Dendrobium findleyanum agglutinin: production, localization, anti-fungal activity and gene characterization

Abstract

The recently reported Dendrobium findleyanum agglutinin (DFA) was identified and determined in different parts of D. findleyanum pseudobulbs by using Western blot analysis, LC-MS/MS, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and histochemical procedure. Western blot analysis of crude protein extract with horseradish peroxidase (HRP), a mannose-rich glycoprotein, showed only one band at 14.5 kDa, which had the same molecular mass as DFA. This band was a major band when the membrane was stained with Coomassie Brilliant Blue. The protein profiles from SDS-PAGE showed higher band intensity of the 14.5 kDa mannose-binding protein in nearly mature and mature stages, compared to very young and young stages of the orchid. In addition, the band intensity was to a great extent different between the swollen and the non-swollen internode of the pseudobulb. Using LC-MS/MS, the sequence tags of the 14.5-kDa protein bands from the node, swollen internode and non-swollen internode revealed that the protein was DFA. Histochemical procedure in the transverse section of the pseudobulbs demonstrated major HRP binding sites, which reflected the location of DFA, in periphery of parenchymal cells. The purified DFA showed anti-fungal activity against Alternaria alternata and Collectotrichum sp. Using reverse transcription polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of the DFA precursor revealed 94% homology with a lectin precursor from D. officinale. N-terminal sequencing demonstrated the processing site between residues 24 and 25 of the DFA precursor.

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

“…2). The deduced amino acid sequence of DPA precursor contains 24 amino acids long as N-terminal signal peptide which was in good agreement with DOA (Chen et al 2005b) and DFA precursors (Sattayasai et al 2009). The presence of sequence TGT at C-terminal part of GNA-related lectin precursors which was proposed to be the cleavage site between G and T (Chen et al 2005b;Upadhyay et al 2011) cannot be applied to DPA precursor since one of the sequence tags of the mature protein detected by LC-MS/MS was DGNLVLYSKPIFATGTNR (Table 1; Fig.…”

“…9). Increased amount of DPA should be advantage for defensive system of D. pendulum since GNA-related lectins were reported to recognize high mannose-type glycans of foreign microorganisms or plant predators (Barre et al 2001;Chen et al 2005a;Sudmoon et al 2008, Sattayasai et al 2009). …”

“…GNA-related lectins which were investigated in Orchidaceae species exhibited interesting properties. 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).…”

“…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.…”

Inhibition of initial adhesion of oral bacteria through a lectin fromBauhinia variegataL. var. variegata expressed inEscherichia coli

“…The interaction between lectins and sugars plays an important role in several biological processes and mediates a broad range of biological activities, including inhibition of bacterial and fungal growth (Ngai and Ng ; Sattayasai et al . ; Alizadeh et al . ; Nunes et al .…”