Functional analysis of the CXXC motif using phage antibodies that cross-react with protein disulphide-isomerase family proteins
Functional analysis of the CXXC motif using phage antibodies that cross-react with protein disulphide-isomerase family proteins
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DOI：
10.1042/bj20040116
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发表时间：
2004-08-15
影响因子：
4.1
通讯作者：
Kikuchi, M
中科院分区：
文献类型：
--
作者：
Kimura, T;Nishida, A;Kikuchi, M
针对特定PDI（蛋白二硫异构酶）家族蛋白的多克隆抗体不与其他PDI家族蛋白发生交叉反应。为了避免对PDI家族蛋白中存在的重要自基序cys - xha - xha - cys的免疫耐受，我们使用了由Griffiths、Williams、Hartley、Tomlinson、Waterhouse、Crosby、Kontennann、Jones、Low、Allison等人建立的噬菌体展示文库[。]（1994） EMBO J. 13, 3245-3260]成功分离出能与人类和牛PDI、人类P5、人类PDI相关蛋白和酵母PDI交叉反应的噬菌体抗体。通过测量scFv（可变区单链抗体片段）与合成肽和PDI家族蛋白突变体的结合，我们在表面等离子体共振装置中鉴定出能够识别含有CGHC基序列或CGHCK序列的克隆。通过使用分离的噬菌体抗体，我们首次证明了CXXC基序后的赖氨酸残基可显著提高PDI家族蛋白的异构酶活性。此外，我们证明了分离的scFvs对突变PDI家族蛋白的亲和力与其活性位点的异构酶活性成正比。
Polyclonal antibodies that had been raised against particular PDI (protein disulphide-isomerase) family proteins did not cross-react with other PDI family proteins. To evade immune tolerance to the important self-motif Cys-Xaa-Xaa-Cys, which is present in PDI family proteins, we used the phage display library [established by Griffiths, Williams, Hartley, Tomlinson, Waterhouse, Crosby, Kontennann, Jones, Low, Allison et at. (1994) EMBO J. 13, 3245-3260] to isolate successfully the phage antibodies that can cross-react with human and bovine PDIs, human P5, human PDI-related protein and yeast PDI By measuring the binding of scFv (single-chain antibody fragment of variable region) to synthetic peptides and to mutants of PDI family proteins in a surface plasmon resonance apparatus, we identified clones that recognized sequences containing the CGHC motif or the CGHCK sequence. By using the isolated phage antibodies, we demonstrated for the first time that a lysine residue following the CXXC motif significantly increases the isomerase activities of PDI family proteins. Moreover, we demonstrated that the affinity of isolated scFvs for mutant PDI family proteins is proportional to the isomerase activities of their active sites.