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 控制它们与肽相互作用的分子及其作用 在确定对来自人的特异性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链中的位点特异性突变，以引入第一个 和/或第三高变区序列从I-Ab转化为I-Ab。 将这些突变构建体引入两种细胞系中， 以及这些突变对抗原的功能影响。 将在体外确定呈递和免疫应答 和体内研究。 该项目的一个附属部分将导致 I-Au转基因小鼠的产生。 这些老鼠将被杂交 用NZB小鼠来检验MHC连锁遗传的假设， NZW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.） 连接的I-Aa u /I-Abu异二聚体。 这将允许 通过磷酸盐的作用分离可溶性I-Au分子 C.这些看起来大部分是空的分子将被用来 研究MBP Ac 1 -11及其类似物与I-Au的相互作用， 测定分离的I-Aau和I-Ab u肽结合 MBPAc 1 -11，并试图生产大量的I-Au， 其中大部分或全部分子结合相同的肽， 即MBP Ac 1 -11。 然后将与以下方面合作进行尝试： 斯坦福大学的结构生物学系来确定 这些分子能够形成晶体。 3)生产 一系列基于MBP Ac 1 -11序列的肽，其中所有的 除了位置5、6和10或2、5、6和10之外的残基被替换 L-丙氨酸。 这种原型肽与I-Au结合的能力 将受到考验。 如果如预测的那样，这种肽结合， 将合成相关肽，其中L-丙氨酸 在从N末端开始的序列中被D-丙氨酸取代， C末端前面的第二序列和C末端前面的第三序列， 其中D-丙氨酸将在每三个丙氨酸处取代L-丙氨酸， 残余物 这些肽应允许确定是否存在 MBP 1-11肽作为α-螺旋肽或作为延伸肽结合，和 还应允许确定I-Au 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， 由具有不变链的金属硫蛋白启动子组成 基因组编码序列。 该结构应允许高水平 不变链的表达式。 这些老鼠将与 I-Abk和I-Aak/I-Abk转基因小鼠，以挽救它们在 B细胞发育。 如果实验成功， 表示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)为了进一步测试汇编中不变链的功能， 运输和功能的I-A和I-E分子，我们将产生 恒定链失活纯合子转基因小鼠 基因在胚胎干细胞系中同源重组， 然后将这些细胞导入囊胚。 这些小鼠 应该无法表达不变链，并且应该具有 II类的组装、转运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， 将使用人CD 4基因产生。 这些转基因 然后将小鼠与表达HLA-DR 4或 HLA DQw 2、6、7或8。 在存在人CD 4的情况下，这些HLA DR和DQ 分子应该在抗原呈递中起作用， 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.