Regulation of production and secretion of defensive molecules in fish skin

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Fish skin mucus contains diverse array of defensive molecules like lysozyme, anti-bacterial peptides, lectins, and antibody. Although our knowledge of biochemical nature and biological functions of those molecules are being increased, how production and secretion of those are regulated is nearly unknown. In this research, we investigated stimulants for skin mucus lectins in conger eel, Conger myriaster.First, we established a method to isolate and culture epidermal cells of conger eel with high survival rate. Next, using this method, we stimulated the isolated cells with various bacteria-originated substances including LPS, peptideglycan, lipoteichoic acid. Supernatant of those cultures were harvested and congerin, a galectin secreted from conger epidermis, was measured by ELISA. Semi-quantitative analysis of gene expression of congerin I, II, and con CL-S (C-type lectin) by RT PCR was also done. Unexpectedly, no obvious change was seen during the stimulation. This suggests that conger skin lectins are produced rather constitutively.We found some glycoproteins localized in cytoplasm of club cells and cell surface of mucus cells of conger skin during observation of lectin-stained sections and cells. When skin extract was applied to SDS-PAGE and lectin blotting using peanuts agglutinin shows there exist some PNA-reactive glycoproteins in skin extracts. One of those molecules were purified and its cDNA was cloned. This molecule was a lectin of about 25kDa, which shows high similarity with carp egg lectin. In addition, this lectin showed high similarity with lectins of sponge and hydra, which suggests that this lectin retains some essential roles throughout metazoan. Other glycoproteins detected lectin blotting, which may be related to specialized functions of club cells or mucus cells, remain to be investigated.