Dendrobium findleyanum agglutinin: production, localization, anti-fungal activity and gene characterization.
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.
Sattayasai
Nison
N
Department of Biochemistry, Faculty of Science, Khon Kaen University, Muang, Khon Kaen, 40002, Thailand. nison@kku.ac.th
Sudmoon
Runglawan
R
Nuchadomrong
Suporn
S
Chaveerach
Arunrat
A
Kuehnle
Adelheid R
AR
Mudalige-Jayawickrama
Rasika G
RG
Bunyatratchata
Wandee
W
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000378:metabolism*; D001483:Base Sequence; D031667:Dendrobium / Q000235:genetics / Q000378:metabolism*; D037601:Mannose-Binding Lectin / Q000096:biosynthesis* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D037121:Plant Lectins / Q000096:biosynthesis* / Q000302:isolation & purification; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry
D000935:Antifungal Agents; D037601:Mannose-Binding Lectin; D037121:Plant Lectins; D018749:RNA, Plant