Regulation of production and secretion of defensive molecules in fish skin

鱼皮粘液含有多种防御分子，如溶菌酶、抗菌肽、凝集素和抗体。虽然我们对这些分子的生物化学性质和生物学功能的了解正在增加，但这些分子的产生和分泌是如何调节的几乎是未知的。本研究以海鳗（Conger myriaster）为研究对象，探索海鳗皮肤粘液凝集素的刺激剂。然后，使用这种方法，我们用各种细菌来源的物质刺激分离的细胞，包括LPS，肽聚糖，脂磷壁酸。收获这些培养物的上清液，并通过ELISA测量从海鳗表皮分泌的半乳糖凝集素--海鳗蛋白。用RT PCR半定量分析了congerin I、II和con CL-S（C型凝集素）的基因表达。出乎意料的是，在刺激过程中没有看到明显的变化。我们在对海鳗皮肤凝集素染色切片和细胞的观察中，发现了一些糖蛋白定位于俱乐部细胞的细胞质和粘液细胞的细胞表面。用花生凝集素对皮提取物进行SDS-PAGE和凝集素印迹分析，结果表明皮提取物中含有与PNA反应的糖蛋白。其中一个分子被纯化并克隆了其cDNA。该分子是一个分子量约为25 kDa的凝集素，与鲤鱼卵凝集素有很高的相似性。此外，该凝集素与海绵和水螅的凝集素具有很高的相似性，这表明该凝集素在后生动物中具有重要的作用。凝集素印迹法检测到的其他糖蛋白，可能与俱乐部细胞或粘液细胞的专门功能有关，仍有待研究。

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.