Schaffeld Michael, Höffling Simon, Haberkamp Mark, Conrad Matthias, Markl Jürgen
Institute of Zoology, Johannes Gutenberg University, 55099 Mainz, Germany.
Differentiation. 2002 Aug;70(6):282-91. doi: 10.1046/j.1432-0436.2002.700606.x.
Five different type I keratins from a teleost fish, the rainbow trout Oncorhynchus mykiss, have been sequenced by cDNA cloning and identified at the protein level by peptide mass mapping using MALDI-MS. This showed that the entire range of type I keratins detected biochemically in this fish has now been sequenced. Three of the keratins are expressed in the epidermis (subtype Ie), whereas the other two occur in simple epithelia and mesenchymal cells (subtype Is). Among the Is keratins is an ortholog of human K18; the second Is polypeptide is clearly distinct from K18. We raised a new monoclonal antibody (F1F2, subclass IgG1) that specifically recognizes trout Is keratins, with negative reactions on zebrafish. A phylogenetic tree has been constructed from a multiple alignment of the rod domains of the new sequences together with type I sequences from other vertebrates such as shark, zebrafish, and human; a recently sequenced lamprey Is keratin was applied as outgroup. This tree shows one branch defining the K18 orthologs and a second branch containing all other type I keratins (mostly subtype Ie). Within this second branch, the teleost keratins form a separate, highly bootstrap-supported twig. This tree leaves little doubt that the teleost Ie keratins diversified independently from the mammalian Ie keratins.
通过cDNA克隆对硬骨鱼虹鳟（Oncorhynchus mykiss）的五种不同的I型角蛋白进行了测序，并使用基质辅助激光解吸电离质谱（MALDI-MS）通过肽质量图谱在蛋白质水平上进行了鉴定。这表明现在已经对在这种鱼中通过生化方法检测到的I型角蛋白的整个范围进行了测序。其中三种角蛋白在表皮中表达（Ie亚型），而另外两种存在于单层上皮细胞和间充质细胞中（Is亚型）。在Is角蛋白中有一种人类K18的直系同源物；第二种Is多肽明显不同于K18。我们制备了一种新的单克隆抗体（F1F2，IgG1亚类），它能特异性识别虹鳟的Is角蛋白，对斑马鱼无反应。根据新序列的杆状结构域与其他脊椎动物如鲨鱼、斑马鱼和人类的I型序列的多序列比对构建了系统发育树；最近测序的七鳃鳗Is角蛋白用作外群。这棵树显示出一个分支定义了K18直系同源物，另一个分支包含所有其他I型角蛋白（主要是Ie亚型）。在这第二个分支中，硬骨鱼角蛋白形成了一个单独的、得到高度自展支持的小分支。这棵树毫无疑问地表明硬骨鱼Ie角蛋白是与哺乳动物Ie角蛋白独立分化的。
