Molecular mechanism of the susceptibility difference between HLA-B*27:02/04/05 and HLA-B*27:06/09 to ankylosing spondylitis: substitution analysis, MD simulation, QSAR modelling, and in vitro assay

The human leukocyte antigen HLA-B27 is directly involved in the disease pathogenesis of ankylosing spondylitis (AS). HLA-B27 has a high degree of genetic polymorphism, with 105 currently known subtypes; the presence of aspartic acid at residue 116 (Asp116) has been found to play an essential role in AS susceptibility. Here, we systematically investigated the molecular mechanism of the susceptibility difference between the AS-associated subtypes HLA-B*27:02/04/05 and AS-unassociated subtypes HLA-B*27:06/09 to AS at sequence, structure, energetic and dynamic levels. In total seven variable residues were identified among the five studied HLA-B27 subtypes, in which Asp116 can be largely stabilized by a spatially vicinal, positively charged His114 through a salt bridge, while five other variable residues seem to have only a marginal effect on AS susceptibility. We also employed a quantitative structure–activity relationship approach to model the statistical correlation between peptide structure and affinity to HLA-B*27:05, a genetic ancestor of all other HLA-B27 subtypes and associated strongly with AS. The built regression predictor was verified rigorously through both internal cross-validation and external blind validation, and was then employed to identify potential HLA-B*27:05 binders from >20,000 cartilage-derived self-peptides. Subsequently, the binding potency of the top five antigenic peptides to HLA-B*27:05 was assayed in vitro using a FACS-based MHC stabilization experiment. Consequently, two (QRVGSDEFK and LRGAGTNEK) out of the five peptides were determined to have high affinity (BL50 = 5.5 and 15.8 nM, respectively) and, as expected, both of them possess positively charged Lys at the C-terminus.

人类白细胞抗原（human leukocyte antigen, HLA）-B27直接参与强直性脊柱炎（ankylosing spondylitis, AS）的致病进程。HLA-B27具有高度遗传多态性，目前已发现105种亚型；研究证实，116位残基处的天冬氨酸（aspartic acid, Asp116）在AS易感性中发挥核心作用。本研究从序列、结构、能量及动态多个层面，系统探究了与AS相关的HLA-B*27:02/04/05亚型与AS无关的HLA-B*27:06/09亚型之间的易感性差异分子机制。在所研究的5种HLA-B27亚型中，共鉴定出7个可变残基。其中，Asp116可通过与空间邻近的带正电的组氨酸（histidine, His）114形成盐桥而得到显著稳定；其余5个可变残基对AS易感性的影响则较为微弱。本研究还采用定量构效关系（quantitative structure–activity relationship）方法，对肽结构与HLA-B*27:05的亲和力之间的统计相关性进行建模。HLA-B*27:05是其余所有HLA-B27亚型的遗传祖先，且与AS呈强相关性。所构建的回归预测器经内部交叉验证与外部盲法验证得到了严格的性能验证，随后被用于从超过20000种软骨来源的自身肽中筛选潜在的HLA-B*27:05结合肽。随后，研究人员采用基于荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）的主要组织相容性复合体（major histocompatibility complex, MHC）稳定实验，对排名前五的抗原肽与HLA-B*27:05的结合效力开展了体外检测。结果显示，5种肽中的2种（QRVGSDEFK与LRGAGTNEK）具有高亲和力（半数有效浓度BL50分别为5.5 nM与15.8 nM）；正如预期，二者的C端均带有带正电的赖氨酸（lysine, Lys）。