PURIFICATION AND CHARACTERIZATION OF A LECTIN, BRYOHEALIN, INVOLVED IN THE PROTOPLAST FORMATION OF A MARINE GREEN ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA) ¹

Abstract

When the coenocytic green alga Bryopsis plumosa (Huds.) Ag. was cut open and the cell contents were expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. Aggregation of cell organelles in seawater was mediated by a lectincarbohydrate complementary system. Two sugars, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine inhibited aggregation of cell organelles. The presence of these sugars on the surface of chloroplasts was verified with their complementary fluorescein isothiacyanate-labeled lectins. An agglutination assay using human erythrocytes showed the presence of lectins specific for N-acetyl-D-galactosamine and N-acetyl-D-glucosamine in the crude extract. One-step column purification using N-acetyl-D-glucosamine-agarose affinity chromatography yielded a homogeneous protein. The protein agglutinated the cell organelles of B. plumosa, and its agglutinating activity was inhibited by the above sugars. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed that this protein might be composed of two identical subunits crosslinked by two disulfide bridges. Enzyme and chemical deglycosylation experiments showed that this protein is deficient in glycosylation. The molecular weight was determined as 53.8 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The N-terminal 15 amino acid sequence of the lectin was Ser-Asp-Leu-Pro-Thr-X-Asp-Phe-Phe-His-Ile-Pro-Glu-Arg-Tyr, and showed no sequence homology to those of other reported proteins. These results suggest that this lectin belongs to a new class of lectins. We named this novel lectin from B. plumosa ''bryohealin.''

Molecular characterization of a lectin, BPL-4, from the marine green alga Bryopsis plumosa (Chlorophyta)

“…For the successful aggregation of cell organelles during protoplast formation in B. plumosa, the existence of additional lectins was suggested (Kim et al 2006, Han et al 2011. In this study, we describe the biochemical and molecular characterization of another lectin from B. plumosa, BPL-4, which might also be involved in the protoplast regeneration of this species.…”

“…First, a degenerate forward primer [BPL4-NDF1, 5′-CA(A/G) plants. Possible involvement of lectins in the aggregation of cell organelles was previously suggested (Pak et al 1991, Kim et al 2001, 2002, Klotchkova et al 2003 and three lectins (Bryohealin, BPL-2, and BPL-3) were isolated in this species (Yoon et al 2008, 2011.For the successful aggregation of cell organelles during protoplast formation in B. plumosa, the existence of additional lectins was suggested (Kim et al 2006, Han et al 2011. In this study, we describe the biochemical and molecular characterization of another lectin from B. plumosa, BPL-4, which might also be involved in the protoplast regeneration of this species.…”

“…posed for the aggregation mechanism of the extruded cell organelles in seawater (Kim et al 2006). So far, three lectins (Bryohealin, BPL-2, and BPL-3) have been isolated in this species (Yoon et al 2008, 2011.…”

Affinity Chromatography of Lectins

“…Recently, isolation of lectins from various sources has been described using numerous sugars, such as lactose (Almanza et al, 2004;Braga et al, 2006;Fujii et al, 2011;Hamako et al, 2007;Kawsar et al, 2009;Mendonça-Franqueiro et al, 2011;Naeem et al, 2007b;Pohleven et al, 2009;Rocha et al, 2009;Silva et al, 2007), sucrose and glucose (Pohleven et al, 2011), galactose (Konami et al, 1991;Lavanya Latha et al, 2006;Mo et al, 2000;Moreira et al, 1998;Naeem et al, 2007b;Nagata, 2005), mannose (Andon et al, 2003;Ooi et al, 2010;Suseelan et al, 2002), fucose (Cammarata et al, 2007;Mansour & Abdul-Salam, 2009), L-rhamnose (Jimbo et al, 2007;Watanabe et al, 2008), and sugars with 2'-acetamido groups N-acetylgalactosamine (Gerlach et al, 2005;Perçin et al, 2009;Qureshi et al, 2006), N-acetylglucosamine (Kaur et al, 2005;Kim et al, 2006;Maheswari et al, 2002;Wang & Ng, 2003), and N,N'-diacetylchitobiose (Konami et al, 1991;Koyama et al, 2002).…”

“…Commercially available agarose matrices pre-coupled with carbohydrates or glycoproteins have also been used, such as agarose beads with immobilized lactose (EY Laboratories) (Hamako et al, 2007), galactose (Pierce) (Trindade et al, 2006), mannose (Sigma-Aldrich) (Ooi et al, 2002;Suseelan et al, 2002), N-acetylgalactosamine (Gerlach et al, 2005;Qureshi et al, 2006), N-acetylglucosamine (Sigma-Aldrich) (Kim et al, 2006;Wang & Ng, 2003), N,N'-diacetylchitobiose (Sigma-Aldrich) (Koyama et al, 2002), mannan (Sigma-Aldrich) (Naeem et al, 2007a;Ourth et al, 2005), and fetuin (Sigma-Aldrich) (Ooi et al, 2002;Rittidach et al, 2007).…”