Proteolytic Activity of Lectins from the Nitrogen-Fixing Bacterium Bacillus polymyxa

Two lectins (LI and LII) stripped from the surface of Bacillus polymyxa1460 cells were found to possess proteolytic activity, which was associated with their hemagglutinating activity. The inhibition of the hemagglutinating activity of lectins by specific carbohydrate haptens changed their enzyme activities. The inhibition of hemagglutinating activity by glucuronic acid or fructose 1,6-diphosphate decreased the proteolytic activities of both lectins, whereas the blocking of this activity with D-glucosamine or D-galactosamine increased the proteolytic activity of LII. The molecules of the B. polymyxalectins are suggested to contain two active centers responsible for hemagglutinating and proteolytic activities.     

Biological Role of Lectins from Bacillus polymyxa during Interaction with Surface Carbohydrates of Wheat Roots

The data obtained demonstrate the capacity of lectins LI and LII from the soil nitrogen-fixing bacteria Bacillus polymyxa 1460 to change their proteolytic activity after interaction with surface carbohydrates of wheat seedling roots.     

Proteolytic activity of lectins from the nitrogen-fixing bacterium Bacillus polymyxa

Two lectins (LI and LII) stripped from the surface of Bacillus polymyxa 1460 cells were found to possess proteolytic activity, which was associated with their hemagglutinating activity. The inhibition of the hemagglutinating activity of lectins by specific carbohydrate haptens changed their enzyme activities. The inhibition of hemagglutinating activity by glucuronic acid or fructose 1,6-diphosphate decreased the proteolytic activities of both lectins, whereas the blocking of this activity with D-glucosamine or D-galactosamine increased the proteolytic activity of LII. The molecules of the B. polymyxa lectins are suggested to contain two active centers responsible for hemagglutinating and proteolytic activities.     

Biological Role of Lectins from the Nitrogen-Fixing <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> Strain 1460 During Bacterial–Plant-Root Interactions

Enzyme-lectins LI and LII from Paenibacillus polymyxa 1460, when incubated with the carbohydrate moiety of the wheat-root exocomponent fraction, showed an increase in their proteolytic activity. This increase may be associated with the presence of lectin-specific carbohydrates in the root fraction. The lectins of the nitrogen-fixing paenibacilli enhance cellulose degradation in the plant cell, thus increasing the activity of -glucosidase in the wheat-root cell wall.     

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.     

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     

The Role of Cell-Surface Lectins in the Aggregation of Azospirilla

The mutant strain Azospirillum brasilenseSp7.2.3 with impaired lectin activity exhibited poorer cell aggregation than its parent strain A. brasilenseSp7(S) both in the exponential and stationary growth phases. The pretreatment of bacterial cells with the specific haptens (L-fucose and D-galactose) of a lectin located at the cell surface of the mutant strain was found to inhibit the aggregation of azospirilla. The specific binding of the A. brasilenseSp7(S) lectin to the extracellular polysaccharide-containing complexes of this strain was revealed by dot immunoblotting on nitrocellulose membrane filters. The interaction of the lectins of A. brasilense75, A. brasilenseSp7, and A. lipoferum59b with the polysaccharide-containing complexes that were isolated from these strains was not specific. No interstrain cross-interaction between the exopolysaccharides and lectins of azospirilla was found. A coflocculation of A. brasilenseSp7 cells with Bacillus polymyxa1460 cells was shown. The involvement of autogenous lectins in the aggregation of bacterial cells is discussed.     

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.     

Occurrence of natural lectin with bacterial agglutination property in the serum of lepidopteran pest,Parasa lepida

Insects depend on lectins for non‐self recognition and clearance of invading pathogens. Naturally occurring lectin showing specificity for galactose was purified from the serum of lepidopteran pest Parasa lepida by affinity chromatography using Sepharose 6B coupled with galactose as a gel matrix. Preliminary studies on crude serum agglutinin revealed that the agglutinin molecule showed varying degrees of specificity to avian and mammalian red blood cells tested. Among them, the highest titer of 128 was recorded against rabbit red blood cell type. The agglutinin molecule in the crude serum was stable up to 60°C and at pH between 6 and 9. Also, the hemagglutinating activity was neither dependent on divalent cations nor sensitive to ethylenediaminetetraacetic acid treatment. Galactose inhibited the hemagglutinating activity at minimum inhibitory concentration of 12.5 mM and hence it was used as a ligand for affinity chromatography. Native polyacrylamide gel electrophoresis analysis revealed a single band and the molecular weight of the lectin was found to be approximately 90 kDa. Bacterial agglutination activity of the purified lectin with two significant toxin bacteria, namely Salmonella typhi and Bacillus thuringiensis, was observed.     

Site of hemolymph lectin production and its activation in vitro by 20-hydroxyecdysone

To identify the tissues which produce hemolymph lectin in larvae of Bombyx mori, ovary, testis, fat body, and hemocytes from 5th-instar larvae were cultured in vitro and the culture medium was partially purified and assayed for hemagglutinating activity. Among the tissues tested, hemocytes appeared to be a major source of the hemolymph lectins. Ovary produced lectins to about one-tenth of the amount observed for the hemocytes, whereas testis and fat body were not productive. To study the hormonal control of hemolymph lectin production by hemocytes, hemocytes from 4th-instar larvae were cultured in vitro. Hemagglutinating activity in the hemolymph of 4th-instar larvae was immunostainable with the monoclonal antibody raised against 350,000 dalton lectin found in the 5th-instar hemolymph, but their molecular sizes were larger than the 5th-instar hemolymph lectins. When 20-hydroxyecdysone was added into the medium, production of the lectin by the hemocytes was remarkably enhanced, depending upon the hormone concentration.     

First report of an arabinose-specific fungal lectin

To date, arabinose-binding lectins have been reported only from the human opportunistic pathogen Pseudomonas aeruginosa, the plant aggressive pathogen Ralstonia solanacearum, and the sponge Pellina semitubulosa. An arabinose-binding lectin with mitogenic activity toward splenocytes and a high specific hemagglutinating activity was isolated in the present study from a wild discomycete mushroom, Peziza sylvestris. The maximal mitogenic activity was induced by a lectin concentration of 8 microM. The lectin was a single-chained protein with a molecular mass of 20 kDa. Its N-terminal sequence showed only slight resemblance to other mushroom lectins. It was adsorbed on both diethylaminoethyl-cellulose and carboxymethyl-cellulose. Unlike previously reported mushroom lectins, the hemagglutinating activity of the lectin was inhibited by arabinose, but not by a large variety of other carbohydrates. The lectin activity was adversely affected in the presence of 0.05 M NaOH or 0.025 M HCl, and when the ambient temperature was elevated above 35 degrees C.     

A Calcium Ion-Dependent Dimeric Bean Lectin with Antiproliferative Activity Toward Human Breast Cancer MCF-7 Cells

In this study, a 60.8-kDa dimeric lectin was isolated from the Phaseolus vulgaris cv. jade bean and characterized. The lectin was bound on Blue Sepharose 6 and Q Sepharose and was finally purified by size exclusion chromatography on Superdex 200. Its hemagglutinating activity toward rabbit erythrocytes was dependent on divalent cations, especially calcium ions. Various carbohydrates tested were devoid of any effect on the hemagglutinating activity. The lectin was stable at pH between 4.5 and 9.4 and temperatures between 30 and 70 °C. It did not exert antifungal activity toward Valsa mali, Setosphaeria turcica, Mycosphaerella arachidicola, Fusarium oxysporum and Bipolaris maydis. The IC₅₀ of the antiproliferative activity of the lectin toward MCF-7 human breast cancer cells was 174 μM. It did not inhibit proliferation of WRL-68 human normal embryonic hepatocytes. The lectin was dependent on calcium ions for hemagglutinating activity and possessed a blocked N-terminus. These two characteristics make the lectin unique among Phaseolus lectins.     

Purification and cDNA cloning of a lectin and a lectin-like protein from Apios americana Medikus tubers

An Apios americana lectin (AAL) and a lectin-like protein (AALP) were purified from tubers by chromatography on Butyl-Cellulofine, ovomucoid-Cellulofine, and DEAE-Cellulofine columns. AAL showed strong hemagglutinating activity toward chicken and goose erythrocytes, but AALP showed no such activity toward any of the erythrocytes tested. The hemagglutinating activity of AAL was not inhibited by mono- or disaccharides, but was inhibited by glycoproteins, such as asialofetuin and ovomucoid, suggesting that AAL is an oligosaccharide-specific lectin. The cDNAs of AAL and AALP consist of 1,093 and 1,104 nucleotides and encode proteins of 302 and 274 amino acid residues, respectively. Both amino acid sequences showed high similarity to known legume lectins, and those of their amino acids involved in carbohydrate and metal binding were conserved.     

Purification,characterization and partial sequence of a pro‐inflammatory lectin from seeds of Canavalia oxyphylla Standl. & L. O. Williams

Recent studies have shown that lectins are promising tools for use in various biotechnological processes, as well as studies of various pathological mechanisms, isolation, and characterization of glycoconjugates and understanding the mechanisms underlying pathological mechanisms conditions, including the inflammatory response. This study aimed to purify, characterize physicochemically, and predict the biological activity of Canavalia oxyphylla lectin (CoxyL) in vitro and in vivo. CoxyL was purified by a single‐step affinity chromatography in Sephadex® G‐50 column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the pure lectin consists of a major band of 30 kDa (α‐chain) and two minor components (β‐chain and γ‐chain) of 16 and 13 kDa, respectively. These data were further confirmed by electrospray ionization mass spectrometry, suggesting that CoxyL is a typical ConA‐like lectin. In comparison with the average molecular mass of α‐chain, the partial amino acid sequence obtained corresponds to approximately 45% of the total CoxyL sequence. CoxyL presented hemagglutinating activity that was specifically inhibited by monosaccharides (D‐glucose, D‐mannose, and α‐methyl‐D‐mannoside) and glycoproteins (ovalbumin and fetuin). Moreover, CoxyL was shown to be thermostable, exhibiting full hemagglutinating activity up to 60°C, and it was pH‐sensitive for 1 h, exhibiting maximal activity at pH 7.0. CoxyL caused toxicity to Artemia nauplii and induced paw edema in rats. This biological activity highlights the importance of lectins as important tools to better understand the mechanisms underlying inflammatory responses. Copyright © 2014 John Wiley & Sons, Ltd.     

Bioinformatics analyses of the mannose-binding lectins from Polygonatum cyrtonema,Ophiopogon japonicus and Liparis noversa with antiproliferative and apoptosis-inducing activities

In the present study, three typical monocot mannose-binding lectins (e.g., Polygonatum cyrtonema lectin [PCL], Ophiopogon japonicus lectin [OJL] and Liparis noversa lectin [LNL]), were reported to possess a similar tertiary structure with three mannose-binding sites and a close phylogenetic relationship. Subsequently, these lectins were found to bear remarkable inhibitory effects on the growth of MCF-7 cells. Further experiments confirmed that there is a link among the hemagglutinating activity, antiproliferative activity and mannose-binding activity. In addition, these lectins were shown to induce MCF-7 cell apoptosis and caspase was found to be involved in this apoptotic pathway. In conclusion, these findings demonstrate that the different antiproliferative effects may be due to the conserved motifs of mannose-binding sites. Furthermore, our results demonstrate that these lectins induce apoptosis in MCF-7 cells via a caspase-dependent pathway.     

Effect of Azospirillum Lectins on the Activities of Wheat-root Hydrolytic Enzymes

This work studied the effect of two cell-surface lectins isolated from the nitrogen-fixing soil bacterium Azospirillum brasilense Sp7 and from its mutant defective in hemagglutinating activity, A. brasilense Sp7.2.3, on the activities of α-glucosidase, β-glucosidase and β-galactosidase in the exocomponent, membrane and apoplast fractions of wheat-seedling roots. Lectin (40 μg mL⁻¹) incubation for 1 h of the plant fractions increased the enzymes’ activities; both wild-type and mutant lectins were most stimulatory to the activities of all the exocomponent-fraction enzymes studied and to the apoplast-fraction β-glucosidase. Pretreatment of the lectins with their carbohydrate hapten, L-fucose, lowered the effect. The observed differences in the lectins’ ability to influence enzyme catalytic activity are explained by change in the antigenic properties of the mutant lectin.     

BUL: A novel lectin from Bauhinia ungulata L. seeds with fungistatic and antiproliferative activities

A new galactose-binding lectin, termed BUL, has been purified from seeds of Bauhinia ungulata L. (Caesalpinoideae) by precipitation with solid ammonium sulfate followed by agarose–lactose affinity chromatography. B. ungulata lectin strongly agglutinated rabbit erythrocytes, both native and treated with proteolytic enzymes, and was inhibited by d-galactose and d-galactose-derived sugars, especially N-acetyl-d-galactosamine. BUL was shown to be a stable glycoprotein, maintaining its hemagglutinating activity after incubation at wide ranges of temperature and pH, but not after incubation with EDTA. By SDS-PAGE analysis, purified BUL showed an electrophoretic profile consisting of a single band with apparent molecular mass of 30 kDa. BUL showed intrinsic fluorescence typical of folded globular proteins, and CD spectra of lectin in the native state showed a predominance of β-sheet secondary structure. The N-terminal amino acid sequence of 19 residues showed a high sequential similarity to other galactose-specific lectins from the Bauhinia genus. In addition, BUL showed antifungal activity against phytopathogenic species and showed in vitro antiproliferative activity against the HT-29 cell line of human colon adenocarcinoma in a dose-dependent manner.     

Comparative studies of seed proteins of the genusArtocarpus with respect to lectins

Proteins from two species of the genusArtocarpus (A. integrifolia L. andA. incisa L.) were compared by ammonium sulphate fractionation, molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis, with special attention to the lectins. The protein content and hemagglutinating activity were markedly different in the two seeds. The protein pattern obtained by both molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis were quite different. The only similarities found were the elution volume of the lectins in the Sephadex G-100 column and the lectin bands (11 500 and 15 000 daltons) in SDS-polyacrylamide gel electrophoresis.     

Purification,characterization and induction of a C-type lectin in the freshwater planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

Lectins are important components of the immune defense system of invertebrates. Given their important functions, numerous investigations have been carried out on the characterization and function of lectins in invertebrates. However, lectin studies with the freshwater planarian, an evolutionarily important animal, are rare. In this paper, we demonstrate agglutination of glutaraldehyde treated erythrocytes by a lectin with preference for rabbit erythrocytes. The result of hemagglutinating activity inhibition assays with several carbohydrates showed the most potent inhibitor was maltose. A natural lectin from the crude homogenates of freshwater planarian Dugesia japonica was purified by single step affinity chromatography using amylose-coupled agarose. The purified protein appeared as one band with a molecular mass of 350 kDa in PAGE, and as one band, approximately 56 kDa, in SDS-PAGE. The purified lectin showed dependence on calcium. The activity of the purified lectin was inhibited at temperatures greater than 50°C and showed a pH optimum between 5–8. The purified lectin also has binding activity to the Gram-negative bacteria E. coli, and the Gram-positive bacteria B. subtilis. Furthermore, the purified lectin obtained from injured and bacteria-induced planarians showed increased agglutinating activity against rabbit erythrocytes. These results suggest that the purified lectin may play an important role in the innate immunity of the freshwater planarian.