Insights into the Structural Features, Conformational Stability and Functional Activity of the Olneya tesota PF2 Lectin

Background: The O. tesota lectin PF2 is a tetrameric protein with subunits of 33 kDa that recognizes only complex carbohydrates, resistant to proteolytic enzymes and has insecticidal activity against Phaseolus beans pest. Objective: To explore PF2 lectin features at different protein structural levels and to evaluate the effect of temperature and pH on its functionality and conformational stability. Methods: PF2 lectin was purified by affinity chromatography. Its primary structure was resolved by mass spectrometry and analyzed by bioinformatic tools, including its tertiary structure homology modeling. The effect of temperature and pH on its conformational traits and stability was addressed by dynamic light scattering, circular dichroism, and intrinsic fluorescence. The hemagglutinating activity was evaluated using a suspension of peripheral blood erythrocytes. Results: The proposed PF2 folding comprises a high content of beta sheets. At pH 7 and 25 °C, the hydrodynamic diameter (Dh) was found to be 12.3 nm which corresponds to the oligomeric native state of PF2 lectin. Dh increased under the other evaluated pH and temperature conditions, suggesting protein aggregation. At basic pH, PF2 exhibited low conformational stability. The native PF2 (pH 7) retained its full hemagglutinating activity up to 45 °C and exhibited one transition state with a melting temperature of 76.8 °C. Conclusion: PF2 showed distinctive characteristics found in legume lectins. The pH influences the functionality and conformational stability of the protein. PF2 lectin displayed a relatively narrow thermostability to the loss of secondary structure and hemagglutinating activity.

The B Subunit of PirABvp Toxin Secreted from Vibrio parahaemolyticus Causing AHPND Is an Amino Sugar Specific Lectin

Vibrio parahaemolyticus (Vp) is the etiological agent of the acute hepatopancreatic necrosis disease (AHPND) in Penaeus vannamei shrimp. Vp possesses a 63–70 kb conjugative plasmid that encodes the binary toxin PirAvp/PirBvp. The 250 kDa PirABvp complex was purified by affinity chromatography with galactose-sepharose 4B and on a stroma from glutaraldehyde-fixed rat erythrocytes column, as a heterotetramer of PirAvp and PirBvp subunits. In addition, recombinant pirB (rPirBvp) and pirA (rPirAvp) were obtained. The homogeneity of the purified protein was determined by SDS-PAGE analysis, and the yield of protein was 488 ng/100 μg of total protein of extracellular products. The PirABvp complex and the rPirBvp showed hemagglutinating activity toward rat erythrocytes. The rPirAvp showed no hemagglutinating capacity toward the animal red cells tested. Among different mono and disaccharides tested, only GalNH2 and GlcNH2 were able to inhibit hemagglutination of the PirABvp complex and the rPirBvp. Glycoproteins showed inhibitory specificity, and fetuin was the glycoprotein that showed the highest inhibition. Other glycoproteins, such as mucin, and glycosaminoglycans, such as heparin, also inhibited the activity. Desialylation of erythrocytes enhanced the hemagglutinating activity. This confirms that Gal or Gal (β1,4) GlcNAc are the main ligands for PirABvp. The agglutinating activity of the PirABvp complex and the rPirBvp is not dependent on cations, because addition of Mg2+ or Ca2+ showed no effect on the protein capacity. Our results strongly suggest that the PirBvp subunit is a lectin, which is part of the PirA/PirBvp complex, and it seems to participate in bacterial pathogenicity.

Two-Step Isolation, Purification, and Characterization of Lectin from Zihua Snap Bean (Phaseolus vulgaris) Seeds

A two-step method based on an aqueous two-phase system and Sephadex G-75 was used to separate and purify lectin from the seeds of the Zihua snap bean. The preliminary properties and bioactivity of the Zihua snap bean lectin were characterized by different instrumental methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), liquid chromatography-nano electrospray ionization mass spectrometry (Nano LC-ESI-MS/MS), and Fourier transform infrared spectroscopy (FTIR). The hemagglutinating activity of the Zihua snap bean lectin could not be inhibited by glucose, N-acetyl-d-glucosamine, d-galactose, N-acetyl-d-galactosamine, fructose, sucrose, d-maltose, d-trehalose, and lactose. It was found that the hemagglutinating activity of the lectin showed strong dependence on Mn2+ and Ca2+. The thermal and pH stability of the Zihua snap bean lectin was studied by FTIR and fluorescence spectroscopy. Relatively good stability was observed when the temperature was not higher than 70 °C, as well as in the pH range of 2.0 to 10.0. Digestive stability in vitro was investigated. The untreated lectin was relatively stable to pepsin and trypsin activity, but heat treatment could significantly reduce the digestive stability in vitro. Moreover, the lectin showed an inhibitory effect on the tested bacteria (Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Bacillus subtilis (B. subtilis)), and it also showed a certain inhibitory effect on the growth of Phytophthora infestans (P. infestans) at higher concentrations.

Investigation of Echinacea purpurea Root Proteins with Hemagglutinating Activity

Proteins were extracted and fractionated from Echinacea purpurea L. (Moench) (purple coneflower) roots, to evaluate their hemagglutinating activity. Our data suggests that the ~ 25 kDa glycosylated protein is responsible for the hemagglutinating activity and might be worth for its investigation for other biological activities.