Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics

Biomolecules

Keisuke Soga²  Hirohito Abo²  Sheng-Ying Qin²  Takuya Kyoutou²  Keiko Hiemori¹  Hiroaki Tateno¹  Naoki Matsumoto²  Jun Hirabayashi¹  Kazuo Yamamoto² 

[1] Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki 305-8568, Japan; E-Mails:;Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Chiba 277-8562, Japan; E-Mails:

关键词: leguminous lectin;    cell surface display;    carbohydrate-binding specificity;    molecular engineering;    scaffold;   

DOI  :  10.3390/biom5031540

来源: mdpi

【 摘 要 】

Leguminous lectins have a conserved carbohydrate recognition site comprising four loops (A–D). Here, we randomly mutated the sequence and length of loops C and D of peanut agglutinin (PNA) and expressed the proteins on the surface of mouse green fluorescent protein (GFP)-reporter cells. Flow cytometry, limiting dilution, and cDNA cloning were used to screen for several mutated PNAs with distinct properties. The mutated PNA clones obtained using NeuAcα2-6(Galβ1-3)GalNAc as a ligand showed preference for NeuAcα2-6(Galβ1-3)GalNAc rather than non-sialylated Galβ1-3GlcNAc, whereas wild-type PNA binds to Galβ1-3GlcNAc but not sialylated Galβ1-3GalNAc. Sequence analyses revealed that for all of the glycan-reactive mutated PNA clones, (i) loop C was eight amino acids in length, (ii) loop D was identical to that of wild-type PNA, (iii) residue 127 was asparagine, (iv) residue 125 was tryptophan, and (v) residue 130 was hydrophobic tyrosine, phenylalanine, or histidine. The sugar-binding ability of wild-type PNA was increased nine-fold when Tyr125 was mutated to tryptophan, and that of mutated clone C was increased more than 30-fold after His130 was changed to tyrosine. These results provide an insight into the relationship between the amino acid sequences of the carbohydrate recognition site and sugar-binding abilities of leguminous lectins.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.