Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
Toxins (Basel). 2013 Jan 30;5(2):224-48. doi: 10.3390/toxins5020224.
Vitetta and colleagues identified and characterized a putative vascular leak peptide (VLP) consensus sequence in recombinant ricin toxin A-chain (RTA) that contributed to dose-limiting human toxicity when RTA was administered intravenously in large quantities during chemotherapy. We disrupted this potentially toxic site within the more stable RTA1-33/44-198 vaccine immunogen and determined the impact of these mutations on protein stability, structure and protective immunogenicity using an experimental intranasal ricin challenge model in BALB/c mice to determine if the mutations were compatible. Single amino acid substitutions at the positions corresponding with RTA D75 (to A, or N) and V76 (to I, or M) had minor effects on the apparent protein melting temperature of RTA1-33/44-198 but all four variants retained greater apparent stability than the parent RTA. Moreover, each VLP(-) variant tested provided protection comparable with that of RTA1-33/44-198 against supralethal intranasal ricin challenge as judged by animal survival and several biomarkers. To understand better how VLP substitutions and mutations near the VLP site impact epitope structure, we introduced a previously described thermal stabilizing disulfide bond (R48C/T77C) along with the D75N or V76I substitutions in RTA1-33/44-198. The D75N mutation was compatible with the adjacent stabilizing R48C/T77C disulfide bond and the T(m) was unaffected, whereas the V76I mutation was less compatible with the adjacent disulfide bond involving C77. A crystal structure of the RTA1-33/44-198 R48C/T77C/D75N variant showed that the structural integrity of the immunogen was largely conserved and that a stable immunogen could be produced from E. coli. We conclude that it is feasible to disrupt the VLP site in RTA1-33/44-198 with little or no impact on apparent protein stability or protective efficacy in mice and such variants can be stabilized further by introduction of a disulfide bond.
维泰塔及其同事在重组蓖麻毒素 A 链（RTA）中鉴定并描述了一个假定的血管渗漏肽（VLP）共识序列，当在化疗中静脉内大剂量给予 RTA 时，该序列导致了剂量限制性的人类毒性。我们在更稳定的 RTA1-33/44-198 疫苗免疫原内破坏了这个潜在的毒性位点，并使用 BALB/c 小鼠的实验性鼻内蓖麻毒素挑战模型来确定这些突变对蛋白质稳定性、结构和保护性免疫原性的影响，以确定这些突变是否相容。在与 RTA D75（至 A 或 N）和 V76（至 I 或 M）对应的位置上的单个氨基酸取代对 RTA1-33/44-198 的表观蛋白熔点温度只有较小影响，但所有四个变体的表观稳定性都比亲本 RTA 更高。此外，测试的每个 VLP(-)变体都提供了与 RTA1-33/44-198 相当的保护，可防止鼻内蓖麻毒素超致死性挑战，如动物存活率和几个生物标志物所示。为了更好地了解 VLP 取代和 VLP 位点附近的突变如何影响表位结构，我们在 RTA1-33/44-198 中引入了先前描述的热稳定二硫键（R48C/T77C）以及 D75N 或 V76I 取代。D75N 突变与相邻的稳定 R48C/T77C 二硫键相容，T(m)不受影响，而 V76I 突变与涉及 C77 的相邻二硫键不太相容。RTA1-33/44-198 R48C/T77C/D75N 变体的晶体结构表明，免疫原的结构完整性基本得以保留，可以从大肠杆菌中生产出稳定的免疫原。我们得出结论，在 RTA1-33/44-198 中破坏 VLP 位点是可行的，对小鼠中的表观蛋白稳定性或保护效力几乎没有影响，并且通过引入二硫键可以进一步稳定这些变体。
