Falnes P O, Choe S, Madshus I H, Wilson B A, Olsnes S
Department of Biochemistry, Norwegian Radium Hospital, Montebello, Oslo.
J Biol Chem. 1994 Mar 18;269(11):8402-7.
Fragment A of diphtheria toxin is translocated to the cytosol when the toxin is presented to receptor-positive cells. The toxin binds to cell surface receptors through its B-fragment, and after endocytotic uptake, the low endosomal pH triggers translocation of the A-fragment across the membrane. Translocation can also be induced at the level of the plasma membrane by exposure to low pH medium. Based on the diphtheria toxin crystal structure, we made five double cysteine mutants of the A-fragment, each expected to form an intramolecular disulfide bond. Four of the double cysteine mutants efficiently formed an intramolecular disulfide bridge, and these same mutants showed a strong reduction in their translocating ability. The inhibition of translocation was observed both when the toxin was endocytosed and when translocation was induced by exposing surface-bound toxin to low pH. The data indicate that extensive unfolding of the A-fragment is required for translocation.
当白喉毒素作用于受体阳性细胞时，其A片段会转移至胞质溶胶。毒素通过其B片段与细胞表面受体结合，经内吞摄取后，内体的低pH值会促使A片段跨膜转移。暴露于低pH值介质中时，质膜水平也可诱导转移。基于白喉毒素的晶体结构，我们构建了A片段的五个双半胱氨酸突变体，每个突变体都预期会形成分子内二硫键。其中四个双半胱氨酸突变体有效形成了分子内二硫桥，且这些相同的突变体转移能力大幅降低。无论是毒素被内吞时，还是通过将表面结合的毒素暴露于低pH值来诱导转移时，均观察到转移受到抑制。数据表明，A片段的广泛解折叠是转移所必需的。
