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Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

机译：多杀性巴氏杆菌有丝分裂毒素功能域的定位

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The locations of the catalytic and receptor-binding domains of thePasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed α/β domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.
机译：研究了多杀性巴氏杆菌毒素（PMT）的催化和受体结合域的位置。克隆PMT的N和C端片段，并表达为具有亲和标签的融合蛋白。在合适的测定中评估纯化的融合蛋白的催化活性和细胞结合能力。 C末端片段（氨基酸681至1285）具有催化活性。当显微注射到静止的Swiss 3T3细胞中时，它会诱导毒素处理细胞典型的细胞形态变化，并刺激DNA合成。在C末端带有His标签的N末端片段（第1至506位氨基酸）与全长毒素竞争结合表面受体，因此含有细胞结合域。在此测定法中，包含C末端附近（C1165S）突变的无活性突变体也与细胞结合。产生至N末端PMT区的多克隆抗体可有效结合全长天然毒素，表明N末端位于表面。将针对PMT C末端的抗体微注射到细胞中，并抑制随后添加到培养基中的毒素的活性，从而确认C末端包含活性位点。对PMT序列的分析预测了一个推定的跨膜结构域，在与细胞毒性坏死因子同源的区域上，在N末端附近具有预测的疏水性和两亲性螺旋。预测PMT的C末端为混合的α/β域，这是催化域中常见的结构。对已知结构的蛋白质的同源性和线程计算支持了这些任务。

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