Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics.
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.
Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Chiba 277-8562, Japan. keisuke.soga@gmail.com.
Keisuke
K
Soga
Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Chiba 277-8562, Japan. 137303@ib.k.u-tokyo.ac.jp.
Hirohito
H
Abo
Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Chiba 277-8562, Japan. qinshengying@gmail.com.
Sheng-Ying
SY
Qin
Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Chiba 277-8562, Japan. kyoutou.takuya@sysmex.co.jp.
Takuya
T
Kyoutou
Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki 305-8568, Japan. keiko-hiemori@aist.go.jp.
Keiko
K
Hiemori
Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki 305-8568, Japan. h-tateno@aist.go.jp.
Hiroaki
H
Tateno
Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Chiba 277-8562, Japan. nmatsu@k.u-tokyo.ac.jp.
Naoki
N
Matsumoto
Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki 305-8568, Japan. jun-hirabayashi@aist.go.jp.
Jun
J
Hirabayashi
Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Chiba 277-8562, Japan. yamamoto@k.u-tokyo.ac.jp.
Kazuo
K
Yamamoto
D000818:Animals; D015723:Gene Library; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D019887:Peanut Agglutinin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D009844:Oligosaccharides; D019887:Peanut Agglutinin; D011993:Recombinant Fusion Proteins; C062923:N-acetylneuraminosyl(alpha2-6)lactosamine
carbohydrate-binding specificity; cell surface display; leguminous lectin; molecular engineering; scaffold