杂交毒素毒性影响因素的研究。不同靶细胞受体分布及毒素结合链的作用。

Study on the variables affecting toxicity of hybrid toxins. The effect of different target cell receptor distribution and toxin binding chain.

作者信息

Chang T M, Chang C H

机构信息

Biochemistry Laboratory, Isaac Gordon Center for Digestive Diseases and Nutrition, Genesee Hospital Rochester, NY 14607.

出版信息

Biochim Biophys Acta. 1989 Oct 9;1013(3):239-46. doi: 10.1016/0167-4889(89)90141-9.

DOI:10.1016/0167-4889(89)90141-9

PMID:2804085

Abstract

Disulfide conjugates of diphtheria toxin (DT) and its fragment A (DTA) to asialoorosomucoid (ASOR) were prepared. The toxicity of the conjugates were compared with DT in isolated rat, rabbit and guinea pig hepatocytes containing different concentration of asialoglycoprotein receptors (Biochim. Biophys, Acta 942, 57, 1988). In rat hepatocytes DTA-ASOR was highly toxic with half-maximal inhibitory concentration (IC50) of protein synthesis occurring at 4 +/- 3.10(-11) M (n = 7) which was much lower than that of DT DT (7.8 +/- 9.8.10(-9) M, n = 7). In rabbit hepatocytes toxicity of the conjugate (IC50 = 5.4 +/- 4.9.10(-10) M, n = 7) was higher than that of DT (IC50 = 5 +/- 4.10(-11) M, n = 7). In guinea pig hepatocytes, DTA-ASOR was not toxic at concentration below 10(-8) M, although DT was highly toxic (IC50 = 1.8 +/- 1.4.10(-10), n = 3). In the presence of 5 microM colchicine, the toxicity of DTA-ASOR in rat and rabbit hepatocytes increased by 10-fold, while in guinea pig hepatocytes it became detectable with an IC50 of 1.2 +/- 0.8.10(-9) M (n = 3). The toxicity of DT in the rat cells was also enhanced 10-fold by colchicine, but not at all in either the rabbit or the guinea pig cells. Addition of isolated diphtheria toxin fragment B (DTB) did not affect significantly the toxicity of DTA-ASOR in all three hepatocytes and that of DT in rat hepatocytes, but reduced toxicity of DT more than 20-fold in the rabbit and guinea pig cells. Toxicity of DT-ASOR in rat hepatocytes was the same as DTA-ASOR both in the absence and presence of colchicine, and abolished completely by excess ASOR, but not by DTB. Toxicity of DT-ASOR in rabbit hepatocytes was 40-times higher than DTA-ASOR, enhanced 10-fold by cochicine and reduced more than 30-fold by excess ASOR, but only slightly by DTB. These results indicate that entry of DTA from DTA-ASOR involve a DTB-independent translocation mechanism which can be as efficient as the DTB-dependent mechanism used by DT in the rabbit and guinea pig cells. The entry of both conjugates appeared to be mediated by the asialoglycoprotein receptors. However, the DTB moiety of DT-ASOR could function only in the DT-sensitive cells indicating the lack of a DTB-mediated translocation in the DT-resistant cells.

摘要

制备了白喉毒素（DT）及其A片段（DTA）与去唾液酸糖蛋白（ASOR）的二硫键缀合物。将这些缀合物的毒性与DT在含有不同浓度去唾液酸糖蛋白受体的分离大鼠、兔和豚鼠肝细胞中的毒性进行了比较（《生物化学与生物物理学报》942, 57, 1988）。在大鼠肝细胞中，DTA - ASOR具有高毒性，蛋白质合成的半数抑制浓度（IC50）在4 ± 3×10⁻¹¹ M时出现（n = 7），这远低于DT的IC50（7.8 ± 9.8×10⁻⁹ M，n = 7）。在兔肝细胞中，缀合物的毒性（IC50 = 5.4 ± 4.9×10⁻¹⁰ M，n = 7）高于DT的毒性（IC50 = 5 ± 4×10⁻¹¹ M，n = 7）。在豚鼠肝细胞中，DTA - ASOR在浓度低于10⁻⁸ M时无毒，而DT具有高毒性（IC50 = 1.8 ± 1.4×10⁻¹⁰，n = 3）。在5 μM秋水仙碱存在下，DTA - ASOR在大鼠和兔肝细胞中的毒性增加了10倍，而在豚鼠肝细胞中变得可检测到，IC50为1.2 ± 0.8×10⁻⁹ M（n = 3）。秋水仙碱也使DT在大鼠细胞中的毒性增强了10倍，但在兔或豚鼠细胞中根本没有增强。添加分离的白喉毒素片段B（DTB）对DTA - ASOR在所有三种肝细胞中的毒性以及DT在大鼠肝细胞中的毒性没有显著影响，但在兔和豚鼠细胞中使DT的毒性降低了20倍以上。DT - ASOR在大鼠肝细胞中的毒性在秋水仙碱存在和不存在时与DTA - ASOR相同，并且被过量的ASOR完全消除，但不被DTB消除。DT - ASOR在兔肝细胞中的毒性比DTA - ASOR高40倍，被秋水仙碱增强10倍，被过量的ASOR降低30倍以上，但仅被DTB轻微降低。这些结果表明，DTA从DTA - ASOR的进入涉及一种不依赖DTB的转运机制，该机制可以与DT在兔和豚鼠细胞中使用的依赖DTB的机制一样有效。两种缀合物的进入似乎都是由去唾液酸糖蛋白受体介导的。然而，DT - ASOR的DTB部分仅在对DT敏感的细胞中起作用，表明在对DT耐药的细胞中缺乏DTB介导的转运。