肉品中PNPLA3水解肌内磷脂的机制研究

Flavor of meat is closely correlated with lipolysis of intramuscular phospholipids. Our previous studies on intramuscular phospholipids hydrolyzed by phospholipases discovered the patatin like phospholipase 3 (PNPLA3). The expression level of PNPLA3 in meat was found highest among kinds of phospholipases. In the process of meat processing, PNPLA3 maintained high concentration and showed stable activity, suggesting its important role in lipolysis of intramuscular phospholipids. However, the lipolysis mechanism of PNPLA3 on phospholipids remains unknown. In the present project, PNPLA3 with high purity will be prepared in vitro, and its activity and the optimal conditions will be determined. The phospholipid binding site will be identified with the aid of surface plasmon resonance (SPR) and Fourier Transform Infrared Spectrometer (FITR). The phospholipid cleavage site will also be determined by analyzing the hydrolysis products of PNPLA3 using HPLC-MS and gas chromatography. The three-dimensional structure of PNPLA3 and phospholipid complex will be determined by X ray diffraction analysis; with the structural data and SPR analysis, the substrate-binding domain of PNPLA3 and its allosteric activation mechanism will be determined; the catalytic domain will be confirmed by the site-specific mutagenesis based on the structure and sequence analysis, and following biochemistry experiments such as ITC. Subsequently, co-expression of active domains of PNPLA3 will be employed to verify the enzymatic activity. The mechanism will be further validated in intramuscular environment. The result will provide a new approach for studies on meat flavor, as well as a theoretical basis for the regulation of meat flavor.

肉品风味与肌内磷脂的水解密切相关。申请者前期研究肌内磷脂水解时发现Patatin样磷脂酶3（PNPLA3），相较于其它磷脂酶在肌肉中表达量最高，且在加工过程中保持较高含量和活力，在肌内磷脂水解中发挥重要作用。但PNPLA3如何在肉品中发挥水解作用未见报道。本项目首先通过体外表达纯化，制备PNPLA3纯品，分析其活性和影响因素；采用表面等离子共振和红外光谱分析PNPLA3结合磷脂的常数，确定其与磷脂的结合位点；运用液质联用和气相色谱分析PNPLA3水解磷脂的产物，推断其水解位点；通过X射线衍射解析PNPLA3-磷脂复合物的晶体结构，确定其磷脂结合结构域；通过结构比较和序列比对，设计突变体并用等温滴定量热法检测，明确PNPLA3的催化结构域；融合表达PNPLA3的2个功能结构域，进行功能验证，阐明其水解肌内磷脂的分子机制，为肉品风味控制提供新思路和理论依据。

肉品风味品质与肌内磷脂酶解密切相关。Patatin样磷脂酶3（PNPLA3）在肌肉中表达量最高，且在板鸭加工过程保持较高含量和水解活力，稳定性强，在肌内磷脂降解中发挥重要作用。在前期研究基础上，本项目首先通过体外表达纯化制备高纯度的PNPLA3，用荧光光谱法测定其水解磷脂的活性和最佳反应条件；采用蛋白互作仪分析其磷脂底物的结合常数，并用傅里叶变换红外光谱法（FITR）研究PNPLA3结合在磷脂的极性端还是非极性端；运用液质联用明确其水解磷脂的靶点；通过建模预测PNPLA3与的三维结构，结合序列比对分析，设计定点突变，明确PNPLA3的催化活性位点；在此基础上研究PNPLA3在肌肉模拟体系中验证水解肌内磷脂的活性。结果表明，PNPLA3催化磷脂水解的最佳反应温度为37℃，最适反应pH为6。HPLC-MS分析显示PNPLA3水解磷脂的位点sn-2位，首次确定了其磷脂酶A2活性。FITR结果分析显示PNPLA3与磷脂结合的位点在磷脂的非极性端。结构分析和同源序列比对，结合定点突变证明了D134是PNPLA3催化活性位点。在肌肉模拟体系中也证明了PNPLA3的磷脂酶A2活性调控肌内磷脂水解的功能。本项目通过阐明PNPLA3水解肌内磷脂的分子机理，为贮藏和加工过程中肉品风味控制提供新的思路和理论依据。