INTERACTIONS OF PEPTIDES W/ CLASS II MAJOR HISTOCOMPATIBILITY COMPLEX MOLECULES

这项研究项目未来五年的目标是 继续分析第二类MHC的结构特征 控制它们与多肽相互作用的分子及其作用 在确定对特定多肽表位的免疫应答中 外来分子和自身分子。具体目标是：1)完成 I-Ab S链上的位点特异性突变首次引入 和/或从I-ABU到I-AbS的第三高变区序列。 这些突变结构将被引入细胞系和 以及这些突变对抗原的功能影响 呈现和免疫反应性将在体外确定 以及活体研究。该项目的一个附属部分将导致 I-Au转基因小鼠的制备。这些老鼠将会被杂交 用NZB小鼠来验证MHC与基因 NZW株在NZB×NZW狼疮性肾炎发病中的作用 模型是I-Au分子。2)磷脂酰肌醇(P.I.) CHO细胞中连接的I-AAu/I-Au异源二聚体。这将是允许的 磷脂膜作用分离可溶性I-Au分子 C.这些看起来大部分是空的分子将被使用 MBP AC1-11及其类似物与I-Au相互作用的研究 分离的I-AAU和I-Abu多肽结合能力的测定 MBP AC1-11，并试图在 其中大多数或所有分子都与相同的多肽结合， 即MBP AC1-11。然后将与以下合作进行尝试 斯坦福大学结构生物学系确定 这些分子能够形成晶体。3)制作 基于MBP AC1-11序列的一系列肽，其中所有的 取代第5、6和10或2、5、6和10位以外的残基 作者：L-丙氨酸。这一原型多肽与I-Au结合的能力 然后将接受测试。如果如预测的那样，这种多肽结合了一系列 将合成相关的多肽，其中L-丙氨酸是 在从N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第三个序列 其中D-丙氨酸将取代L-丙氨酸每3个丙氨酸 残留物。这些多肽应该可以确定是否有 MBP 1-11肽以Alpaheliy或延伸肽的形式结合，以及 还应允许确定I-AU在多大程度上 结合部位在结合前区分多肽的手性。 4)将产生表达构建物的转基因小鼠。 由具有不变链的金属硫蛋白启动子组成 基因组编码序列。此构造应允许高级别 不变链的表达式。这些老鼠将与 I-Abk和I-AA k/I-Abk转基因小鼠修复缺陷的实验研究 B细胞发育。如果这个实验成功了，它将 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)为了进一步测试装配中不变链的功能， I-A和I-E分子的运输和功能，我们将产生 转基因小鼠不变链失活的纯合 基因在胚胎干细胞系中的同源重组， 然后将这些细胞引入囊胚。这些老鼠 应无法表示不变链，并应具有 第II类分子在组装、运输和细胞表面表达方面的缺陷 MHC分子。6)促进人类白细胞抗原-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 将使用人类的CD4基因来产生。这些转基因生物 然后将小鼠与表达人类白细胞抗原-DR4或 HLADQw2、6、7或8。在人类CD4存在的情况下，这些HLADR和DQ 分子应该在阳性和阳性的抗原提呈中起作用 胸腺中的负选择，从而允许仔细分析 由这些分子呈递给T细胞的多肽表位。

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.