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.''
When a coenocytic cell of the green alga Bryopsis plumosa (Hudson) C. Agardh was cut open and the cell contents expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. This process was mediated by a lectin, Bryohealin. The full sequence of the cDNA encoding Bryohealin was obtained, which consisted of 1,101 base pairs (bp), with 24 bp of 5' untranslated region (UTR) and 201 bp of 3' UTR. It had an open reading frame (ORF) of 771 bp encoding 257 amino acid residues. A signal peptide consisted of 22 amino acids presented before the start codon of Bryohealin, indicating that this lectin was a vacuolar (storage) protein. The C-terminal sequence of Bryohealin was composed of antibiotic domains, suggesting that this lectin could perform two functions: (i) aggregation of cell organelles in seawater and (ii) protection from bacterial contamination for successful protoplast regeneration. The BLAST search result showed that Bryohealin had little sequence homology with any known plant lectins, but rather resembled animal lectins with fucolectin domains. The expression of recombinant Bryohealin (rBryohealin) was obtained in the Escherichia coli system.
A D-mannose specific lectin was purified from the green marine alga, Bryopsis plumosa (Huds.) Ag. The lectin agglutinated horse and sheep erythrocytes. Matrix assisted laser desorption/ionization time of flight mass spectrometry, size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two dimensional gel electrophoresis (2DE) results showed that the lectin was a monomer with molecular weight of 17 kDa and pI 7.3. The agglutinating activity was inhibited by D-mannose (1 mM), a-methyl-D-mannose (4 mM) and L-fucose (8 mM). D-glucose (125 mM) showed weak inhibition. The lectin did not need divalent cations for agglutinating activity. N-terminal amino acid sequence of the lectin was analyzed. As the lectin was novel, we named it BPL-2 (Bryopsis plumosa lectin 2). Full cDNA sequence of BPL-2 was obtained using cDNA library. It was comprised of 624 bp of open reading frame and 167 bp/ 57 bp of 3′/5′ untranslated regions as well as N-terminal signal peptide. No antimicrobial activity of BPL-2 was observed in four bacteria strains tested.
A low temperature up-regulated gene was isolated from Spirogyra varians using the differentially expressed gene method. The full cDNA sequence consisted of 1798 bp with an open reading frame of 1560 bp encoding 520 amino acid residues, which had neither a cleavage site nor a signal peptide. The deduced amino acid sequence was highly similar to the bi-functional 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHQ/SDH) gene of higher plants. As the gene was cold regulated, we named it SVCR3 (Spirogyra varians cold regulated gene). SVCR3 had two conserved domains of DHQase I and SDH (AroE), as well as a shikimate-binding site as found in other DHQ/SDH genes. Northern blot analysis showed that SVCR3 was up-regulated at 4°C. When combined with light (>50 μmol photon m-2 s-1) the expression of SVCR3 increased more at 4°C, but the expression was not affected by light intensity alone up to 200 μmol photon m-2 s-1 at temperatures higher than 10°C. Using the zymogram method (which is a visualization method for gel areas containing specific enzymes based on chromogenic reactions), we showed that there were three isozymes of DHQ/SDH in Spirogyra varians. This is the first report of DHQ/SDH-like gene regulation by cold stress.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.