Identifying relevant HLA-F ligands

项目摘要/摘要 ·我们的基本假设是，与人类经典和其他非经典MHC I类蛋白不同，HLA-1是一种免疫调节蛋白。 F配体识别集中在聚糖上，无论是作为寡糖、蛋白聚糖/肽聚糖的组分，还是作为蛋白聚糖/肽聚糖的组分， 糖蛋白/糖肽-代表了当前理解的范式转变。这个假设是基于 严格重新评估最近的，否则开创性的晶体学和质谱结果（1）。 ·响应FOA PA-19 - 066，我们的长期总体目标是"表征抗原加工和呈递 [...]新的肽和非肽配体提出的[HLA-F]，并确定这些贡献 独特的抗原配体：对感染性病原体和/或疫苗的保护性免疫应答;病原体- 相关的免疫发病机制;和/或在免疫介导的疾病的诱导/进展或预防中， 轻松”。我们在R21中实现这一目标的第一步将是：（a）从生理上确定- HLA-F相关配体/s使用严格的生物化学方法，重点是糖肽和糖缀合物， 门;和（B）确定用于生长衍射质量共晶体的条件以支持未来的晶体学 结构确定。这些结果将包括必要的初步结果，以支持后续补助金 在结构上、生物学上和功能上验证候选HLA-F糖配体的应用。 ·我们将通过以下具体目标实现这些目标，并严格检验我们的假设： 使用我们新的ARTEMIS质谱肽发现平台，聚糖阵列和片段筛选应用程序， 方法，以充分解析HLA-F配体特异性的糖基化配体。候选配体将用于筛选 结晶条件，以支持后续的晶体学研究，最终进行结构表征 识别机制，以充分解析特异性。最终通过仔细的生化反应 和结构验证，并成功的可能性将最大限度地纳入多个，平行的ap- 研究和实验方法进入拟议的范围，以克服潜在的混淆和陷阱。 ·意义（来自PA-19 - 066）："对抗原加工和呈递的经典理解始于 与MHC分子结合的蛋白质片段（肽）。[...]这些长期被接受的范式认为- 免疫抗原的加工和呈递以及T细胞识别是不完全的。大约10%的抗原 肽可以来自非常规来源[.]。此外，触发非- 经典的MHC I限制性CD8 T细胞和非常规或先天性T细胞包括脂质和小分子 代谢产物，但没有得到很好的表征。[...]这项研究计划的目的是促进发现 独特的抗原配体（肽和非肽），并了解对这些配体的免疫反应： 由非经典MHC分子呈递的肽和非肽配体：[HLA-F]。

Project Abstract/Summary · Our underlying hypothesis is that, unlike human classical and other non-classical MHC class I proteins, HLA- F ligand recognition is focused on glycans, either as components of oligosaccharides, proteo/peptidoglycans, or glycoproteins/glycopeptides – representing a paradigm shift in current understanding. This hypothesis is based on a rigorous reevaluation of recent, otherwise seminal crystallographic and mass-spec results (1). · Echoing FOA PA-19-066, our long-term overall goal is “to characterize antigen processing and presentation […] of novel peptidic and non-peptidic ligands presented by [HLA-F], and to determine the contribution of these unique antigenic ligands to: protective immune responses to infectious pathogens and/or vaccines; pathogen- associated immune pathogenesis; and/or in the induction/progression or prevention of immune-mediated dis- eases.” Our first steps towards accomplishing this goal in this R21 will be to: (a) identify physiologically- relevant ligand/s for HLA-F using rigorous biochemical approaches, focusing on glycopeptides and glycoconju- gates; and (b) determine conditions for growing diffraction-quality co-crystals to support future crystallographic structure determinations. These results will comprise the necessary preliminary results to support follow-on grant applications to structurally, biologically, and functionally validate candidate HLA-F glycoligands. · We will achieve these goals, and rigorously test our hypothesis, through the following Specific Aim: we will use our novel ARTEMIS mass-spec peptide discovery platform, glycan arrays, and fragment screening ap- proaches, to fully parse HLA-F ligand specificity for glycosylated ligands. Candidate ligands will be used to screen crystallization conditions to support follow-on crystallographic studies to eventually structurally characterize recognition mechanisms to fully parse specificity. Rigor will ultimately be achieved through careful biochemical and structural validation, and the likelihood of success will be maximized by incorporating multiple, parallel ap- proaches and experimental methods into the proposed scope to overcome potential confounders and pitfalls. · Significance (from PA-19-066): “The classic understanding of antigen processing and presentation begins with a protein fragment (peptide) associating with MHC molecules. […] These long-accepted paradigms regard- ing antigen processing and presentation and T cell recognition are incomplete. Approximately 10% of antigenic peptides can be derived from unconventional sources […]. Additionally, the antigen components that trigger non- classical MHC I restricted CD8 T cells and unconventional or innate T cells include lipids and small-molecule metabolites but are not well characterized. […] The objective of this research program is to promote the discovery of unique antigenic ligands (peptidic and non-peptidic) and understand the immune responses to these ligands: peptidic and non-peptidic ligands presented by non-classical MHC molecules: [HLA-F].”