Fugu, Takifugu ruberipes, mucus keratins act as defense molecules against fungi.

Keratin is a cytoskeletal protein that constitutes the intermediate filament. Its distribution is restricted to epithelial tissues in mammals, but is wider in fish. An interesting feature of fish keratin is that it is abundant in the cutaneous mucus. However, the biological function of keratin in the mucus has not been explored. In the present study, we hypothesized that mucus keratins of fugu Takifugu rubripes function as antimicrobial molecules. To verify this hypothesis, we first identified all of the keratins expressed in the epidermis and present in mucus. Five of 15 keratins including Tr-K4 expressed in the epidermis were identified in the mucus. Subsequently, we examined the interaction of keratin molecules present in fugu mucus with yeast. Affinity chromatography using yeast as a carrier and subsequent LC-MS/MS analysis revealed that three types of keratin were bound to the yeast. Furthermore, yeast incubated with fugu mucus was agglutinated, and this was inhibited by anti-recombinant Tr-K4 (rTr-K4) antibody. Immunohistochemical analysis also revealed that keratin was attached to the surface of agglutinated yeasts. These findings indicate that mucus keratin agglutinates yeast. Furthermore, we found insoluble clumps in fugu mucus, which were mainly comprised of keratin. After incubation of yeast with soluble mucus fraction, insoluble clumps incorporating yeast were formed. This observation suggests that fugu mucus keratin sequesters microbes into insoluble clumps, which are eventually eliminated from the mucus. Here, we present our finding of the novel function of keratin as a defense molecule in fish mucus.

Sacciform cells in the epidermis of fugu (Takifugu rubripes) produce and secrete kalliklectin, a novel lectin found in teleosts.

Kalliklectin is a novel lectin identified in the skin mucus and blood plasma of teleosts, including fugu (Takifugu rubripes). It has been found to exhibit sequence similarity to mammalian plasma kallikrein and coagulation factor XI. The objective of the present study was to clarify the cellular localization of kalliklectin using an antiserum specific to fugu kalliklectin. Immunohistochemical analysis showed that positive reactions were observed in the skin and liver, but not in other tested tissues. Several types of epidermal cells were stained by the antiserum; sacciform cells were one of the types of cells most densely stained by the antiserum in adult fugu skin, whereas mucous cells showed negative staining results. RT-PCR demonstrated that the kalliklectin gene was transcribed in the mucous cell-poor region of adult fugu skin, where sacciform cells were present. These results indicated that epidermal cells, including sacciform cells, produce kalliklectin and secrete it into the mucus.