title: Immobilized glycosylated Fmoc-amino acid for SPR: comparative studies of lectin-binding to linear or biantennary diLacNAc structures.
abstract: A method to immobilize glycan-linked amino acids with protected α-amino groups, which are key intermediates to produce the desired neoglycoprotein, to a Biacore sensor chip was developed and its utility for interaction analyses was demonstrated. Two types of diN-acetyllactosamine (diLacNAc)-containing glycans, a core 2 hexasaccharide involving linear diLacNAc that is O-linked to N-(9-fluorenyl)methoxycarbonyl (Fmoc)-Thr and a biantennary diLacNAc that is N-linked to Fmoc-Asn, were used as ligands. For immobilization, the free carboxyl groups of the amino acid residues were activated with EDC/NHS, then reacted with the ethylenediamine-derivatized carboxymethyldextran sensor chip to obtain the desired ligand concentrations. Interactions of the ligands with five plant lectins were analyzed by surface plasmon resonance, and the bindings were compared. The resonance unit of each lectin was corrected by subtracting that of the reference cell on which the Fmoc-Thr-core 1 or Fmoc-Asn was immobilized as a ligand. The carbohydrate specificities of interactions were verified by preincubating lectins with their respective inhibitory sugar before injection. By steady state analysis, the Lycopersicon esculentum lectin showed a 27-fold higher affinity to linear diLacNAc than to biantennary diLacNAc, while Datura stramonium and Solanum tuberosum lectins both showed low Ka,apps of 10(6)M(-1) for these two ligands. In contrast, Ricinus communis agglutinin-120 showed a 3.2-fold higher Ka,app to biantennary LacNAc than to linear diLacNAc. A lectin purified from Pleurocybella porrigens mushroom interacted at the high affinity of 10(8)M(-1) with both linear and biantennary diLacNAcs, which identified it as a unique probe. This method provides a useful and sensitive system to analyze interactions by simulating the glycans on the cell surface.
authors.affiliation: Graduate School of Humanities and Sciences and Glycoscience Institute, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan. Electronic address: kosnakamura@nihs.go.jp.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Nakamura
authors.forename: Hiromi
authors.initials: H
authors.lastname: Sakagami
authors.forename: Kimie
authors.initials: K
authors.lastname: Asanuma-Date
authors.forename: Nao
authors.initials: N
authors.lastname: Nagasawa
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Nakahara
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Akiyama
authors.forename: Haruko
authors.initials: H
authors.lastname: Ogawa
mesh_terms: D000363:Agaricales / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D000606:Amino Sugars / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods; D002240:Carbohydrate Sequence; D003911:Dextrans / Q000737:chemistry; D005029:Ethylenediamines / Q000737:chemistry; D005449:Fluorenes / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D055767:Immobilized Proteins / Q000737:chemistry; D037102:Lectins / Q000032:analysis / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D037121:Plant Lectins / Q000032:analysis / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D000596:Amino Acids; D000606:Amino Sugars; D003911:Dextrans; D005029:Ethylenediamines; D005449:Fluorenes; D055767:Immobilized Proteins; D037102:Lectins; D008024:Ligands; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D037121:Plant Lectins; C017707:Ricinus communis agglutinin-1; C034981:tomato lectin; C000458:N-acetyllactosamine; C031234:ethylenediamine; C014392:carboxymethyl dextran
keywords: (GlcNAc)(5–6); 10mM Tris-buffered saline containing 10mM Tris, 137mM NaCl, and 2.7mM KCl (pH 7.5); Carbohydrate-binding specificity; Cell-surface simulating; DSA; Datura stramonium lectin; Fmoc; Gal-specific Pleurocybella porrigens lectin; Glycosylated Fmoc-amino acid; LEA; LacNAc; Lycopersicon esculentum lectin; N-(9-fluorenyl)methoxycarbonyl; N-Acetyllactosamine-binding lectin; N-acetyllactosamine; PPL II; Polylactosaminoglycan; RCA I; Ricinus communis agglutinin-120; SPR; STA; Solanum tuberosum lectin; TBS; a core 2 hexasaccharide O-linked to Fmoc-Thr; biantennary diLacNAc; biantennary lactosaminoglycan N-linked to Fmoc-Asn; linear diLacNAc; oligo N-acetyl chitooligosaccharides; surface plasmon resonance