinaris, all of which have mannose binding properties, wereantigenically identical. The antiserum reacted with the analogouslectins from V.faba, V. hirsuta, and V. angustifolia, but formationof a spur in the diffusion assay showed that they were slightlydifferent from V. tetrasperma lectin. (Received December 24, 1985; Accepted March 12, 1986)     

Alkaline phosphatase from pig kidney. Microheterogeneity and the role of neuraminic acid

Several alkaline phosphatases (EC 3.1.3.1) could be obtained from pig kidney brush-border membrane on extraction with butan-1-ol. Three of the multiple forms were separated by DEAE-cellulose chromatography and further purified. They form a regular series with different degrees of glycosylation (mainly owing to N-acetylneuraminic acid), of charge, of molecular weight, of stability to temperature, to pH and to urea, of minimal requirement for Mg²⁺ and of extractability by butan-1-ol. In contrast, the detectable antigenic sites, the inhibition by amino acids and the pH-dependency of K_m and V_max. were identical for these multiple forms. On treatment with neuraminidase, the multiple forms became identical in all their properties. It was therefore concluded that the microheterogeneity of alkaline phosphatase is due to different degrees of glycosylation at polypeptide chains which appear to be otherwise identical.     

Separation and biological properties of Phaseolus vulgaris isolectins

Two isolectins (L₄E₀-PHA and L₀E₄-PHA) from red kidney beans (Phaseolus vulgaris) were isolated by affinity chromatography on immobilized thyroglobulin and by chromatography on hydroxyapatite. The L₄E₀-PHA siolectin was not retarded on the affinity column and was eluted from the hydroxyapatite column with a 0.1 M Phosphate buffer. This isolectin had no erythroagglutinating activity but had high lymphoagglutinating and lymphocyte stimulating activities. The L₀E₄-PHA isolectin was adsorbed on the affinity column and was eluted from the hydroxyapatite column with a 0.25 M Phosphate buffer. This isolectin has a high erythroagglutinating activity, a very low lymphoagglutinating activity and no lymphocyte stimulating activity. These two isolectins are shown however to be closely related with respect to their oligomeric structure and reactivity towards anti-PHA antibodies. The lack of mitogenic activity of the L₀E₄-PHA isolectin suggests that in the other isolectins, the E monomer is not responsible for their mitogenic activity and that the membrane glycoproteins, which contain the E monomer — specific oligosaccharide, are not involved in the process inducing mitosis.     

Mannose-specific isolectins with different hemagglutinating potencies isolated from Chinese daffodil (Narcissus tazetta var. chinensis) leaves

Three mannose-specific lectins exhibiting considerable similarities in NH₂-terminal amino acid sequence were isolated from leaves of the Chinese daffodil Narcissus tazetta (Family Amaryllidaceae). The purification protocol involved extraction with an aqueous buffer, anion exchange chromatography on DEAE-cellulose using stepwise elution with increasing salt concentrations, affinity chromatography on mannose-agarose, and FPLC-gel filtration on Superose 12. From the peak unadsorbed on DEAE-cellulose, and two peaks adsorbed on the ion exchanger and eluted respectively with 0.2 M Tris-HCl buffer and 0.5 M NaCl, were prepared fractions which yielded isolectins 1, 2, and 3 after adsorption on mannose-agarose and FPLC-gel filtraton. All three isolectins were homodimers with a molecular weight of 26 kDa. The lectin unadsorbed on DEAE-cellulose had the lowest, while the most strongly adsorbed lectin had the highest hemagglutinating activity.     

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

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

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.