Kitova Elena N, Mulvey George L, Dingle Tanis, Sinelnikov Igor, Wee Stefanie, Griener Thomas P, Armstrong Glen D, Klassen John S
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Biochemistry. 2009 Jun 16;48(23):5365-74. doi: 10.1021/bi9003155.
A systematic investigation into the assembly and stability of native and modified subunits of the Shiga toxins (Stx) in vitro is described. Analysis of the assembly of native and modified B subunits of Stx1 and Stx2 in solution, carried out using electrospray ionization mass spectrometry (ES-MS), suggests that the lower thermodynamic stability of the B subunit homopentamer of Stx2, compared to that of Stx1, is due to the presence of a repulsive interaction involving Asp70 of the Stx2 B subunit. In Stx1 B, the corresponding (spatially) residue is Arg. Using temperature-controlled ES-MS, it is shown that the Stx1 and Stx2 holotoxins exhibit differences in their resistance to temperature- and acid-induced dissociation. However, both Stx1 and Stx2 are fully assembled at pH >3.5 and 37 degrees C. This finding has several important biological implications. First, it argues against the likelihood that the difference in Stx1 and Stx2 toxicity arises from differential dissociation of the toxins during the intracellular trafficking steps of the cellular intoxication process. Second, it implies that the activation of the A subunits of Stx1 and Stx2 by enzymatic cleavage must occur while the A subunit is assembled with the B subunit homopentamer. It is, therefore, proposed that the differential toxicities of Stx1 and Stx2 reflect the relative efficiencies of intracellular activation of the A subunits.
本文描述了对志贺毒素（Stx）天然亚基和修饰亚基在体外的组装及稳定性的系统研究。利用电喷雾电离质谱（ES-MS）对溶液中Stx1和Stx2的天然B亚基及修饰B亚基的组装情况进行分析，结果表明，与Stx1相比，Stx2的B亚基同五聚体热力学稳定性较低是由于Stx2 B亚基的Asp70存在排斥相互作用。在Stx1 B中，相应的（空间上的）残基是Arg。利用温控ES-MS表明，Stx1和Stx2全毒素在对温度和酸诱导解离的抗性方面存在差异。然而，在pH>3.5和37℃时，Stx1和Stx2均能完全组装。这一发现具有几个重要的生物学意义。首先，这表明Stx1和Stx2毒性差异不太可能源于细胞中毒过程细胞内运输步骤中毒素的差异解离。其次，这意味着Stx1和Stx2的A亚基通过酶切激活必须在A亚基与B亚基同五聚体组装时发生。因此，有人提出，Stx1和Stx2的毒性差异反映了A亚基细胞内激活的相对效率。
