Identification of keratins and analysis of their expression in carp and goldfish: comparison with the zebrafish and trout keratin catalog

Abstract

With more than 50 genes in human, keratins make up a large gene family, but the evolutionary pressure leading to their diversity remains largely unclear. Nevertheless, this diversity offers a means to examine the evolutionary relationships among organisms that express keratins. Here, we report the analysis of keratins expressed in two cyprinid fishes, goldfish and carp, by two-dimensional polyacrylamide gel electrophoresis, complementary keratin blot binding assay, and immunoblotting. We further explore the expression of keratins by immunofluorescence microscopy. Comparison is made with the keratin expression and catalogs of zebrafish and rainbow trout. The keratins among these fishes exhibit a similar range of molecular weights and isoelectric points, with a similar overall pattern on two-dimensional gels. In addition, immunofluorescence microscopy studies of goldfish and carp tissues have revealed the expression of keratins in both epithelial and mesenchymally derived tissues, as reported previously for zebrafish and trout. We conclude that keratin expression is qualitatively similar among these fishes, with goldfish and carp patterns being more similar to each other than to zebrafish, and the cyprinid fishes being more similar to each other than to the salmonid trout. Because of the detected similarity of keratin expression among the cyprinid fishes, we propose that, for certain experiments, they are interchangeable. Although the zebrafish distinguishes itself as being a developmental and genetic/genomic model organism, we have found that the goldfish, in particular, is a more suitable model for both biochemical and histological studies of the cytoskeleton, especially since goldfish cytoskeletal preparations seem to be more resistant to degradation than those from carp or zebrafish.

A novel and ancient group of type I keratins with members in bichir, sturgeon and gar

“…2a"). It showed a positive reaction with the anti-trout-keratin antiserum GPpoly (data not shown), which we previously introduced as a general keratin marker in fish [8,10-15,19]. The two-dimensional position of this protein spot fits the theoretical values calculated from the sequence of AbaK14 (see above and Table 1).…”

“…Compared to our recent analyses [14-18,28], we have now included additional type I keratin sequences from zebrafish and Xenopus ; therefore, in this study the K18 branch shows a more complex branching pattern, not allowing a clear identification of the authentic K18 counterparts in the different vertebrates solely on the basis of their position in the tree. However, our previous identification of K18 in shark, bichir, sturgeon, trout and zebrafish was additionally based on its typical occurrence in simple epithelia, such as liver hepatocytes or intestinal mucosal epithelium, corresponding to the situation in man and other tetrapods [10,12-16,19,29]. Nevertheless, these "functional K18 counterparts" identified in the different vertebrate groups have to be considered as paralogous protein sequences.…”

“…Our investigations of the keratin systems in lamprey, shark, trout, zebrafish, carp, goldfish and lungfish have so far revealed that type I and type II keratins are apparently present in all classes of vertebrates and that the various keratins can generally be subdivided into the "E" keratins, expressed in epidermal keratinocytes and other stratified epithelia, and those appearing in cells forming simple epithelia, thus named "S" keratins [8-19]. Nevertheless, our data based on cDNA sequence analysis, followed by thorough phylogenetic analyses [13-18] as well as the studies based on the recently available genome data from man and teleost fish [1,2,5-7], strongly support the view of largely independent origins of the keratin genes found in fish and man.…”

Genes coding for intermediate filament proteins closely related to the hagfish “thread keratins (TK)” α and γ also exist in lamprey, teleosts and amphibians

“…Club cells containing similarly packed filaments have also been described for the skin of a variety of teleost fish, including cyprinids, catfish, eels and morays [52][53][54]. Our investigations of keratins in carp showed that the club cells (Leydig cells) in this fish are intensively stained by several, monoclonal, keratin-specific antibodies, including antibody 164.4 [22]. But, evidence for thread-like structures similar to those detected in the hagfish gland thread cells is currently not available for any other fish species.…”

“…Type I and II keratins have so far been found in all classes of vertebrates [10]. Our surveys of keratin expression in lower vertebrates, notably lamprey, shark, trout, zebrafish, carp, goldfish and lungfish, showed that vertebrates generally express at least two different subsets of keratins: one typical for the single-layered, internal epithelia ("S" keratins) and a second typical for the stratified, external epithelia ("E" keratins) [10][11][12][13][14][15][16][17][18][19][20][21][22]. The phylogenetic analyses of the available "E" and "S" keratin sequences so far suggest that the typical "S" keratins K8 and K18 are the only keratins present in all classes of vertebrates.…”