鸡卵泡膜细胞中膜联蛋白A2互作细胞蛋白的筛选及其功能分析

【目的】从鸡卵泡膜细胞蛋白中筛选和鉴定与膜联蛋白A2（ANXA2）相互作用的细胞蛋白并进行功能分析，为深入研究ANXA2调控鸡卵泡发育的作用机制提供理论依据。【方法】制备开产后30周龄贵州黄鸡的卵泡膜细胞，提取卵泡膜总蛋白后利用His Pull-Down联合质谱技术（LC-MS/MS）从卵泡膜细胞中筛选出与鸡ANXA2互作的细胞蛋白，然后通过GO数据库和KEEG数据库分别进行GO功能富集分析及KEEG信号通路注释分析，并利用STRING Version 11.0绘制蛋白互作网络图。【结果】通过His Pull-Down联合LC-MS/MS共鉴定获得41个鸡ANXA2互作细胞蛋白，GO功能富集分析发现这些互作细胞蛋白在分子功能、生物学进程和细胞组成均发挥作用。其中，在分子功能方面主要涉及蛋白结合（占58.06%）、催化活性（占19.35%）、核糖体结构（占16.13%）及细胞骨架结构组成（占6.45%），在生物学进程方面主要参与细胞骨架（占19.35%）、刺激反应（占19.35%）、翻译（占16.13%）、代谢过程（占12.90%）、细胞迁移（占12.90%）、蛋白折叠（占9.68%）和蛋白运输（占9.68%），而细胞组分显示以定位于细胞膜的蛋白为主（占32.26%）。鸡ANXA2蛋白互作细胞蛋白参与的KEEG信号通路主要有应激反应、代谢、翻译、信号转导、免疫系统和蛋白定位等。鸡ANXA2互作细胞蛋白互作网络可分为3条，即CNN2-FN1-MYH9-MYH10-ACTN1-CSRP1、ANXA1-ANXA2-ENO1-PRDX4-GPI-ATP5B-PRDX3-HSPA8-TUBB2A和CCT7-CCT4-GNB2L1-ATP5A1-RPS3-RPS3A-RPL23A-RPL22-RPS7；互作细胞蛋白间存在复杂的互作关系，其中又以膜联蛋白A1（ANXA1）与烯醇化酶-1（ENO1）及ANXA2的互作关系最明显。【结论】鸡ANXA2互作细胞蛋白主要参与细胞骨架形成、应对刺激和翻译等生物学过程，涉及应激反应、代谢、翻译、信号转导、免疫及蛋白定位等信号通路。其中，PRDX3、PRDX4、MYH9和TCSC可能通过与ANXA2蛋白相互作用而参与鸡卵巢相关疾病的发生，而ANXA1与ANXA2相互作用可能在鸡卵泡的发育及排卵过程中发挥重要调节作用。

Screening and functional analysis of cellular proteins interacting with annexin A2 in chicken theca cells

【Objective】The aim of this study was to screen，identify and analyze the functions of cellular proteins interacting with annexin A2（ANXA2）in chicken theca cells，which provided theoretical basis for further studying the mechanism of ANXA2 regulating chicken follicular development.【Method】The chicken theca cells of 30-week-old Guizhou yellow laying hens were prepared and then used to extract the total proteins. The chicken ANXA2 recombinant protein His-ANXA2 and His tag protein were used as bait proteins，respectively，to screen the cellular proteins interacting with His-ANXA2 and His by His Pull-Down combined with mass-spectrometric technique（LC-MS/MS）method. The GO function annotation，KEGG signaling pathway and protein-protein interaction network analysis of the identified proteins was performed using the bioinformatics online software. Protein interaction networks were drawn using STRING Version 11.0.【Result】A total of 41 cellular proteins that interacted with chicken ANXA2 were identified by His Pull-Down combined with LC-MS/MS. GO annotation analysis showed that these interacting cellular proteins played roles in molecular function，biological processes，and cell composition. As for the molecular function analysis，these proteins were involved in protein binding（58.06%），catalytic activity（19.35%），structural consituent of ribosome（16.13%），and structural constitute of cytoskeleton（6.45%）. The results of biological processes analysis revealed that these proteins mainly participated in cytoskeleton organization（19.35%），to stimulus response（19.35%），translation（16.13%），metabolic process （12.90%），cell migration（12.90%），protein folding（9.68%），and protein transport（9.68%）. However，the distribution of cell membrane（32.26%）was the main localization pattern in cell composition analysis. Meanwhile，KEGG signaling pathway analysis showed that these proteins were related to stress response，metabolism，translation，signal transduction， immune system，and protein localization. The protein-protein interaction networks were divided into three ways，including CNN2-FN1-MYH9-MYH10-ACTN1-CSRP1，ANXA1-ANXA2-ENO1-PRDX4-GPI-ATP5B-PRDX3-HSPA8-TUBB2A， CCT7-CCT4 -GNB2L1-ATP5A1-RPS3-RPS3A-RPL23A-RPL22-RPS7，which showed the complex interactions among them. Of which annexin A1（ANXA1）and enolase -1（ENO1）had the most obvious interactions with ANXA2.【Conclusion】The identified cellular proteins interacting with chicken ANXA2 are mainly involved in the biological processes of cytoskeleton formation，response to stimulationand translation，and the signaling pathways of stress response，metabolism，translation，signal transduction，immunity and protein localization. PRDX3，PRDX4，MYH9，and TCSC possibly participate in the occurrence of chicken ovaries related diseases by interacting with ANXA2，while ANXA1 interacting with ANXA2 might regulate the follicular development and ovulation in chickens.