IL-1 Epitope Mapping Using Site-Directed Mutagenesis and Hydrogen-Deuterium Exchange Mass Spectrometry Analysis
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- 作者：Jirong Lu ; Derrick R. Witcher ; Melissa A. White ; Xiliang Wang ; Lihua Huang ; Radhakrishnan Rathnachalam ; John M. Beals ; and Stuart Kuhstoss
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：August 23, 2005
- 年：2005
- 卷：44
- 期：33
- 页码：11106 - 11114
- 全文大小：502K
- 年卷期：v.44,no.33(August 23, 2005)
- ISSN：1520-4995
文摘
Hu007, a humanized IgG1 monoclonal antibody, binds and neutralizes human, cynomolgus,and rabbit IL-1
but only weakly binds to mouse and rat IL-1. Biacore experiments demonstrated thatHu007 and the type-I IL-1 receptor competed for binding to IL-1. Increasing salt concentrations decreasethe association rate with only moderate effects on the dissociation rate, suggesting that long-rangeelectrostatics are critical for formation of the initial complex. To understand the ligand-binding specificityof Hu007, we have mapped the critical residues involved in the recognition of IL-1. Selected residuesin cynomolgus IL-1 were mutated to the corresponding residues in mouse IL-1, and the effects of thechanges on binding were evaluated by surface plasmon resonance measurements using Biacore. Specifically,substitution of F150S decreased binding affinity by 100-fold, suggesting the importance of hydrophobicinteractions in stabilizing the antibody/antigen complex. Substitution of three amino acids near the N-and C-terminal regions of cIL-1 with those found in mouse IL-1 (V3I/S5Q/F150S) decreased the bindingaffinity of Hu007 to IL-1 by about 1000-fold. Conversely, mutating the corresponding residues in mouseIL-1 to the human sequence resulted in an increase in binding affinity of about 1000-fold. Hydrogen-deuterium exchange/mass spectrometry analysis confirmed that these regions of IL-1 were protectedfrom exchange because of antibody binding. The results from this study demonstrate that Hu007 binds toa region located in the open end of the -barrel structure of IL-1 and blocks binding of IL-1 to itsreceptor.
but only weakly binds to mouse and rat IL-1. Biacore experiments demonstrated thatHu007 and the type-I IL-1 receptor competed for binding to IL-1. Increasing salt concentrations decreasethe association rate with only moderate effects on the dissociation rate, suggesting that long-rangeelectrostatics are critical for formation of the initial complex. To understand the ligand-binding specificityof Hu007, we have mapped the critical residues involved in the recognition of IL-1. Selected residuesin cynomolgus IL-1 were mutated to the corresponding residues in mouse IL-1, and the effects of thechanges on binding were evaluated by surface plasmon resonance measurements using Biacore. Specifically,substitution of F150S decreased binding affinity by 100-fold, suggesting the importance of hydrophobicinteractions in stabilizing the antibody/antigen complex. Substitution of three amino acids near the N-and C-terminal regions of cIL-1 with those found in mouse IL-1 (V3I/S5Q/F150S) decreased the bindingaffinity of Hu007 to IL-1 by about 1000-fold. Conversely, mutating the corresponding residues in mouseIL-1 to the human sequence resulted in an increase in binding affinity of about 1000-fold. Hydrogen-deuterium exchange/mass spectrometry analysis confirmed that these regions of IL-1 were protectedfrom exchange because of antibody binding. The results from this study demonstrate that Hu007 binds toa region located in the open end of the -barrel structure of IL-1 and blocks binding of IL-1 to itsreceptor.