Changing the Antigen Binding Specificity by Single Point Mutations of an Anti-p24 (HIV-1) Antibody1
Changing the Antigen Binding Specificity by Single Point Mutations of an Anti-p24 (HIV-1) Antibody1
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通过抗 p24 (HIV-1) 抗体的单点突变改变抗原结合特异性1
作者：
K. Winkler;A. Kramer;G. Küttner;M. Seifert;C. Scholz;H. Wessner;J. Schneider;W. Höhne
针对p24（HIV-1）产生的鼠mAb CB 4 -1识别HIV-1衣壳蛋白的线性表位。此外，CB 4 -1表现出与表位同源肽的交叉反应性结合和与表位非同源肽的多特异性反应。晶体结构表明，表位肽（e-pep）和非同源肽在CB 4 -1的结合区域内采用不同的构象。使用CB 4 -1的单链（sc）Fv构建体进行片段可变（Fv）区的定点诱变，以分析单个氨基酸侧链对e-pep和对一个表位非同源肽的结合贡献。与Ag直接接触的Ab氨基酸侧链的突变对两种肽的结合表现出相反的影响。尽管e-pep对CB 4 -1 scFv突变体重链可变区Tyr 32Ala的亲和力降低了250倍，但非同源肽的结合保持不变。相比之下，突变轻链可变区Phe 94 Ala使非同源肽的亲和力比其对e-pep的亲和力降低10倍。因此，通过单个氨基酸交换可以观察到特异性的实质性变化。通过肽的取代分析对scFv突变体的进一步表征表明突变的影响不限于接触残基。该方法还揭示了非同源肽的反向补偿性氨基酸交换，其将对scFv突变体轻链可变区Phe 94 Ala的亲和力增加至对野生型scFv的e-pep亲和力的水平。
The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein. Additionally, CB4-1 exhibits cross-reactive binding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides. Crystal structures demonstrate that the epitope peptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1. Site-directed mutagenesis of the fragment variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutations of Ab amino acid side chains, which are in direct contact with the Ag, show opposite influences on the binding of the two peptides. Whereas the affinity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr32Ala is decreased 250-fold, the binding of the nonhomologous peptide remains unchanged. In contrast, the mutation light chain variable region Phe94Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep. Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the scFv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method also reveals an inverse compensatory amino acid exchange for the nonhomologous peptide which increases the affinity to the scFv mutant light chain variable region Phe94Ala up to the level of the e-pep affinity to the wild-type scFv.
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通过定点体外诱变表征抗地高辛抗体结合位点。
影响因子：
3.6
作者：
Near,RI;Mudgett-Hunter,M;Novotny,J;Bruccoleri,R;Ng,SC
通讯作者：
Ng,SC
重链连接氨基酸多样性对偶氮苯胂酸特异性抗体中抗体亲和力的贡献。
DOI：
--
发表时间：
1996
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Parhami-Seren,B;Margolies,MN
通讯作者：
Margolies,MN
影响因子：
7.8
作者：
Kelsoe, G
通讯作者：
Kelsoe, G
影响因子：
2.9
作者：
DallAcqua, W;Goldman, ER;Mariuzza, RA
通讯作者：
Mariuzza, RA
抗体结合位点的寡核苷酸定向诱变。
影响因子：
5
作者：
Sharon,J;Kao,CY;Sompuram,SR
通讯作者：
Sompuram,SR