Thermostable Lectin Research Articles

Purification, Characterization, and Assessment of Antimicrobial Activity and Toxicity of Portulaca elatior Leaf Lectin (PeLL).

Lectins are carbohydrate-binding proteins with several bioactivities, including antimicrobial properties. Portulaca elatior is a species found at Brazilian Caatinga and data on the biochemical composition of this plant are scarce. The present work describes the purification of P. elatior leaf lectin (PeLL) as well as the assessment of its antimicrobial activity and toxicity. PeLL, isolated by chromatography on a chitin column, had native liquid charge and subunit composition evaluated by electrophoresis. Hemagglutinating activity (HA) of PeLL was determined in the presence of carbohydrates or divalent cations, as well as after heating and incubation at different pH values. Changes in the lectin conformation were monitored by evaluating intrinsic tryptophan fluorescence and using the extrinsic probe bis-ANS. Antimicrobial activity was evaluated against Pectobacterium strains and Candida species. The minimal inhibitory (MIC), bactericidal (MBC), and fungicidal (MFC) concentrations were determined. Finally, PeLL was evaluated for in vitro hemolytic activity in human erythrocytes and in vivo acute toxicity in mice (5 and 10mg/kg b.w. per os). PeLL (pI 5.4; 20kDa) had its HA was inhibited by mannose, galactose, Ca2+, Mg2+, and Mn2+. PeLL HA was resistant to heating at 100°C, although conformational changes were detected. PeLL was more active in the acidic pH range, in which no conformational changes were observed. The lectin presented MIC and MBC of 0.185 and 0.74μg/mL for all Pectobacterium strains, respectively; MIC of 1.48μg/mL for C. albicans, C. tropicalis, and C. krusei; MIC and MFC of 0.74 and 2.96μg/mL for C. parapsilosis. No hemolytic activity or signs of acute toxicity were observed in the mice. In conclusion, a new, low-toxic, and thermostable lectin was isolated from P. elatior leaves, being the first plant compound to show antibacterial activity against Pectobacterium.

Purification of a lectin from Cratylia mollis crude extract seed by a single step PEG/phosphate aqueous two-phase system

The partitioning and purification of lectins from the crude extract of Cratylia mollis seeds (Cramoll 1,4) was investigated in aqueous two-phase systems (ATPS). A factorial design model (24) was used to evaluate the influence of polyethylene glycol (PEG) molar mass (1500–8000 g/mol), PEG concentration (12.5–17.5% w/w), phosphate (10–15% w/w) concentration, and pH (6–8) on the differential partitioning, purification factor, and yield of the lectin. Polymer and salt concentration were the most important variables affecting partition of lectin and used to find optimum purification factor by experimental Box–Behnken design together with the response surface methodology (RSM). ATPS showed best conditions composed by 13.9% PEG1500, 15.3% phosphate buffer at pH 6, which ensured purification factor of 4.70. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed a single band of protein with 26.1 kDa. Furthermore, results demonstrated a thermostable lectin presenting activity until 60 °C and lost hemagglutinating activity at 80 °C. According to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of lectins.

DdeL, a novel thermostable lectin from Dypsis decaryi seeds: Biological properties

Abstract A novel thermostable lectin, called DdeL, was obtained from the extract of Dypsis decaryi seeds. The extract of the tegument had the highest specific hemagglutination activity (196.03 HA/mg) when subjected to 70% acetone precipitation and purification on Superdex G-75 and exhibited a single peak (38.8 kDa). DdeL, a glycoprotein, had agglutinated blood types and was inhibited only by glycoproteins (albumin, azoalbumin and azocasein). Biochemical properties showed higher activity at acid pH (pH 5–6) than alkaline pH and activity was observed at all temperatures (up to 100 °C). DdeL exhibited bacteriostatic activity against Providencia stuartii and Staphylococcus aureus ATCC 29213, which presented minimum inhibitory concentration (MIC) of 750.0 μg/mL. When tested against fungi Sporotrichum schenckii and Candida albicans showed excellent MIC50 at 0.52 and 2.06 μg/mL, respectively. DdeL showed no cytotoxic effect on macrophages (J774.A1) and renal embryonic cells (HEK-293). in vitro acaricide activity of DdeL against Rhipicephalus showed high activity in low concentration. Therefore, we conclude that DdeL may be of interest for potential biotechnological applications.

Isolation and characterization of a galactose-specific lectin (EantH) with antimicrobial activity from Euphorbia antiquorum L. latex

A homodimeric 75 kDa lectin with hemagglutination activity (HA) was purified from the crude latex of Euphorbia antiquorum L. by two types of chromatography, on cation exchange (HiTrap SP FF) and hydrophobic HiTrap Phenyl FF (high sub) columns. The purified protein was designated EantH, and is classified as a galactose-specific thermostable lectin. The HA of EantH was stable at pH values of 5–9 and temperature 5–65 °C. The lectin had bacteriostatic action on the Gram-positive bacteria Staphylococcus aureus and S. epidermidis, with a minimum inhibitory concentration (MIC) of 2000 μg/ml and on a Gram-negative bacterium Samonella typhimurium, with a MIC of 1000 μg/ml. EantH inhibited the growth of Propionibacterium acnes and Streptococcus agalactiae with MIC of 125 μg/ml and 250 μg/ml, respectively. EantH killed P. acnes and S. agalactiae with a minimum microbicidal concentration (MMC) of 1000 μg/ml and 2000 μg/ml, respectively. Scanning electron microscopy indicated that binding of EantH to the carbohydrates in the cell walls of P. acnes and S. typhimurium drastically altered the bacterial cells, and led to inhibition of growth and/or cell death. The antimicrobial activity of EantH could be neutralized by d‑galactose, indicating that its bactericidal action involves binding to galactose in the cell wall.

CasuL: A new lectin isolated from Calliandra surinamensis leaf pinnulae with cytotoxicity to cancer cells, antimicrobial activity and antibiofilm effect

This work describes the isolation of a lectin (CasuL) from the leaf pinnulae of Calliandra surinamensis and the evaluation of its cytotoxic, antimicrobial and antibiofilm properties. Proteins from pinnulae extract were precipitated with ammonium sulphate (60% saturation) and submitted to Sephadex G-75 chromatography, which yielded isolated CasuL (purification factor: 113). Native CasuL is an acidic protein (pI 5.82) with a relative molecular mass of 48kDa. This lectin is also an oligomeric protein composed of three subunits and mass spectrometry revealed similarities with a Sorghum bicolor protein. CasuL did not undergo unfolding when heated but changes in conformation and hemagglutinating activity were detected at basic pH. CasuL did not reduce the viability of human peripheral blood mononuclear cells but was toxic to leukemic K562 cells (IC50 67.04±5.78μg/mL) and breast cancer T47D cells (IC50: 58.75±2.5μg/mL). CasuL (6.25–800μg/mL) only showed bacteriostatic effect but was able to reduce biofilm formation by Staphylococcus saprophyticcus and Staphylococcus aureus (non-resistant and oxacillin-resistant isolates). CasuL showed antifungal activity against Candida krusei causing alterations in cell morphology and damage to cell wall. In conclusion, the pinnulae of C. surinamensis leaves contain a thermo-stable lectin with biotechnological potential as cytotoxic, antibiofilm, and antifungal agent.

An alpha-glucosidase inhibitory activity of thermostable lectin protein from Archidendron jiringa Nielsen seeds

Inhibitors of α-glucosidase from natural resources that inhibit the digestion of carbohydrate polymers into monosaccharides in the gut are used in the treatment of insulin-independent diabetes mellitus type 2. Archidendron jiringa belongs to pea family of leguminous plants, some of which are a source of interesting bioactivities, including α-glucosidase inhibitory (GI) activity. A novel GI lectin was enriched from the seeds of the Djenkol bean, A. jiringa , to apparent homogeneity by 90% saturation ammonium sulfate precipitation and Con A-Sepharose affinity column chromatography. This lectin had an IC 50 value for GI activity of 0.031 „b 0.02 mg/ml, an estimated molecular mass of 35.7 kDa, of which 15.8% was carbohydrate, was thermostable up to 80¢XC for 70 min, showed an optimum activity within the pH range of 8.0 to 10.0 and a high activity with some divalent cations such as copper (Cu 2+ ) and high levels (50 to 100 mM) of zinc (Zn 2+ ) and iron (Fe 2+ ). The sequence of an internal 16 amino acid fragment of the protein showed 100% identity to the mannose-glucose specific lectin precursor of Dioclea guainensis. The GI lectin had a high specific interaction with α-glucosidase (affinity constant = 9.3773 „e 10-7 s -1 , Ks = 0.0241 s -1 , Ka = 2.39 „e 103 s -1 M -1 and Kd = 0.0117 M). Key words: Archidendron jiringa, α-glucosidase inhibitors, lectin.

Isolation and characterization of a novel thermostable lectin from the wild edible mushroom Agaricus arvensis

A thermostable novel lectin with a molecular weight of 30.4 kDa was isolated from dried fruiting bodies of Agaricus arvensis. It was a dimer made up of two 15.2 kDa subunits. The lectin was unadsorbed on DEAE-cellulose in 10 mM phosphate buffer (pH 7.5), subsequently adsorbed on CM-cellulose in 10 mM NaAc buffer (pH 4.6) and then on SP-Sepharose in 10 mM NaAc buffer (pH 4.6), and finally purified by fast protein liquid chromatography-gel filtration on Superdex 75. The hemagglutinating activity of the lectin was stable at temperatures up to 90 °C. The activity was preserved in concentrations of NaOH solution up to 50 mM, but was sensitive to HCl and declined to 12.5% in 12.5 mM HCl. The activity was unaffected by Ca(2+), Mn(2+), Zn(2+) and Mg(2+) ions, but was activated by Al(3+) and Fe(3+) ions. Among the carbohydrates tested, only inulin could inhibit the hemagglutinating activity of the lectin. It did not exhibit anti-HIV reverse transcriptase activity. Proliferation of HepG2 and MCF7 tumor cells was inhibited by the lectin with an IC(50) of 1.64 and 0.82 μM, respectively. The lectin was devoid of antifungal activity. The lectin has a remarkable thermostablity and a unique N-terminal amino acid sequence, TYAVLNFVYG. The present report is the first report on a lectin from wild mushroom Agaricus arvensis.