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

被引：5

作者：

Nakamura, Kosuke ^{[1
,2
]}

Sakagami, Hiromi ^{[1
,2
]}

Asanuma-Date, Kimie ^{[1
,2
]}

Nagasawa, Nao ^{[1
,2
]}

Nakahara, Yoshiaki ^{[3
]}

Akiyama, Hiroshi ^{[4
]}

Ogawa, Haruko ^{[1
,2
]}

机构：

[1] Ochanomizu Univ, Grad Sch Humanities & Sci, Bunkyo Ku, Tokyo 1128610, Japan

[2] Ochanomizu Univ, Glycosci Inst, Bunkyo Ku, Tokyo 1128610, Japan

[3] Tokai Univ, Dept Appl Biochem, Hiratsuka, Kanagawa 2591292, Japan

[4] Natl Inst Hlth Sci, Div Novel Foods & Immunochem, Setagaya Ku, Tokyo 1588501, Japan

来源：

CARBOHYDRATE RESEARCH | 2013年 / 382卷

关键词：

Glycosylated Fmoc-amino acid; Carbohydrate-binding specificity; SPR; N-Acetyllactosamine-binding lectin; Polylactosaminoglycan; Cell-surface simulating; SOLID-PHASE SYNTHESIS; SURFACE-PLASMON RESONANCE; N-ACETYLLACTOSAMINE; GLYCANS; OLIGOSACCHARIDES; GLYCOPEPTIDE; AFFINITY; CELLS; VITRONECTIN;

D O I：

10.1016/j.carres.2013.10.003

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

A method to immobilize glycan-linked amino acids with protected a-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,app)s of 10(6) M-1 for these two ligands. In contrast, Ricinus communis agglutinin-120 showed a 3.2-fold higher K-a,K-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. (C) 2013 Elsevier Ltd. All rights reserved.

引用

页码：77 / 85

页数：9