PROPERTIES OF DOMINANT ANTIGENIC EPITOPES INFLUENCING MHC BINDING

MHC呈现抗原的多肽片段 T细胞的等位基因特异性方式。从一个巨大的水池中 在处理过程中生成的潜在多肽，仅选择一个子集 在细胞表面以表位的形式呈现，因此具有 有可能引发T细胞反应。所选的主要多肽 来自HEL的I-AK由氨基酸HEL 48-62组成。之前的数据已经 推测HEL48-62表位的主锚点为D52。 这一点得到了实验的支持，在这些实验中， 残基极大地降低了多肽与 我-AK。已经进行了一系列初步实验，以 确定A52 HEL的主要表位，并表明 48-62A52肽检测不到。相反，44胜61负的A52已经被 经二维鉴定为A52HEL的优势I-AK表位 高效液相色谱和质谱学分析。初始I-AK竞争性结合 实验表明，44-61A52的优势表位来源于 A52HEL对I-AK的相对亲和力高于突变体48-62 A52肽。44-61A52肽的相对亲和力相等 与野生型占优势的HEL 48-62多肽的同源性。在这 我们正在努力开发各种方法来降低 二维液相色谱分离抗原肽混合物的一维性 一种是离子交换层析，另一种是毛细管反相高效液相色谱法。

The MHC presents peptide fragments of antigens in an allele-specific manner to T cells. Out of an enormous pool of potential peptides generated during processing, only a select subset are presented as epitopes on the cell surface, and therefore have the potential to elicit T-cell responses. The dominant peptide selected from HEL by I-Ak comprises amino acids HEL 48-62. Previous data have suggested that the primary anchor of the HEL48-62 epitope is D52. This is supported by experiments in which alanine substitution of this residue drastically decreased the binding affinity of the peptide for I-Ak. A number of preliminary experiments have been performed to identify the major epitopes of A52 HEL and have indicated that the 48-62 A52 peptide is undetectable. Instead, 44-61 A52 has been identified as the predominant I-Ak epitope derived from A52 HEL by 2-D HPLC and mass spectrometry. Initial I-Ak competitive binding experiments show that the dominant 44-61 A52 epitope d eriv ed from A52HEL has a greater relative affinity for I-Ak than the mutant 48-62 A52 peptide. The relative affinity of the 44-61 A52 peptide is equal to that of the dominant wildtype processed HEL 48-62 peptide. In this project we are trying to develop methods to reduce the complexity of the antigenic peptide mixtures by using 2-D HPLC, the first dimension being ion-exchange chromatography and the second, capillary RP-HPLC.