Haussmann D, Figueroa J
Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile.
Vet Immunol Immunopathol. 2011 Feb 15;139(2-4):119-27. doi: 10.1016/j.vetimm.2010.09.005. Epub 2010 Sep 22.
Glandular Kallikrein is a serine-protease with trypsin-like activity and is able to generate bioactive peptides from inactive precursors. We have evaluated the presence of this protease in the different organs of the Atlantic salmon (Salmo salar). The results clearly indicate that GK and PRL are generated in the same pituitary cells based on a co-localization by confocal microscopy. Based on probed cross-reactivity between C. striata and C. carpio glandular anti-GK antibodies, we used a homologous antibody to detect the presence of GK in several salmon tissues. We have evaluated the GK expression in healthy and defied fish. P. salmonis and V. ordalii. The GK immunoreaction in organs such as leukocytes, gills and skin is considerably increased in defied fish compared to healthy fish. This increase was present in the cells of the excretory kidney and in the intercellular tissue, where the development of hematopoietic and lymphocytic lines in fish take place. One of the most interesting organs to study was the skin, bearing in mind that this is a primary barrier to all pathogens. The skin of the defied fish exhibited an increase in immunoreactivity for glandular kallikrein similar to the protease found in mucus. An immunoreactive tissue kallikrein-like protein was identified and partially separated by perfusion chromatography. Enzymatic activity of salmon muscle prokallikrein was determined before and after trypsin activation. Kallikrein activity was characterized with respect to their ability to cleave the chromogenic leaving group, p-nitroanilide, from the peptidyl kallikrein and trypsin substrate. These findings constitute a important contribution to reveal the role of kallikrein in the innate immune system of fish.
腺激肽释放酶是一种具有胰蛋白酶样活性的丝氨酸蛋白酶，能够从无活性前体生成生物活性肽。我们评估了这种蛋白酶在大西洋鲑（Salmo salar）不同器官中的存在情况。结果清楚地表明，基于共聚焦显微镜下的共定位，GK和PRL是在同一垂体细胞中产生的。基于条纹鲈和鲤鱼腺抗GK抗体之间的交叉反应性，我们使用同源抗体检测了几种鲑鱼组织中GK的存在。我们评估了健康鱼和感染鱼（感染杀鲑气单胞菌和奥氏弧菌）中GK的表达。与健康鱼相比，感染鱼的白细胞、鳃和皮肤等器官中的GK免疫反应显著增加。这种增加出现在排泄肾细胞和细胞间组织中，鱼类造血和淋巴细胞系在这些部位发育。最值得研究的器官之一是皮肤，因为它是抵御所有病原体的主要屏障。感染鱼的皮肤对腺激肽释放酶的免疫反应性增加，类似于在黏液中发现的蛋白酶。通过灌注色谱法鉴定并部分分离出一种免疫反应性组织激肽释放酶样蛋白。测定了鲑鱼肌肉前激肽释放酶在胰蛋白酶激活前后的酶活性。根据激肽释放酶从肽基激肽释放酶和胰蛋白酶底物上裂解发色离去基团对硝基苯胺的能力，对其活性进行了表征。这些发现为揭示激肽释放酶在鱼类先天免疫系统中的作用做出了重要贡献。
