Thermostable mannose-binding lectin from <italic>Dendrobium findleyanum</italic> with activities dependent on sulfhydryl content

Abstract

A mannose-binding lectin was purified from Dendrobium (D.) findleyanum pseudobulb using mannan-agarose column chromatography. After heating in the presence of SDS with or without 2-mercaptoethanol on SDS-PAGE with a continuous gradient of 8%−20% acrylamide, the purified lectin showed only one protein band with a molecular mass of 14.5 kDa. Without heating, two bands were seen on the gel at the positions of 14.5 kDa and 53.7 kDa, but a higher amount of the 53.7 kDa protein was observed in the presence of 2-mercaptoethanol. Protein identification of both protein bands by liquid chromatography-tandem mass spectrometry showed three peptide fragments identical to parts of a lectin precursor from D. officinale; the lectin was named D. findleyanum agglutinin (DFA). Using various concentrations of native-PAGE and Ferguson plot, only one protein band revealed a molecular mass of 56.2 kDa, indicating four 14.5 kDa polypeptide subunits in the DFA. Isoelectric focusing revealed that the DFA had three conformational forms with an isoelectric point of 5.18, 4.87 and 4.72, whereas 2-mercaptoethanoltreated DFA showed only one band with an isoelectric point of 5.18. DFA exhibited specificity towards mannose using the solid-phase method. The binding activity, anti-fungal activity and hemagglutination activity of DFA were not affected by heat, but were increased by free sulfhydryl groups.

The Pepper Mannose-Binding Lectin Gene CaMBL1 Is Required to Regulate Cell Death and Defense Responses to Microbial Pathogens

“…Gastrodianin-like proteins also inhibit the mycelial growth of Botrytis cinerea, Gibberella zeae, Ganoderma lucidum, R. solani, and Valsa ambients (Cox et al, 2006). Dendrobium findleyanum agglutinin inhibits the growth of Alternaria alternata (Sudmoon et al, 2008).…”

The first trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae was found in Dendrobium pendulum: purification, characterization, and effects of stress factors

“…Crude protein extraction was performed according to the procedures reported by Sudmoon et al (2008) with some modifications. A 0.4 g of treated or untreated pseudobulb was ground in 0.5 ml of extraction buffer (100 mM TrisHCl, 20 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0, containing 5 mM 4-aminobenzamidine dihydrochloride, and 1 mM phenylmethylsulfonyl fluoride) by mortar and pestle at 4°C.…”

“…Mannan-agarose (Sigma; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) column chromatography was used to purify mannose-binding protein from the crude protein extract as described by Sudmoon et al (2008) with slight modification. The eluted protein was immediately adjusted to pH 7 with 1 M Tris base.…”

“…Almost all plant lectins can be grouped into 12 families based on their structure, sugar specificity, and sequence homology (Van Damme et al 2008;Lannoo and Van Damme 2010). Among these lectin families, Galanthus nivalis agglutinin (GNA)-related lectin family, previously called monocot mannose-binding lectins family (Van Damme et al 2008;Lannoo and Van Damme 2010), has increased attention from the researchers because of their antitumor (Wu and Bao 2013), antiviral (Balzarini et al 2004;Wu and Bao 2013), insecticidal (Vandenborre et al 2011;Gupta and Das 2012), and antifungal activities (Xu et al 1998;Chen et al 2005a;Sudmoon et al 2008;Sattayasai et al 2009). Previous studies presumed that the GNA-related lectins were found only in monocots.…”

“…Gastrodia antifungal protein (GAFP-1) (Xu et al 1998), Dendrobium findleyanum agglutinin (DFA) (Sattayasai et al 2009), and Dendrobium officinale agglutinin2 (DOA2) (Chen et al 2005a) showed antifungal properties while Listera ovata agglutinin (LOA), Cymbidium hybrid agglutinin, and Epipactis helleborine agglutinin (EHA) showed inhibitory effects against human immunodeficiency virus (HIV) (Balzarini et al 1992;Balzarini 2006). In addition to the antifungal activity, DFA showed three conformational forms and its activities were increased by 2-mercaptoethanol (Sudmoon et al 2008). Because of the interesting properties of DFA and specific morphological traits of D. findleyanum pseudobulb, we decided to investigate the lectins from other Dendrobium species with similar pseudobulb morphology.…”