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.     

Transgenic Tobacco Plants with Two Consecutive Oxidation Reactions Catalyzed by Hyoscyamine 6β-Hydroxylase

Among mushrooms of Pleurotus cornucopiae two kinds of fruit bodies, lectin (PCL)-containing and -deficient, were found. The PCL-deficient fruit body was found to contain a 16.5-kDa protein, which was purified to homogeneity and crystallized. Properties of the protein were investigated in comparison with PCL. The protein consisted of four identical subunits each having a molecular mass of 16.5 kDa, which was close to that of PCL. Partial amino acid sequences of the two proteins were analyzed and some sequence homology was found, although the protein did not cross-react with anti-PCL serum and it was devoid of binding activity for mucin, an inhibitor of PCL. The 16.5-kDa protein was not found in vegetatively growing mycelia, indicating that the synthesis of the protein was developmentally regulated as was PCL. The results suggest that the 16.5-kDa protein was related to PCL.     

Azolla fern lectins that specifically recognize endosymbiotic cyanobacteria

Lectins were extracted from whole fern grindings ofAzolla pinnata (AP) andAzolla filiculoides (AF) by precipitation with ammonium sulfate to 20% of saturation. At high pH both lectins dissociate into inactive subunits (5000 mol wt) which reassociate into active aggregates (>500,000 mol wt) following concentration by ammonium sulfate precipitation or freezing and thawing. Although amino sugars inhibited hemagglutinating activity of both AP and AF lectins,d-fructose was inhibitory only to the AP lectin hemagglutinating activity, andd-galactose was slightly inhibitory to the AP lectin but not to the AF lectin. Both lectins exhibited specificity for freshly extracted cyanobionts from homologous fern species: AP lectin agglutinated cyanobiont filaments from AP, but not from AF; AF lectin agglutinated cyanobiont filaments from AF, but not AP. Neither lectin reacted with cultured cyanobionts from either fern species. Hemagglutinating titers were likewise reduced by adsorption of these lectins to homologous cyanobiont cells. This report provides strong suggestive evidence for specificity in this N-fixing symbiosis between aquatic fern and cyanobacterium.     

Comparative study of the post-translational processing of the mannose-binding lectins in the bulbs of garlic (Allium sativum L.) and ramsons (Allium ursinum L.)

The biosynthesis and processing of the homodimeric and heterodimeric lectins from the bulbs of garlic (Allium sativum) and ramsons (wild garlic;Allium ursinum) were studied using pulse and pulse-chase labelling experiments on developing bulbs. By combining the results of thein vivo biosynthesis studies and the cDNA cloning of the respective lectins, the sequence of events leading from the primary translation products into the mature lectin polypeptides could be reconstructed. From this it is demonstrated that garlic and ramsons use different schemes of post-translational modifications in order to synthesize apparently similar lectins from totally different precursors. Both the homomeric garlic lectin (ASAII) and its homologue in ramsons (AUAII) are synthesized on the endoplasmic reticulum (ER) as nonglycosylated 13.5 kDa precursors, which, after their transport out of the ER are converted into the mature 12.0 kDa lectin polypeptides by the cleavage of a C-terminal peptide. The heterodimeric garlic lectin ASAI is synthesized on the ER as a single glycosylated precursor of 38 kDa, which after its transport out of the ER undergoes a complex processing which gives rise to two mature lectin subunits of 11.5 and 12.5 kDa. In contrast, both subunits of the heterodimeric ramsons lectin AUAI are synthesized separately on the ER as glycosylated precursors, which after their transport out of the ER are deglycosylated and further processed into the mature lectin polypeptides by the cleavage of a C-terminal peptide.     

Intracellular lectins of <Emphasis Type="Italic">Lentinus edodes</Emphasis> at various developmental stages of the fungus

A number of lectins varying in polypeptide composition and carbohydrate specificity were isolated from Lentinus edodes at different stages of its morphogenesis: nonpigmented mycelium, brown mycelium film, and fruiting body. Three lectins were identified at the nonpigmented mycelium stage, two of them being dimers consisting of 16 and 45 kDa and 16 and 42 kDa subunits; the third is a tetramer of 16, 39, 42, and 45 kDa subunits. The fractions with lectin activity obtained at the brown mycelium film stage contained polypeptides of 24, 30, and 38 kDa, characteristic of this morphological structure. The fruiting body was shown to contain two lectins of 43 and 55 kDa. All of the isolated lectins expressed the highest affinity towards L,D-melibiose, D-lactose, and D-galactose.     

Isolation and characterization of a jacalin-related mannose-binding lectin from salt-stressed rice (Oryza sativa) plants

A novel plant lectin was isolated from salt-stressed rice (Oryzasativa L.) plants and partially characterized. The lectin occurs as a natural mixture of two closely related isoforms consisting of two identical non-covalently linked subunits of 15 kDa. Both isoforms are best inhibited by mannose and exhibit potent mitogenic activity towards T-lymphocytes. Biochemical analyses and sequence comparisons further revealed that the rice lectins belong to the subgroup of mannose-binding jacalin-related lectins. In addition, it could be demonstrated that the lectins described here correspond to the protein products of previously described salt-stress-induced genes. Our results not only identify the rice lectin as a stress protein but also highlight the possible importance of protein-carbohydrate interactions in stress responses in plants. Received: 27 July 1999 / Accepted: 11 November 1999     

Intracellular lectins of Lentinus edodes at various developmental stages of the fungus

A number of lectins varying in polypeptide composition and carbohydrate specificity were isolated from Lentinus edodes at different stages of its morphogenesis: nonpigmented mycelium, brown mycelium film, and fruiting body. Three lectins were identified at the nonpigmented mycelium stage, two of them being dimers consisting of 16 and 45 kDa and 16 and 42 kDa subunits; the third is a tetramer of 16, 39, 42, and 45 kDa subunits. The fractions with lectin activity obtained at the brown mycelium film stage contained polypeptides of 24, 30, and 38 kDa, characteristic of this morphological structure. The fruiting body was shown to contain two lectins of 43 and 55 kDa. All of the isolated lectins expressed the highest affinity towards L,D-melibiose, D-lactose, and D-galactose.     

Expression of the 14 kDa and 16 kDa galactoside-binding lectins during differentiation of the chick yolk sac

The gastrulating chick embryo expresses two galactoside-binding lectins of 14 kDa and 16 kDa. These lectins are present in the area pellucida and area opaca, and in the latter are concentrated in the endoderm. Since the area opaca is the progenitor of the yolk sac, we studied the galactose-binding lectins during the development of this extraembryonic organ. In the yolk sac, lectin expression surges between 2 and 4 days, and thereafter remains constant throughout development. Using monoclonal antibodies (mAbs) specific to the 16 kDa yolk sac lectin, and a panel of polyclonal antibodies to the 14 kDa and 16 kDa lectins we studied lectin expression. The mAbs inhibit the hermagglutinating activity of extracts from chick yolk sac, embryonic pectoral muscle, and adult liver, but have no effect on the hemagglutinating activity of extracts from the adult intestine. Immunolocalization studies with the mAbs and polyclonal antibodies indicate that in the less differentiated endodermal cells of the area vitellina the 16 kDa lectin is present in discrete lectin-rich inclusions. In contrast, within the maturing endodermal epithelium of area vasculosa the 16 kDa lectin is present around the intracellular yolk platelets, and is associated with the cytoplasmic matrix. The 16 kDa lectin is also found at the apical cell surface of the yolk sac epithelium, in some regions closely associated with the plasma membrane. The 14 kDa lectin is distributed intracellularly surrounding the yolk platelets of the maturing yolk sac endoderm. The surge in expression of the 16 kDa lectin at the time of expansion of the area opaca suggests that it may be involved in the spreading of this area. Our findings also indicate that as the yolk sac endoderm differentiates into an epithelium intracellular lectin expression changes from predominantly organelle associated to cytoplasm associated. The association of both lectins with yolk suggest that the lectins may also be involved in the processing of intracellular and extracellular yolk proteins. These results, in con junction with previous findings indicating the presence of these lectins in the extracellular matrix (Didier et al., Histochemistry 100:485, 1993; Zalik et al., Intl J Dev Biol 38:55–68, 1994) indicate that these lectins play multiple roles in embryonic development.     

The seed lectins of black locust (robinia pseudoacacia) are encoded by two genes which differ from the bark lectin genes

Two lectins were isolated from Robinia pseudoacacia (black locust) seeds using affinity chromatography on fetuin-agarose, and ion exchange chromatography on a Neobar CS column. The first lectin, R. pseudoacacia seed agglutinin I, referred to as RPsAI, is a homotetramer of four 34 kDa subunits whereas the second lectin, referred to as RPsAII, is composed of four 29 kDa polypeptides. cDNA clones encoding the polypeptides of RPsAI and RPsAII were isolated and their sequences were determined. Both polypeptides are translated from mRNAs of ca. 1.2 kb encoding a precursor carrying a signal peptide. Alignment of the deduced amino acid sequences of the different clones indicates that the 34 and 29 kDa seed lectin polypeptides show 95% sequence identity. In spite of this striking homology, the 29 kDa polypeptide has only one putative glycosylation site whereas the 34 kDa subunit has four of these sites. Carbohydrate analysis revealed that the 34 kDa possesses three carbohydrate chains whereas the 29 kDa polypeptide is only partially glycosylated at one site. A comparison of the deduced amino acid sequences of the two seed and three bark lectin polypeptides demonstrated unambiguously that they are encoded by different genes. This implies that five different genes are involved in the control of the expression of the lectins in black locust.Abbreviations LECRPAs cDNA clone encoding Robinia pseudoacacia seed lectin - LoLI Lathyrus ochrus isolectin I - PsA Pisum sativum agglutinin - RPbAI Robinia pseudoacacia bark agglutinin I - RPbAII Robinia pseudoacacia bark agglutinin II - RPsAI Robinia pseudoacacia seed agglutinin I - RPsAII Robinia pseudoacacia seed agglutinin II     

Purification and characterization of two major lectins fromVigna mungo (blackgram)

Blackgram (Vigna mungo L. Hepper)seeds contain two galactose-specific lectins, BGL-I and BGL-II. BGL-I was partially purified into two monomeric lectins which were designated as BGL-I-1 (94 kDa) and BGL-I-2 (89 kDa). BGL-II is a monomeric lectin of 83 kDA. The purified lectins were associated with galactosidase activities. BGL-I-1 and BGL-II were copurified with α-galactosidase activity while BGL-I-2 was largely associated with β-galactosidase activity. These lectins agglutinate trypsin treated rabbit erythrocytes, but not the human erythrocytes of A, B or O groups. They were stable between pH 3·5 and 7·5 for their agglutination. The lectins did not show any metalion requirement. They were inactivated at 50°C. The lectin activity was inhibited by D-galactose (0·1 mM). The Scatchard plots of galactose binding to these lectins are nonlinear and biphasic curves indicative of multiple binding sites. The data show that the monomeric lectins have both lectin and galactosidase activities suggestive of a bifunctional protein.     

A latex lectin from <Emphasis Type="Italic">Euphorbia trigona</Emphasis> is a potent inhibitor of fungal growth

In this study we identified and characterized a major latex lectin — designated as EtLLH — with antimicrobial activity from the succulent African milk tree Euphorbia trigona. The lectin is highly concentrated in the latex of E. trigona and appears to be composed of at least two subunits with a molecular mass of 32 kDa. EtLLH shares significant similarities to known plant lectins — ricin from Ricinus communis and agglutinin from Viscum album coloratum — which specifically bind D-galactose and N-acetyl-D-glucosamine, the major building blocks of many fungal cell walls. Antimicrobial activity assays revealed an impact of EtLLH on three phytopathogenic filamentous ascomycetes. The germination of the conidiospores and the hyphal growth of Aspergillus niger and Fusarium graminearum were severely inhibited by EtLLH already at concentrations below 0.1 mg cm⁻³, while the effect on germination of the melanized conidiospores of Botrytis cinerea was less significant.     

PURIFICATION AND CHARACTERIZATION OF A LECTIN,BRYOHEALIN, INVOLVED IN THE PROTOPLAST FORMATION OF A MARINE GREEN ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA) 1

When the coenocytic green alga Bryopsis plumosa (Huds.) Ag. was cut open and the cell contents were expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. Aggregation of cell organelles in seawater was mediated by a lectin–carbohydrate complementary system. Two sugars, N‐acetyl‐d ‐glucosamine and N‐acetyl‐d ‐galactosamine inhibited aggregation of cell organelles. The presence of these sugars on the surface of chloroplasts was verified with their complementary fluorescein isothiacyanate‐labeled lectins. An agglutination assay using human erythrocytes showed the presence of lectins specific for N‐acetyl‐d ‐galactosamine and N‐acetyl‐d ‐glucosamine in the crude extract. One‐step column purification using N‐acetyl‐d ‐glucosamine‐agarose affinity chromatography yielded a homogeneous protein. The protein agglutinated the cell organelles of B. plumosa, and its agglutinating activity was inhibited by the above sugars. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed that this protein might be composed of two identical subunits cross‐linked by two disulfide bridges. Enzyme and chemical deglycosylation experiments showed that this protein is deficient in glycosylation. The molecular weight was determined as 53.8 kDa by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The N‐terminal 15 amino acid sequence of the lectin was Ser–Asp–Leu–Pro–Thr–X–Asp–Phe–Phe–His–Ile–Pro–Glu–Arg–Tyr, and showed no sequence homology to those of other reported proteins. These results suggest that this lectin belongs to a new class of lectins. We named this novel lectin from B. plumosa“bryohealin.”     

The occurrence of a premature form of egg-specific protein in vitellogenic follicles ofBombyx mori

Summary Analysis of yolk proteins of the silkworm,Bombyx mori, by SDS-polyacrylamide gel electrophoresis and immunoblotting showed that there was a developmental change in subunit composition of egg-specific protein; egg-specific protein consisting of 72 kDa subunits alone (premature form) was found in vitellogenic follicles, whereas the protein in mature eggs was composed of 72 kDa and 64 kDa subunits (mature form). The premature form of egg-specific protein was purified from young ovaries to homogeneity using a high performance liquid chromatography system. The purified protein had an apparent molecular mass of 225 kDa which could not be distinguished from that of the mature form. By circular dichroism analysis, both egg-specific proteins were estimated to have about 30% -helix and 20% -sheet, but the mature form showed a relatively rigid conformation in the aromatic region. The premature egg-specific protein purified from vitellogenic ovaries, consisted of three 72 kDa subunits, whereas mature egg-specific protein was composed of two 72 kDa subunits and one 64 kDa subunit. All of these subunits showed the same immunoreactivity towards antiserum raised against the mature form. An identical NH₂-terminal amino acid sequence was found in both 72 kDa polypeptides and 64 kDa polypeptide for the initial 10 amino acids.Abbreviations SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - PAGE polyacrylamide gel electrophoresis - HPLC high performance liquid chromatography - ESP egg-specific protein - Vtn vitellin     

Isolation and Characterization of Isolectins from Talisia esculenta Seeds

Four isolectins (TEL-I, TEL-II, TEL-III and TEL-IV) were isolated from seeds of Talisia esculenta by reverse-phase high-performance liquid chromatography. RP-HPLC was performed on a u-Bondapack C18 column (0.78 cm × 30 cm) (Waters 991-PDA system) at room temperature. Rechromatography of the four fractions on a C18 column under the same conditions yielded lectins with two dissimilar subunits (M_r 20 kDa and 40 kDa) bound noncovalently. The isolectins showed very similar characteristics, such as molecular masses, N-terminal sequences, and hemagglutinating activity, but differed in their isoelectric points and in inhibition by carbohydrates.     

Isolation of a novel plant lectin with an unusual specificity from Calystegia sepium

A novel plant lectin has been isolated from the rhizomes of Calystegia sepium (hedge bindweed) and partially characterized. The lectin is a dimeric protein composed of two identical non-covalently linked subunits of 16kDa. Hapten inhibition studies indicate that the novel lectin is best inhibited by maltose and mannose and hence exhibits a sugar binding specificity that differs in some respects from that of all previously isolated plant lectins. Mitogenicity tests have shown that the Calystegia lectin is a powerful T-cell mitogen. Affinity purification of human, plant and fungal glycoproteins on immobilized C. sepium lectin demonstrates that this novel lectin can be used for the isolation of glycoconjugates from various sources. Moreover, it can be expected that by virtue of its distinct specificity, the new lectin will become an important tool in glycobiology. Abbreviations: Calsepa, lectin isolated from Calystegia sepium; ConA, concanavalin A; LPS, lipopolysaccharide; PBS, phosphate buffered saline (1.5 mMKH2PO4, 10 mM Na2HPO4, 3 mM KCl, 140 mM NaCl, pH 7.4) This revised version was published online in November 2006 with corrections to the Cover Date.     

Characterization of Acid-Cyclodextrin Glycosyltransferase of an Alkalophilic Bacillus sp.

Three kinds of lectins (LOL-I, II and III) were isolated from seeds of Lathyrus odoratus (sweet pea) in a homogenous form. The three fractions agglutinated the erythrocytes of laying hens, and the agglutination was strongly inhibited by α-methyl d-mannoside and d-mannose. However, they did not agglutinate those of the males and nonlyaing hens, differing from concanavalin A which showed a similar binding specificity for monosaccharide to LOL and agglutinated all types of erythrocytes derived from chicken in this study. LOL–I and II had a molecular weight of 52,000 and both consisted of two large (20,000 daltons) and two small subunits (6000 daltons). LOL–III had a molecular weight of 55,000, and its subunit structure was different from those of LOL–I and II. The amino acid compositions of the three fractions were very similar. They contained large amounts of aspartic acid, threonine, serine and valine, but no cysteine or methionine. Circular dichroism measurements indicated that β-structure was a major secondary structure of these lectins. The addition of α-methyl d-mannoside or d-mannose had significant effects on the CD spectra in the near-ultraviolet region, but no detectable change was observed in the 200~250 nm region. LOL–I had two binding sites for d-mannose, and the association constant was about 1000 liters per mol.     

Lectin activity in pollen from plants representative of thirty orders of spermatophyta

Summary Pollen extracts from a variety of species representative of thirty orders of spermatophyta, including gymnosperms, dicotyledons and monocotyledons, were examined for the presence of lectin activity by means of a hemagglutination assay. Hemagglutinating activity (HA) was detected in the pollen extracts of all the species examined, indicating that lectins are generally present in the pollen of spermatophyta. The response of this pollen hemagglutinating activity to the sugars and glycoproteins tested as potential inhibitors was identical in all species examined. Moreover, the hemagglutinating activity of pollen extracts from eight species which had been selected as representative of the gymnosperms and both subclasses of angiosperms exhibited similar properties (e.g. distribution by differential centrifugation, stability to heat, response to bivalent ions). The bulk of the hemagglutinating activity was always recovered in the pellet after centrifugation at 1000 g for 5 min. Although sequential treatments with 1% Triton X-100 and 1 M KCl were ineffective, subsequent incubation of the pellet with saline phosphate buffer released hemagglutinating activity. The solubilized hemagglutinating activity was destroyed by protease treatment, indicating that the substance(s) responsible for the activity is (are) protein in nature and, consequently, might be considered to be a lectin. The sugar specifity of the pollen lectin activity from wheat, potato and bean was compared with that of wheat germ agglutinin (WGA), potato agglutinin and bean agglutinin — the lectins present in sporophytic tissues of these plants. For all three plants, the response of the pollen lectin activity to sugars and glycoproteins was different from that shown by the lectin from sporophytic tissues.Abbreviations HA Hemagglutinating activity - PBS 150 mM Na-phosphate buffer (pH 7.2) containing 0.9% NaCl - PHA Phaseolus vulgaris agglutinin - STA Solanum tuberosum agglutinin - WGA wheat germ agglutinin     

Isolation and characterization of the potential receptor for wheat germ agglutinin from human neutrophils

Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.