Advertisement

A Lectin from the Mussel Mytilus galloprovincialis Has a Highly Novel Primary Structure and Induces Glycan-mediated Cytotoxicity of Globotriaosylceramide-expressing Lymphoma Cells*

  • Yuki Fujii
    Footnotes
    Affiliations
    Laboratory of Glycobiology and Marine Biochemistry, Department of Life and Environmental System Science, Graduate School of NanoBio Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan

    Divisions of Functional Morphology and Microbiology, Department of Pharmacy, Faculty of Pharmaceutical Science, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan
    Search for articles by this author
  • Naoshi Dohmae
    Footnotes
    Affiliations
    Biomolecular Characterization Team, RIKEN Advanced Science Institute, Saitama 351-0198, Japan
    Search for articles by this author
  • Koji Takio
    Affiliations
    Biomolecular Characterization Team, RIKEN Advanced Science Institute, Saitama 351-0198, Japan
    Search for articles by this author
  • Sarkar M.A. Kawsar
    Affiliations
    Laboratory of Glycobiology and Marine Biochemistry, Department of Life and Environmental System Science, Graduate School of NanoBio Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan

    Laboratory of Carbohydrate and Protein Chemistry, Department of Chemistry, Faculty of Science, University of Chittagong, Chittagong-4331, Bangladesh
    Search for articles by this author
  • Ryo Matsumoto
    Affiliations
    Laboratory of Glycobiology and Marine Biochemistry, Department of Life and Environmental System Science, Graduate School of NanoBio Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan
    Search for articles by this author
  • Imtiaj Hasan
    Affiliations
    Laboratory of Glycobiology and Marine Biochemistry, Department of Life and Environmental System Science, Graduate School of NanoBio Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan

    Department of Biochemistry and Molecular Biology, Faculty of Science, Rajshahi University, Rajshahi-6205, Bangladesh
    Search for articles by this author
  • Yasuhiro Koide
    Affiliations
    Laboratory of Glycobiology and Marine Biochemistry, Department of Life and Environmental System Science, Graduate School of NanoBio Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan
    Search for articles by this author
  • Robert A. Kanaly
    Affiliations
    Laboratory of Glycobiology and Marine Biochemistry, Department of Life and Environmental System Science, Graduate School of NanoBio Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan
    Search for articles by this author
  • Hidetaro Yasumitsu
    Affiliations
    Laboratory of Glycobiology and Marine Biochemistry, Department of Life and Environmental System Science, Graduate School of NanoBio Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan
    Search for articles by this author
  • Yukiko Ogawa
    Affiliations
    Divisions of Functional Morphology and Microbiology, Department of Pharmacy, Faculty of Pharmaceutical Science, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan
    Search for articles by this author
  • Shigeki Sugawara
    Affiliations
    Division of Cell Recognition Study, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
    Search for articles by this author
  • Masahiro Hosono
    Affiliations
    Division of Cell Recognition Study, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
    Search for articles by this author
  • Kazuo Nitta
    Affiliations
    Division of Cell Recognition Study, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
    Search for articles by this author
  • Jiharu Hamako
    Affiliations
    Department of Biology, Fujita Health University, Toyoake, Aichi 470-1192, Japan
    Search for articles by this author
  • Taei Matsui
    Affiliations
    Department of Biology, Fujita Health University, Toyoake, Aichi 470-1192, Japan
    Search for articles by this author
  • Yasuhiro Ozeki
    Correspondence
    To whom correspondence should be addressed. Tel.: 81-45-787-2221; Fax: 81-45-787-2413
    Affiliations
    Laboratory of Glycobiology and Marine Biochemistry, Department of Life and Environmental System Science, Graduate School of NanoBio Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science and Japanese Association for Marine Biology from the Ministry of Education, Culture, Sports, Science, and Technology Japan.The protein sequence data reported in this paper will appear in the UniProt Knowledgebase under the accession number B3EWR1 for MytiLec.
    1 Both authors contributed equally to this work and should be considered first authors.
Open AccessPublished:December 28, 2012DOI:https://doi.org/10.1074/jbc.M112.418012
      A novel lectin structure was found for a 17-kDa α-d-galactose-binding lectin (termed “MytiLec”) isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of ∼50 amino acids each having 45–52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Galα1–4Galβ1–4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an α-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.

      Introduction

      Mollusks are important aquatic and scientific resources. The Mediterranean mussel (Mytilus galloprovincialis; family Mytilidae) is an invasive species that originated in the Mediterranean and has been introduced to intertidal and near-shore habitats in many parts of the world, including the coasts of Japan. These marine bivalves are filter feeders that filter large amounts of debris, often including pathogenic microorganisms or heavy metals, and have evolved tolerance and defense mechanisms that help them adapt to diverse environments. Recent genomic research on bivalves has led to the establishment of expressed sequence tag libraries (
      • Kinoshita S.
      • Wang N.
      • Inoue H.
      • Maeyama K.
      • Okamoto K.
      • Nagai K.
      • Kondo H.
      • Hirono I.
      • Asakawa S.
      • Watabe S.
      Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster.
      ,
      • Venier P.
      • De Pittà C.
      • Bernante F.
      • Varotto L.
      • De Nardi B.
      • Bovo G.
      • Roch P.
      • Novoa B.
      • Figueras A.
      • Pallavicini A.
      • Lanfranchi G.
      MytiBase. A knowledge base of mussel (M. galloprovincialis) transcribed sequences.
      ,
      • Venier P.
      • Varotto L.
      • Rosani U.
      • Millino C.
      • Celegato B.
      • Bernante F.
      • Lanfranchi G.
      • Novoa B.
      • Roch P.
      • Figueras A.
      • Pallavicini A.
      Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis.
      ). One of these libraries has been useful for the identification of key genes that regulate pearl formation in pearl oysters (
      • Kinoshita S.
      • Wang N.
      • Inoue H.
      • Maeyama K.
      • Okamoto K.
      • Nagai K.
      • Kondo H.
      • Hirono I.
      • Asakawa S.
      • Watabe S.
      Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster.
      ). Another expressed sequence tag library, MytiBase, developed from studies of M. galloprovincialis, provides a valuable bioinformatics tool for investigating mechanisms involved in development, differentiation, and defense in this species. The MytiBase library has been useful in the elucidation of novel genes expressed in hemocytes and related to innate immunity in studies of Vibrio bacterial infection (
      • Venier P.
      • Varotto L.
      • Rosani U.
      • Millino C.
      • Celegato B.
      • Bernante F.
      • Lanfranchi G.
      • Novoa B.
      • Roch P.
      • Figueras A.
      • Pallavicini A.
      Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis.
      ).
      Lectins are glycan-binding proteins that function in the recognition of a wide variety of glycan structures and can be used to decipher the glyco-codes that determine the composition of oligosaccharides in glycoconjugates (glycosphingolipids, glycoproteins, and proteoglycans). Lectins have been isolated from almost all animal and plant phyla. Certain lectin-coding genes in bivalves have been found to be up- or down-regulated in association with infection by pathogenic microorganisms, indicating that the lectins are able to respond to external/environmental stimuli (
      • Venier P.
      • Varotto L.
      • Rosani U.
      • Millino C.
      • Celegato B.
      • Bernante F.
      • Lanfranchi G.
      • Novoa B.
      • Roch P.
      • Figueras A.
      • Pallavicini A.
      Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis.
      ,
      • Kim Y.M.
      • Park K.I.
      • Choi K.S.
      • Alvarez R.A.
      • Cummings R.D.
      • Cho M.
      Lectin from the Manila clam Ruditapes philippinarum is induced upon infection with the protozoan parasite Perkinsus olseni.
      ). Several lectins with characteristic structures have been described in bivalves, including C-type lectins and galectins (
      • Pales Espinosa E.
      • Perrigault M.
      • Allam B.
      Identification and molecular characterization of a mucosal lectin (MeML) from the blue Mytilus edulis and its potential role in particle capture.
      ,
      • Tasumi S.
      • Vasta G.R.
      A galectin of unique domain organization from hemocytes of the Eastern oyster (Crassostrea virginica) is a receptor for the protistan parasite Perkinsus marinus.
      ), fibrinogen-type (
      • Gorbushin A.M.
      • Iakovleva N.V.
      A new gene family of single fibrinogen domain lectins in Mytilus.
      ), C1q-type (
      • Li C.
      • Yu S.
      • Zhao J.
      • Su X.
      • Li T.
      Cloning and characterization of sialic acid binding lectins (SABL) from Manila clam Venerupis philippinarum.
      ), and F-type lectins (fucolectin) (
      • Chen J.
      • Xiao S.
      • Yu Z.
      F-type lectin involved in defense against bacterial infection in the pearl oyster (Pinctada martensii).
      ). A Gal/GalNAc-binding lectin (information on primary structure unavailable) was reported in Crenomytilus, a genus related to Mytilus (
      • Belogortseva N.I.
      • Molchanova V.I.
      • Kurika A.V.
      • Skobun A.S.
      • Glazkova V.E.
      Isolation of characterization of new GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus.
      ). A lectin domain of the sea urchin egg lectin (SUEL)
      The abbreviations used are:
      SUEL
      sea urchin egg lectin
      FACT
      frontal affinity chromatography technology
      FT-ICR MS
      Fourier-transform ion cyclotron resonance mass spectrometry
      Gb3
      globotriaosylceramide
      GPC
      gel permeation chromatography
      MytiLec
      Mytilus galloprovincialis α-d-galactose-binding lectin
      PA
      pyridylamino
      Sup
      supernatant
      HSA
      human serum albumin
      SPR
      surface plasmon resonance.
      -type that was originally reported in a deuterostome (sea urchin) (
      • Ozeki Y.
      • Matsui T.
      • Suzuki M.
      • Titani K.
      Amino acid sequence and molecular characterization of a d-galactoside-specific lectin purified from sea urchin (Anthocidaris crassispina) eggs.
      ) was later identified in the bivalve Pteria penguin (
      • Naganuma T.
      • Ogawa T.
      • Hirabayashi J.
      • Kasai K.
      • Kamiya H.
      • Muramoto K.
      Isolation, characterization, and molecular evolution of a novel pearl shell lectin from a marine bivalve, Pteria penguin.
      ). These findings suggest that bivalves, including mytilids, are interesting subjects for studies of characteristic lectins and glycan-dependent phenomena. Glycobiological investigation of these animal will help elucidate their biochemical and physiological mechanisms.
      A variety of methodologies using advanced equipment has been developed to elucidate the glycan binding specificities of lectins, to identify the specific oligosaccharides involved, and to better understand the molecular interactions between lectins and glycans (
      • Blixt O.
      • Head S.
      • Mondala T.
      • Scanlan C.
      • Huflejt M.E.
      • Alvarez R.
      • Bryan M.C.
      • Fazio F.
      • Calarese D.
      • Stevens J.
      • Razi N.
      • Stevens D.J.
      • Skehel J.J.
      • van Die I.
      • Burton D.R.
      • Wilson I.A.
      • Cummings R.
      • Bovin N.
      • Wong C.-H.
      • Paulson J.C.
      Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.
      ,
      • Angeloni S.
      • Ridet J.L.
      • Kusy N.
      • Gao H.
      • Crevoisier F.
      • Guinchard S.
      • Kochhar S.
      • Sigrist H.
      • Sprenger N.
      Glycoprofiling with micro-arrays of glycoconjugates and lectins.
      ,
      • Tateno H.
      • Uchiyama N.
      • Kuno A.
      • Togayachi A.
      • Sato T.
      • Narimatsu H.
      • Hirabayashi J.
      A novel strategy for mammalian cell surface glycome profiling using lectin microarray.
      ,
      • Hirabayashi J.
      Concept, strategy, and realization of lectin-based glycan profiling.
      ,
      • Hirabayashi J.
      • Hashidate T.
      • Arata Y.
      • Nishi N.
      • Nakamura T.
      • Hirashima M.
      • Urashima T.
      • Oka T.
      • Futai M.
      • Muller W.E.
      • Yagi F.
      • Kasai K.
      Oligosaccharide specificity of galectins. A search by frontal affinity chromatography.
      ,
      • Hirabayashi J.
      • Arata Y.
      • Kasai K.
      Frontal affinity chromatography as a tool for elucidation of sugar recognition properties of lectins.
      ). The precise glycan binding specificities of lectins have been identified using sophisticated glycome procedures such as glycan microarrays (
      • Blixt O.
      • Head S.
      • Mondala T.
      • Scanlan C.
      • Huflejt M.E.
      • Alvarez R.
      • Bryan M.C.
      • Fazio F.
      • Calarese D.
      • Stevens J.
      • Razi N.
      • Stevens D.J.
      • Skehel J.J.
      • van Die I.
      • Burton D.R.
      • Wilson I.A.
      • Cummings R.
      • Bovin N.
      • Wong C.-H.
      • Paulson J.C.
      Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.
      ,
      • Angeloni S.
      • Ridet J.L.
      • Kusy N.
      • Gao H.
      • Crevoisier F.
      • Guinchard S.
      • Kochhar S.
      • Sigrist H.
      • Sprenger N.
      Glycoprofiling with micro-arrays of glycoconjugates and lectins.
      ) and frontal affinity chromatography technology (FACT) (
      • Tateno H.
      • Uchiyama N.
      • Kuno A.
      • Togayachi A.
      • Sato T.
      • Narimatsu H.
      • Hirabayashi J.
      A novel strategy for mammalian cell surface glycome profiling using lectin microarray.
      ,
      • Hirabayashi J.
      Concept, strategy, and realization of lectin-based glycan profiling.
      ,
      • Kasai K.
      • Ishii S.
      Affinity chromatography of trypsin and related enzymes. V. Basic studies of quantitative affinity chromatography.
      ,
      • Kasai K.
      • Ishii S.
      Studies on the interaction of immobilized trypsin and specific ligands by quantitative affinity chromatography.
      ,
      • Hirabayashi J.
      • Hashidate T.
      • Arata Y.
      • Nishi N.
      • Nakamura T.
      • Hirashima M.
      • Urashima T.
      • Oka T.
      • Futai M.
      • Muller W.E.
      • Yagi F.
      • Kasai K.
      Oligosaccharide specificity of galectins. A search by frontal affinity chromatography.
      ,
      • Hirabayashi J.
      • Arata Y.
      • Kasai K.
      Frontal affinity chromatography as a tool for elucidation of sugar recognition properties of lectins.
      ). In FACT analysis, a column-immobilized lectin is connected to an HPLC pump, and fluorescence detector and pyridylamino (PA)-labeled oligosaccharides are injected onto the column. The affinity of glycan binding to the lectin is assessed based on the delaying elution volume of the oligosaccharides. Studies using FACT analysis have revealed that the diversity of glycan-binding profiles for d-Gal-binding lectins in aquatic animals is much greater than was previously suspected (
      • Naganuma T.
      • Ogawa T.
      • Hirabayashi J.
      • Kasai K.
      • Kamiya H.
      • Muramoto K.
      Isolation, characterization, and molecular evolution of a novel pearl shell lectin from a marine bivalve, Pteria penguin.
      ,
      • Kawsar S.M.
      • Fujii Y.
      • Matsumoto R.
      • Ichikawa T.
      • Tateno H.
      • Hirabayashi J.
      • Yasumitsu H.
      • Dogasaki C.
      • Hosono M.
      • Nitta K.
      • Hamako J.
      • Matsui T.
      • Ozeki Y.
      Isolation, purification, characterization and glycan-binding profile of a d-galactoside specific lectin from the marine sponge, Halichondria okadai.
      ,
      • Kawsar S.M.
      • Takeuchi T.
      • Kasai K.
      • Fujii Y.
      • Matsumoto R.
      • Yasumitsu H.
      • Ozeki Y.
      Glycan-binding profile of a d-galactose binding lectin purified from the annelid, Perinereis nuntia ver. vallata.
      ,
      • Kawsar S.M.
      • Matsumoto R.
      • Fujii Y.
      • Matsuoka H.
      • Masuda N.
      • Chihiro I.
      • Yasumitsu H.
      • Kanaly R.A.
      • Sugawara S.
      • Hosono M.
      • Nitta K.
      • Ishizaki N.
      • Dogasaki C.
      • Hamako J.
      • Matsui T.
      • Ozeki Y.
      Cytotoxicity and glycan-binding profile of a d-galactose-binding lectin from the eggs of a Japanese sea hare (Aplysia kurodai).
      ,
      • Matsumoto R.
      • Shibata T.F.
      • Kohtsuka H.
      • Sekifuji M.
      • Sugii N.
      • Nakajima H.
      • Kojima N.
      • Fujii Y.
      • Kawsar S.M.
      • Yasumitsu H.
      • Hamako J.
      • Matsui T.
      • Ozeki Y.
      Glycomics of a novel type-2 N-acetyllactosamine-specific lectin purified from the feather star, Oxycomanthus japonicus Pelmatozoa: Crinoidea).
      ,
      • Matsumoto R.
      • Fujii Y.
      • Kawsar S.M.
      • Kanaly R.A.
      • Yasumitsu H.
      • Koide Y.
      • Hasan I.
      • Iwahara C.
      • Ogawa Y.
      • Im C.H.
      • Sugawara S.
      • Hosono M.
      • Nitta K.
      • Hamako J.
      • Matsui T.
      • Ozeki Y.
      Cytotoxicity and glycan-binding properties of an 18-kDa lectin isolated from the marine sponge Halichonderia okadai.
      ).
      In this study, we purified an α-Gal-binding lectin from the mantle of M. galloprovincialis. Protein chemical analysis and glycome procedures revealed that the primary structure of the lectin is extremely novel and has no similarity to previously described structures. The unique glycan-binding profile of the lectin involves a specific affinity with a neutral glycan in the glycosphingolipid Gb3 (Galα1–4Galβ1–4Glc). The lectin displayed Gb3-dependent cytotoxicity against Burkitt lymphoma cells that express the glycan.

      DISCUSSION

      The complete primary structure determined for MytiLec, a lectin isolated from the mussel M. galloprovincialis, is unique among the structures of other known animal lectins. It consists of 149 amino acids without similarity to other known structures. MytiLec has a triple tandem-repeat motif of 50 amino acids. The three motifs show >50% similarity with each other. The basic amino acid residues of Lys, His, and Arg are highly conserved throughout the domains of the motifs, whereas the acidic amino acid residues of Asp and Glu are located mainly in the carboxyl-terminal side. Other characteristic features of the MytiLec structure are one Trp, 12 His, and no Cys residues involved in the polypeptide. The high number of conserved amino acid residues (His, Asp, and Arg) in the polypeptide is interesting in that these residues are found as essential carbohydrate-binding amino acids in many lectins (
      • Naismith J.H.
      • Field R.A.
      Structural basis of trimannoside recognition by concanavalin A.
      ,
      • Transue T.R.
      • Smith A.K.
      • Mo H.
      • Goldstein I.J.
      • Saper M.A.
      Structure of benzyl T-antigen disaccharide bound to Amaranthus caudatus agglutinin.
      ,
      • Kasai K.
      • Hirabayashi J.
      Galectins. A family of animal lectins that decipher glycocodes.
      ,
      • Weis W.I.
      • Taylor M.E.
      • Drickamer K.
      The C-type lectin superfamily in the immune system.
      ,
      • Vakonakis I.
      • Langenhan T.
      • Prömel S.
      • Russ A.
      • Campbell I.D.
      Solution structure and sugar-binding mechanism of mouse latrophilin-1 RBL. A 7TM receptor-attached lectin-like domain.
      ). Acetylation at the amino-terminal Thr was the only observed post-translational modification in MytiLec; there was neither glycosylation nor phosphorylation. Results from gel permeation chromatography indicate that MytiLec is present as a monomer, suggesting that each of its polypeptide motifs has hemagglutinating activity. It is interesting that a motif consisting of 50 amino acids could have carbohydrate binding ability; well known animal lectin families such as galectins and C-type lectins require >130 amino acids to function as a carbohydrate-recognition domain (
      • Pales Espinosa E.
      • Perrigault M.
      • Allam B.
      Identification and molecular characterization of a mucosal lectin (MeML) from the blue Mytilus edulis and its potential role in particle capture.
      ,
      • Weis W.I.
      • Taylor M.E.
      • Drickamer K.
      The C-type lectin superfamily in the immune system.
      ). Structural biological studies of MytiLec will provide additional information regarding the glycan binding properties of the polypeptide subdomains. The highly novel primary structure of MytiLec has no known homologues at present, but we anticipate that our finding will lead to future studies that reveal such homologues. In analogy, the primary structure of d-galactoside-binding lectin isolated from SUEL had no structural homologues when we first reported it in 1991 (
      • Ozeki Y.
      • Matsui T.
      • Suzuki M.
      • Titani K.
      Amino acid sequence and molecular characterization of a d-galactoside-specific lectin purified from sea urchin (Anthocidaris crassispina) eggs.
      ); however, >1000 structural homologues with SUEL-type lectin domains have been found during the 20 years since then. This structure has been observed even in the lectin domain of a neurotoxin receptor (latrophilin-1) in mammalian brain (
      • Vakonakis I.
      • Langenhan T.
      • Prömel S.
      • Russ A.
      • Campbell I.D.
      Solution structure and sugar-binding mechanism of mouse latrophilin-1 RBL. A 7TM receptor-attached lectin-like domain.
      ,
      • Ogawa T.
      • Watanabe M.
      • Naganuma T.
      • Muramoto K.
      Diversified carbohydrate-binding lectins from marine resources.
      ) and in plant β-galactosidase (
      • Kotake T.
      • Dina S.
      • Konishi T.
      • Kaneko S.
      • Igarashi K.
      • Samejima M.
      • Watanabe Y.
      • Kimura K.
      • Tsumuraya Y.
      Molecular cloning of a β-galactosidase from radish that specifically hydrolyzes {β}-(1–3)-and {β}-(1–6)-galactosyl residues of Arabidnogalactan protein.
      ). As an example of a new structural domain in animal lectins, MytiLec is of interest in and will promote the field of glycobiology.
      Our next interest will be a survey of α-galactosides in other animal species. An α-galactoside was specifically recognized by MytiLec in FACT analysis, although the occurrence of these sugars in mussels is not yet clear. Certain Gal-containing oligosaccharides in glycoproteins have been recently found in some mollusks by mass spectrometric analysis (
      • Stepan H.
      • Pabst M.
      • Altmann F.
      • Geyer H.
      • Geyer R.
      • Staudacher E.
      O-Glycosylation of snails.
      ,
      • Velkova L.
      • Dolashka P.
      • Lieb B.
      • Dolashki A.
      • Voelter W.
      • Van Beeumen J.
      • Devreese B.
      Glycan structures of the structural subunit (HtH1) of Haliotis tuberculata hemocyanin.
      ,
      • Gutternigg M.
      • Bürgmayr S.
      • Pöltl G.
      • Rudolf J.
      • Staudacher E.
      Neutral N-glycan patterns of the gastropods Limax maximus Cepaea hortensis Planorbarius corneus Arianta arbustorum and Achatina fulica.
      ). MytiLec was obtained together with the haptenic saccharide Gal and therefore bound to mussel tissues. Isolation of the endogenous ligands of MytiLec will help elucidate the physiological roles of Gal-binding lectins isolated from other mollusk species.
      FACT analysis showed that MytiLec specifically recognizes Gb3. Other lectins in marine organisms have been previously found to recognize α-galactosides (
      • Ozeki Y.
      • Matsui T.
      • Suzuki M.
      • Titani K.
      Amino acid sequence and molecular characterization of a d-galactoside-specific lectin purified from sea urchin (Anthocidaris crassispina) eggs.
      ,
      • Naganuma T.
      • Ogawa T.
      • Hirabayashi J.
      • Kasai K.
      • Kamiya H.
      • Muramoto K.
      Isolation, characterization, and molecular evolution of a novel pearl shell lectin from a marine bivalve, Pteria penguin.
      ,
      • Kawsar S.M.
      • Matsumoto R.
      • Fujii Y.
      • Matsuoka H.
      • Masuda N.
      • Chihiro I.
      • Yasumitsu H.
      • Kanaly R.A.
      • Sugawara S.
      • Hosono M.
      • Nitta K.
      • Ishizaki N.
      • Dogasaki C.
      • Hamako J.
      • Matsui T.
      • Ozeki Y.
      Cytotoxicity and glycan-binding profile of a d-galactose-binding lectin from the eggs of a Japanese sea hare (Aplysia kurodai).
      ). Because of the characteristic glycan binding property of MytiLec in recognizing the α-galactoside Gb3, it selectively aggregated and directly killed human Burkitt lymphoma Raji cells, which express Gb3 ceramide in the glycosphingolipid-enriched microdomain of the cell membrane through glycan-lectin interaction. FACS analysis indicated that MytiLec was associated with late-stage apoptosis and induced both cell membrane inversion and the loss of membrane integrity. It will be interesting to investigate how transduction of the lectin signal kills cells that specifically express Gb3 in glycosphingolipid-enriched microdomain. We showed recently that another Gb3-binding lectin with a SUEL-type lectin domain, isolated from catfish eggs, reduced the expression of mRNA coding a multidrug-resistant transporter in Raji cells (
      • Fujii Y.
      • Sugawara S.
      • Araki D.
      • Kawano T.
      • Tatsuta T.
      • Takahashi K.
      • Kawsar S.M.
      • Matsumoto R.
      • Kanaly R.A.
      • Yasumitsu H.
      • Ozeki Y.
      • Hosono M.
      • Miyagi T.
      • Hakomori S.-I.
      • Takayanagi M.
      • Nitta K.
      MRP1 expressed on Burkitt lymphoma cells was depleted by catfish egg lectin through Gb3-glycosphinogolipid and enhanced cytotoxic effect of drugs.
      ). MytiLec and SAL both have a triple-tandem structure but differ in terms of multivalency; MytiLec is structured as a monomer (containing three carbohydrate-recognition domains in total), whereas SAL is a trimer (containing nine carbohydrate-recognition domains in total) under physiological conditions. If it is found that the same ligand is recognized by lectins that have a different affinity constant and multimerization, we could hypothesize that independent signal pathways are being activated, with results differing from those of other regulatory pathways in the same cells. Subsequent studies will determine the targeting signal transduction molecules that are stimulated by MytiLec and tissue localization of MytiLec during mussel development. Various types of molecules are known to modulate signal transduction from glycosphingolipid-enriched microdomain in cell membranes. Molecular recognition between MytiLec and Gb3 may play an important role in regulating the fate of cells.

      Acknowledgments

      We thank the City of Yokohama “Blue Carbon Project” and Yachiyo Engineering Co., Ltd., for their support. Naoko Masuda and Chihiro Iwahara are specially thanked for assistance with experiments. We thank Dr. Stephen Anderson for the English editing of the manuscript.

      References

        • Kinoshita S.
        • Wang N.
        • Inoue H.
        • Maeyama K.
        • Okamoto K.
        • Nagai K.
        • Kondo H.
        • Hirono I.
        • Asakawa S.
        • Watabe S.
        Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster.
        PLoS ONE. 2011; 6: e21238
        • Venier P.
        • De Pittà C.
        • Bernante F.
        • Varotto L.
        • De Nardi B.
        • Bovo G.
        • Roch P.
        • Novoa B.
        • Figueras A.
        • Pallavicini A.
        • Lanfranchi G.
        MytiBase. A knowledge base of mussel (M. galloprovincialis) transcribed sequences.
        BMC Genomics. 2009; 10: 72
        • Venier P.
        • Varotto L.
        • Rosani U.
        • Millino C.
        • Celegato B.
        • Bernante F.
        • Lanfranchi G.
        • Novoa B.
        • Roch P.
        • Figueras A.
        • Pallavicini A.
        Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis.
        BMC Genomics. 2011; 12: 69
        • Kim Y.M.
        • Park K.I.
        • Choi K.S.
        • Alvarez R.A.
        • Cummings R.D.
        • Cho M.
        Lectin from the Manila clam Ruditapes philippinarum is induced upon infection with the protozoan parasite Perkinsus olseni.
        J. Biol. Chem. 2006; 281: 26854-26864
        • Pales Espinosa E.
        • Perrigault M.
        • Allam B.
        Identification and molecular characterization of a mucosal lectin (MeML) from the blue Mytilus edulis and its potential role in particle capture.
        Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2010; 156: 495-501
        • Tasumi S.
        • Vasta G.R.
        A galectin of unique domain organization from hemocytes of the Eastern oyster (Crassostrea virginica) is a receptor for the protistan parasite Perkinsus marinus.
        J. Immunol. 2007; 179: 3086-3098
        • Gorbushin A.M.
        • Iakovleva N.V.
        A new gene family of single fibrinogen domain lectins in Mytilus.
        Fish Shellfish Immunol. 2011; 30: 434-438
        • Li C.
        • Yu S.
        • Zhao J.
        • Su X.
        • Li T.
        Cloning and characterization of sialic acid binding lectins (SABL) from Manila clam Venerupis philippinarum.
        Fish Shellfish Immunol. 2011; 30: 1202-1206
        • Chen J.
        • Xiao S.
        • Yu Z.
        F-type lectin involved in defense against bacterial infection in the pearl oyster (Pinctada martensii).
        Fish Shellfish Immunol. 2011; 30: 750-754
        • Belogortseva N.I.
        • Molchanova V.I.
        • Kurika A.V.
        • Skobun A.S.
        • Glazkova V.E.
        Isolation of characterization of new GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus.
        Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 1998; 119: 45-50
        • Ozeki Y.
        • Matsui T.
        • Suzuki M.
        • Titani K.
        Amino acid sequence and molecular characterization of a d-galactoside-specific lectin purified from sea urchin (Anthocidaris crassispina) eggs.
        Biochemistry. 1991; 30: 2391-2394
        • Naganuma T.
        • Ogawa T.
        • Hirabayashi J.
        • Kasai K.
        • Kamiya H.
        • Muramoto K.
        Isolation, characterization, and molecular evolution of a novel pearl shell lectin from a marine bivalve, Pteria penguin.
        Mol. Divers. 2006; 10: 607-618
        • Blixt O.
        • Head S.
        • Mondala T.
        • Scanlan C.
        • Huflejt M.E.
        • Alvarez R.
        • Bryan M.C.
        • Fazio F.
        • Calarese D.
        • Stevens J.
        • Razi N.
        • Stevens D.J.
        • Skehel J.J.
        • van Die I.
        • Burton D.R.
        • Wilson I.A.
        • Cummings R.
        • Bovin N.
        • Wong C.-H.
        • Paulson J.C.
        Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.
        Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 17033-17038
        • Angeloni S.
        • Ridet J.L.
        • Kusy N.
        • Gao H.
        • Crevoisier F.
        • Guinchard S.
        • Kochhar S.
        • Sigrist H.
        • Sprenger N.
        Glycoprofiling with micro-arrays of glycoconjugates and lectins.
        Glycobiology. 2005; 15: 31-41
        • Tateno H.
        • Uchiyama N.
        • Kuno A.
        • Togayachi A.
        • Sato T.
        • Narimatsu H.
        • Hirabayashi J.
        A novel strategy for mammalian cell surface glycome profiling using lectin microarray.
        Glycobiology. 2007; 17: 1138-1146
        • Hirabayashi J.
        Concept, strategy, and realization of lectin-based glycan profiling.
        J. Biochem. 2008; 144: 139-147
        • Kasai K.
        • Ishii S.
        Affinity chromatography of trypsin and related enzymes. V. Basic studies of quantitative affinity chromatography.
        J. Biochem. 1978; 84: 1051-1060
        • Kasai K.
        • Ishii S.
        Studies on the interaction of immobilized trypsin and specific ligands by quantitative affinity chromatography.
        J. Biochem. 1978; 84: 1061-1069
        • Hirabayashi J.
        • Hashidate T.
        • Arata Y.
        • Nishi N.
        • Nakamura T.
        • Hirashima M.
        • Urashima T.
        • Oka T.
        • Futai M.
        • Muller W.E.
        • Yagi F.
        • Kasai K.
        Oligosaccharide specificity of galectins. A search by frontal affinity chromatography.
        Biochim. Biophys. Acta. 2002; 1572: 232-254
        • Hirabayashi J.
        • Arata Y.
        • Kasai K.
        Frontal affinity chromatography as a tool for elucidation of sugar recognition properties of lectins.
        Methods Enzymol. 2003; 362: 353-368
        • Kawsar S.M.
        • Fujii Y.
        • Matsumoto R.
        • Ichikawa T.
        • Tateno H.
        • Hirabayashi J.
        • Yasumitsu H.
        • Dogasaki C.
        • Hosono M.
        • Nitta K.
        • Hamako J.
        • Matsui T.
        • Ozeki Y.
        Isolation, purification, characterization and glycan-binding profile of a d-galactoside specific lectin from the marine sponge, Halichondria okadai.
        Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2008; 150: 349-357
        • Kawsar S.M.
        • Takeuchi T.
        • Kasai K.
        • Fujii Y.
        • Matsumoto R.
        • Yasumitsu H.
        • Ozeki Y.
        Glycan-binding profile of a d-galactose binding lectin purified from the annelid, Perinereis nuntia ver. vallata.
        Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2009; 152: 382-389
        • Kawsar S.M.
        • Matsumoto R.
        • Fujii Y.
        • Matsuoka H.
        • Masuda N.
        • Chihiro I.
        • Yasumitsu H.
        • Kanaly R.A.
        • Sugawara S.
        • Hosono M.
        • Nitta K.
        • Ishizaki N.
        • Dogasaki C.
        • Hamako J.
        • Matsui T.
        • Ozeki Y.
        Cytotoxicity and glycan-binding profile of a d-galactose-binding lectin from the eggs of a Japanese sea hare (Aplysia kurodai).
        Protein J. 2011; 30: 509-519
        • Matsumoto R.
        • Shibata T.F.
        • Kohtsuka H.
        • Sekifuji M.
        • Sugii N.
        • Nakajima H.
        • Kojima N.
        • Fujii Y.
        • Kawsar S.M.
        • Yasumitsu H.
        • Hamako J.
        • Matsui T.
        • Ozeki Y.
        Glycomics of a novel type-2 N-acetyllactosamine-specific lectin purified from the feather star, Oxycomanthus japonicus Pelmatozoa: Crinoidea).
        Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2011; 158: 266-273
        • Matsumoto R.
        • Fujii Y.
        • Kawsar S.M.
        • Kanaly R.A.
        • Yasumitsu H.
        • Koide Y.
        • Hasan I.
        • Iwahara C.
        • Ogawa Y.
        • Im C.H.
        • Sugawara S.
        • Hosono M.
        • Nitta K.
        • Hamako J.
        • Matsui T.
        • Ozeki Y.
        Cytotoxicity and glycan-binding properties of an 18-kDa lectin isolated from the marine sponge Halichonderia okadai.
        Toxins. 2012; 4: 323-338
        • Gourdine J.P.
        • Cioci G.
        • Miguet L.
        • Unverzagt C.
        • Silva D.V.
        • Varrot A.
        • Gautier C.
        • Smith-Ravin E.J.
        • Imberty A.
        High affinity interaction between a bivalve C-type lectin and a biantennary complex-type N-glycan revealed by crystallography and microcalorimetry.
        J. Biol. Chem. 2008; 283: 30112-30120
        • Smith P.K.
        • Krohn R.I.
        • Hermanson G.T.
        • Mallia A.K.
        • Gartner F.H.
        • Provenzano M.D.
        • Fujimoto E.K.
        • Goeke N.M.
        • Olson B.J.
        • Klenk D.C.
        Measurement of protein using bicinchoninic acid.
        Anal. Biochem. 1985; 150: 76-85
        • Wiechelman K.J.
        • Braun R.D.
        • Fitzpatrick J.D.
        Investigation of the bicinchoninic acid protein assay. Identification of the groups responsible for color formation.
        Anal. Biochem. 1988; 175: 231-237
        • Laemmli U.K.
        Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
        Nature. 1970; 227: 680-685
        • Masaki T.
        • Tanabe M.
        • Nakamura K.
        • Soejima M.
        Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1. I. Purification and some enzymatic properties.
        Biochim. Biophys. Acta. 1981; 660: 44-50
        • Gross E.
        Cleavage of peptide chains. The cyanogen bromide reaction.
        Methods Enzymol. 1967; 11: 238-255
        • Tahirov T.H.
        • Oki H.
        • Tsukihara T.
        • Ogasahara K.
        • Yutani K.
        • Ogata K.
        • Izu Y.
        • Tsunasawa S.
        • Kato I.
        Crystal structure of methionine aminopeptidase from hyperthermophile, Pyrococcus furiosus.
        J. Mol. Biol. 1998; 284: 101-124
        • Titani K.
        • Narita K.
        Amino acid sequences of 18 peptides isolated from the tryptic hydrolysate of Baker's yeast cytochrome c.
        J. Biochem. 1964; 56: 241-256
        • Simpson R.J.
        • Neuberger M.R.
        • Liu T.Y.
        Complete amino acid analysis of proteins from a single hydrolysate.
        J. Biol. Chem. 1976; 251: 1936-1940
        • Hewick R.M.
        • Hunkapiller M.W.
        • Hood L.E.
        • Dreyer W.J.
        A gas-liquid solid phase peptide and protein sequenator.
        J. Biol. Chem. 1981; 256: 7990-7997
        • Altschul S.F.
        • Lipman D.J.
        Protein database searches for multiple alignments.
        Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 5509-5513
        • Altschul S.F.
        • Madden T.L.
        • Schäffer A.A.
        • Zhang J.
        • Zhang Z.
        • Miller W.
        • Lipman D.J.
        Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
        Nucleic Acids Res. 1997; 25: 3389-3402
        • Xu N.
        • Huang Z.-H.
        • Watson J.T.
        • Gage D.A.
        Mercaptobenzothiazoles. A new class of matrices for laser desorption ionization mass spectrometry.
        J. Am. Soc. Mass Spectrom. 1997; 8: 116-124
        • Vukelić Z.
        • Zamfir A.D.
        • Bindila L.
        • Froesch M.
        • Peter-Katalinić J.
        • Usuki S.
        • Yu R.K.
        Screening and sequencing of complex sialylated and sulfated glycosphingolipid mixtures by negative ion electrospray Fourier transform ion cyclotron resonance mass spectrometry.
        J. Am. Soc. Mass Spectrom. 2005; 16: 571-580
        • Shinohara Y.
        • Kim F.
        • Shimizu M.
        • Goto M.
        • Tosu M.
        • Hasegawa Y.
        Kinetic measurement of the interaction between an oligosaccharide and lectins by a biosensor based on surface plasmon resonance.
        Eur. J. Biochem. 1994; 223: 189-194
        • Kawano T.
        • Sugawara S.
        • Hosono M.
        • Tatsuta T.
        • Ogawa Y.
        • Fujimura T.
        • Taka H.
        • Murayama K.
        • Nitta K.
        Globotriaosylceramide-expressing Burkitt lymphoma cells are committed to early apoptotic status by rhamnose-binding lectin from catfish eggs.
        Biol. Pharm. Bull. 2009; 32: 345-353
        • Kawano T.
        • Sugawara S.
        • Hosono M.
        • Tatsuta T.
        • Nitta K.
        Alteration of gene expression induced by Silurus asotus lectin in Burkitt lymphoma cells.
        Biol. Pharm. Bull. 2008; 31: 998-1002
        • Sugawara S.
        • Hosono M.
        • Ogawa Y.
        • Takayanagi M.
        • Nitta K.
        Catfish egg lectin causes rapid activation of multidrug resistance 1 P-glycoprotein as a lipid translocase.
        Biol. Pharm. Bull. 2005; 28: 434-441
        • Sugawara S.
        • Sasaki S.
        • Ogawa Y.
        • Hosono M.
        • Nitta K.
        Catfish (Silurus asotus) lectin enhances the cytotoxic effects of doxorubicin.
        Yakugaku Zasshi. 2005; 125: 327-334
        • Tennant J.R.
        Evaluation of the trypan blue technique for determination of cell viability.
        Transplantation. 1964; 2: 685-694
        • Ishiyama M.
        • Miyazono Y.
        • Sasamoto K.
        • Ohkura Y.
        • Ueno K.
        A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability.
        Talanta. 1997; 44: 1299-1305
        • Pepper C.
        • Thomas A.
        • Tucker H.
        • Hoy T.
        • Bentley P.
        Flow cytometric assessment of three different methods for the measurement of in vivo apoptosis.
        Leuk. Res. 1998; 22: 439-444
        • Naismith J.H.
        • Field R.A.
        Structural basis of trimannoside recognition by concanavalin A.
        J. Biol. Chem. 1996; 271: 972-976
        • Transue T.R.
        • Smith A.K.
        • Mo H.
        • Goldstein I.J.
        • Saper M.A.
        Structure of benzyl T-antigen disaccharide bound to Amaranthus caudatus agglutinin.
        Nat. Struct. Biol. 1997; 4: 779-783
        • Kasai K.
        • Hirabayashi J.
        Galectins. A family of animal lectins that decipher glycocodes.
        J Biochem. 1996; 119: 1-8
        • Weis W.I.
        • Taylor M.E.
        • Drickamer K.
        The C-type lectin superfamily in the immune system.
        Immunol. Rev. 1998; 163: 19-34
        • Vakonakis I.
        • Langenhan T.
        • Prömel S.
        • Russ A.
        • Campbell I.D.
        Solution structure and sugar-binding mechanism of mouse latrophilin-1 RBL. A 7TM receptor-attached lectin-like domain.
        Structure. 2008; 16: 944-953
        • Ogawa T.
        • Watanabe M.
        • Naganuma T.
        • Muramoto K.
        Diversified carbohydrate-binding lectins from marine resources.
        J. Amino Acids. 2011; 2011: 838914
        • Kotake T.
        • Dina S.
        • Konishi T.
        • Kaneko S.
        • Igarashi K.
        • Samejima M.
        • Watanabe Y.
        • Kimura K.
        • Tsumuraya Y.
        Molecular cloning of a β-galactosidase from radish that specifically hydrolyzes {β}-(1–3)-and {β}-(1–6)-galactosyl residues of Arabidnogalactan protein.
        Plant Physiol. 2005; 138: 1563-1576
        • Stepan H.
        • Pabst M.
        • Altmann F.
        • Geyer H.
        • Geyer R.
        • Staudacher E.
        O-Glycosylation of snails.
        Glycoconj. J. 2012; 29: 189-198
        • Velkova L.
        • Dolashka P.
        • Lieb B.
        • Dolashki A.
        • Voelter W.
        • Van Beeumen J.
        • Devreese B.
        Glycan structures of the structural subunit (HtH1) of Haliotis tuberculata hemocyanin.
        Glycoconj. J. 2011; 28: 385-395
        • Gutternigg M.
        • Bürgmayr S.
        • Pöltl G.
        • Rudolf J.
        • Staudacher E.
        Neutral N-glycan patterns of the gastropods Limax maximus Cepaea hortensis Planorbarius corneus Arianta arbustorum and Achatina fulica.
        Glycoconj. J. 2007; 24: 475-489
        • Fujii Y.
        • Sugawara S.
        • Araki D.
        • Kawano T.
        • Tatsuta T.
        • Takahashi K.
        • Kawsar S.M.
        • Matsumoto R.
        • Kanaly R.A.
        • Yasumitsu H.
        • Ozeki Y.
        • Hosono M.
        • Miyagi T.
        • Hakomori S.-I.
        • Takayanagi M.
        • Nitta K.
        MRP1 expressed on Burkitt lymphoma cells was depleted by catfish egg lectin through Gb3-glycosphinogolipid and enhanced cytotoxic effect of drugs.
        Protein J. 2012; 31: 15-26