Larsen Anett K, Kristiansen Kurt, Sylte Ingebrigt, Seternes Ole-Morten, Bang Berit E
Department of Occupational- and Environmental Medicine, University Hospital North Norway, Tromsø, Norway.
BMC Res Notes. 2013 Jul 20;6:281. doi: 10.1186/1756-0500-6-281.
Salmon trypsin is shown to increase secretion of the pro-inflammatory cytokine interleukin (IL)-8 from human airway epithelial cells through activation of PAR-2. Secretion of IL-8 induced by king crab trypsin is observed in a different concentration range compared to salmon trypsin, and seems to be only partially related to PAR-2 activation. This report aim to identify differences in the molecular structure of king crab trypsin (Paralithodes camtschaticus) compared to salmon (Salmo salar) and bovine trypsin (Bos taurus) that might influence the ability to activate protease-activated receptor-2 (PAR-2).
During purification king crab trypsin displayed stronger binding capacity to the anionic column used in fast protein liquid chromatography compared to fish trypsins, and was identified as a slightly bigger molecule. Measurements of enzymatic activity yielded no obvious differences between the trypsins tested. Molecular modelling showed that king crab trypsin has a large area with strong negative electrostatic potential compared to the smaller negative areas in bovine and salmon trypsins. Bovine and salmon trypsins also displayed areas with strong positive electrostatic potential, a feature lacking in the king crab trypsin. Furthermore we have identified 3 divergent positions (Asp196, Arg244, and Tyr247) located near the substrate binding pocket of king crab trypsin that might affect the binding and cleavage of PAR-2.
These preliminary results indicate that electrostatic interactions could be of importance in binding, cleavage and subsequent activation of PAR-2.
研究表明，鲑鱼胰蛋白酶可通过激活蛋白酶激活受体-2（PAR-2）来增加人呼吸道上皮细胞促炎细胞因子白细胞介素（IL）-8的分泌。与鲑鱼胰蛋白酶相比，帝王蟹胰蛋白酶诱导的IL-8分泌出现在不同的浓度范围内，且似乎仅部分与PAR-2激活有关。本报告旨在确定帝王蟹胰蛋白酶（堪察加拟石蟹）与鲑鱼（大西洋鲑）和牛胰蛋白酶（牛）在分子结构上的差异，这些差异可能会影响激活PAR-2的能力。
在纯化过程中，与鱼类胰蛋白酶相比，帝王蟹胰蛋白酶对快速蛋白质液相色谱中使用的阴离子柱表现出更强的结合能力，并且被鉴定为分子稍大。所测试的胰蛋白酶之间的酶活性测量未产生明显差异。分子建模显示，与牛和鲑鱼胰蛋白酶中较小的负电区域相比，帝王蟹胰蛋白酶有一个具有强负静电势的大面积区域。牛和鲑鱼胰蛋白酶也显示出具有强正静电势的区域，而帝王蟹胰蛋白酶缺乏这一特征。此外，我们已经确定了帝王蟹胰蛋白酶底物结合口袋附近的3个不同位置（Asp196、Arg244和Tyr247），这些位置可能会影响PAR-2的结合和切割。
这些初步结果表明，静电相互作用在PAR-2的结合、切割和随后的激活中可能很重要。
