期刊
CARBOHYDRATE RESEARCH
卷 382, 期 -, 页码 77-85出版社
ELSEVIER SCI LTD
DOI: 10.1016/j.carres.2013.10.003
关键词
Glycosylated Fmoc-amino acid; Carbohydrate-binding specificity; SPR; N-Acetyllactosamine-binding lectin; Polylactosaminoglycan; Cell-surface simulating
资金
- Ministry of Education, Culture, Sports, Science, and Technology [22570111]
- Grants-in-Aid for Scientific Research [25440016, 22570111] Funding Source: KAKEN
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.
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