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

桥粒斑蛋白总cDNA的克隆及其分子分析和染色体定位

• 批准号: 02454280
• 负责人: HASHIMOTO Takashi
• 金额: $ 4.22万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454280/
• 关键词: Epidermis; Cell adhesion; Desmosome; Desmoyokin; Molecular biology; Biochemistry

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.

我们已经分离出一种单克隆抗体，它最初被认为与桥粒斑蛋白I和II（一种主要的桥粒斑蛋白）反应。然而，在进一步详细表征该单克隆抗体后，发现该单克隆抗体与桥粒的一种新鉴定的680 kD噬斑蛋白--桥粒yokin反应。通过免疫筛选我们的小鼠角质形成细胞库的单克隆抗体，我们已经分离出几个阳性的cDNA克隆，其中之一是进一步的特点。cDNA插入片段（DY 6，3693 bp）与来自人或小鼠角质形成细胞的极大mRNA杂交。DY 6产生的重组蛋白不仅与用于免疫筛选的单克隆抗体反应，而且还与抗桥粒连接蛋白单克隆抗体反应。DY 6的核苷酸序列和推导的氨基酸序列与已报道的桥粒其他组分的序列没有明显的同源性。氨基酸序列分析表明，DY 6由大约10个高度同源的重复序列组成，每个重复序列长约128个残基，每个重复序列包含4个不同的片段。此外，128个残基的重复序列表现出准7个残基的子结构，然而，从根本上不同于在α-纤维蛋白质的杆域中看到的七肽重复序列。二级结构预测表明，所采用的构象不是α-螺旋，但最有可能是一个反平行的β-折叠结构，一面非极性，另一面包含酸性和碱性残基的周期性分布。这些和其他结果表明，桥粒细胞因子具有独特的结构和功能的桥粒组分，DY 6可能编码的C-末端球状结构域的桥粒细胞因子的主要部分。

项目成果

橋本 隆: "皮膚科における遺伝子工学の応用:デスモソ-ム蛋白の分子生物学" 日本皮膚科学会雑誌. 101. 1681-1686 (1991)

Takashi Hashimoto：“基因工程在皮肤病学中的应用：桥粒蛋白的分子生物学”日本皮肤病学会杂志 101。1681-1686（1991）。

Hashimoto T et al: "Cloning and characterization of a mouse cDNA for clesmoyokin,a 680 KD desmosomal plaque pratein" J.Cell Biol.

Hashimoto T 等人：“clesmoyokin（一种 680 KD 桥粒斑块蛋白）的小鼠 cDNA 的克隆和表征”J.Cell Biol。

Hashimoto T: "Applications of gene technology in Dermatology. Molecular biology in desmosomal proteins" Jap J Dermatol. 101. 1681-1686 (1991)

Hashimoto T：“基因技术在皮肤病学中的应用。桥粒蛋白的分子生物学”Jap J Dermatol。

Hashimoto T.et al: "Cloning and characterization of a mouse cDNA for desmoyokin,a 680 kD desmosomal plaque protein" J.Cell Biol.

Hashimoto T. 等人：“桥粒蛋白（一种 680 kD 桥粒斑蛋白）的小鼠 cDNA 的克隆和表征”J.Cell Biol。