Marine Biotechnology

, Volume 19, Issue 1, pp 49–64 | Cite as

Purification, Biochemical Characterization, and Amino Acid Sequence of a Novel Type of Lectin from Aplysia dactylomela Eggs with Antibacterial/Antibiofilm Potential

  • Rômulo Farias Carneiro
  • Renato Cézar Farias Torres
  • Renata Pinheiro Chaves
  • Mayron Alves de Vasconcelos
  • Bruno Lopes de Sousa
  • André Castelo Rodrigues Goveia
  • Francisco Vassiliepe Arruda
  • Maria Nágila Carneiro Matos
  • Helena Matthews-Cascon
  • Valder Nogueira Freire
  • Edson Holanda Teixeira
  • Celso Shiniti Nagano
  • Alexandre Holanda SampaioEmail author
Original Article


A new lectin from Aplysia dactylomela eggs (ADEL) was isolated by affinity chromatography on HCl-activated Sepharose™ media. Hemagglutination caused by ADEL was inhibited by several galactosides, mainly galacturonic acid (Ka = 6.05 × 106 M−1). The primary structure of ADEL consists of 217 residues, including 11 half-cystines involved in five intrachain and one interchain disulfide bond, resulting in a molecular mass of 57,228 ± 2 Da, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. ADEL showed high similarity with lectins isolated from Aplysia eggs, but not with other known lectins, indicating that these lectins could be grouped into a new family of animal lectins. Three glycosylation sites were found in its polypeptide backbone. Data from peptide-N-glycosidase F digestion and MS suggest that all oligosaccharides attached to ADEL are high in mannose. The secondary structure of ADEL is predominantly β-sheet, and its tertiary structure is sensitive to the presence of ligands, as observed by CD. A 3D structure model of ADEL was created and shows two domains connected by a short loop. Domain A is composed of a flat three-stranded and a curved five-stranded β-sheet, while domain B presents a flat three-stranded and a curved four-stranded β-sheet. Molecular docking revealed favorable binding energies for interactions between lectin and galacturonic acid, lactose, galactosamine, and galactose. Moreover, ADEL was able to agglutinate and inhibit biofilm formation of Staphylococcus aureus, suggesting that this lectin may be a potential alternative to conventional use of antimicrobial agents in the treatment of infections caused by Staphylococcal biofilms.


Sea hare Lectin Galacturonic acid Biofilm Mass spectrometry 



This work was supported by the Brazilian agencies CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), FUNCAP (Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico), and FINEP (Financiadora de Estudos e Projetos). The authors thank CETENE for access to the mass spectrometer. The authors are especially grateful to Dr. Julia Campos for performing the MALDI-TOF experiments. The authors are grateful to Professor David Martin for helping in the English writing. A.H.S., C.S.N., and E.H.T. are senior investigators of CNPq.

Supplementary material

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Supplementary Figure 1

(GIF 132 kb)

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High resolution image (TIFF 1138 kb)
10126_2017_9728_MOESM2_ESM.docx (19 kb)
ESM 2 (DOCX 19 kb)


  1. Alpuche J, Pereyra A, Mendoza-Hernández G, Agundis C, Rosas C, Zenteno E (2010) Purification and partial characterization of an agglutinin from Octopus maya serum. Comp Biochem Physiol B 156:1–5CrossRefPubMedGoogle Scholar
  2. Bradford MM (1976) A rapid and sensitive method for quatitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal Biochem 72:248–534CrossRefPubMedGoogle Scholar
  3. Bulgakov A, Park KI, Choi KS, Lim HK, Cho M (2004) Purification and characterisation of a lectin isolated from the Manila clam Ruditapes philippinarum in Korea. Fish Shellfish Immunol 16:487–499CrossRefPubMedGoogle Scholar
  4. Carneiro RF, Melo AA, Almeida AS, Moura RM, Chaves RP, Sousa BL, Nascimento KS, Sampaio SS, Lima JP, Cavada BS, Nagano CS, Sampaio AH (2013) H-3, a new lectin from the marine sponge Haliclona caerulea: purification and mass spectrometric characterization. Int J Biochem Cell Biol 45:2864–2873CrossRefPubMedGoogle Scholar
  5. Carneiro RF, Teixeira CS, Melo AA, Almeida AS, Cavada BS, Sousa OV, Rocha BAM, Nagano CS, Sampaio AH (2015) L-rhamnose-binding lectin from eggs of the Echinometra lucunter: amino acid sequence and molecular modeling. Int J Biol Macromol 78:180–188CrossRefPubMedGoogle Scholar
  6. Chen J, Xiao S, Yu Z (2011) F-type lectin involved in defense against bacterial infection in the pearl oyster (Pinctada martensii). Fish Shellfish Immunol 30:750–754CrossRefPubMedGoogle Scholar
  7. Cheung RC, Wong JH, Pan W, Chan YS, Yin C, Dan X, Ng TB (2015) Marine lectins and their medicinal applications. Appl Microbiol Biotechnol 99:3755–3773CrossRefPubMedGoogle Scholar
  8. Davis IW, Murray LW, Richardson JS, Richardson DC (2004) MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes. Nucleic Acids Res 32:615–619CrossRefGoogle Scholar
  9. Ding S, Li Y, Shi Z, Yan S (2014) A protein structural classes prediction method based on predicted secondary structure and PSI-BLAST profile. Biochimie 97:60–65Google Scholar
  10. Ferre F, Clote P (2005) DiANNA: a web server for disulfide connectivity prediction. Nucleic Acids Res 33:230–232CrossRefGoogle Scholar
  11. Flemming HC, Wingender J (2010) The biofilm matrix. Nat Rev Microbiol 8:623–633PubMedGoogle Scholar
  12. Fujii Y, Kawsar SM, Matsumoto R, Yasumitsu H, Ishizaki N, Dogasaki C, Hosono M, Nitta K, Hamako J, Taei M, Ozeki Y (2011) A D-galactose-binding lectin purified from coronate moon turban, turbo (Lunella) coreensis, with a unique amino acid sequence and the ability to recognize lacto-series glycosphingolipids. Comp Biochem Physiol C 158:30–37CrossRefGoogle Scholar
  13. Fujii Y, Dohmae N, Takio K, Kawsar SM, Matsumoto R, Hasan I, Koide Y, Kanaly RA, Yasumitsu H, Ogawa Y, Sugawara S, Hosono M, Nitta K, Hamako J, Matsui T, Ozeki Y (2012) A lectin from the mussel Mytilus galloprovincialis has a highly novel primary structure and induces glycan-mediated cytotoxicity of globotriaosylceramide-expressing lymphoma cells. J Biol Chem 287:44772–44783CrossRefPubMedPubMedCentralGoogle Scholar
  14. Gasparini F, Franchi N, Spolaore B, Ballarin L (2008) Novel rhamnose-binding lectins from the colonial ascidian Botryllus schlosseri. Dev Comp Immunol 32:1177–1191CrossRefPubMedGoogle Scholar
  15. Ghazarian H, Idoni B, Oppenheimer SB (2011) A glycobiology review: carbohydrates, lectins and implications in cancer therapeutics. Acta Histochem 113:236–247CrossRefPubMedGoogle Scholar
  16. Gilboa-Garber N, Sudakevitz D (2001) Usage of Aplysia lectin interactions with T antigen and poly-N-acetyllactosamine for screening of E. coli strains which bear glycoforms cross-reacting with cancer-associated antigens. FEMS Immunol Med Microbiol 30:235–240Google Scholar
  17. Gilboa-Garber N, Susswein AJ, Mizrahi L, Avichezer D (1985) Purification and characterization of the gonad lectin of Aplysia depilans. FEBS Lett 181:267–270CrossRefPubMedGoogle Scholar
  18. Greenfield NJ (2006) Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat Protoc 1:2527–2535CrossRefPubMedPubMedCentralGoogle Scholar
  19. Greenfield NJ (2007) Determination of the folding of proteins as a function of denaturants, osmolytes or ligands using circular dichroism. Nat Protoc 1:2733–2741CrossRefGoogle Scholar
  20. He X, Zhang Y, Yu F, Yu Z (2011) A novel sialic acid binding lectin with anti-bacterial activity from the Hong Kong oyster (Crassostrea hongkongensis). Fish Shellfish Immunol 31:1247–1250CrossRefPubMedGoogle Scholar
  21. Hirabayashi J, Dutta SK, Kasai KI (1998) Novel galactose-binding proteins in Annelida: characterization of 29-kDa tandem repeat-type lectins from the earthworm Lumbricus terrestris. J Biol Chem 273:14450–14460CrossRefPubMedGoogle Scholar
  22. Ito S, Shimizu M, Nagatsuka M, Kitajima S, Honda M, Tsuchiya T, Kanzawa N (2011) High molecular weight lectin isolated from the mucus of the giant African snail Achatina fulica. Biosci Biotechnol Biochem 75:20–25CrossRefPubMedGoogle Scholar
  23. Jimbo M, Yanohara T, Koike K, Koike K, Sakai R, Muramoto K, Kamiya H (2000) The D-galactose-binding lectin of the octocoral Sinularia lochmodes: characterization and possible relationship to the symbiotic dinoflagellates. Comp Biochem Physiol B 125:227–236CrossRefPubMedGoogle Scholar
  24. Kawsar SMA, Matsumoto R, Fujii Y, Yasumitsu H, Dogasaki C, Hosono M, Nitta K, Hamako J, Matsui T, Kojima N, Ozeki Y (2009) Purification and biochemical characterization of a D-galactose binding lectin from Japanese sea hare (Aplysia kurodai) eggs. Biochemistry (Mosc) 74:709–716CrossRefGoogle Scholar
  25. Kawsar SMA, Matsumoto R, Fujii Y, Matsuoka H, Masuda N, Chihiro I, Yasumitsu H, Kanaly RA, Sugawara S, Hosono M, Nitta K, Ishizaki N, Dogasaki C, Hamako J, Matsui T, Ozeki Y (2011) Cytotoxicity and glycan-binding profile of a D-galactose-binding lectin from the eggs of a Japanese sea hare (Aplysia kurodai). Protein J 30:509–519CrossRefPubMedGoogle Scholar
  26. Kukuruzinska MA, Lennon K (1998) Protein N-glycosylation: molecular genetics and functional significance. Crit Rev Oral Biol Med 9:415–448CrossRefPubMedGoogle Scholar
  27. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680–683CrossRefPubMedGoogle Scholar
  28. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291CrossRefGoogle Scholar
  29. Liljemark WF, Bloomquist CG, Germaine GR (1981) Effect of bacterial aggregation on the adherence of oral streptococci to hydroxyapatite. Infect Immun 31:935–941PubMedPubMedCentralGoogle Scholar
  30. Matthews-Cascon H, Rocha-Barreira CA, Meirelles CAO (2011) Egg masses of some Brazilian mollusk. Expressão Gráfica e Editora, FortalezaGoogle Scholar
  31. Melo VMM, Duarte ABG, Carvalho AFFU, Siebra EA, Vasconcelos IM (2000) Purification of a novel antibacterial and haemagglutinating protein from the purple gland of the sea hare, Aplysia dactylomela rang, 1828. Toxicon 38:1415–1427CrossRefPubMedGoogle Scholar
  32. Melo AA, Carneiro RF, Melo WS, Moura RM, Silva GC, Sousa OV, Saboya JPS, Nascimento KS, Saker-Sampaio S, Nagano CS, Cavada BS, Sampaio AH (2014) HGA-2, a novel galactoside-binding lectin from the sea cucumber Holothuria grisea binds to bacterial cells. Int J Biol Macromol 64:435–442CrossRefPubMedGoogle Scholar
  33. Morris G, Huey H (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30:2785–2791CrossRefPubMedPubMedCentralGoogle Scholar
  34. Moura RM, Queiroz AFS, Fook JMSLL, Dias ASF, Monteiro NKV, Ribeiro JKC, Moura GE, Macedo LL, Santos EA, Sales MP (2006) CvL, a lectin from the marine sponge Cliona varians: isolation, characterization and its effects on pathogenic bacteria and Leishmania promastigotes. Comp Biochem Physiol B 145:517–523CrossRefGoogle Scholar
  35. Mu C, Chen L, Zhao J, Wang C (2014) Molecular cloning and expression of a C-type lectin gene from Venerupis philippinarum. Mol Biol Rep 41:139–144CrossRefPubMedGoogle Scholar
  36. Naganuma T, Ogawa T, Hirabayashi J, Kasai K, Kamiya H, Muramoto K (2006) Isolation, characterization and molecular evolution of a novel pearl shell lectin from a marine bivalve, Pteria penguin. Mol Divers 10:607–618CrossRefPubMedGoogle Scholar
  37. Pales-Espinosa E, Perrigault M, Allam B (2010) Identification and molecular characterization of a mucosal lectin (MeML) from the blue mussel Mytilus edulis and its potential role in particle capture. Comp Biochem Physiol A Mol Integr Physiol 156:495–501CrossRefPubMedGoogle Scholar
  38. Roué M, Quévrain E, Domart-Coulon I, Bourguet-Kondracki ML (2012) Assessing calcareous sponges and their associated bacteria for the discovery of new bioactive natural products. Nat Prod Rep 29:739–751CrossRefPubMedGoogle Scholar
  39. Sampaio AH, Rogers DJ, Barwell CJ (1998) A galactose-specific lectin from the red marine alga Ptilota filicina. Phytochemistry 48:765–769CrossRefPubMedGoogle Scholar
  40. Shen M, Sali A (2006) Statistical potential for assessment and prediction of protein structures. Protein Sci 15:2507–2524CrossRefPubMedPubMedCentralGoogle Scholar
  41. Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860CrossRefPubMedGoogle Scholar
  42. Slomiany DL, Meyer K (1972) Isolation and structural studies of sulphated glycoproteins of hog gastric mucin. J Biol Chem 247:5062–5070PubMedGoogle Scholar
  43. Song X, Zhang H, Wang L, Zhao J, Mu C, Song L, Qiu L, Liu X (2011) A galectin with quadruple-domain from bay scallop Argopecten irradians is involved in innate immune response. Dev Comp Immunol 35:592–602CrossRefPubMedGoogle Scholar
  44. Sun J, Wang L, Wang B, Guo Z, Liu M, Jiang K, Luo Z (2007) Purification and characterisation of a natural lectin from the serum of the shrimp Litopenaeus vannamei. Fish Shellfish Immunol 23:292–299CrossRefPubMedGoogle Scholar
  45. Takahashi KG, Kuroda T, Muroga K (2008) Purification and antibacterial characterization of a novel isoform of the Manila clam lectin (MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum. Comp Biochem Physiol B Biochem Mol Biol 150:45–52CrossRefPubMedGoogle Scholar
  46. Trott O, Olson AJ (2009) Software news and update Autodock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455–461Google Scholar
  47. Uchida T, Yamasaki T, Eto S, Sugawara H, Kurisu G, Nakagawa A, Kusunoki M, Hatakeyama T (2004) Crystal structure of the hemolytic lectin CEL-III isolated from the marine invertebrate Cucumaria echinata: implications of domain structure for its membrane pore-formation mechanism. J Biol Chem 135:37133–37141CrossRefGoogle Scholar
  48. Van Stokkum IHM, Spoelder HJW, Bloemendal M, Van Grondelle R, Groen FCA (1990) Estimation of protein secondary structure and error analysis from CD spectra. Anal Biochem 191:110–118CrossRefPubMedGoogle Scholar
  49. Vasconcelos MA, Arruda FV, Carneiro VA, Silva HC, Nascimento KS, Sampaio AH, Cavada BS, Teixeira EH, Henriques M, Pereira MO (2014) Effect of algae and plant lectins on planktonic growth and biofilm formation in clinically relevant bacteria and yeasts. Biomed Res Int. doi: 10.1155/2014/365272 Google Scholar
  50. Vasta GR, Marchalonis JJ (1986) Galactosyl-binding lectins from the tunicate Didemnum candidum. Carbohydrate specificity and characterization of the combining site. J Biol Chem 261:9182–9186PubMedGoogle Scholar
  51. Vasta GR, Ahmed H, Odom EW (2004) Structural and functional diversity of lectin repertoires in invertebrates, protochordates and ectothermic vertebrates. Curr Opin Struct Biol 14:617–630CrossRefPubMedGoogle Scholar
  52. Wang Y, Xiao Y, Suzek TO, Zhang J, Wang J, Bryant SH (2009) PubChem: a public information system for analyzing bioactivities of small molecules. Nucleic Acids Res 37:1–11CrossRefGoogle Scholar
  53. Webb B, Sali A (2014) Comparative protein structure modeling using MODELLER. Curr Protoc Bioinformatics. doi: 10.1002/0471250953.bi0506s47
  54. Weidenmaier C, Peschel A (2008) Teichoic acids and related cell-wall glycopolymers in gram-positive physiology and host interactions. Nat Rev Microbiol 6:276–287CrossRefPubMedGoogle Scholar
  55. Whitmore L, Wallace BA (2008) Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 89:392–400CrossRefPubMedGoogle Scholar
  56. Wittmann V, Pieters RJ (2013) Bridging lectin binding sites by multivalent carbohydrates. Chem Soc Rev 42:4492–4503CrossRefPubMedGoogle Scholar
  57. Xu D, Zhang Y (2013) Ab initio structure prediction for Escherichia coli: towards genome-wide protein structure modeling and fold assignment. Sci Rep. doi: 10.1038/srep01895 Google Scholar
  58. Yang J, Zhang Y (2015) I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res 43:174–181CrossRefGoogle Scholar
  59. Yang J, Roy A, Zhang Y (2013) Protein-ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment. Bioinformatics 29:2588–2595CrossRefPubMedPubMedCentralGoogle Scholar
  60. Zelensky AN, Gready JE (2005) The C-type lectin-like superfamily. FEBS J 272:6179–61217CrossRefPubMedGoogle Scholar
  61. Zhang J, Qiu R, Hu Y (2014) HdhCTL1 is a novel C-type lectin of abalone Haliotis discus hannai that agglutinates gram-negative bacterial pathogens. Fish Shellfish Immunol 41:466–472CrossRefPubMedGoogle Scholar
  62. Zheng P, Wang H, Zhao J, Song L, Qiu L, Dong C, Wang B, Gai Y, Mu C, Li C, Ni D, Xing K (2008) A lectin (CfLec-2) aggregating staphylococcus haemolyticus from scallop Chlamys farreri. Fish Shellfish Immunol 24:286–293CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Rômulo Farias Carneiro
    • 1
  • Renato Cézar Farias Torres
    • 1
  • Renata Pinheiro Chaves
    • 1
  • Mayron Alves de Vasconcelos
    • 2
  • Bruno Lopes de Sousa
    • 3
  • André Castelo Rodrigues Goveia
    • 1
  • Francisco Vassiliepe Arruda
    • 2
  • Maria Nágila Carneiro Matos
    • 1
  • Helena Matthews-Cascon
    • 4
  • Valder Nogueira Freire
    • 3
  • Edson Holanda Teixeira
    • 2
  • Celso Shiniti Nagano
    • 1
  • Alexandre Holanda Sampaio
    • 1
    Email author
  1. 1.Laboratório de Biotecnologia Marinha – BioMar-Lab, Departamento de Engenharia de PescaUniversidade Federal do CearáFortalezaBrazil
  2. 2.Laboratório Integrado de Biomoléculas - LIBS, Departamento de Patologia e Medicina LegalUniversidade Federal do CearáFortalezaBrazil
  3. 3.Departamento de FísicaUniversidade Federal do CearáFortalezaBrazil
  4. 4.Laboratório de Invertebrados Marinhos do Ceará – LIMCE, Departamento de BiologiaUniversidade Federal do CearáFortalezaBrazil

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