Immobilized glycosylated Fmoc-amino acid for SPR: comparative studies of lectin-binding to linear or biantennary diLacNAc structures

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• 作者：Kosuke Nakamura ; Hiromi Sakagami ; Kimie Asanuma-Date ; Nao Nagasawa ; Yoshiaki Nakahara ; Hiroshi Akiyama ; Haruko Ogawa
• 关键词：Fmoc ; N-(9-fluorenyl)methoxycarbonyl ; LacNAc ; N-acetyllactosamine ; linear diLacNAc ; a core 2 hexasaccharide O-linked to Fmoc-Thr ; biantennary diLacNAc ; biantennary lactosaminoglycan N-linked to Fmoc-Asn ; STA ; Solanum tuberosum lectin ; DSA ; Datura stramonium lectin ; LEA ; Lycopersicon esculentum lectin ; RCA I ; Ricinus communis agglutinin-120 ; PPL II ; Gal-specific Pleurocybella porrigens lectin ; TBS ; 10  ; mM Tris-buffered saline containing 10  ; mM Tris ; 137  ; mM NaCl ; and 2.7  ; mM KCl (
• 刊名：Carbohydrate Research
• 出版年：15 December, 2013
• 年：2013
• 卷：382
• 期：Complete
• 页码：77-85
• 全文大小：985 K

文摘

A method to immobilize glycan-linked amino acids with protected 伪-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 K_a,apps of 10⁶ M^鈭? for these two ligands. In contrast, Ricinus communis agglutinin-120 showed a 3.2-fold higher K_a,app to biantennary LacNAc than to linear diLacNAc. A lectin purified from Pleurocybella porrigens mushroom interacted at the high affinity of 10⁸ M^鈭? 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.