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: 7723049
• 负责人: BRADFORD S POWELL
• 金额: $ 0.39万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723049
• 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 炭疽芽孢杆菌的保护性抗原（PA）蛋白通过促进毒性因子转运到靶宿主细胞中而在炭疽的病理学中起重要作用。由于重组PA（rPA）蛋白本身具有高度免疫原性和无毒性，因此也被提议用于改进的人炭疽疫苗的药物物质。自发现以来，已知PA包含多电荷同种型，但PA中异质性的原因尚未得到具体的结构描述。 两种主要同种型在表达期间早期在体内出现，并且代表纯化后的主要组分。 尽管与药物产品相比，两种亚型在动物中引起等同的保护性免疫，但rPA的结构异质性需要进一步的化学和生物学表征，因为其预期最终用于人类。此外，异构体在体外处理不当后重新出现;因此，需要更好地定义这种降解的来源和性质。 使用液相色谱-串联质谱法（LC-ESI-MS/MS），验证氨基酸修饰的自动分配，我们最近证明了药用级rPA在68个总天冬酰胺（Asn）残基中的6个残基处含有可测量的脱酰胺作用。异构体的复杂性和百分比脱酰胺之间的直接相关性，观察到各种等级和治疗的rPA，以及异构体之间的凝胶电泳纯化，这样，都降低了纯度和蛋白质老化增加。在生化和生物学功能方面，具有更多异构体和更大脱酰胺的rPA显示出较低的体外七聚化、全毒素形成和巨噬细胞毒性活性。然而，任何给定亚型的总体复杂性或同一性均与任何观察位点的脱酰胺百分比无关。位置N537在所有分析的样品中始终显示出最高的修饰，即使它通过理论建模方法（可在www.deamidation.org上查看）排名第10，该方法在从确定的晶体结构预测蛋白质和肽脱酰胺方面非常准确。 由于在rPA中观察到的其他五个位点也与脱酰胺水平的预测排序不一致，因此rPA似乎表现出尚未纳入流行建模算法的特性。 正在编写一份出版物。