Purification and characterization of a lectin, Bryohelion, involved in the protoplast formation of a marine green alga Bryopsis plumosa (Chlorophyta)

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 lectin-carbohydrate 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 cross-linked 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.'