Krell Tino, Greco Frédéric, Nicolaï Marie-Claire, Dubayle Jean, Renauld-Mongénie Geneviève, Poisson Nadège, Bernard Isabelle
Aventis Pasteur, 1541 avenue Marcel Mérieux, 69280 Marcy l'etoile, France.
Biotechnol Appl Biochem. 2003 Dec;38(Pt 3):241-51. doi: 10.1042/BA20030089.
Tetanus neurotoxin (TeNT), pertussis toxin (PT) and pertussis filamentous haemagglutinin (FHA) are major virulence factors of Clostridium tetani and Bordetella pertussis, which are the causative agents of tetanus and whooping cough respectively. Inactivated forms of these virulence factors are the protein components of vaccines against these diseases. Here we report microcalorimetric studies to characterize these proteins. The microcalorimetric titration curves of TeNT with micelles of gangliosides GD1b, GT1b and GQ1b were biphasic. For these gangliosides a high-affinity binding site (KD 45-277 nM) can be distinguished from a lower-affinity binding event (KD 666-1190 nM). This is direct evidence for multiple binding sites for gangliosides of the 1b series at TeNT as proposed by Emsley et al. [Emsley, Fotinou, Black, Fairweather, Charles, Watts, Hewitt and Isaacs (2000) J. Biol. Chem. 275, 8889-8894]. In agreement with previous reports, no binding was observed for gangliosides GM1, GM2, GM3 and GD2. The thermal denaturation of TeNT was characterized by two unfolding transitions centred around 57.4 and 62.4 degrees C. The conversion of TeNT into the toxoid form by formaldehyde treatment was accompanied by a large increase in Tm (the midpoint of protein unfolding transition, that is, the temperature at which half the protein is denatured and the other half is still present in its native form). Fetuin and asialofetuin bound to PT with similar affinities (KD 420 and 335 nM respectively). Binding was largely enthalpy-driven and counterbalanced by an unfavourable entropy change, indicating a loss of conformational flexibility. The latter could account for the observed inhibition of ATP binding after binding to fetuin. Furthermore, the molecular limits of mature PT subunit S5 were defined by MS and N-terminal peptide sequencing. The differential-scanning-calorimetry thermogram of FHA shows four well-resolved unfolding transitions, a finding consistent with the sequential denaturation of four structural domains.
破伤风神经毒素（TeNT）、百日咳毒素（PT）和百日咳丝状血凝素（FHA）分别是破伤风梭菌和百日咳博德特氏菌的主要毒力因子，这两种细菌分别是破伤风和百日咳的病原体。这些毒力因子的灭活形式是针对这些疾病的疫苗的蛋白质成分。在此，我们报告了用于表征这些蛋白质的微量热分析研究。TeNT与神经节苷脂GD1b、GT1b和GQ1b的胶束进行微量热滴定曲线呈双相。对于这些神经节苷脂，可区分出一个高亲和力结合位点（KD 45 - 277 nM）和一个低亲和力结合事件（KD 666 - 1190 nM）。这是Emsley等人[Emsley, Fotinou, Black, Fairweather, Charles, Watts, Hewitt和Isaacs（2000）J. Biol. Chem. 275, 8889 - 8894]所提出的TeNT上1b系列神经节苷脂存在多个结合位点的直接证据。与先前报道一致，未观察到神经节苷脂GM1、GM2、GM3和GD2的结合。TeNT的热变性以两个围绕57.4和62.4摄氏度的解折叠转变为特征。通过甲醛处理将TeNT转化为类毒素形式伴随着Tm（蛋白质解折叠转变的中点，即一半蛋白质变性而另一半仍以天然形式存在时的温度）的大幅升高。胎球蛋白和去唾液酸胎球蛋白以相似的亲和力（分别为KD 420和335 nM）与PT结合。结合主要由焓驱动，并被不利的熵变所抵消，表明构象灵活性丧失。后者可以解释与胎球蛋白结合后观察到的ATP结合抑制。此外，通过质谱和N端肽测序确定了成熟PT亚基S5的分子界限。FHA的差示扫描量热法热谱图显示出四个分辨率良好的解折叠转变，这一发现与四个结构域的顺序变性一致。
