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Biomolecules 2015, 5(3), 1540-1562; doi:10.3390/biom5031540

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
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)
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Abstract

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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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

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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