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Mouse Toxicity and Cytokine Release by Verotoxin 1 B Subunit Mutants

机译：Verotoxin 1 B亚基突变体的小鼠毒性和细胞因子释放。

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The crystal structure of the verotoxin 1 (VT1) B subunit complexed with a globotriaosylceramide (Gb3) analogue showed the presence of three receptor binding sites per monomer. We wished to study the effects of altering the three sites, singly or in combination, on animal toxicity and cytokine induction in vitro. We found that while the site 1 and 2 mutants were modestly (two- to sevenfold) reduced in their ability to cause disease in BALB/c mice, the site 3 mutant, W34A, was as toxic as VT1. However, all the double-mutant proteins, irrespective of which two sites were mutated, exhibited approximately a 100-fold reduction in their 50% lethal doses for mice. These results suggest that multivalent receptor binding is important in vivo and that all three binding sites make a similar contribution to the latter process. The triple-mutant holotoxin, F30A G62T W34A, administered intraperitoneally without adjuvant, stimulated a strong antibody response in BALB/c mice, and the immune sera neutralized the activity of VT1 in vitro. Induction of tumor neurosis factor alpha release from differentiated human monocytes (THP-1 cells) was relatively impaired for site 1 and site 2 but not site 3 mutants, suggesting an auxiliary role for the latter site in mediation of cytokine release in vitro. Cytotoxicity assays on undifferentiated THP-1 cells have also demonstrated the importance of sites 1 and 2 and the relatively small role played by site 3 in causing cell death. These data suggest an association between the cytotoxicity of the protein and its ability to induce cytokine release.
机译：Vergloxinosylceramide（Gb 3 ）类似物的维毒素1（VT1）B亚基的晶体结构表明每个单体存在三个受体结合位点。我们希望研究单独或组合改变三个位点对动物毒性和体外细胞因子诱导的影响。我们发现，虽然第1位和第2位突变体在引起BALB / c小鼠疾病的能力中有所降低（2至7倍），但第3位突变体W34A的毒性与VT1一样。但是，所有的双突变蛋白，无论哪个位点都突变，都对小鼠的50％致死剂量降低了约100倍。这些结果表明，多价受体结合在体内是重要的，并且所有三个结合位点均对后一过程做出相似的贡献。三突变体全毒素，F30A G62T W34A，无佐剂腹膜内给药，在BALB / c小鼠中刺激了强烈的抗体反应，并且免疫血清在体外中和了VT1的活性。诱导分化的人类单核细胞（THP-1细胞）的肿瘤神经症因子α对于位点1和位点2相对受损，但对于位点3突变体则相对受损，表明后者在体外介导细胞因子释放中具有辅助作用。对未分化的THP-1细胞的细胞毒性测定也证明了位点1和2的重要性以及位点3在引起细胞死亡中所起的相对较小的作用。这些数据表明该蛋白质的细胞毒性与其诱导细胞因子释放的能力之间存在关联。

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