Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics.
Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics.
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DOI：
10.3390/biom5031540
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发表时间：
2015-07-20
期刊：
影响因子：
5.5
通讯作者：
Yamamoto K
中科院分区：
文献类型：
--
作者：
Soga K;Abo H;Qin SY;Kyoutou T;Hiemori K;Tateno H;Matsumoto N;Hirabayashi J;Yamamoto K
豆科植物凝集素具有包含四个环（A-D）的保守的碳水化合物识别位点。在这里，我们随机突变的花生凝集素（PNA）的C和D环的序列和长度，并在小鼠绿色荧光蛋白（GFP）报告细胞的表面上表达的蛋白。使用流式细胞术、有限稀释和cDNA克隆来筛选具有不同性质的几种突变的PNA。使用NeuAcα2-6（Galβ1-3）GalNAc作为配体获得的突变PNA克隆显示出对NeuAcα2-6（Galβ1-3）GalNAc的偏好，而不是非唾液酸化Galβ1-3GlcNAc，而野生型PNA结合Galβ1- 3GlcNAc，但不结合唾液酸化Galβ1-3GalNAc。序列分析显示，对于所有聚糖反应性突变的PNA克隆，（i）环C的长度为8个氨基酸，（ii）环D与野生型PNA的环D相同，（iii）残基127是天冬酰胺，（iv）残基125是色氨酸，以及（v）残基130是疏水性酪氨酸、苯丙氨酸或组氨酸。当Tyr 125突变为色氨酸时，野生型PNA的糖结合能力增加了9倍，而突变的克隆C在His 130改变为酪氨酸后增加了30倍以上。这些结果提供了一个深入了解的碳水化合物识别位点的氨基酸序列和糖的豆科凝集素的结合能力之间的关系。
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.