Pruijn G J, Schoute F, Thijssen J P, Smeenk R J, van Venrooij W J
Department of Biochemistry, University of Nijmegen, The Netherlands.
J Autoimmun. 1997 Apr;10(2):127-36. doi: 10.1006/jaut.1996.0123.
In this study we have used a number of monoclonal antibodies with various anti-Sm specificities originating from MRL/lpr mice to map B cell epitopes of the Sm-B/B' and Sm-D1 proteins. Selection of Sm-B subfragments reactive with the Sm-B/B'-specific monoclonal antibody ANA125 from a DNaseI fragment expression library revealed that the epitope recognized by this monoclonal antibody is located between amino acids 146 and 158: GRGTVAAAAAAAT. The epitopes recognized by two distinct Sm-D1-specific monoclonal antibodies, 7.13 and ANA127, appeared to be located in the carboxy-terminal region of the protein as revealed by immunoprecipitation of in vitro translated deletion mutants of Sm-D1. These epitopes are probably identical and not simply composed of a GR repeat, which is a characteristic feature of this part of the protein. Immunoprecipitation of in vitro translated deletion mutants of both Sm-B and Sm-D1 was also employed to determine the sequence requirements for recognition by two monoclonal antibodies that are cross-reactive with several Sm proteins, Y12 and ANA128. The epitope recognized by these two monoclonal antibodies is probably also identical and composed by the juxtaposition of several regions in the folded protein. The low, but significant, level of immunoprecipitation of truncated versions of both Sm-B and Sm-D1, suggests that the Sm domain, which is shared by all Sm proteins, in particular the amino-terminal part of the Sm1 motif of Sm-B and Sm-D1, plays an important role in formation of the cross-reactive epitope and might contribute to cross-reactivity with other Sm proteins. The results of immunoprecipitation experiments with cellular extracts show that the epitopes recognized by all anti-Sm monoclonal antibodies used in this study are accessible in the assembled snRNPs.
在本研究中，我们使用了多种源自MRL/lpr小鼠、具有不同抗Sm特异性的单克隆抗体，来绘制Sm-B/B'和Sm-D1蛋白的B细胞表位图谱。从DNA酶I片段表达文库中筛选出与Sm-B/B'特异性单克隆抗体ANA125反应的Sm-B亚片段，结果显示该单克隆抗体识别的表位位于氨基酸146至158之间：GRGTVAAAAAAAT。对Sm-D1体外翻译缺失突变体进行免疫沉淀分析表明，两种不同的Sm-D1特异性单克隆抗体7.13和ANA127识别的表位似乎位于该蛋白的羧基末端区域。这些表位可能相同，并非简单地由GR重复序列组成，而GR重复序列是该蛋白这一部分的特征。我们还利用Sm-B和Sm-D1体外翻译缺失突变体的免疫沉淀实验，来确定两种与多种Sm蛋白交叉反应的单克隆抗体Y12和ANA128识别表位的序列要求。这两种单克隆抗体识别的表位可能也相同，由折叠蛋白中几个区域并列组成。Sm-B和Sm-D1截短版本的免疫沉淀水平较低但具有统计学意义，这表明所有Sm蛋白共有的Sm结构域，特别是Sm-B和Sm-D1的Sm1基序的氨基末端部分，在交叉反应表位的形成中起重要作用，可能有助于与其他Sm蛋白发生交叉反应。对细胞提取物进行免疫沉淀实验的结果表明，本研究中使用的所有抗Sm单克隆抗体识别的表位在组装好的核小核糖核蛋白中均可接近。
