The Function of Epiplakin - in vitro, in vivo Analysis and Gene Structure of Mouse Epiplakin

Epiplakin最初被鉴定为450-k Da的人表皮自身抗原，其与来自具有表皮下起泡疾病的个体的血清反应。该蛋白的结构分析表明，它是斑蛋白家族的一个非常独特的成员（Fujiwara et al.：J Biol Chem 276：13340-13347，2001）。该基因为一个外显子，覆盖epiplakin的整个开放阅读框架。将该基因定位于染色体8q24.3带，与plectin基因定位在同一位点。基因定位结果表明，该基因集中在一个位点上，排除了我们分离的基因组克隆是加工过的假基因的可能性。我们以HeLa细胞DNA为模板，通过聚合酶链反应扩增了5个羧基末端同源重复序列的编码区，并将这些序列与HeLa细胞的cDNA序列进行了比较。cDNA与分离的基因之间有30个核苷酸的差异，其中19个可能导致氨基酸的改变。3^* 强保守区的cDNA与扩增产物之间的核苷酸差异有40个位点，其中30个位点可能导致氨基酸的改变。个体间的基因多态性，特别是在最后5个同源重复序列中的三核苷酸（GCC）重复数。结果表明，在15例日本人中，有5、8、9个GCC重复序列发生变异，部分重复序列由GCC变为ACC，导致氨基酸发生变化（丙氨酸变为苏氨酸），经聚合酶链反应和直接测序后，检测了3^* 强保守区第一个重复序列中GCC重复序列的数目。8 GCC重复是最常见的检测，虽然五个和九个重复也被发现在少数群体。这些数据表明epiplakin基因多态性的存在。

Epiplakin was originally identified as a 450-k Da human epidermal autoantigen that reacted with the serum from an individual with a subepidermal blistering disease. The structural analysis of this protein showed that it is a highly unique member of the plakin family (Fujiwara et al.: J Biol Chem 276: 13340-13347, 2001).In this study, we have isolated human epiplakin gene (EPPK 1) by screening of leukocyte genomic library. The gene was one exon which covers entire open reading frame of epiplakin. The gene was mapped to chromosomal band 8q24.3, which was the same locus of plectin. The gene converged into one locus in mapping excluded the possibility that the genomic clones we isolated are processed pseudogene(s). We amplified the coding region of five COOH-terminal homologous repeats with polymerase chain reaction using DNA from HeLa cells as template and these sequences were compared with those of cDNA derived from HeLa cells. The numbers of different nucleotides between cDNA and the isolated gene were 30 sites, 19 of which might lead amino acid change. The numbers of different nucleotides between cDNA and the amplified product of 3^* strongly conserved region were 40 sites, 30 of which might lead amino acid change. Gene polymorphism between individuals was detected especially in number of trinucleotide (GCC) repeats in the last five homologous repeats. Five, eight or nine GCC repeats-variations and the change of GCC to ACC in some repeats which leads amino acid change (alanine to threonine) were found. After polymerase chain reaction followed by direct sequencing using DNA isolated from nonrelated 15 Japanese, the numbers of GCC repeats in the first repeat of 3^* strongly conserved region were examined. Eight GCC repeats were most frequently detected though five and nine repeats were also found in minor populations. This data indicated the presence of polymorphism of epiplakin gene.