Thermostable mannose-binding lectin from Dendrobium findleyanum with activities dependent on sulfhydryl content.
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-mercaptoethanol-treated 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.
Sudmoon
Runglawan
R
Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand.
Sattayasai
Nison
N
Bunyatratchata
Wandee
W
Chaveerach
Arunrat
A
Nuchadomrong
Suporn
S
D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D031667:Dendrobium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007525:Isoelectric Focusing; D037601:Mannose-Binding Lectin / Q000302:isolation & purification / Q000378:metabolism*; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D037121:Plant Lectins / Q000737:chemistry* / Q000378:metabolism*; D013438:Sulfhydryl Compounds; D053719:Tandem Mass Spectrometry
D037601:Mannose-Binding Lectin; D037121:Plant Lectins; D013438:Sulfhydryl Compounds; D008623:Mercaptoethanol