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Open AccessArticle

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

1
Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Chiba 277-8562, Japan
2
Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki 305-8568, Japan
*
Author to whom correspondence should be addressed.
Academic Editor: Hans Vliegenthart
Biomolecules 2015, 5(3), 1540-1562; https://doi.org/10.3390/biom5031540
Received: 17 May 2015 / Revised: 16 June 2015 / Accepted: 18 June 2015 / Published: 20 July 2015
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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. View Full-Text
Keywords: leguminous lectin; cell surface display; carbohydrate-binding specificity; molecular engineering; scaffold leguminous lectin; cell surface display; carbohydrate-binding specificity; molecular engineering; scaffold
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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.

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