Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics
AbstractLeguminous 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. View Full-Text
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Soga, K.; Abo, H.; Qin, S.-Y.; Kyoutou, T.; Hiemori, K.; Tateno, H.; Matsumoto, N.; Hirabayashi, J.; Yamamoto, K. Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics. Biomolecules 2015, 5, 1540-1562.
Soga K, Abo H, Qin S-Y, Kyoutou T, Hiemori K, Tateno H, Matsumoto N, Hirabayashi J, Yamamoto K. Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics. Biomolecules. 2015; 5(3):1540-1562.Chicago/Turabian Style
Soga, Keisuke; Abo, Hirohito; Qin, Sheng-Ying; Kyoutou, Takuya; Hiemori, Keiko; Tateno, Hiroaki; Matsumoto, Naoki; Hirabayashi, Jun; Yamamoto, Kazuo. 2015. "Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics." Biomolecules 5, no. 3: 1540-1562.