DEAMIDATION AND ISOASPARTIC ACID FORMATION IN PROTECTIVE ANTIGEN

保护性抗原中的脱酰胺和异天冬氨酸的形成

• 批准号: 7602043
• 负责人: BRADFORD S POWELL
• 金额: $ 0.65万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-03 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602043
• 关键词: Aging; Algorithms; Amino Acids; Animals; Anthrax Vaccines; Anthrax disease; Antigens; Asparagine; Bacillus anthracis; Biochemical; Biological; Biological Process; Cells; Charge; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Exhibits; Funding; Grant; Heterogeneity; Human; Immunity; In Vitro; Institution; Isoaspartic Acid; Liquid Chromatography; Measurable; Methods; Modeling; Modification; Nature; Pathology; Peptides; Pharmacologic Substance; Play; Positioning Attribute; Property; Protein Isoforms; Proteins; Publications; Recombinants; Research; Research Personnel; Resources; Role; Sampling; Site; Source; Structure; Theoretical model; Toxic effect; United States National Institutes of Health; deamidation; gel electrophoresis; holotoxins; immunogenic; improved; in vivo; macrophage; tandem mass spectrometry

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 炭疽芽孢杆菌的保护性抗原 (PA) 蛋白通过促进有毒因子易位到目标宿主细胞中，在炭疽病理学中发挥着重要作用。由于重组PA（rPA）蛋白本身具有高度免疫原性和无毒性，因此它也是改进的人炭疽疫苗的拟议药物物质。自从发现 PA 以来，我们就知道它包含多电荷异构体，但 PA 异质性的原因一直没有具体的结构描述。 两种主要同工型在表达早期出现在体内，并代表纯化后的主要成分。 尽管与药品相比，这两种亚型在动物中引起同等的保护性免疫力，但 rPA 的结构异质性需要进一步的化学和生物学表征，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，需要更好地界定这种退化的根源和性质。 使用液相色谱-串联质谱 (LC-ESI-MS/MS) 并验证氨基酸修饰的自动分配，我们最近证明药物级 rPA 在 68 个总天冬酰胺 (Asn) 残基中的 6 个含有可测量的脱酰胺作用。在不同等级和处理的 rPA 中，以及通过凝胶电泳纯化的同工型之间，观察到同工型复杂性和脱酰胺百分比之间存在直接相关性，因此两者都随着纯度而降低，并随着蛋白质老化而增加。在生化和生物功能方面，具有更多亚型和更大脱酰胺作用的rPA表现出较低的七聚化、全毒素形成和巨噬细胞毒性的体外活性。然而，任何给定亚型的总体复杂性和特性均与任何观察位点的脱酰胺百分比无关。 N537 位置在所有分析的样品中始终显示出最高的修饰，尽管通过理论建模方法（可在 www.deamidation.org 上查看）排名第 10，该方法在从定义的晶体结构预测蛋白质和肽脱酰胺方面非常准确。 由于 rPA 中观察到的其他五个位点也与脱酰胺水平的预测排名顺序不一致，因此 rPA 似乎表现出尚未纳入流行建模算法的特性。 正在准备一份出版物。