Identification of a 450-kDa human epidermal autoantigen as a new member of the plectin family.
Identification of a 450-kDa human epidermal autoantigen as a new member of the plectin family.
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DOI：
10.1111/1523-1747.ep12340171
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发表时间：
1996-05
期刊：
影响因子：
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通讯作者：
S. Fujiwara;K. Kohno;A. Iwamatsu;I. Naito;H. Shinkai
中科院分区：
文献类型：
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作者：
S. Fujiwara;K. Kohno;A. Iwamatsu;I. Naito;H. Shinkai
来自患有表皮下水疱病的个体的血清先前被证明识别450 kDa的表皮自身抗原。通过用患者血清筛选人角质形成细胞cDNA文库来研究该抗原的分子结构。一个克隆，与276 bp的cDNA插入，编码的抗原决定簇识别的血清被分离出来。针对相应的融合蛋白制备的兔多克隆抗体识别450-kDa表皮抗原并染色人表皮中的基底角质形成细胞。该克隆用于进一步筛选原始角质形成细胞和HeLa细胞cDNA文库。分离出两种不同但密切相关的0.8-和2.0-kb cDNA，其推导的氨基酸序列表明编码的蛋白质属于plectin家族。用这些cDNA插入片段作为探针对来自人角质形成细胞的总RNA进行北方印迹分析，检测到约12-13 kb的RNA。0.8 kb的cDNA杂交的多聚腺苷酸化的RNA物种从人骨骼肌，心脏，肺，肾，而2.0 kb的cDNA杂交的转录本只在肾和肺。从人胎盘的基因组DNA的Southern印迹分析显示类似的，但不相同，与0.8-和2.0-kb的cDNA杂交模式。数据表明，0.8和2.0 kb的cDNA编码两种不同的蛋白质，但来自同一基因。
The serum from an individual with a subepidermal blistering disease was previously shown to recognized a 450-kDa epidermal autoantigen. The molecular structure of this antigen was investigated by screening a human keratinocyte cDNA library with the patient's serum. One clone, with a 276-bp cDNA insert, that encoded an epitope recognized by the serum was isolated. Rabbit polyclonal antibodies that were prepared against the corresponding fusion protein recognized the 450-kDa epidermal antigen and stained the basal keratinocytes in human epidermis. This clone was used for further screening of the original keratinocyte and HeLa cell cDNA libraries. Two different, but closely related, 0.8- and 2.0-kb cDNAs were isolated, and their deduced amino acid sequences indicated that the encoded proteins belonged to the plectin family. Northern blot analysis of total RNA from human keratinocytes with these cDNA inserts as probes detected RNAs of approximately 12-13 kb. The 0.8-kb cDNA hybridized to polyadenylated RNA species from human skeletal muscle, heart, lung, and kidney, whereas the 2.0-kb cDNA hybridized to transcripts present only in kidney and lung. Southern blot analysis of genomic DNA from the human placenta revealed similar, but not identical, patterns of hybridization with the 0.8- and 2.0-kb cDNAs. Data suggest that the 0.8- and 2.0-kb cDNAs encode two different proteins but are derived from the same gene.