Purification and Characterization of a Lectin from Green Split Peas (Pisum sativum).

Lectins have captured the attention of a large number of researchers on account of their various exploitable activities, including antitumor, immunomodulatory, antifungal, as well as HIV reverse transcriptase inhibitory activities. A mannose/glucose-specific lectin was isolated from green split peas (a variety of Pisum sativum) and characterized. The purification step involved anion-exchange chromatography on a DEAE-cellulose column, cation-exchange chromatography on an SP-Sepharose column, and gel filtration by fast protein liquid chromatography (FPLC) on Superdex 200. The purified lectin had a native molecular mass of around 50 kDa as determined by size exclusion chromatography. It appeared as a heterotetramer, composed of two distinct polypeptide bands with a molecular mass of 6 and 19 kDa, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal sequence of green split pea lectin shows some degree of homology compared to lectins from other legume species. Its hemagglutinating activity was inhibited by glucose, mannose, and sucrose, and attenuated at pH values higher than 12 or lower than 3. Hemagglutinating activity was preserved at temperatures lower than 80 °C. The lectin did not show antifungal activity toward fungi including Fusarium oxysporum, Botrytis cinerea, and Mycosphaerella arachidicola. Green split pea lectin showed a mitogenic effect toward murine splenocytes and could inhibit the activity of HIV-1 reverse transcriptase.

Purification, characterization, and mitogenic potential of a mucin-specific mycelial lectin from Aspergillus sparsus.

Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which is responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Due to their carbohydrate specificity, lectins have been used for purification and characterization of glycoproteins like antibodies, cytokines, tumor-associated glycoproteins, hormones, inhibitors, growth factors, various enzymes, membrane proteins (receptors), or even toxins and viruses. In the present study, a mycelial lectin from Aspergillus sparsus was purified, characterized, and evaluated for its mitogenic potential. Lectin could be effectively purified 17.8-fold in a single-step using affinity chromatography on mucin-sepharose column. Lectin migrated as a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass of 100.2 kDa. Lectin showed pH optima of 6.5-8.0, and optimum temperature was determined to be 20-30 °C. Lectin was stable within a pH range of 5.5-10.0 and showed fairly good thermostability. Lectin activity was unaffected in the presence of EDTA, while activity reduced upon interaction with denaturants. MTT assay revealed strong mitogenic potential of A. sparsus lectin at a concentration up to 100 µg/ml.

Characterisation of a haemagglutinin from Hokkaido red bean (Phaseolus vulgaris cv. Hokkaido red bean).

BACKGROUND: A haemagglutinin was purified from Japanese Hokkaido red beans (Phaseolus vulgaris cv. Hokkaido red bean) with a procedure that included three chromatographic media. RESULTS: Haemagglutinating activity was adsorbed on DEAE cellulose, Affi-gel blue gel and Mono S. The pure haemagglutinin was a homodimer and each subunit was around 30 kDa in molecular mass. The haemagglutinating activity of this agglutinin could not be inhibited by a variety of simple sugars at 200 mmol L(-1) concentration including alpha-L-fucose, D(+)-galactose, D(+)-glucose, D(+)-glucosamine, D(-)galactosamine, galacturonic acid, (+)-lactose, D(+)-melibose, L(-)-mannose, D(+)-mannose, D-mannosamine, D(+)-raffinose, L-rhamnose, (+)-xylose and galacturonic acid. The haemagglutinating activity was fully retained at pH 4-11 and at 0-80 degrees C, but was completely lost at extreme pH values (0-2 and 13-14) and at very high temperatures (90 degrees C and 100 degrees C). The haemagglutinin exhibited a weak mitogenic activity toward mouse splenocytes, a stronger anti-proliferative activity than Con A toward HepG2 (human hepatoma) cells and inhibited >80% of HIV-1 reverse transcriptase inhibitory activity at 3.3 micromol L(-1). It was devoid of anti-fungal activity. CONCLUSION: Hokkaido red bean haemagglutinin possesses a potent anti-proliferative effect on HepG2 cells.

A novel lectin with highly potent antiproliferative and HIV-1 reverse transcriptase inhibitory activities from cicada (Cicada flammata).

A dimeric lectin with a molecular weight of 60 kDa and high hemagglutinating activity was isolated from dried cicadas. It was adsorbed on Q-Sepharose and unadsorbed on Affi-Gel Blue gel. Its hemagglutinating activity was stable up to 55 degrees C and between pH 2 and 13. The activity was inhibited by glucuronic acid and raffinose, K(+) ions, and Mg(2+) ions. Cicada lectin potently inhibited proliferation of HepG2 hepatoma and MCF 7 breast cancer cells, with an IC(50) value of 0.76 and 0.49 microM, respectively. It potently inhibited HIV-1 reverse transcriptase activity with an IC(50) of 0.36 microM but was devoid of mitogenic activity on spleen cells. Its N-terminal sequence exhibited slight similarity to a conserved hypothetical protein from Culex quinquefasciatus and a gene product from transcript GH19834-RA of Drosophila grimshawi, but there was no resemblance to lectins from other insects, including Drosophila, Sarcophaga, Glossina, and Aedes species.

Purification and characterization of a lectin from Phaseolus vulgaris cv. (Anasazi beans).

A lectin has been isolated from seeds of the Phaseolus vulgaris cv. "Anasazi beans" using a procedure that involved affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The lectin was comprised of two 30-kDa subunits with substantial N-terminal sequence similarity to other Phaseolus lectins. The hemagglutinating activity of the lectin was stable within the pH range of 1-14 and the temperature range of 0-80 degrees C. The lectin potently suppressed proliferation of MCF-7 (breast cancer) cells with an IC(50) of 1.3 microM, and inhibited the activity of HIV-1 reverse transcriptase with an IC(50) of 7.6 microM. The lectin evoked a mitogenic response from murine splenocytes as evidenced by an increase in [3H-methyl]-thymidine incorporation. The lectin had no antifungal activity. It did not stimulate nitric oxide production by murine peritoneal macrophages. Chemical modification results indicated that tryptophan was crucial for the hemagglutinating activity of the lectin.

Antiproliferative effect of T/Tn specific Artocarpus lakoocha agglutinin (ALA) on human leukemic cells (Jurkat, U937, K562) and their imaging by QD-ALA nanoconjugate.

T/Tn specificity of Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha (Moraceae) fruit and a heterodimer (16 kD and 12 kD) of molecular mass 28 kD, was further confirmed by SPR analysis using T/Tn glycan containing mammalian glycoproteins. N-terminal amino acid sequence analysis of ALA showed homology at 15, 19-21, 24-27, and 29 residues with other lectin members of Moraceae family viz., Artocarpus integrifolia (jacalin) lectin, Artocarpus hirsuta lectin, and Maclura pomifera agglutinin. It is mitogenic to human PBMC and the maximum proliferation was observed at 1 ng/ml. It showed an antiproliferative effect on leukemic cells, with the highest effect toward Jurkat cells (IC(50) 13.15 ng/ml). Synthesized CdS quantum dot-ALA nanoconjugate was employed to detect the expression of T/Tn glycans on Jurkat, U937, and K562 leukemic cells surfaces as well as normal lymphocytes by fluorescence microscopy. No green fluorescence was observed with normal lymphocytes indicating that T/Tn determinants, which are recognized as human tumor associated structures were cryptic on normal lymphocyte surfaces, whereas intense green fluorescent dots appeared during imaging of leukemic cells, where such determinants were present in unmasked form. The above results indicated that QD-ALA nanoconjugate is an efficient fluorescent marker for identification of leukemic cell lines that gives rise to high quality images.

Mitogenic component in polar lipid-enriched Anaplasma phagocytophilum membranes.

Human granulocytic anaplasmosis is an emerging tick-borne disease caused by Anaplasma phagocytophilum. A. phagocytophilum cells activate Toll-like receptor 2 signaling and possess mitogenic activity, and A. phagocytophilum infection in vivo activates NKT cells unrelated to major surface protein 2 (Msp2) hypervariable region expression. Thus, we hypothesized that lipoprotein or glycolipid components of A. phagocytophilum membranes could be important triggers of the innate immune response and immunopathology. A. phagocytophilum membranes depleted of Msp2 and protein antigens enhanced the proliferation of naïve mouse splenocytes beyond that of untreated membranes. Protein-depleted and polar lipid-enriched membranes from low-passage A. phagocytophilum cultures enhanced naïve splenocyte lymphoproliferation to a much greater degree than did these fractions from high-passage cultures of bacterial membranes (1.8- to 3.7-fold for protein-depleted fractions and 4.8- to > or =17.7-fold for polar lipid-enriched fractions). These results support the hypothesis that components that are enriched among polar lipids in the A. phagocytophilum membrane stimulate innate immune cell proliferation, possibly activating NKT cells that link innate and adaptive immunity, and immunopathology.

A mitogenic defensin from white cloud beans (Phaseolus vulgaris).

A peptide, with a molecular mass of 7458 Da, was purified from the seeds of white cloud beans (Phaseolus vulgaris cv. 'white cloud bean'). This peptide was isolated using a simple protocol consisting of affinity chromatography on Affi-gel blue gel and gel filtration on Superdex 75. The peptide had both antifungal and antibacterial activities. It reduced the activity of HIV-1 reverse transcriptase and it also inhibited translation in a cell-free rabbit reticulocyte lysate system. Its antifungal activity was retained after incubation with trypsin but was reduced when the ambient ionic strength was raised. The peptide elicited a mitogenic response from mouse splenocytes but did not stimulate nitric oxide production in mouse macrophages.

Purification and characterization of a galactose-specific lectin with mitogenic activity from pinto beans.

A galactose-specific dimeric lectin from pinto beans was purified using a procedure that involved affinity chromatography on Affi-gel blue gel, anion exchange chromatography on Q-Sepharose, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The molecular mass of this homodimeric lectin was 62 kDa and that of each of its subunits was 31 kDa. The hemagglutinating activity of pinto bean lectin was stable within the pH range of 3-12 and the temperature range of 0-70 degrees C. By using the [3H-methyl]-thymidine incorporation assay, it was shown that the lectin had the ability to evoke a mitogenic response from murine splenocytes but it did not inhibit proliferation of L1210 leukemia cells. The pinto bean lectin inhibited HIV-1 reverse transcriptase with an IC50 of 3 microM.

Two novel lectins from Parkia biglandulosa and Parkia roxburghii: isolation, physicochemical characterization, mitogenicity and anti-proliferative activity.

Two mannose/glucose specific seed lectins were isolated from Parkia biglandulosa and Parkia roxburghii and were characterized w.r.t various physicochemical properties. Unlike other Parkia lectins a comparison of native and subunit molecular mass showed that both Parkia lectins were heterotetramers. Parkia biglandulosa lectin was found to be T-cell mitogen as revealed by IL-2 bioassay. These lectins showed anti-proliferative effect on two murine macrophage cancer cell lines i.e. P 388DI (50%) and J774 (70%). In addition Parkia roxburghii also inhibited proliferation of HB98 (65.47%), a B-cell hybridoma cell line.

Novel lectins from rhizomes of two Acorus species with mitogenic activity and inhibitory potential towards murine cancer cell lines.

Two novel lectins were purified from rhizomes of two sweet flag species, namely Acorus calamus (Linn.) and Acorus gramineus (Solandin Ait.) by affinity chromatography on mannose linked epoxy-activated Sepharose 6B. The apparent molecular mass of the lectins, as determined by gel filtration chromatography, was 56 kDa for ACL and 55 kDa for AGL. In SDS-PAGE, pH 8.3, both lectins migrated with a subunit molecular mass of 13.6 kDa and 13.5 kDa, respectively, under reducing and non-reducing conditions thus indicating the absence of disulphide linkages. Acorus lectins readily agglutinated rabbit, rat and guinea pig erythrocytes. Both ACL and AGL also reacted with RBCs from sheep, goat and human ABO blood groups after neuraminidase treatment. ACL and AGL were inhibited by mannose/glucose and their derivatives. The most effective inhibitor was methyl-alpha-D-mannopyranoside. Acorus lectins were stable up to 55 degrees C, did not require metal ions for their activity and were also affected by high concentrations of denaturants like urea, thiourea and guanidine-HCl. These lectins showed potent mitogenic activity towards mouse splenocytes and human lymphocytes. Both ACL and AGL also significantly inhibited the growth of J774, a murine macrophage cancer cell-line and to lesser extent WEHI-279, a B-cell lymphoma.

Mitogenic activity of tracheal effluents from premature infants with chronic lung disease.

Lung injury alters the expression and release of growth factors that disrupt postnatal pulmonary development in newborns and causes chronic lung disease (CLD). The effect of these factors, released into the airways of newborns with CLD, on cell proliferation and collagen production was characterized in vitro. Human fetal lung fibroblast and alveolar-epithelial-like cell lines (FHs 738Lu and A549, respectively) were exposed to tracheal effluents from infants with CLD (mean gestation, 24.7 +/- 0.9 wk; birth weight, 666 +/- 85 g; postnatal age, 0-62 d). In both cell types, proliferation was assessed by measuring [(3)H]-thymidine uptake; in fibroblasts, collagen production was analyzed by measuring [(3)H]-proline incorporation. The activity of specific growth factors in effluents was determined using anti-growth factor antibodies and the growth factors themselves. Growth factors in tracheal effluents promoted proliferation in a dose-dependent manner and caused up to a 10.2- and 3.1-fold increase in thymidine uptake by fibroblasts and epithelial cells, respectively. Collagen production by fibroblasts increased dose dependently, peaking at 177% of baseline. Antibody against transforming growth factor beta-1 (TGF-beta(1)) inhibited proliferation and the increase in collagen production by 31% (p = 0.01) and 14% (p = 0.045), respectively. Antibody against hepatocyte growth factor (HGF) inhibited proliferation of epithelial cells (25%, p = 0.039). The effects of exogenous TGF-beta(1) on fibroblasts and HGF on epithelial cells resembled those of tracheal effluents. Potent mitogenic and differentiating substances are released into the tracheal effluents of newborns with CLD. TGF-beta(1) may worsen CLD by inducing fibrosis whereas HGF may favor resolution by promoting epithelialization.

Phospholipids and their derivatives as mitogen and motogen of budding tunicates.

In order to discover novel invertebrate cytokines from the budding tunicate, Polyandrocarpa misakiensis, we treated the water-insoluble fraction of tunicate homogenates with trypsin. The extracts showed remarkable activities to promote the growth and motility of tunicate cells. The activities were heat-stable and proteinase K-resistant. After anion exchange chromatography, the activities were eluted with detergents such as 0.1% deoxycholic acid. The Fourier transform infrared spectrum indicated large amounts of fatty acids and phospholipids instead of polypeptides in the extracts. Consistently, the activities were extractable with organic solvents such as chloroform. Long chains of n-3 polyunsaturated free fatty acids (FFA), phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) were the major components in the lipid-soluble fraction. A cDNA for FFA-releasing enzyme phospholipase A(2) (PLA(2)) was cloned. The expression of this gene could be seen in epidermal cells during budding. The recombinant protein, as in the case of the authentic PLA2, preferred PC and PE as substrates, followed by PS and PI. The resultant FFAs only promoted cell growth, while the remaining lysophospholipids stimulated cell motility. The former contained unsaturated fatty acids (C18:1, C20:5, and C22:6) while the latter did not, suggesting that unsaturated fatty acids are responsible for mitogenic activity in tunicate cells. These results show for the first time that phospholipids and their derivatives are bio-mediators promoting cell growth and cell motility in invertebrates.

A mushroom (Ganoderma capense) lectin with spectacular thermostability, potent mitogenic activity on splenocytes, and antiproliferative activity toward tumor cells.

An 18-kDa lectin, with an N-terminal sequence displaying slight similarity to some lectins and fungal immunomodulatory proteins, was isolated from the mushroom Ganoderma capense (Lloyd) Teng. It exhibited more potent mitogenic activity than that of concanavalin A toward mouse splenocytes, and antiproliferative activity toward leukemia (L1210 and M1) cells and hepatoma (HepG2) cells. The isolation procedure entailed ion exchange chromatography on Q-Sepharose, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 75. D(+)-galactose and D(+)-galactosamine specifically inhibited the hemagglutinating activity of the lectin. The hemagglutinating activity of the lectin was not affected over the temperature range 0-100 degrees C and after exposure to 100 degrees C for 60min. The activity was stable in the pH range of 4-11, and after incubation with solutions of various chlorides (from 3.125 to 50mM) including NaCl, KCl, CaCl(2), MgCl(2), ZnCl(2), MnCl(2), and AlCl(3). However, it was potentiated by 12.5-50mM FeCl(3). The lectin was devoid of HIV-1 reverse transcriptase inhibitory and antifungal activities.

Isolation of vulgin, a new antifungal polypeptide with mitogenic activity from the pinto bean.

An antifungal polypeptide bearing an N-termnial sequence with some homology to chitinases was purified from an extract of pinto beans. The polypeptide, designated vulgin, exerted antifungal activity toward Mycosphaerella arachidicola, Coprinus cornatus, Fusarium oxysporum and Botrytis cinerea. Vulgin inhibited translation in a rabbit reticulocyte lysate system with an IC50 of 4.3 microM and HIV-1 reverse transcriptase activity with an IC50 of 58 microM. Vulgin stimulated in vitro incorporation of methyl [3H] thymidine into mouse splenocytes.

Fishing for bioactive substances from scorpionfish and some sea urchins.

Venom proteins from the dorsal spine of two scorpionfish, Hypodytes rubripinnis and Synanceia verrucosa were assayed for mitogenicity and cytotoxicity. The two venoms had both mitogenic and cytotoxic activity on murine splenocytes and murine P388 leukemic cells. In H. rubripinnis, the second gel chromatographic fraction showed cytotoxic activity on P388 leukemic cells. On native PAGE, the glycoprotein isolated by concavalin A sepharose chromatography appeared to have a molecular mass of 110 kDa. In addition, two D-galactose-binding lectins (SUL-I and SUL-II) and a heparin-binding lectin (TGL-I) were purified from the globiferous pedicellariae of the toxopneustid sea urchins, Toxopneustes pileolus and Tripneustes gratilla, respectively. SUL-I (Nakagawa et al., 1999a) had mitogenic activity and cytotoxic activity but SUL-II and TGL-I did not. SUL-I did not show sequence homology to SUL-II. A hemolytic lectin with a molecular mass of 29 kDa was isolated from the coelomic fluid of T. gratilla. The hemolytic activity of the lectin was dependent on Ca2+ concentration and inhibited by lactose. The present results suggest that some species of scorpionfish and sea urchins may be novel sources for biologically active substances such as anti-tumor compounds or new lectins.

Isolation of lilin, a novel arginine- and glutamate-rich protein with potent antifungal and mitogenic activities from lily bulbs.

From the dried bulbs of the lily (Lilium brownii), a protein with strong antifungal and mitogenic activities was isolated. It also exhibited an inhibitory action on the activity of HIV-1 reverse transcriptase. The protein was single-chained and possessed a molecular weight of 14.4 kDa and an N-terminal sequence distinct from chitinases and antimicrobial proteins of garlic, leek and onion which belong to a family closely related to lily. However, there was a small degree of resemblance to cyclophilins and a considerable extent of identity to the 6.5 kDa arginine/glutamate-rich polypeptide from Luffa cylindrica seeds. A nearly homogeneous preparation was obtained after the extract was fractionated on DEAE-cellulose and Affi-gel Blue gel since subsequent chromatography on Mono S and Superdex 75 both yielded a single peak.

Identification of an endocrine disrupting agent from corn with mitogenic activity.

A mitogenic agent in corncob bedding and fresh corn products disrupts sexual behavior and estrous cyclicity in rats. The mitogenic activity resides in an isomeric mixture of linoleic acid derivatives with a tetrahydrofuran ring and two hydroxyl groups (THF-diols) that include 9, (12)-oxy-10,13-dihydroxystearic acid and 10, (13)-oxy-9,12-dihydroxystearic acid. Synthetic THF-diols stimulated breast cancer cell proliferation in vitro and disrupted the estrous cycle in female rats at oral doses of approximately 0.30 mg/kg body weight/day. Exposure to THF-diols may disrupt endocrine function in experimental animals at doses approximately 200 times lower than classical phytoestrogens, promote proliferation of breast or prostate cancer, and adversely affect human health.

Identification of an angiogenic mitogen selective for endocrine gland endothelium.

The known endothelial mitogens stimulate growth of vascular endothelial cells without regard to their tissue of origin. Here we report a growth factor that is expressed largely in one type of tissue and acts selectively on one type of endothelium. This molecule, called endocrine-gland-derived vascular endothelial growth factor (EG-VEGF), induced proliferation, migration and fenestration (the formation of membrane discontinuities) in capillary endothelial cells derived from endocrine glands. However, EG-VEGF had little or no effect on a variety of other endothelial and non-endothelial cell types tested. Similar to VEGF, EG-VEGF possesses a HIF-1 binding site, and its expression is induced by hypoxia. Both EG-VEGF and VEGF resulted in extensive angiogenesis and cyst formation when delivered in the ovary. However, unlike VEGF, EG-VEGF failed to promote angiogenesis in the cornea or skeletal muscle. Expression of human EG-VEGF messenger RNA is restricted to the steroidogenic glands, ovary, testis, adrenal and placenta and is often complementary to the expression of VEGF, suggesting that these molecules function in a coordinated manner. EG-VEGF is an example of a class of highly specific mitogens that act to regulate proliferation and differentiation of the vascular endothelium in a tissue-specific manner.