丝氨酸蛋白酶参与鱼类肌肉软化基质金属蛋白酶活化机理研究

Large yellow croaker is a type of local characteristic fish cultured in FuJian province. It is often transported and sold in form of chilled fish or frozen fish, due to the limitation of preservation technology and cost. The degradation of muscle protein induced by endogenous proteinase from fish muscle is in correspondence to the post-mortem softening. Among them, collagen hydrolysis by matrix metalloproteinase (MMP) was considered as a more important reason. In vivo, zymogen (pro-MMP) must be activated to produce mature MMP by endogenous proteinase. In mammalians, endogenous serine proteinase (SP) widely involved in the activation of pro-MMPs, revealed they have a close relationship. But, no much information is available for enzyme properties of SP involveded in pro-MMPs activation from fish muscle and mechanism of pro-MMPs activation induced by it. In the present study, combining technologies of enzymology, biotechnology and food chemistry, SP which may involve in MMPs activation will be purified to homogeneity to clarify MMPs activation mechanism and dynamic variation from large yellow croaker (Pseudosciaena crocea) muscle. The mechanism of endogenous SP involving in MMPs activation will be elucidated, with a purpose to reveal collagen metabolic regularity and textural properties of fish muscle caused by endogenous MMPs. The results of the present work will be beneficial for adding more novel information in fish muscle softening during post-mortem period and providing theoretical basis for processing and preservation of marine fish.

大黄鱼是福建省特色经济鱼种，因保活技术和成本的局限，大黄鱼常常以冷藏形式消费。在贮藏期，内源性蛋白酶导致的肌肉蛋白水解是鱼肉软化的主要原因。其中，基质金属蛋白酶（MMP）水解胶原蛋白认为是更重要的原因，而机体中分泌的基质金属蛋白酶原（pro-MMP）必须被内源性蛋白酶活化为成熟MMP后才具有生物活性。在哺乳动物中，内源性丝氨酸蛋白酶（SP）广泛地参与pro-MMPs活化过程，两者有密切的关系。但鱼类肌肉中是否有类似的SP及其参与MMPs酶原活化的机理尚不清楚。因此，本项目拟结合酶学、分子生物学和食品化学等相关技术，从大黄鱼肌肉中分离SP，分析其酶学特性和结构特点，解析SP参与MMPs酶原活化的内在机理，探索SP活化MMPs的动态规律，揭示由MMPs介导的鱼类胶原蛋白代谢特性和规律及其与鱼体肌肉软化的内在联系。该研究将有助于鱼类死后肌肉软化重要机理的阐明，也可为海水鱼加工与贮藏提供理论依据。

本项目旨在通过蛋白酶学、分子生物学和食品化学等相关技术手段，解析大黄鱼SP参与MMPs酶原活化的内在机理，探索SP活化MMPs的动态规律，揭示由MMPs介导的鱼类胶原蛋白代谢特性和规律及其与鱼体肌肉软化的内在机理。项目的研究计划主要包括：1）大黄鱼肌肉MMPs活化相关内源性蛋白酶的分析和鉴定；2）内源性SP的分离纯化、性质研究及抗体制备；3）大黄鱼肌肉中内源性SP与pro-MMPs活化的内在机理研究；4）大黄鱼肌肉SP的基因和蛋白水平的变化规律。在三年的研究工作中，本课题按照计划开展了以下工作：1）分离制备得到大黄鱼肌肉的I型胶原蛋白与V型胶原蛋白，并对其理化特性进行研究；2）通过硫酸铵盐析、阴离子交换柱层析等方法，从大黄鱼肌肉中分离纯化得到内源性丝氨酸蛋白酶与基质金属蛋白酶，并对其酶学性质进行研究，同时揭示了内源性SP的蛋白表达规律；3）基于大黄鱼全基因组数据，克隆表达得到大黄鱼pro-MMP-2的部分蛋白片段，从蛋白水平揭示了SP激活pro-MMP-2的内在规律。此外，本项目还克隆得到了黄鳍鲷肌肉弹性蛋白的全基因片段，并进行了生物信息学分析；克隆并表达了真鲷肌肉内源性基质金属蛋白酶抑制剂TIMP-2a，阐明了二硫键的形成规律，初步探讨TIMP-2a对MMPs的抑制作用。这些研究系统的分析了胶原蛋白、SP、MMP和TIMP间的内在关联，解析鱼类肌肉基质蛋白、水解酶和内源性抑制剂三者间的作用机制，阐明了鱼类肌肉软化内在分子机理，对海水鱼肌肉的贮藏与加工具有一定的理论指导意义。