Mechanistic Insights into How the Single Point Mutation Change the Autoantibody Repertoire

IF 1.9 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY
Zhong Ni, Fangyuan Song, Huimin Zhou, Ying Xu, Zhiguo Wang, Dongfeng Chen
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引用次数: 0

Abstract

A recent study showed that just one point mutation F33 to Y in the complementarity-determining region 1 of heavy chain (H-CDR1) could lead to the auto-antibody losing its DNA binding ability. However, the potential molecular mechanisms have not been well elucidated. In this study, we investigated how the antibody lost the DNA binding ability caused by mutation F33 to Y in the H-CDR1. We found that the electrostatic force was not the primary driving force for the interaction between anti-DNA antibodies and the antigen single strand DNA (ssDNA), and that the H-CDR2 largely contributed to the binding of antigen ssDNA, even larger than H-CDR1. The H-F33Y mutation could increase the hydrogen-bond interaction but impair the pi-pi stacking interaction between the antibody and ssDNA. We further found that F33H, W98H and Y95L in the wiletype antibody could form the stable pi-pi stacking interaction with the nucleotide bases of ssDNA. However, the Y33 in mutant could not form the parallel sandwich pi-pi stacking interaction with the ssDNA. To further confirm the importance of pi-pi stacking, the wildtype antibody and the mutants (F33YH, F33AH, W98AH and Y95AL) were experimentally expressed in CHO cells and purified, and the results from ELISA clearly showed that all the mutants lost the ssDNA binding ability. Taken together, our findings may not only deepen the understanding of the underlying interaction mechanism between autoantibody and antigen, but also broad implications in the field of antibody engineer.

Abstract Image

单点突变如何改变自身抗体汇集的机制启示
最近的一项研究表明,只要重链互补决定区 1(H-CDR1)中的一个点突变 F33 变为 Y,就会导致自身抗体失去与 DNA 结合的能力。然而,潜在的分子机制尚未得到很好的阐明。在本研究中,我们研究了抗体是如何因 H-CDR1 中的 F33 突变为 Y 而失去 DNA 结合能力的。我们发现,静电力并不是抗DNA抗体与抗原单链DNA(ssDNA)相互作用的主要驱动力,H-CDR2在很大程度上促进了抗原ssDNA的结合,甚至大于H-CDR1。H-F33Y突变可增加抗体与抗原单链DNA之间的氢键相互作用,但会损害抗体与抗原单链DNA之间的π-π堆积相互作用。我们进一步发现,Wile 型抗体中的 F33H、W98H 和 Y95L 可以与 ssDNA 的核苷酸碱基形成稳定的 pi-pi 堆叠作用。然而,突变体中的 Y33 不能与 ssDNA 形成平行的三明治 pi-pi 堆叠作用。为了进一步证实 pi-pi 堆叠的重要性,我们在 CHO 细胞中实验表达并纯化了野生型抗体和突变体(F33YH、F33AH、W98AH 和 Y95AL),ELISA 的结果清楚地表明所有突变体都失去了与 ssDNA 结合的能力。综上所述,我们的发现不仅可以加深对自身抗体与抗原之间相互作用机制的理解,而且在抗体工程师领域具有广泛的意义。
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来源期刊
The Protein Journal
The Protein Journal 生物-生化与分子生物学
CiteScore
5.20
自引率
0.00%
发文量
57
审稿时长
12 months
期刊介绍: The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.
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