鱼类脯氨酰内肽酶的结构、功能及其对胶原肽的降解机理

In our previous study, we have found that matrix metalloproteinases (MMPs) play critical roles in the degradation of type I and type V collagens and which are major reasons for the softening of fish muscle after death. Since the final products of collagens after degradation by MMPs are peptides while collagen peptides contain high contents of -imino acids, it was thus proposed that further degradation of collagen peptides should be performed by specific prolyl endopeptidase (PEP). However, study on PEPs from fish has just started. Their primary structures, tissue distribution, especially the limit of peptide length and cleavage sites still remain unknown. .In the present study, we try to use marine fish sea bass (Lateolabrax japonicas) and freshwater fish silver carp (Hypophthalmichthys molitrix) as research targets. PEPs from these two species of fish will be purified and their biochemical properties and characteristics in food science will be investigated. The full-length sequences of PEP will be cloned and expressed in vitro and the crystal structures of PEPs will be determined. Based on this study, the enzymatic properties as well as the primary sequences of PEPs from both marine and freshwater fish will be elucidated. The possible involvement of PEP in the metabolism of collagens will be determined. Our present study results will surely beneficial for the basic research on fisheries products processing and also deliver some theoretical evidence for the application of PEP in food industry.

前期研究中我们发现，基质金属蛋白酶(MMP)对I型和V型胶原蛋白的分解是导致鱼体死后肌肉软化的主要原因。MMP对蛋白降解产生的胶原小肽富含-亚氨基的脯氨酸残基，其进一步被降解为胶原超小肽应由特异分解脯氨酸的脯氨酰内肽酶（PEP）来完成。但是，对鱼类PEP的研究刚刚起步。PEP的一级结构、组织分布、对底物的长度要求、特异性等性质仍未解明。本项目拟以海水鲈鱼和淡水鲢鱼为研究对象，获得两种鱼的PEP并详细分析对比它们的生物化学和食品学特性、利用分子克隆手段获得全序列并完成体外表达、实现PEP的蛋白结晶和结构解析。通过本研究，将能解明鱼类PEP的酶学特性及其一级结构并从原理上揭示由PEP参与的肌肉胶原蛋白新陈代谢等生化特性与规律，海水和淡水鱼类PEP的蛋白结构将首次得到确定并实现比较分析。研究结果有助于弥补我国在水产食品加工利用基础理论研究方面的不足，也可为PEP在食品工业中的应用提供理论依据。

本研究以海水鲈鱼、淡水鲢鱼以及皱纹盘鲍为研究对象，采用现代生化分离技术分别纯化得到天然的脯氨酰内肽酶（prolyl endopeptidase, PEP）；同时，通过 5’-RACE 和3’-RACE 技术，克隆得到鲈鱼、鲢鱼以及鲍鱼PEP的全长基因序列，可溶表达具有较高酶活力的鲍鱼PEP重组蛋白，制备获得多克隆抗体，检测PEP在不同器官组织中的分布情况，并对这三种水生动物来源的PEP酶学性质与结构特点进行了分析比较；在此基础之上，对鲈鱼、鲢鱼以及鲍鱼PEP进行结晶条件的筛选及优化，并结合X-射线衍射技术解明了重组鲍鱼PEP蛋白的晶体结构。通过比较三种来源PEP的三级结构，揭示了PEP结构的高度相似性，其催化三联体被β-折叠形成的螺旋桨区域的中心通道所覆盖，能将富含脯氨酸的胶原肽进行降解，从PEP的结构功能性解析了其与基质金属蛋白酶等一起参与富含脯氨酸残基的动物肌肉胶原蛋白的新陈代谢以及死后的自溶软化的作用机理。此外，采用分子动力学模拟PEP与抑制剂相互作用的动态过程，发现PEP结构中具有结构柔性的区域Loop A（193-209），删除该区域能使底物更容易进入酶的催化活性中心，得到活性更高的突变蛋白酶。.本研究结合了蛋白质化学、酶学和现代生物技术等相关领域的技术手段，研究结果有助于丰富对水生动物PEP结构和功能的研究。突变体PEP的获得可为今后开发新型酶制剂提供理论依据。