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，人类白细胞抗原- F配体识别主要集中在低聚糖、蛋白质/肽多聚糖的组成成分上，或者 糖蛋白/糖肽--代表了当前理解的范式转变。这一假设是有根据的 基于对最近的，其他方面的开创性的结晶学和质谱学结果的严格重新评估(1)。 ·与FOA PA-19-066相呼应，我们的长期总体目标是“表征抗原的处理和呈递 […][HLA-F]提出的新的多肽和非肽配体，并确定这些配体的贡献 独特的抗原配体：对传染性病原体和/或疫苗的保护性免疫反应；病原体- 相关的免疫发病机制；和/或在诱导/进展或预防免疫介导性疾病方面 放松点。“我们在R21中实现这一目标的第一步将是：(A)从生理上识别- 相关配体/S使用严格的生化方法，重点是糖肽和糖结合- 以及(B)确定生长衍射级共晶的条件以支持未来的结晶学 结构确定。这些结果将包括必要的初步结果，以支持后续赠款 应用于从结构、生物学和功能上验证候选的人类白细胞抗原-F糖配体。 ·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：我们将 使用我们的新型Artemis质谱肽发现平台、糖链阵列和片段筛选AP- 来全面分析糖基化配体的人类白细胞抗原-F配体的特异性。候选配体将用于筛选 支持后续结晶学研究以最终确定结构特征的结晶条件 识别机制，以充分解析特异性。严谨最终将通过仔细的生化反应来实现 和结构验证，成功的可能性将通过合并多个并行AP- 进入建议范围的方法和实验方法，以克服潜在的混杂因素和陷阱。 ·意义(摘自PA-19-066)：“对抗原处理和呈递的经典理解开始了 具有与MHC分子相关联的蛋白质片段(肽)。[…]这些长期被接受的范例涉及- ING抗原的处理和提呈以及T细胞识别不完整。大约10%的抗原性 多肽可以从非传统来源中提取[…]。此外，能够触发非免疫的抗原成分 经典MHC I限制性CD8 T细胞和非常规或天然T细胞包括脂类和小分子 代谢物，但没有得到很好的描述。[…]这项研究计划的目的是促进这一发现 了解独特的抗原配体(多肽和非多肽)，并了解对这些配体的免疫反应： 由非经典MHC分子提出的多肽和非肽配体：[HL A-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].”