An attempt to form mutant CRM197 proteins of diphtheria toxin with neutralizing activity to heregulin.

我们发现白喉毒素（DT）结合到白喉毒素受体的egf样结构域/膜锚定形式的肝素结合egf样生长因子（proHB-EGF）。DT或其无毒突变体CRM197抑制HB-EGF与EGF受体的结合，从而抑制HB-EGF的有丝分裂活性。这项工作的目的是产生白喉毒素的CRM197的突变形式，具有中和活性的调节蛋白。我们得到了以下结果。(1) HB-EGF的egf样结构域上的dt结合位点分析为了形成heregin -binding CRM197，我们精确确定了HB-EGF的egf样结构域上的dt结合位点。我们在egf样结构域中引入了10个氨基酸的单点突变。F115、L127和E141位点的突变显著降低了HB-EGF的dt结合活性，而其余7个氨基酸位点的突变影响不大。(2) HB-EGF类EGF结构域三维结构模型的构建基于EGF和TGFalpha的NMR数据，构建HB-EGF类EGF结构域三维结构模型。模型显示，氨基酸残基F115、L127和E141存在于分子的同一侧，表明HB-EGF与DT在这一侧相互作用。这些结果对于构建调节蛋白结合的CRM197至关重要。进一步的研究正在进行中。

We have found that diphtheria toxin (DT) bind to the EGF-like domain of diphtheria toxin receptor/membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF). DT,or its non-toxic mutant CRM197, inhibits the binding of HB-EGF to EGF receptor, resulting in inhibition of mitogenic activity of HB-EGF.The aim of this work is to produce mutant forms of CRM197 of diphtheria toxin with neutralizing activity to heregulin. We have obtained the following results.(1) Analysis of the DT-binding site on the EGF-like domain of HB-EGFTo form heregulin-binding CRM197, DT-binding site on EGF-like domain of HB-EGF was precisely determined. We introduced single point mutations for 10 amino acids within the EGF-like domain. Mutation at F115, L127 or E141 greatly decreased the DT-binding activityof HB-EGF,but mutations for the remaining 7 amino acids did not show large effect.(2) Construction of three-dimentional structure model of the EGF-like domain of HB-EGFThree-dimentional structure model of the EGF-like domain of HB-EGF was constructed based on the NMR data of EGF and TGFalpha. The model indicated that amino acid residues F115, L127 and E141 exist on the same side of the molecule, suggesting that HB-EGF interacts with DT at this side.These results is critical to construct heregulin-binding CRM197. Further studies are in progress.