牛卵泡颗粒细胞CART相互作用蛋白鉴定及受体筛选

旨在筛选牛卵泡发育关键负调控因子可卡因-苯丙胺调节转录肽（cocaine and amphetamine regulated transcript peptide,CART）的相互作用蛋白及其受体。本研究采集3头健康母牛双侧卵巢,分离卵泡后刮取颗粒细胞（granulosa cells,GCs）混样;采用跨膜蛋白Extraction试剂盒提取膜蛋白,运用Protein G琼脂糖磁珠与重组CART蛋白、Anti-CART抗体共孵育（n=3）;洗脱CART-蛋白复合物进行二级质谱（LC-MS/MS）分析;获得的蛋白经可信度过滤,利用生物信息学技术对其进行功能聚类分析,通过CELLO V2.5软件对蛋白进行亚细胞定位和跨膜区分析;获得7次跨膜的G蛋白偶联受体（G-protein-coupled receptors,GPCRs）,经ProtScale软件分析其胞内外结构区段特点,符合神经肽与受体结合结构特点的GPCR作为CART的候选受体。抽提的膜蛋白经免疫亲和层析（n=3）后,分别鉴定出149、557和298个相互作用蛋白,经筛选剔除重复蛋白,共鉴定出620个蛋白。多数据库集功能富集分析表明,CART相互作用蛋白包含参与信号传导活性的GNAS、GNG8和JUP,参与类固醇生物合成的HSD3B,参与细胞凋亡或增殖调节的OPA1和S100A11,参与Notch信号通路的ERH,这些蛋白或信号通路均与牛卵泡发育密切相关。亚细胞定位和跨膜区分析共获得膜蛋白156个,占总蛋白数的20.53%;其中7次跨膜的GPCRs有8个。经TMHMM分析表明,8个GPCRs中仅ZMPSTE24、HM13和GPR108的N端在胞外,C端在胞内,与神经肽受体的结构特征一致;经PredictProtein结合位点分析表明,ZMPSTE24在5~6跨膜区间的胞内环存在G蛋白结合的位点。ZMPSTE24作为CART的候选受体,有待于分子互作和功能试验进一步加以验证。本研究为牛卵泡CART受体的鉴定奠定了基础,对丰富牛卵泡发育调控理论具有重要意义。

Identification and Receptor Screening of CART Interacting Proteins in Bovine Follicular Granulosa Cells

The aim of this study was to screen the interaction proteins and receptor of CART (cocaine and amphetamine regulated transcript peptide), which was a key negative regulatory protein of follicular development in bovine. The both ovaries of 3 healthy cows were collected, after sepa-rating follicles, the GCs (granulosa cells) were scraped and mixed. The membrane proteins were extracted by transmembrane protein extraction kit. The protein G agarose magnetic beads were used to incubate with recombinant CART protein and Anti-CART antibody (n=3). CART-protein complex was eluted and analyzed by LC-MS/MS. The obtained proteins were filtered, and function cluster was analyzed by bioinformatics technology, subcellular localization and transmembrane region analysis of these proteins were performed by CELLO V2.5 software. The intracellular and extracellular structural segments of GPCRs (G-protein-coupled receptors) with 7 times transmembrane were obtained and analyzed by ProtScale software. The GPCRs were used as candidate receptors of CART, their structural characteristics were consistent with the structural characteristics of neuropeptide and receptor binding. The 149, 557 and 298 interaction proteins were identified by immunoaffinity chromatography (n=3), respectively, and total 620 proteins were identified after screening and elimating repetitive proteins. Functional enrichment analysis of multiple database sets showed that GNAS, GNG8 and JUP involved in signal transduction activity, HSD3B involved in steroid biosynthesis, OPA1 and S100A11 involved in regulation of cell apoptosis or proliferation, and ERH involved in Notch signaling pathway, they were interaction proteins and closely related to follicular development in bovine. A total of 156 membrane proteins were obtained by subcellular localization and transmembrane domain analysis, which was 20.53% of total protein. Out of 156, 8 proteins with 7 times transmembrane were considered as GPCRs. The TMHMM analysis showed that the characteristics of ZMPSTE24, HM13 and GPR108 was consistent with the structural characteristics of neuropeptide receptor, their N-terminal was outside and C-terminal was inside. The analysis of PredictProtein binding site showed that there was a G-protein binding site in the intracellular loop of 5-6 transmembrane region of ZMPSTE24. As a candidate receptor for CART, ZMPSTE24 needs to be further verified by molecular interaction and functional tests. This study has laid a foundation for the identification of CART receptors in bovine follicles, and was of great significance to enrich the theory of regulation of follicular development in bovine.