Mukku P V, Passmore D, Phan D, Nag B
Anergen, Inc., Redwood City, CA 94063, USA.
Mol Immunol. 1995 Jun;32(8):555-64. doi: 10.1016/0161-5890(95)00030-i.
Major histocompatibility complex (MHC) class II molecules are cell surface glycoproteins and are known to display processed antigens on the surface of antigen presenting cells (APC). Within the APC, the loading of processed antigenic peptides to MHC class II molecules is known to take place in the endosomal compartment at acidic pH environment. The present study describes the in vitro effect of pH on binding of four biotinylated myelin basic protein (MBP) peptides to affinity purified HLA-DR2 containing a mixture of DRB11501 and DRB50101 beta chain. The binding affinity of the selected peptides are in the order of MBP(83-102)Y83 > MBP(124-143) > MBP(143-168) > MBP(1-14). Most of these peptides in association with HLA-DR2 are considered as immunodominant epitopes for human multiple sclerosis autoimmune disorder. One epitope, MBP(1-14), had almost no affinity to purified HLA-DR2 and was used as a control peptide in all binding assays. The quantitation of the bound peptide at various pH was carried out by antibody capture of complexes followed by avidin-alkaline phosphatase detection system. Among four peptides tested, only the highest affinity MBP(83-102)Y83 peptide showed maximum binding to purified HLA-DR2 at acidic pH. Two other epitopes, MBP(124-143) and MBP(143-168), showed maximum binding at basic and neutral pH values, respectively. The binding of only high affinity peptides, MBP(83-102)Y83 and MBP(124-143), was significantly affected by changing the pH of the binding buffer. Such alteration in pH of the binding buffer resulted in 100% occupancy of DR2 with both high affinity MBP peptides. In contrast, no significant increase in binding of the low affinity MBP(143-168) peptide was observed at altered pH values. The specificity of the increased binding of high affinity peptides to HLA-DR2 at optimum pH was demonstrated by competitive binding assays using non-biotinylated peptides. Finally, the stability of various MBP peptide bound complexes was tested at 4 degrees, 25 degrees and 37 degrees C which correlates well with their affinity to HLA-DR2. These results suggest that pH plays an important role in in vitro binding of antigenic peptides and such manipulation of binding conditions can be utilized in generating 100% loaded MHC class II with high affinity antigenic peptides. Since high affinity peptides are generally considered as major immunodominant epitopes, the in vitro pH dependent binding can be utilized in screening immunodominant epitopes of various autoantigens and generating complexes of defined composition.
主要组织相容性复合体（MHC）II类分子是细胞表面糖蛋白，已知其在抗原呈递细胞（APC）表面展示加工后的抗原。在APC内，加工后的抗原肽加载到MHC II类分子上的过程已知是在酸性pH环境的内体区室中进行的。本研究描述了pH对四种生物素化髓鞘碱性蛋白（MBP）肽与亲和纯化的含有DRB11501和DRB50101β链混合物的HLA - DR2结合的体外影响。所选肽的结合亲和力顺序为MBP(83 - 102)Y83 > MBP(124 - 143) > MBP(143 - 168) > MBP(1 - 14)。这些肽中的大多数与HLA - DR2结合被认为是人类多发性硬化自身免疫性疾病的免疫显性表位。一个表位MBP(1 - 14)对纯化的HLA - DR2几乎没有亲和力，在所有结合试验中用作对照肽。通过抗体捕获复合物，然后用抗生物素蛋白 - 碱性磷酸酶检测系统对不同pH下结合的肽进行定量。在测试的四种肽中，只有亲和力最高的MBP(83 - 102)Y83肽在酸性pH下与纯化的HLA - DR2表现出最大结合。另外两个表位MBP(124 - 143)和MBP(143 - 168)分别在碱性和中性pH值下表现出最大结合。仅高亲和力肽MBP(83 - 102)Y83和MBP(124 - 143)的结合受到结合缓冲液pH变化的显著影响。结合缓冲液pH的这种改变导致两种高亲和力MBP肽使DR2的占有率达到100%。相比之下，在改变的pH值下未观察到低亲和力MBP(143 - 168)肽的结合有显著增加。使用非生物素化肽的竞争性结合试验证明了高亲和力肽在最佳pH下与HLA - DR2结合增加的特异性。最后，在4℃、25℃和37℃测试了各种MBP肽结合复合物的稳定性，其与它们对HLA - DR2的亲和力密切相关。这些结果表明，pH在抗原肽的体外结合中起重要作用，这种结合条件的操纵可用于产生100%负载高亲和力抗原肽的MHC II类分子。由于高亲和力肽通常被认为是主要的免疫显性表位，体外pH依赖性结合可用于筛选各种自身抗原的免疫显性表位并生成特定组成的复合物。
