title: Algal lectin binding to core (α1-6) fucosylated N-glycans: structural basis for specificity and production of recombinant protein.
abstract: We determined the specificity of BTL, a lectin from the red marine alga Bryothamnion triquetrum, toward fucosylated oligosaccharides. BTL showed a strict specificity for the core α1,6-fucosylation, which is an important marker for cancerogenesis and quality control of therapeutical antibodies. The double fucosylation α1,6 and α1,3 was also recognized, but the binding was totally abolished in the sole presence of the α1,3-fucosylation. A more detailed analysis of the specificity of BTL showed a preference for bi- and tri-antennary nonbisected N-glycans. Sialylation or fucosylation at the nonreducing end of N-glycans did not affect the recognition by the lectin. BTL displayed a strong affinity for a core α1,6-fucosylated octasaccharide with a Kd of 12 μM by titration microcalorimetry. The structural characterization of the interaction between BTL and the octasaccharide was obtained by STD-NMR. It demonstrated an extended epitope for recognition that includes the fucose residue, the distal GlcNAc and one mannose residue. Recombinant rBTL was obtained in Escherichia coli and characterized. Its binding properties for carbohydrates were studied using hemagglutination tests and glycan array analysis. rBTL was able to agglutinate rabbit erythrocytes with strong hemagglutination activity only after treatment with papain and trypsin, indicating that its ligands were not directly accessible at the cell surface. The hemagglutinating properties of rBTL confirm the correct folding and functional state of the protein. The results show BTL as a potent candidate for cancer diagnosis and as a reagent for the preparation and quality control of antibodies lacking core α1,6-fucosylated N-glycans.
authors.affiliation: CERMAV, UPR5301, CNRS and Université Grenoble Alpes, 38041 Grenoble, France Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, Brazil.
authors.forename: Antônia S F
authors.initials: AS
authors.lastname: do Nascimento
authors.affiliation: Glycotechnology Laboratory, CICbiomaGUNE, 20009 San Sebastian, Spain.
authors.forename: Sonia
authors.initials: S
authors.lastname: Serna
authors.affiliation: Glycotechnology Laboratory, CICbiomaGUNE, 20009 San Sebastian, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Beloqui
authors.affiliation: Chemical and Physical Biology, Centro de Investigaciones Biologicas, CSIC, 28040 Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Arda
authors.affiliation: Laboratório de Biotecnologia Marinha-BioMar-Lab, Departamento de Engenharia de Pesca, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.
authors.forename: Alexandre H
authors.initials: AH
authors.lastname: Sampaio
authors.affiliation: Bioorganische Chemie, Gebäude NW1, Universität Bayreuth, 95440 Bayreuth, Germany.
authors.forename: Janika
authors.initials: J
authors.lastname: Walcher
authors.affiliation: Bioorganische Chemie, Gebäude NW1, Universität Bayreuth, 95440 Bayreuth, Germany.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Ott
authors.affiliation: Bioorganische Chemie, Gebäude NW1, Universität Bayreuth, 95440 Bayreuth, Germany.
authors.forename: Carlo
authors.initials: C
authors.lastname: Unverzagt
authors.affiliation: Glycotechnology Laboratory, CICbiomaGUNE, 20009 San Sebastian, Spain CIBER-BBN, Paseo Miramon 182, 20009 San Sebastian, Spain.
authors.forename: Niels-Christian
authors.initials: NC
authors.lastname: Reichardt
authors.affiliation: Chemical and Physical Biology, Centro de Investigaciones Biologicas, CSIC, 28040 Madrid, Spain.
authors.forename: Jesus
authors.initials: J
authors.lastname: Jimenez-Barbero
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, Brazil.
authors.forename: Kyria S
authors.initials: KS
authors.lastname: Nascimento
authors.affiliation: CERMAV, UPR5301, CNRS and Université Grenoble Alpes, 38041 Grenoble, France imberty@cermav.cnrs.fr bscavada@gmail.com varrot@cermav.cnrs.fr.
authors.forename: Anne
authors.initials: A
authors.lastname: Imberty
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, Brazil imberty@cermav.cnrs.fr bscavada@gmail.com varrot@cermav.cnrs.fr.
authors.forename: Benildo S
authors.initials: BS
authors.lastname: Cavada
authors.affiliation: CERMAV, UPR5301, CNRS and Université Grenoble Alpes, 38041 Grenoble, France imberty@cermav.cnrs.fr bscavada@gmail.com varrot@cermav.cnrs.fr.
authors.forename: Annabelle
authors.initials: A
authors.lastname: Varrot
mesh_terms: D020418:Algal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004912:Erythrocytes / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D005643:Fucose / Q000737:chemistry*; D037102:Lectins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D000461:Rhodophyta / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D020418:Algal Proteins; D037102:Lectins; D011134:Polysaccharides; D011994:Recombinant Proteins; D005643:Fucose
keywords: Bryothamnion triquetrum; algal lectin; carbohydrate-binding specificity; core α1,6-fucose