Cloning of total cDNA for desmoplakin and its molecular analysis and chromosomal mapping

我们已经分离出一种单氯抗体，它最初被认为与桥粒蛋白I和II发生反应，桥粒蛋白I和II是一种主要的桥粒蛋白。然而，在对该单抗进行进一步详细鉴定后，发现该单抗与桥粒的一种新发现的680kD斑块蛋白Desmoyokin发生反应。用该单抗免疫筛选小鼠角质形成细胞文库，获得了几个阳性克隆，其中一个进行了进一步鉴定。该插入片段(DY6,3693bp)与来自人或小鼠角质形成细胞的超大mRNA杂交。DY6表达的重组蛋白不仅能与用于免疫筛选的单抗反应，还能与抗桥粒连接蛋白单抗反应。DY6的核苷酸和推导的氨基酸序列与以前报道的包括其他桥粒成分的序列没有明显的同源性。对氨基酸序列的分析表明，DY6由大约10个高度同源的重复序列组成，每个重复序列由四个不同的片段组成，长约128个残基。此外，128个残基重复序列显示出一个准7个残基的亚结构，然而，这与在α纤维蛋白的杆状结构域中看到的七肽重复序列有根本的不同。二级结构预测表明，所采用的构象不是α螺旋的，而很可能是一种反平行的β-折叠结构，其中一面无极，另一面由酸性和碱性残基组成的周期性分布。这些结果和其他结果表明，桥粒连接蛋白在桥粒成分中具有独特的结构和功能，DY6可能编码桥粒连接蛋白C末端球状结构域的主要部分。

We have isolated a monocloanl antibody that was first thought to react with desmoplakins I and II, a major desmosomal plaque protein. However, after the monoclonal antibody was characterized further in detail, the monoclonal antibody was found to react with desmoyokin, a newly identified 680 kD plaque protein of the desmosomes. By immunoscreening our mouse keratinocyte library with the monoclonal antibody, we have isolated several positive cDNA clones, one of which was further characterized. The cDNA insert(DY6, 3693 bp)hybridized with an extremely large mRNA from either human or mouse keratinocytes. The recombinant protein produced by DY6 was reacted not only with the monoclonal antibody used for immunoscreening but also with anti-desmoyokin monoclonal antibody. Nucleotide and deduced amino acid sequences of DY6 showed no significant homology with previously reported sequences including other desmosomal components. Analysis of the amino acid sequence revealed that DY6 consists of about ten highly homologous repeats about 128 residues long, each comprising four distinct segments. Furthermore, the 128 residue repeats exhibit a quasi seven-residue substructure that is, however, fundamentally different from the heptad repeat seen in the rod domains of alpha-fibrous proteins. Secondary structure prediction shows that the conformation adopted is not alpha-helical, but most probably an antiparallel beta-sheet structure with one face apolar and the other comprising a periodic distribution of acidic and basic residues. These and other results suggest that desmoyokin has a unique structure and function among desmosomal components, and that DY6 may encode a major part of the C-terminal globular domain of desmoyokin.