Schaffeld M, Löbbecke A, Lieb B, Markl J
Institute of Zoology, Johannes Gutenberg University of Mainz, Germany.
Eur J Cell Biol. 1998 Oct;77(2):69-80. doi: 10.1016/S0171-9335(98)80074-5.
We have studied individual keratins of an elasmobranch, the shark Scyliorhinus stellaris (the lesser-spotted dogfish). From various shark tissues, notably skin and stomach, cytoskeletal proteins were isolated and then separated by two-dimensional polyacrylamide gel electrophoresis. Using complementary keratin blot-binding assays and immunoblotting, among these proteins we identified a variety of type I and type II keratins. According to their tissue-specific expression, we distinguished Is and IIs keratins from IE and IIE keratins ("S" and "E" from "simple epithelial" and "epidermal", respectively). Guinea pig antibodies which in immunoblots specifically labeled the entire range of identified shark keratins, and a monoclonal antibody specific for IE keratins were used for immunofluorescence microscopy of a broad range of shark tissues. These experiments demonstrated that in this shark, keratin expression is largely restricted to epithelia and - in contrast to the situation in teleost fishes - is lacking in mesenchymally derived cells and tissues. Peptide mass mapping of the major electrophoretically separated shark keratin spots revealed that the identified Is, IIs and IIE polypeptides are modifications of a single genuine keratin, respectively, whereas there are two different IE keratins. It, therefore, appears that in this shark most (if not all) of the keratin cytoskeleton is constituted by only five different gene products (each present in various modifications): a heterologous pair of "S" and three different "E" keratins. We sequenced three of them (Is, IIs and IIE) via cDNA cloning. Sequence alignments showed that the shark Is keratin (termed SstK18) is an ortholog of human K18, whereas the IIs keratin (termed SstK8) corresponds to human K8. In contrast, the shark IIE keratin (termed SstK1; it is the first known primary structure of a fish IIE keratin) apparently has no direct equivalent in human. On the basis of a phylogenetic tree constructed from 37 aligned keratin sequences, these results are discussed with respect to the evolution of keratin diversity in vertebrates.
我们研究了一种板鳃亚纲动物——星鲨（Scyliorhinus stellaris，即斑点猫鲨）的个体角蛋白。从鲨鱼的各种组织，尤其是皮肤和胃中分离出细胞骨架蛋白，然后通过二维聚丙烯酰胺凝胶电泳进行分离。利用互补角蛋白印迹结合分析和免疫印迹技术，我们在这些蛋白质中鉴定出了多种I型和II型角蛋白。根据它们的组织特异性表达，我们将Is和IIs角蛋白与IE和IIE角蛋白区分开来（“S”和“E”分别来自“简单上皮”和“表皮”）。在免疫印迹中能特异性标记所鉴定的全部鲨鱼角蛋白的豚鼠抗体，以及一种对IE角蛋白特异的单克隆抗体，被用于对多种鲨鱼组织进行免疫荧光显微镜检查。这些实验表明，在这种鲨鱼中，角蛋白的表达主要局限于上皮组织，并且——与硬骨鱼类的情况相反——间充质来源的细胞和组织中不存在角蛋白。对主要通过电泳分离的鲨鱼角蛋白斑点进行肽质量图谱分析表明，所鉴定的Is、IIs和IIE多肽分别是单一原始角蛋白的修饰形式，而有两种不同的IE角蛋白。因此，在这种鲨鱼中，似乎大多数（如果不是全部）角蛋白细胞骨架仅由五种不同的基因产物（每种都有多种修饰形式）构成：一对异源的“S”角蛋白和三种不同的“E”角蛋白。我们通过cDNA克隆对其中三种（Is、IIs和IIE）进行了测序。序列比对显示，鲨鱼的Is角蛋白（称为SstK18）是人类K18的直系同源物，而IIs角蛋白（称为SstK8）对应于人类K8。相比之下，鲨鱼的IIE角蛋白（称为SstK1；它是已知的首个鱼类IIE角蛋白的一级结构）在人类中显然没有直接对应的角蛋白。基于由37个比对后的角蛋白序列构建的系统发育树，我们就脊椎动物角蛋白多样性的进化对这些结果进行了讨论。
