A novel lectin specific to N-acetyl-D-galactosamine as well as N-acetyl-D-glucosamine was isolated from Bryopsis plumosa and named as BPL-4. Sodium dodecyl sulfate polyacrylamide gel electrophorese (SDS-PAGE) and matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry data showed that this lectin was a monomeric protein with molecular weight 12.9 kDa. The N-terminal amino acid sequences of the lectin were determined by Edman degradation and the full cDNA sequence encoding this lectin was obtained using the degenerate primers designed from the amino acid sequence. The size of the cDNA was 414 bp containing single open reading frame (ORF) encoding the lectin precursor. The homology analysis showed that this lectin might belong to H lectin group. BPL-4 showed high sequence similarity (60.6%) to BPL-3, which is a previously reported lectin from the same species. The comparative analysis on the lectin's primary structure showed two conserved domains including one possible active domain of H lectin group.Key Words: active domain; BPL-4; Bryopsis plumosa; lectin; N-acetyl-D-galactosamine
INTRODUCTIONLectins have the capacity to serve as recognition molecules between cells or organisms because of their noncatalytic sugar binding properties (Sharon 2008). Lectins are proteins that bind reversibly to carbohydrates, agglutinate cells, or precipitate polysaccharides and glycoproteins (Goldstein et al. 1980). Carbohydrate-binding properties of lectins have been applied in the fields of immunology, cell biology, cancer research, and genetic engineering (e.g., Sharon and Lis 2004).Recently, the medical use of lectins has been studied extensively and some lectins exhibiting high anti-HIV (Sato et al. 2007) and antibiotic activities (Liao et al. 2003) were isolated. The use of lectins as pesticide agents (Wellman-Labadie et al. 2008) and potential drug delivery systems were also explored (e.g., Jung et al. 2010). Lectinbased cell-cell recognition systems have been proposed in many algal groups and in some cases the corresponding lectins have been isolated. However, the number of characterized lectins is still considered too small (Yoon et al. 2008, Han et al. 2011.Marine green alga Bryopsis plumosa (Hudson) Agardh possesses unique ability to regenerate new functional cells from small droplets of protoplasm extruded in seawater (Kim et al. 2001). When protoplasm is released in the seawater, the organelles aggregate in vitro and form protoplasts. Then, the protoplasts develop into individual
56Dried proteins were dissolved in 150 μL of rehydration buffer (7 M urea, 2 M thio-urea, 4% CHAPS, 2% ampholyte of pH 4-10) and centrifuged at 12,000 g for 15 min to remove insoluble materials. IPG dry strip (pH 4-10, 7 cm; Bio-Rad, Bedford, CA, USA) was rehydrated with 125 μL of sample in rehydration buffer for overnight at 20°C. Isoelectric focusing (IEF) was performed at 20°C using a PROTEAN IEF Cell (Bio-Rad). The voltage was linearly increased from 250 to 4,000 V during 2 h, followed by constant 4,000...