Tracing keratin evolution: catalog, expression patterns and primary structure of shark (Scyliorhinus stellaris) keratins.

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.

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