Morgan C L, Ruprai A K, Solache A, Lowdell M, Price C P, Cohen S B, Parham P, Madrigal J A, Newman D J
Anthony Nolan Research Institute, The Royal Free Hospital, London NW3 2QG, UK.
Immunogenetics. 1998 Jul;48(2):98-107. doi: 10.1007/s002510050409.
We used an optical biosensor to determine the relative binding affinity of peptides to purified HLA class I molecules. In this assay we monitor beta2-microglobulin (beta2m) exchange within the HLA-A2 molecule, whereby native beta2m in the complex is replaced by beta2m immobilized at the surface of the biosensor. Quantitative kinetic measurements permit us to obtain association rate (kass), dissociation rate (kdiss) and affinity constants (KA) for the beta2m exchange reaction, alone, (control) and in the presence of exogenous peptide. We tested a panel of six peptides which had been designed and synthesized with an HLA-A2 binding motif, and had also been tested by the T2-cell binding assay, along with control peptides. The biosensor results demonstrate that exogenous peptide influences the dynamics of beta2m exchange in a sequence-specific manner. Five of six peptides increased the association rate, decreased the dissociation rate, and significantly increased the affinity (KA=1. 55-1.88x10(9) M-1) of HLA-A2 for immobilized beta2m compared with the control (KA =1.14+/-0.04x10(9)M-1), demonstrating stabilization of the complex. One peptide was unable to stabilize the complex, as also shown in the T2 binding assay. However, analysis of peptide sequences demonstrated that the HLA-A2 secondary motif as well as primary motif residues are required for HLA-A2 stabilization. Further experiments demonstrated that beta2m exchange alone cannot stabilize the HLA class I complex at the cell surface until a peptide of sufficient binding affinity is bound. Hence kinetics equal to or below the control values in our biosensor assay probably represent an unstable complex in vivo. Unlike other methods described for the analysis of peptide stabilization, this approach is significantly faster, provides full kinetic analysis, and is simpler, since it requires no labeling of peptides. Furthermore, this may have important implications in the assessment of peptide vaccines.
我们使用光学生物传感器来确定肽与纯化的HLA I类分子的相对结合亲和力。在该测定中，我们监测HLA - A2分子内的β2 - 微球蛋白（β2m）交换，即复合物中的天然β2m被固定在生物传感器表面的β2m所取代。定量动力学测量使我们能够获得β2m交换反应单独（对照）以及在外源肽存在下的缔合速率（kass）、解离速率（kdiss）和亲和常数（KA）。我们测试了一组六种肽，这些肽是按照HLA - A2结合基序设计和合成的，并且也通过T2细胞结合试验以及对照肽进行了测试。生物传感器结果表明，外源肽以序列特异性方式影响β2m交换的动力学。与对照（KA = 1.14±0.04×10⁹ M⁻¹）相比，六种肽中的五种增加了缔合速率，降低了解离速率，并显著增加了HLA - A2对固定化β2m的亲和力（KA = 1.55 - 1.88×10⁹ M⁻¹），表明复合物得到了稳定。一种肽无法稳定复合物，这在T2结合试验中也得到了证实。然而，肽序列分析表明，HLA - A2稳定需要HLA - A2二级基序以及一级基序残基。进一步的实验表明，在结合具有足够结合亲和力的肽之前，仅β2m交换不能在细胞表面稳定HLA I类复合物。因此，在我们的生物传感器测定中，动力学等于或低于对照值可能代表体内不稳定的复合物。与描述的用于分析肽稳定化的其他方法不同，这种方法明显更快，提供完整的动力学分析，并且更简单，因为它不需要对肽进行标记。此外，这可能对肽疫苗的评估具有重要意义。
