Effects of polymorphism of HLA on binding of antigenic peptides to HLA and recognition of peptides complexed with HLA by T cell receptor

人类白细胞抗原-DR分子是一种高度多态的分子，可与抗原肽结合，将其呈递给CD4+的辅助T细胞。特定的DR等位基因被认为决定了自身免疫性疾病的易感性，因为与健康对照组相比，患者组中特定的DR等位基因的频率增加。因此，DRB1^&lt；**&gt；0405和DRB1^&lt；**&gt；0406仅与DRB1^&lt；**&gt；0405的四个氨基酸残基不同，分别控制类风湿性关节炎和胰岛素自身免疫综合征的易感性。我们研究了DRB1^&lt；**&gt；0405或DRB1^&lt；**&gt；0406分子结合的抗原肽的结构特征。为此，我们分离了与纯化的DRB1^&lt；**&gt；0405分子结合的自体多肽，并用反相高效液相色谱仪进行分离，测定了7个独立多肽的氨基酸序列，其中4个是已知蛋白质的片段。合成了这些自体多肽，并进行了放射性碘标记，研究了标记多肽与DR分子的结合情况。在7个自体多肽中，结合能力最强的是DRB1^&lt；**&gt；0405和DRB1^&lt；**&gt；0406。建立了结合抑制实验，通过测量多肽对标记的最强结合分子与DR分子结合的抑制作用来确定待测肽的结合活性。为了确定与DR结合有关的氨基酸残基，从原始多肽中合成了只有一个氨基酸取代的模拟多肽，并研究了它们与DRB1^&lt；**&gt；0405和DRB1^&lt；**&gt；0406的结合。DR结合肽由9~20个氨基酸组成，与DR分子的3个主要锚定氨基酸残基结合。这三个锚定残基被两个和一个插入氨基酸分开。第一

HLA-DR molecules are highly polymorphic molecules and bind antigenic peptides to present it to CD4 positive helper T cells. The particular DR alleles are thought to determine susceptibility to autoimmune diseases because the frequencies of particular DR allelse are increased in the patients group as compared with those in healthy controls. Thus DRB1 ^<**>0405 and DRB1 ^<**>0406 which differs in only four amino acid residues from DRB1 ^<**>0405 control susceptibility to rheumatoid arthritis and insulin autoimmune syndrome respectively. We have investigated the structural characteristics of antigenic peptides bound to either DRB1 ^<**>0405 or DRB1 ^<**>0406 molecules. For this aim, self peptides bound to purified DRB1 ^<**>0405 molecules were isolated and fractionated by a reversed phase HPLC.Amino acid sequences of seven independent peptides were determined and four of them were identified to be fragments of known proteins. These self peptides were synthesized and radio-iodinated, and binding of labeled peptides to DR molecules were investigated. The strongest binder among seven self peptides was identified and this peptide bound to both DRB1 ^<**>0405 and DRB1 ^<**>0406 equally. The binding inhibition assay, in which binding activity of a peptide to be tested was determined by measuring inhibitory effect of the excess amount of peptide on binding of the labeled strongest binder to DR molecule, was established. In order to determine amino acid residues important for DR binding in the strongest binder, analogue peptides having only one amino acid substitution from the original peptides were synthesized and their binding to both DRB1 ^<**>0405 and DRB1 ^<**>0406 was investigated. DR binding peptides consisted of nine to twenty amino acids were bound at three major anchorage amino acid residues of the peptide to DR molecule. These three anchorage residues were separated by two and one intervening amino acids. The first