0000000000013043
AUTHOR
Michael Schaffeld
Evolution of tissue-specific keratins as deduced from novel cDNA sequences of the lungfish Protopterus aethiopicus.
Lungfishes are possibly the closest extant relatives of the land vertebrates (tetrapods). We report here the cDNA and predicted amino acid sequences of 13 different keratins (ten type I and three type II) of the lungfish Protopterus aethiopicus. These keratins include the orthologs of human K8 and K18. The lungfish keratins were also identified in tissue extracts using two-dimensional polyacrylamide gel electrophoresis, keratin blot binding assays and immunoblotting. The identified keratin spots were analyzed by peptide mass fingerprinting which assigned seven sequences (inclusively Protopterus K8 and K18) to their respective protein spot. The peptide mass fingerprints also revealed the fac…
A novel and ancient group of type I keratins with members in bichir, sturgeon and gar
Abstract 1. Background Vertebrate epithelial cells typically express a specific set of keratins. In teleosts, keratins are also present in a variety of mesenchymal cells, which usually express vimentin. Significantly, our previous studies revealed that virtually all known teleost keratins evolved independently from those present in terrestrial vertebrates. To further elucidate the evolutionary scenario that led to the large variety of keratins and their complex expression patterns in present day teleosts, we have investigated their presence in bichir, sturgeon and gar. 2. Results We have discovered a novel group of type I keratins with members in all three of these ancient ray-finned fish, …
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 identifi…
Genes coding for intermediate filament proteins closely related to the hagfish "thread keratins (TK)" alpha and gamma also exist in lamprey, teleosts and amphibians.
The "thread keratins (TK)" alpha and gamma so far have been considered highly specialized intermediate filament (IF) proteins restricted to hagfish. From lamprey, we now have sequenced five novel IF proteins closely related to TKalpha and TKgamma, respectively. Moreover, we have detected corresponding sequences in EST and genomic databases of teleosts and amphibians. The structure of the TKalpha genes and the positions of their deduced amino acid sequences in a phylogenetic tree clearly support their classification as type II keratins. The genes encoding TKgamma show a structure typical for type III IF proteins, whereas their positions in phylogenetic trees favor a close relationship to the…
cDNA sequences of the authentic keratins 8 and 18 in zebrafish
From the zebrafish Danio rerio, we have cDNA cloned and sequenced a novel type II and a novel type I keratin, termed DreK8 and DreK18, respectively. We identified DreK8/18 as the true orthologs of the human keratin pair K8/18 as follows: (i) MALDI-MS assignment to the biochemically identified K8 and K18 candidates that are co-expressed in simple epithelia and absent in epidermal keratinocytes; (ii) multiple sequence alignments and phylogenetic tree analysis, showing that DreK8, within the phylogenetic tree of type II keratins, forms a highly bootstrap-supported branch together with K8 from goldfish and rainbow trout, whereas DreK18, within the phylogenetic tree of type I keratins, groups wi…
Vimentin and desmin of a cartilaginous fish, the shark Scyliorhinus stellaris: Sequence, expression patterns and in vitro assembly
In the shark Scyliorhinus stellaris we have biochemically identified and cDNA-cloned orthologs of human vimentin and desmin, SstV and SstD, as deduced from immunoblotting and sequence alignment with teleost, frog and human vimentin and desmin, respectively. This allowed us to further clarify the relationship of previously identified lower vertebrate intermediate filament proteins to mammalian vimentin and desmin. Immunofluorescence microscopy with antibodies H5 and VIM13.2 showed vimentin expression in shark eye and brain and absence in epithelia, which resembles the situation in higher vertebrates. In addition, SstV is expressed in many mesenchymal cell types which corresponds to the case …
Sequence, evolution and tissue expression patterns of an epidermal type I keratin from the shark Scyliorhinus stellaris.
From the shark Scyliorhinus stellaris we cloned and sequenced a cDNA encoding a novel type I keratin, termed SstK10. By MALDI-MS peptide mass fingerprinting of cytoskeletal proteins separated on polyacrylamide gels, we assigned SstK10 to a 46-kDa protein which is the major epidermal type I (“IE”) keratin in this fish and is specifically expressed in stratified epithelia. In a phylogenetic tree based on type I keratin sequences and with lamprey keratins applied as outgroup, SstK10 branches off in a rather basal position. This tree strongly supports the concept that teleost keratins and tetrapod keratins resulted from two independent gene radiation processes. The only exception is human K18 b…
Type I keratin cDNAs from the rainbow trout: independent radiation of keratins in fish
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 react…
Type II keratin cDNAs from the rainbow trout: implications for keratin evolution.
From a teleost fish, the rainbow trout Oncorhynchus mykiss, we have cloned and sequenced cDNAs encoding five different type II keratins. The corresponding protein spots, as separated by 2D-PAGE of trout cytoskeletal preparations, have been identified by peptide mass mapping using MALDI mass spectrometry. Three of the sequenced keratins are expressed in the epidermis (subtype IIe), and two in simple epithelia and mesenchymal cells (subtype IIs). The IIs keratins are both orthologs of human K8. This leaves unsequenced only the trace component S3 of the biochemically established trout keratin catalog. A phylogenetic tree has been constructed from a multiple alignment of the rod domains of the …