Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity.
Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity.
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DOI：
10.1371/journal.pone.0135306
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Marks JD
中科院分区：
文献类型：
--
作者：
Fan Y;Geren IN;Dong J;Lou J;Wen W;Conrad F;Smith TJ;Smith LA;Ho M;Pires-Alves M;Wilson BA;Marks JD
麻痹性肉毒中毒是由肉毒毒素神经毒素（BoNT）引起的，这是一种含有锌内肽酶的多结构域蛋白，它可以切割同源的SNARE蛋白，从而阻断乙酰胆碱神经递质的释放。目前用于治疗肉毒杆菌中毒的抗毒素可以中和循环中的BoNT，但不能进入、结合或中和已经进入神经元的BoNT。BoNT血清型A （BoNT/A）的轻链内肽酶结构域（LC）被用于产生单克隆抗体（mab），该单克隆抗体可以逆转BoNT/A中毒引起的瘫痪。在酵母表面显示免疫的人和小鼠的单链可变片段（scFv）文库，并利用荧光活化细胞分选（FACS）分离出19个BoNT/A lc特异性单克隆抗体。单抗对BoNT/A LC的亲和力范围为9.0×10−11 M至3.53×10−8 M（平均KD为5.38×10−9 M，中位数KD为1.53×10−9 M），通过流式细胞术分析确定。11个单抗抑制BoNT/A LC催化活性的IC50值在8.3 ~73×10−9 m之间，选择的单抗也通过丙氨酸扫描诱变绘制了优良的表位，发现抑制单抗结合了远离BoNT/A LC催化中心的α-外源区。结果提供的单克隆抗体可用于中毒后瘫痪的细胞内逆转，并进一步确定可被小分子抑制剂靶向的表位。
The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10−11 M to 3.53×10−8 M (mean KD 5.38×10−9 M and median KD 1.53×10−9 M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10−9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.