Jong Won Han

Purification and characterization of a D-mannose specific lectin from the green marine alga, Bryopsis plumosa

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), α‐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.

PURIFICATION OF A SEX-SPECIFIC LECTIN INVOLVED IN GAMETE BINDING OF AGLAOTHAMNION CALLOPHYLLIDICOLA (RHODOPHYTA)1

Egg and sperm binding and correct recognition is the first stage for successful fertilization. In red algae, spermatial attachment to female trichogynes is mediated by a specific binding between the lectin(s) distributed on the surface of trichogyne and the complementary carbohydrates on the spermatial surface. A female‐specific lectin was isolated from Aglaothamnion callophyllidicola by agarose‐bound fetuin affinity chromatography. Two proteins, 50 and 14 kDa, eluted from the fetuin column were separated using a native‐polyacrylamide gel electrophoresis method and subjected to a gamete binding assay. The 50 kDa protein, which blocked spermatial binding to female trichogynes, was used for further analysis. Internal amino acid sequence of the 50 kDa protein was analyzed using matrix‐assisted laser desorption/ionization‐mass spectrometry and degenerated primers were designed based on the information. A full‐length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends polymerase chain reaction (PCR). The cDNA was 1552 bp in length and coded for a protein of 450 amino acids with a deduced molecular mass of 50.7 kDa, which agreed well with the protein data. Real‐time PCR analysis showed that this protein was up‐regulated about 10‐fold in female thalli. As the protein was novel and showed no significant homology to any known proteins, it was designated Rhodobindin.

Isolation and Characterization of a Sex-Specific Lectin in a Marine Red Alga, Aglaothamnion oosumiense Itono

ABSTRACT In red algae, spermatial binding to female trichogynes is mediated by a lectin-carbohydrate complementary system. Aglaothamnion oosumiense is a microscopic filamentous red alga. The gamete recognition and binding occur at the surface of the hairlike trichogyne on the female carpogonium. Male spermatia are nonmotile. Previous studies suggested the presence of a lectin responsible for gamete recognition on the surface of female trychogynes. A novel N-acetyl-d-galactosamine-specific protein was isolated from female plants of A. oosumiense by affinity chromatography and named AOL1. The lectin was monomeric and did not agglutinate horse blood or human erythrocytes. The N-terminal amino acid sequence of the protein was analyzed, and degenerate primers were designed. A full-length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends-PCR (RACE-PCR). The cDNA was 1,095 bp in length and coded for a protein of 259 amino acids with a deduced molecular mass of 21.4 kDa, which agreed well with the protein data. PCR analysis using genomic DNA showed that both male and female plants have this gene. However, Northern blotting and two-dimensional electrophoresis showed that this protein was expressed 12 to 15 times more in female plants. The lectin inhibited spermatial binding to the trichogynes when preincubated with spermatia, suggesting its involvement in gamete binding.

Isolation of Total RNA from a Freshwater Green Alga, Zygnema cruciatum, Containing High Levels of Pigments

For the study of gene transcription, obtaining total intact RNA is very important. But it is difficult to get intact RNA from some plants. This is mainly because of high contents of polysaccharides, pigments, and other unidentified compounds in these plants (Wang et al. 2004). This causes problems by interfering in RNA experiments such as a RT-PCR, DD-RT-PCR or Northern blot analysis. Various protocols for RNA isolation have been reported for plants containing high levels of polysaccharides and pigments which overcome the above mentioned limitations (Gao et al. 2001; Tai et al. 2004; Tao et al. 2004; Wang et al. 2004; Meisel et al. 2005; Birtic and Kranner 2006; Salter and conlon 2007). However, some plant materials have not yet been studied using these modified methods. Zygnema spp. are well known freshwater conjugating green algae that form free floating mats in shallow waters (Kim et al. 2007). Zygnema spp. have been reported in every biome from tundra through boreal forest to tropical rain forest and desert chaparral. It is one of few filamentous freshwater algae that lives in the Arctic as well as the Antarctic (Hawes 1989). However, little is known regarding gene transcription and translation in this complex. Because of high pigment amounts and RNA interactive molecules, it has been impossible to isolate and purify mRNA from Zygnema using conventional methods. In this paper we describe the development of a new method of RNA isolation using DEAE-cellulose resin and compare the results with other RNA isolation methods.

Functional Recombinants Designed from a Fetuin/Asialofetuin-Specific Marine Algal Lectin, Rhodobindin

Plant lectins have attracted much attention for biomedical applications including targeted drug delivery system and therapy against tumors and microbial infections. The main problem of using lectins as a biomedical tool is a batch-to-batch variation in isoforms content. The production of lectins using recombination tools has the advantage of obtaining high amounts of proteins with more precise properties, but there are only a handful of functional recombinant lectins presently available. A fetuin/asialo-fetuin specific lectin, Rhodobindin, has unique tandem repeats structure which makes it useful in exploiting for recombinant lectin. We developed three functional recombinant lectins using E. coli expression system: one from full cDNA sequence and two from fragmentary sequences of Rhodobindin. Hemagglutinating activity and solubility of the recombinant lectins were highest at OD 0.7 cell concentration at 20 °C. The optimized process developed in this study was suitable for the quality-controlled production of high amounts of soluble recombinant lectins.

Erratum to: Accumulation of galloyl derivatives in a green alga, Spirogyra varians, in response to cold stress

One of the co-author’s middle initial, family name and affiliation contain an error. The correct name should be Frithjof C. Kupper not Frithjof D. Kupper while the affiliation should be Scottish Association for Marine Science, Scottish Marine Institute, Oban, Argyll PA37 1QA, UK not Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Dunbeg PA371QA, UK. Corrected name and affiliation of the author are shown above and below.