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

本研究利用（MRL/lpr × C3 H/lpr）F_2小鼠进行了全基因组筛选，并对MRL和C3 H型OPN多肽进行了功能分析，确定了MRL/lpr小鼠中OPN为肾小球肾炎易感基因。为了鉴定这些等位基因之间的功能差异的负责结构域，我们产生了突变体Opn肽，其在两个等位基因之间的取代氨基酸中被修饰，然后进行结构和功能分析。同时，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Opn基因位点具有C3 H等位基因的同类小鼠，然后对同类小鼠进行体内和组织病理学分析（等位基因a）和C3 H/HeJ型（等位基因B）OPN肽，然后通过SDS-PAGE和Western印迹分析这些肽。2）同样，使用基于PCR的诱变技术，我们产生了突变的Opn肽， ...更多信息 在等位基因a和等位基因B之间修饰一个取代位点，然后与上述相同地进行分析。3）为了鉴定负责的多态性结构域，我们使用修饰的多态性Opn肽对来源于MRL-Fas^+/+>小鼠的脾细胞和巨噬细胞进行生物测定。这些分析的结果表明，在该系统中，某个取代位点显著地改变了细胞因子诱导的功能差异。4）在MRL/lpr ×（MRL/lpr × C3 H/lpr）N_ F_2小鼠中建立了Opn位点特异的同系近交系MRL/lpr-Opn ^&lt;C3 H/C3 H&gt;<13>。用MRL/lpr-Opn^&lt;C3 H/C3 H&gt;、MRL/lpr-Opn^&lt;MRL/C3 H&gt;和MRL/lpr-Opn ^&lt;MRL/MRL&gt;小鼠在N_F_2<13>小鼠中进行实验，证实MRL/lpr-Opn^&lt;C3 H/C3 H&gt;小鼠肾小球肾炎的发病率明显降低，提示小鼠Opn等位基因多态性中的氨基酸替换可能在MRL/lpr小鼠肾小球肾炎的发生中起重要作用。少

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