The mycotoxin patulin induces intra- and intermolecular protein crosslinks in vitro involving cysteine, lysine, and histidine side chains, and α-amino groups
The mycotoxin patulin induces intra- and intermolecular protein crosslinks in vitro involving cysteine, lysine, and histidine side chains, and α-amino groups
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DOI：
10.1016/s0009-2797(99)00123-4
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发表时间：
1999-11-30
影响因子：
5.1
通讯作者：
Metzler, M
中科院分区：
文献类型：
--
作者：
Fliege, R;Metzler, M
由于以前的研究表明，多亲电：对简单的硫醇亲核试剂的展青霉素（PAT）的反应性，我们有系统地研究了PAT在体外共价交联蛋白质的能力。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳，PAT诱导的分子间蛋白质-蛋白质交联的形成被清楚地证明为牛血清白蛋白含有一个巯基每个分子，但也为无巯基的鸡蛋溶菌酶。通过（1）用N-乙基马来酰亚胺和2-亚氨基硫杂环戊烷调节硫醇基团;（2）与各种已知交联剂（即苯二马来酰亚胺、戊二醛和辛二酰亚胺二甲酯）进行比较，以及（3）使用丹磺酰标记的氨基酸和荧光谷胱甘肽衍生物进行荧光掺入研究，对交联位点进行表征。半胱氨酸的巯基优选用于PAT介导的交联反应，但赖氨酸和组氨酸的侧链以及α-氨基也表现出反应性。PAT既可以作为同双官能交联剂，也可以作为异双官能交联剂。具有硫醇基团的单加合物的初始形成似乎活化PAT用于随后与氨基的反应，但当没有用于交联完成的第二亲核试剂可用时，也导致进一步亲电性质的快速损失。使用微管蛋白作为具有实验可控的四元结构的蛋白质和PAT毒性的拟议细胞靶点的研究强调了在低蛋白浓度下特定空间条件对交联形成的影响。非聚合的微管蛋白，即微管蛋白α，β-二聚体，与PAT形成由分子内交联的β-微管蛋白组成的确定产物，而在用PAT处理之前，鸟苷三磷酸或紫杉醇诱导聚合成微管样四元结构，导致α-和β-微管蛋白之间形成分子间交联。与此相反，变性的微管蛋白没有产生这两种新的蛋白质种类，但只有非特异性的分子内交联和高度交联的聚集体。因此，除了氨基酸组成，蛋白质的三级和四级超结构似乎显着影响其对PAT的反应性。在适当的条件下，在PAT浓度等于或甚至低于蛋白质浓度时，可以容易地观察到蛋白质交联的产生。这些新的反应途径的PAT在体外证明其在体内的毒性机制的相关性仍有待研究。(C)1999爱思唯尔科学爱尔兰有限公司保留所有权利。
As previous studies have indicated a multiple electrophilic: reactivity of patulin (PAT) towards simple thiol nucleophiles, we have methodically investigated the ability of PAT to covalently crosslink proteins in vitro. By means of sodium dodecylsulphate polyacrylamide gel electrophoresis, the formation of PAT-induced intermolecular protein-protein crosslinks was clearly demonstrated for bovine serum albumin containing one thiol group per molecule, but also for the thiol-free hen egg lysozyme. Characterization of the crosslink sites was carried out by (1) modulation of the thiol groups with N-ethylimaleimide and 2-iminothiolane; (2) comparison with various known crosslinking agents, i.e. phenylenedimaleimide, glutardialdehyde, and dimethylsuberimidate, and (3) fluorescence incorporation studies using dansyl-labeled amino acids and a fluorescent glutathione derivative. The thiol group of cysteine was preferred for PAT-mediated crosslink reactions, but the side chains of lysine and histidine, and cc-amino groups also exhibited reactivity. PAT can act both as a homobifunctional as well as a heterobifunctional crosslinking agent. The initial formation of a monoadduct with a thiol group appears to activate PAT for the subsequent reaction with an amino group, but also leads to rapid loss of further electrophilic properties when no second nucleophile for crosslink completion is available. Studies using microtubule proteins as a protein with experimentally controllable quarternary structure and a proposed cellular target for PAT toxicity emphasized the influence of specific sterical conditions on crosslink formation at low protein concentrations. Non-polymerized microtubule proteins, i.e. tubulin alpha,beta-dimers, formed a defined product with PAT consisting of an intramolecularly crosslinked beta-tubulin, whereas guanosine triphosphate- or paclitaxel-induced polymerization to microtubule-like quarternary structures prior to treatment with PAT gave rise to intermolecular crosslink formation between alpha- and beta-tubulin. In contrast, denaturated tubulin yielded none of those two new protein species, but only unspecific intramolecular crosslinks and highly crosslinked aggregates. Thus, in addition to the amino acid composition, the tertiary and quarternary superstructures of proteins appear to markedly influence their reactivity towards PAT. Under appropriate conditions, the generation of protein crosslinks could easily be observed at concentrations of PAT equal to or even below the concentration of the protein. The relevance of these novel reaction pathways of PAT demonstrated in vitro for its in vivo mechanisms of toxicity remains to be investigated. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.