Journal of Applied Phycology

, Volume 22, Issue 6, pp 793–802 | Cite as

Characterization of carbohydrate combining sites of Bryohealin, an algal lectin from Bryopsis plumosa

  • Min Gui Jung
  • Key Pyoung Lee
  • Han-Gu Choi
  • Sung-Ho Kang
  • Tatyana A. Klochkova
  • Jong Won Han
  • Gwang Hoon Kim


Bryohealin is a lectin involved in the wound-healing process of the marine green alga Bryopsis plumosa. In the previous purification study, it has been shown that lectin was composed of two identical subunits of 27 kDa, cross-linked by disulfide bond, and showed binding specificity to N-acetyl-d-glucosamine and N-acetyl-d-galactosamine (GlcNAc and GalNAc, respectively). To determine if the lectin recognize the two different sugars at the same binding domain, the carbohydrate binding sites of Bryohealin was analyzed using chromatography and chemical modification methods. Results showed that the same binding site of the lectin was responsible for the recognition of two sugars, GalNAc as well as GlcNAc. Chemical modification studies showed that hemagglutinating activities of Bryohealin were not affected by modification of histidine, tryptophan, aspartic acid, and glutamic acid. When arginine residues were modified with 1,2-cyclohexanedione, the activity of Bryohealin rapidly decreased. The sugar binding sites remained intact when the lectin was treated with inhibitory sugars (0.2 M GalNAc and/or GlcNAc) prior to 1,2-cyclohexanedione treatment. The sugar binding domain of Bryohealin was predicted from the MALDI-TOF analysis and the full cDNA sequence of the lectin gene.


Bryopsis plumosa Algae Bryohealin Lectin N-acetyl-d-galactosamine N-acetyl-d-glucosamine Sugar binding site Chemical modification 


  1. Ahmed H, Bianchet MA, Amzel LM, Hirabayashi J, K-i K, Giga-Hama Y, Tohda H, Vasta GR (2002) Novel carbohydrate specificity of the 16-kDa galactin from Caenorhabditis elegans: binding to blood group precursor oligosaccharides (type 1, type 2, Tα, and Tβ) and gangliosides. Glycobiology 12:451–461CrossRefPubMedGoogle Scholar
  2. Alvarez-Hernandez S, Lara-Isassi GD, Arreguın-Espinoza R, Arreguın B, Hernandez-Santoyo A, Rodrıguez-Romero A (1999) Isolation and partial characterization of giraffine, a lectin from the Mexican endemic alga Codium giraffa Silva. Bot Mar 42:573–580CrossRefGoogle Scholar
  3. Beisel H-G, Kawabata S-I, Iwanaga S, Huber R, Bode W (1999) Tachylectin-2: crystal structure of a specific GlcNAc/GalNAc-binding lectin involved in the innate immunity host defense of the Japanese horseshoe crab Tachypleus tridentatus. EMBO J 18:2313–2322CrossRefPubMedGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of proteins using the principle of protein-dye binding. Analyt Biochem 72:248–254CrossRefPubMedGoogle Scholar
  5. Calvete JJ, Costa FHF, Saker-Sampaio S, Murciano MPM, Nagano CS, Cavada BS (2000) The amino acid sequence of the agglutinin isolated from the red marine alga Bryothamnion triquetrum defines a novel lectin structure. Cell Mol Life Sci 57:343–350CrossRefPubMedGoogle Scholar
  6. Carraway KL, Koshland DE (1972) Carbodiimide modification of protein. Methods Enzymol 25:616–623CrossRefGoogle Scholar
  7. Carrizo ME, Capaldi S, Perduca M, Irazoqui FJ, Nores GA, Monaco HL (2006) The antineoplastic lectin of the common edible mushroom (Agaricus bisporus) has two binding sites, each specific for a different configuration at a single epimeric hydroxyl. J Biol Chem 280:10614–10623CrossRefGoogle Scholar
  8. Elizabeth F-B, Alisdair BB (2006) The interaction of a carbohydrate-binding module from a Clostridium perfringens N-acetyl-hexosaminidase with its carbohydrate receptor. J Biol Chem 281:37748–37757CrossRefGoogle Scholar
  9. Fabregas J, Llovo AMJ, Carracedo A (1988) Purification and partial characterization of tomentine. An N-acetylglucosamine-specific lectin from the green alga Codium tomentossum (Huds.) Stackh. J Exp Mar Biol Ecol 124:21–30CrossRefGoogle Scholar
  10. Hemmi H, Kuno A, Ito S, Suzuki R, Hasegawa T, Hirabayashi J (2009) NMR studies on the interaction of sugars with the C-terminal domain of an R-type lectin from the earthworm Lumbricus terrestric. FEBS J 276:2095–2105CrossRefPubMedGoogle Scholar
  11. Hori K, Miyazawa K, Ito K (1990) Some common properties of lectins from marine algae. Hydrobiologia 204(205):561–566CrossRefGoogle Scholar
  12. Kamiya H, Shiomi K, Shimizu Y (1980) Marine biopolymers with cell specificity. III. Agglutinins in the red alga Cystoclonium purpureum: isolation and characterization. J Nat Prod 43:36–139CrossRefGoogle Scholar
  13. Kamiya H, Ogata K, Hori K (1982) Isolation and characterization of a new agglutinin in the red alga Palmaria palmata (L.) O. Küntze. Bot Mar 25:537–540CrossRefGoogle Scholar
  14. Kim GH, Klotchkova TA, Kang Y-M (2001) Life without a cell membrane: regeneration of protoplast from disintegrated cells of the marine green alga, Bryopsis plumosa. J Cell Sci 114:2009–2014PubMedGoogle Scholar
  15. Kim GH, Klochkova TA, Yoon KS, Song YS, Lee KP (2006) Purification and characterization of a lectin, Bryohealin, involved in the protoplast formation of a marine green alga Bryopsis plumosa (Chlorophyta). J Phycol 42:86–95CrossRefGoogle Scholar
  16. Klotchkova TA, Chah O-K, West JA, Kim GH (2003) Cytochemical and ultrastructural studies on protoplast formation from disintegrated cells of the marine alga Chaetomorpha aerea (Chlorophyta). Eur J Phycol 38:205–216CrossRefGoogle Scholar
  17. Klochkova TA, Yoon KS, West JA, Kim GH (2005) Experimental hybridization between some marine coenocytic green algae using protoplasms extruded in vitro. Algae 20:239–249CrossRefGoogle Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  19. Leonidas DD, Swamy BM, Hatzopoulos GN, Gonchigar SJ, Chachadi VB, Inamdar SR, Zographos SE, Oikonomakos NG (2007) Structural basis for the carbohydrate recognition of the Sclerotium rolfsii lectin. J Mol Biol 368:1145–1161CrossRefPubMedGoogle Scholar
  20. Miles EW (1977) Modification of histidyl residues in protein by diethylpyrocarbonate. Methods Enzymol 47:431–442CrossRefPubMedGoogle Scholar
  21. Nagano CS, Moreno FBMB, Bloch JC, Prates MV, Clavete JJ, Saker-Sampaio S, Farias WRL et al (2002) Purification and characterization of a new lectin from the red marine alga Hypnea musciformis. Protein Pep Lett 9:159–165CrossRefGoogle Scholar
  22. Nagano CS, Debray H, Nascimento KS, Pinto VPT, Cavada BS, Saker-Sampio S, Farias WRL, Sampaio AH, Calvete JJ (2005) HCA and HML isolated from the red marine algae Hypnea cervicornis and Hypnea musciformis define a novel lectin family. Protein Sci 14:2167–2176CrossRefPubMedGoogle Scholar
  23. Nakamura S, Yagi F, Totani K, Ito Y, Hirabayashi J (2005) Comparative analysis of carbohydrate-binding properties of two tandem repeat-type Jacalin-related lectins, Castanea crenata agglutinin and Cycas revoluta leaf lectin. FEBS J 272:2784–2799CrossRefPubMedGoogle Scholar
  24. Oliveira SRM, Nascimento AE, Lima MEP, Leite YFMM, Benevides NMB (2002) Purification and characterization of a lectin from the red marine alga Pteocladia capillacea (S. G. Gmel.) Santel. et Hommers. Rev Bras Bot 25:397–403Google Scholar
  25. Pak JY, Solorzano C, Arai M, Nitta T (1991) Two distinct steps for spontaneous generation of sub-protoplasts from a disintegrated Bryopsis cell. Plant Physiol 96:819–825CrossRefPubMedGoogle Scholar
  26. Patnjali SR, Swamy MJ, Anantharam V, Khan MI, Surolia A (1984) Chemical modification studies on Abrus agglutinin. Involvement of tryptophan residues in sugar binding. Biochem J 217:773–781Google Scholar
  27. Privat J-P, Lotan R, Bouchard P, Sharon N, Monsigny M (1976) Chemical modification of the tryptophan residues of wheat-germ agglutinin. Effect on fluorescence and saccharide-binding properties. Eur J Biochem 68:563–572CrossRefPubMedGoogle Scholar
  28. Riordan JF, Wacker WEC, Vallee BL (1965) N-acetylimidazol: a reagent determination of “Free” tyrosyl residues of proteins. Biochemistry 4:1758–1765CrossRefGoogle Scholar
  29. Rogers DJ, Hori K (1993) Marine algal lectins: new developments. Hydrobiologia 260-261:589–593CrossRefGoogle Scholar
  30. Roger DJ, Loveless RW, Balding J (1986) Isolation and characterization of the lectins from sub-species of Codium fragile. Lectins 5:155–160Google Scholar
  31. Sampaio AH, Rogers DJ, Barwell CJ (1998) A galactose-specific lectin from the red marine alga Ptilota filicina. Phytochem 48:765–769CrossRefGoogle Scholar
  32. Sampaio AH, Rogers DJ, Barwell CJ, Saker-Sampaio S, Costa FHF, Ramos MV (1999) A new isolation procedure and further characterisation of the lectin from the red marine alga Ptilota serrata. J Appl Phycol 10:539–546CrossRefGoogle Scholar
  33. Sharon N (2008) Lectins: past, present and future. Biochem Soc Trans 36:1457–1460CrossRefPubMedGoogle Scholar
  34. Sharon N, Lis H (1989) Lectins as cell recognition molecules. Science 177:949–959CrossRefGoogle Scholar
  35. Spande TF, Witkop B (1967) Determination of the tryptophan content of proteins with N-bromosuccinimide. Methods Enzymol 11:498–506CrossRefGoogle Scholar
  36. Shiomi K, Kamiya H, Shimizu Y (1979) Purification and characterization of an agglutinin in the red alga Agardhiella tenera. Biochem Biophys Acta 576:118–127PubMedGoogle Scholar
  37. Shiomi K, Yamanaka H, Kikuchi T (1981) Purification and physicochemical properties of a hemagglutinin (GVA-1) in the red alga Gracilaria verrucosa. Bull Jpn Soc Sci Fish 47:1079–1084Google Scholar
  38. Sultan NAM, Kenoth R, Swamy MJ (2004) Purification, physicochemical characterization, saccharide specificity, and chemical modification of a Gal/GalNAc specific lectin from the seeds of Trichosanthes dioica. Arch Biochem Biophys 432:212–221CrossRefPubMedGoogle Scholar
  39. Vasta GR, Ahmed H, Odom EW (2004) Structural and functional diversity of lectin repertoires in invertebrates, protochordates and ectothermic vertebrates. Curr Opin Struct Biol 14:617–663CrossRefPubMedGoogle Scholar
  40. Wu AM, Song S-C, Chang S-C, Wu JH, Chang KSS, Kabat EA (1997) Further characterization of the binding properties of a GalNAc specific lectin from Codium fragile subspecies tomentosoides. Glycobiology 7:1061–1066CrossRefPubMedGoogle Scholar
  41. Yoon KS, Lee KP, Klochkova TA, Kim GH (2008) Molecular characterization of the lectin, Bryohealin, involved in protoplast regeneration of the marine alga Bryopsis plumosa (Chlorophyta). J Phycol 44:103–112CrossRefGoogle Scholar
  42. ZióLkowska NE, O'keefe BR, Mori T, Zhu C, Giomarelli B, Vojdani F, Palmer KE, McMahon JB, Wlodawer A (2006) Domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding. Structure 7:1127–1135CrossRefGoogle Scholar
  43. ZióLkowska NE, Shenoy SR, O'keefe BR, Wlodawer A (2007) Crystallographic studies of the complexes of antiviral protein griffithsin with glucose and N-acetylglucosamine. Protein Sci 16:1485–1489CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Min Gui Jung
    • 1
  • Key Pyoung Lee
    • 2
  • Han-Gu Choi
    • 1
  • Sung-Ho Kang
    • 1
  • Tatyana A. Klochkova
    • 3
  • Jong Won Han
    • 3
  • Gwang Hoon Kim
    • 3
  1. 1.Korea Polar Research InstituteIncheonKorea
  2. 2.Department of ChemistryKongju National UniversityKongju ChungnamKorea
  3. 3.Department of BiologyKongju National UniversityKongju ChungnamKorea

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