INTERACTIONS OF PEPTIDES W/ CLASS II MAJOR HISTOCOMPATIBILITY COMPLEX MOLECULES

该研究项目未来五年的目标是 II类MHC结构特征的持续分析 控制与肽相互作用的分子及其作用 确定对特定肽表位的免疫反应 外来分子和自身分子。 具体目标是： 1）完成 I-Ab 链中的位点特异性突变引入了第一个 和/或从I-Abu到I-Ab的第三高变区序列。 这些突变构建体将被引入到两种细胞系中并 进入小鼠体内，以及这些突变对抗原的功能影响 表现和免疫反应性将在体外确定 和体内研究。 该项目的一个附属部分将导致 I-Au转基因小鼠的生产。 这些老鼠将被杂交 与 NZB 小鼠一起测试 MHC 与遗传相关的假设 NZW 菌株导致 NZB x NZW 狼疮性肾炎的因素 模型是 I-Au 分子。 2）磷酸肌醇（P.I.）的表达 CHO 细胞中连接的 I-Aa u /I-Abu 异二聚体。 这将允许 通过磷脂酶的作用分离可溶性 I-Au 分子 C. 这些看起来大部分是空的分子将被使用 研究 MBP Ac1-11 及其类似物与 I-Au 的相互作用 确定分离的 I-Aau 和 I-Ab u 肽结合的能力 MBP Ac1-11，并尝试在中生产大量 I-Au 大多数或所有分子都结合相同的肽， 即MBP Ac1-11。 随后将尝试与 斯坦福大学结构生物学系确定是否 这些分子能够形成晶体。 3) 生产 基于 MBP Ac1-11 序列的一系列肽，其中所有 除位置 5、6 和 10 或 2、5、6 和 10 之外的残基被替换 由L-丙氨酸。 该原型肽与 I-Au 结合的能力 然后将被测试。 如果正如预测的那样，该肽结合，一系列 将合成相关肽，其中 L-丙氨酸是 在从 N 末端开始的序列中被 D-丙氨酸取代，a C 末端之前的第二个序列和中的第三个序列 每三个丙氨酸中 D-丙氨酸将取代 L-丙氨酸 残留物。 这些肽应该能够确定是否 MBP 1-11 肽作为 alpaheliy 或作为延伸肽结合，并且 还应该允许确定 I-Au 的程度 结合位点在结合之前区分肽的手性。 4) 将产生表达构建体的转基因小鼠 由金属硫蛋白启动子和不变链组成 基因组编码序列。 该结构应允许高水平 不变链的表达。 这些小鼠将与 I-Abk 和 I-Aa k /I-Ab k 转基因小鼠以挽救其缺陷 B细胞发育。 如果这个实验成功的话 表明不变链的主要功能之一是 促进 I-A 分子的组装和运输到表面。 5）进一步测试不变链在汇编中的功能， I-A 和 I-E 分子的运输和功能，我们将生产 恒定链失活的纯合转基因小鼠 通过胚胎干细胞系中的同源重组基因， 然后将这些细胞引入胚泡中。 这些老鼠 应该无法表达不变链并且预计有 II 类组装、运输和细胞表面表达缺陷 MHC 分子。 6) 促进HLA-DQ和DR的功能测试 在转基因小鼠中表达的分子，转基因小鼠将 所产生的将使用人类CD4基因产生。 这些转基因 然后将小鼠与表达 HLA-DR4 或 HLA DQw2、6、7 或 8。在人类 CD4 存在的情况下，这些 HLA DR 和 DQ 分子应在抗原呈递中发挥积极和 胸腺中的负选择，从而允许仔细分析 这些分子将肽表位呈递给 T 细胞。

The goals of this research project for the next five years are a continuing analysis of the structural characteristics of class II MHC molecules which govern their interactions with peptides and their role in determining immune responsiveness to specific peptide epitopes from foreign and self molecules. The specific aims are: 1) Completion of site specific mutations in the I-Ab s chain to introduce the first and/or third hypervariable region sequences from I-Abu into I-Ab s. These mutant constructs will be introduced into both cell lines and into mice, and the functional effects of these mutations on antigen presentation and immune responsiveness will be determined in in vitro and in vivo studies. A subsidiary part of this project will lead to the production of I-Au transgenic mice. These mice will be crossed with NZB mice to test the hypothesis that the MHC linked genetic factor contributed by the NZW strain to the NZB x NZW lupus nephritis model is the I-Au molecule. 2) Expression of phosphoinositol (P.I.) linked I-Aa u /I-Abu heterodimers in CHO cells. This willpermit isolation of soluble I-Au molecules through the action of phosolipae C. These molecules which appear to be in large part empty will be used to study the interaction of MBP Ac1-11 and its analogs with I-Au to determine the ability of isolated I-Aau and I-Ab u peptides to bind MBP Ac1-11, and to attempt to produce large amounts of the I-Au in which most or all of the molecules are binding the same peptide, namely MBP Ac1-11. Attempts will then be made in collaboration with the department of structural biology at Stanford to determine whether these molecules are capable of forming crystals. 3) Production of a series of peptides based on the MBP Ac1-11 sequence in which all the residues except positions 5, 6 and 10 or 2, 5, 6 and 10 are replaced by L-alanine. The ability of thisprototype peptide to bind to I-Au will then be tested. If, as predicted, this peptide binds, a series of related peptides will be synthesized in which the L-alanines are replaced by D-alanine in a sequence proceeding from the N terminus, a second sequence preceding from the C terminus and a third sequence in which the D-alanine will replace L-alanine at every third alanine residue. These peptides should permit determination of whether the MBP 1-11 peptide binds as a alpaheliy or as an extended peptide, and should also permit a determination of the extent to which the I-Au binding site distinguishes chirality of a peptide prior to binding. 4) Transgenic mice will be produced which express a construct consisting of the metallothionein promoter with the invariant chain genomic coding sequences. This construct should permit high level expression of the invariant chain. These mice will be crossed with the I-Abk and I-Aa k /I-Ab k transgenic mice to rescue their defect in B cell development. If this experiment is successful, it will indicate that one of the main functions of the invariant chain is to facilitate assembly and transport of the I-A molecules to the surface. 5) To further test the function of the invariant chain in assembly, transport and function of the I-A and I-E molecules, we will produce transgenic mice homozygous for inactivation of the invariant chain gene by homologous recombination in an embryonal stem cell line, followed by introduction of these cells into blastoysts. These mice should fail to express the invariant chain and are expected to have defects in assembly, transport and cell surface expression of class II MHC molecules. 6) To facilitate functional testing of HLA-DQ and DR molecules expressed in transgenic mice, transgenic mice will be produced will be produced using the human CD4 gene. These transgenic mice will then be crossed with transgenic lines expressing HLA-DR4 or HLA DQw2, 6, 7or 8. In the presence of human CD4, these HLA DR and DQ molecules should function in antigen presentation in positive and negative selection in the thymus, thus permitting careful analysis of peptide epitopes presented to T cells by these molecules.