Alanine-scanning mutations in domain 4 of anthrax toxin protective antigen reveal residues important for binding to the cellular receptor and to a neutralizing monoclonal antibody
Alanine-scanning mutations in domain 4 of anthrax toxin protective antigen reveal residues important for binding to the cellular receptor and to a neutralizing monoclonal antibody
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DOI：
10.1074/jbc.m301154200
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发表时间：
2003-08-15
影响因子：
4.8
通讯作者：
Leppla, SH
中科院分区：
文献类型：
--
作者：
Rosovitz, MJ;Schuck, P;Leppla, SH
构建了一组炭疽毒素保护性抗原结构域4小环中丙氨酸替代的变体，以确定对与细胞受体以及中和性单克隆抗体14B7相互作用至关重要的单个氨基酸残基。通过细胞毒性试验分析替代的保护性抗原蛋白，并通过等离子体表面共振和分析超速离心测量它们与抗体的相互作用。残基天冬氨酸（683）对细胞结合和毒性最为关键，导致毒性降低约1000倍，但对与14B7的相互作用影响不大。残基酪氨酸（681）、天冬酰胺（682）和脯氨酸（686）的替代也使毒性显著降低，降低了10 - 100倍。其中，只有天冬酰胺（682）和脯氨酸（686）对与14B7的相互作用也至关重要。然而，残基赖氨酸（684）、亮氨酸（685）、亮氨酸（687）和酪氨酸（688）对14B7结合至关重要，但对毒性影响不大。K684A和L685A变体在细胞培养试验中表现出野生型毒性水平；L687A和Y688A变体分别仅降低1.5倍和5倍。
A panel of variants with alanine substitutions in the small loop of anthrax toxin protective antigen domain 4 was created to determine individual amino acid residues critical for interactions with the cellular receptor and with a neutralizing monoclonal antibody, 14B7. Substituted protective antigen proteins were analyzed by cellular cytotoxicity assays, and their interactions with antibody were measured by plasmon surface resonance and analytical ultracentrifugation. Residue Asp(683) was the most critical for cell binding and toxicity, causing an similar to1000-fold reduction in toxicity, but was not a large factor for interactions with 14B7. Substitutions in residues Tyr(681), Asn(682), and Pro(686) also reduced toxicity significantly, by 10-100-fold. Of these, only Asn(682) and Pro(686) were also critical for interactions with 14B7. However, residues Lys(684), Leu(685), Leu(687), and Tyr(688) were critical for 14B7 binding without greatly affecting toxicity. The K684A and L685A variants exhibited wild type levels of toxicity in cell culture assays; the L687A and Y688A variants were reduced only 1.5- and 5-fold, respectively.