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Analyses of responsible domain inducing functional difference of polymorphic murine osteopontin protein

多态性鼠骨桥蛋白功能差异的责任域分析

基本信息

批准号：
15590346
负责人：
MIYAZAKI Tatsuhiko
金额：
$ 2.3万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

We previously identified Opn as a candidate gene susceptible to glomerulonephritis in MRL/lpr mice by genome wide screening using (MRL/lpr x C3H/lpr)F_2 mice, followed by a functional assay of synthetic polymorphic OPN peptides of MRL and C3H type. To identify the responsible domain to the functional difference between these alleles, we generated mutant Opn peptides which were modified in substituted amino acid between two alleles followed by structural and functional analyses. Also, to determine the role of the allelic difference in vivo, we made selective congenic mice of Opn gene locus which have C3H allele Opn in MRL background, and then, carried out in vivo and histopathological analyses on the congenic mice.1)We synthesized MRL/Mp type (allele a) and C3H/HeJ type (allele b) OPN peptide using Cell Free protein synthesis system, then analyzed these peptides by SDS-PAGE and Western blotting.2)Also, using PCR based mutagenesis technique, we generated mutated Opn peptides which were m … More odified a substitution site between allele a and allele b, then analyzed as same as above.3)To identify the responsible polymorphic domain, we carried out bioassays using modified polymorphic Opn peptides on splenic cells and macrophages derived from MRL-Fas^<+/+> mice. As a result of these analyses, we manifested a certain substitution site dramatically modified the functional difference in cytokine inducing in this system. That polymorphic site might seem to be involved in the functional difference of these alleles.4)We established Opn locus specific congenic inbred mice strain MRL/lpr-Opn^<C3H/C3H> in MRL/lpr x (MRL/lpr x C3H/lpr)N_<13>F_2 mice. By using MRL/lpr-Opn^<C3H/C3H>, MRL/lpr-Opn^<MRL/C3H>, and MRL/lpr- Opn^<MRL/MRL> mice in N_<13>F_2 mice, we confirmed that MRL/lpr-Opn^<C3H/C3H> mice had marked reduction in incidence of glomerulonephritis.These results suggest that an amino acid substitution in allelic polymorphism of murine Opn may play a critical role for the development of glomerulonephritis in MRL/lpr mice. Less

我们之前通过（MRL/lpr x C3H/lpr）F_2小鼠基因组筛选，确定了Opn是MRL/lpr小鼠肾小球肾炎的候选基因，随后进行了MRL和C3H型合成多态Opn肽的功能测定。为了确定这些等位基因之间功能差异的责任域，我们在两个等位基因之间的取代氨基酸上进行了修饰，然后进行了结构和功能分析。此外，为了确定等位基因差异在体内的作用，我们在MRL背景下，选择具有C3H等位基因Opn的Opn基因位点的基因小鼠，然后对基因小鼠进行体内和组织病理学分析。1)利用Cell Free蛋白合成系统合成MRL/Mp型（等位基因a）和C3H/HeJ型（等位基因b） OPN肽，并采用SDS-PAGE和Western blotting对其进行分析。2)同样，利用PCR诱变技术，我们生成了突变的Opn多肽，这些突变多肽在等位基因a和等位基因b之间修饰了一个替代位点，然后进行同上分析。3)为了确定相关的多态结构域，我们对MRL-Fas^<+/+>小鼠的脾细胞和巨噬细胞进行了修饰的多态Opn肽生物测定。通过这些分析，我们发现了一个特定的替代位点显著地改变了该系统中细胞因子诱导的功能差异。这个多态位点似乎与这些等位基因的功能差异有关。4)在MRL/lpr x (MRL/lpr x C3H/lpr)N_<13>F_2小鼠中建立了Opn位点特异性同源自交系小鼠MRL/lpr-Opn^<C3H/C3H>。采用MRL/lpr-Opn^<C3H/C3H>、MRL/lpr-Opn^<MRL/C3H>、MRL/lpr-Opn^<MRL/ MRL>对N_<13>F_2小鼠进行研究，证实MRL/lpr-Opn^<C3H/C3H>小鼠肾小球肾炎发生率明显降低。这些结果表明，小鼠Opn等位基因多态性的氨基酸替换可能在MRL/lpr小鼠肾小球肾炎的发展中起关键作用。少

项目成果

期刊论文数量（48）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Pathological diagnostic manual of collagen diseases

胶原病病理诊断手册

DOI：
发表时间：
2005
期刊：
Pathology and Clinical Medicine (Ozaki S., Nose M Ed.) (Bunkodo, Tokyo) (in press)
影响因子：
0
作者：
Miyyazaki T;Ono M;Endo Y;Nose M. et al.;Miyazaki T
通讯作者：
Miyazaki T

Bone marrow transfer of autoimmune diseases in an MRL strain of mice with a deficit in functional Fas ligand : Dissociation of arteritis from glomerulonephritis

功能性 Fas 配体缺陷的 MRL 品系小鼠中自身免疫性疾病的骨髓移植：动脉炎与肾小球肾炎的分离