DEAMIDATION AND ISOASPARTIC ACID FORMATION IN PROTECTIVE ANTIGEN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 炭疽芽孢杆菌的保护性抗原(PA)蛋白通过促进毒素转位到目标宿主细胞，在炭疽病的病理过程中起着至关重要的作用。由于重组PA(RPA)蛋白本身具有高度的免疫原性和无毒性，因此它也是改进的人类炭疽疫苗的建议药材。自从发现PA以来，人们就知道PA由多个带电的异构体组成，但PA的异质性的原因一直没有具体的结构描述。两种主要的异构体在表达早期就出现在体内，纯化后代表了主要成分。虽然这两种异构体在动物中都能引起与药物产品相同的保护性免疫，但RPA的结构异质性需要进一步的化学和生物学特性，因为它最终是供人类使用的。此外，在体外处理不当后，异构体会重新出现；因此，这种降解的来源和性质需要更好的定义。 利用LC-ESI-MS/MS(LC-ESI-MS/MS)并验证氨基酸修饰的自动分配，我们最近证明，在68个天冬酰胺(ASN)残基中，有6个药用级RPA含有可测量的脱酰胺基。不同等级和不同处理的RPA之间以及通过凝胶电泳法纯化的异构体之间存在着直接的相关性，这种关系随着纯度的降低而降低，随着蛋白质老化而增加。在生化和生物学功能方面，异构体越多、脱酰胺程度越大的RPA在体外对肝细胞异构化、全毒素形成和巨噬细胞毒性的活性都较低。然而，任何给定的异构体的总体复杂性或同一性都与任何观察到的部位的脱酰胺百分率无关。在所有被分析的样品中，N537位始终显示出最高的修饰，尽管通过理论建模的方法(可在www.deamidation.org上查看)它排在第10位，该方法在根据定义的晶体结构预测蛋白质和多肽的脱酰胺化方面非常准确。由于RPA中的其他五个观察到的位点也与预测的脱酰胺化水平的排名顺序不一致，RPA似乎显示出尚未纳入流行的建模算法的属性。 一份出版物正在筹备中。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The protective antigen (PA) protein of Bacillus anthracis plays an essential role for the pathology of anthrax by facilitating the translocation of toxic factors into targeted host cells. Since it is highly immunogenic and non-toxic by itself, recombinant PA (rPA) protein is also the proposed pharmaceutical substance for an improved human anthrax vaccine. PA has been known since discovery to comprise multiply charged isoforms, but the cause of heterogeneity in PA has eluded specific structural description. Two primary isoforms appear in vivo early during expression and represent the majority components after purification. Although both isoforms elicit equivalent protective immunity in animals as compared to the pharmaceutical product, structural heterogeneity of rPA requires further chemical and biological characterization since it is intended for eventual human use. Moreover, isoforms re-appear after improper handling in vitro; therefore, the source and nature of this degradation requires better definition. Using liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) with verification of automated assignments for amino acid modification, we have recently demonstrated that pharmaceutical grade rPA contains measurable deamidation at 6 of 68 total asparagine (Asn) residues. A direct correlation between isoform complexity and percent deamidation was observed among various grades and treatments of rPA, as well as between isoforms purified by gel electrophoresis, such that both decreased with purity and increased with protein aging. With respect to biochemical and biological function, rPA with more isoforms and greater deamidation displayed lower in vitro activities for heptamerization, holotoxin formation, and macrophage toxicity. However, neither the overall complexity nor the identity of any given isoform was associated with percent deamidation at any observed site. Position N537 consistently showed the highest modification in all samples analyzed, even though it ranks 10th by a method of theoretical modeling (viewable at www.deamidation.org) which has otherwise been remarkably accurate in predicting protein and peptide deamidation from defined crystal structure. Since five other observed sites in rPA also did not align with predicted rank order for levels of deamidation, rPA appears to exhibit properties not yet incorporated into the popular modeling algorithm. A publication is being prepared.