基于蛋白质组学揭示长牡蛎中汞的富集、形态转化与代谢解毒机制

Proteomics-based investigation into the mechanisms of mercury enrichment, speciation transformation and depuration in Magallana gigas

中文关键词:  长牡蛎  汞  蛋白质组学  生物标志物  分子机制

英文关键词:Magallana gigas  mercury  proteomics  biomarkers  molecular mechanisms

中文摘要:

      目的 探究牡蛎对汞的富集、形态转化及代谢净化机制, 阐明MeHg的形成与清除动力学。方法 采用双箱动力学模型模拟汞在长牡蛎体内的累积、转化与净化过程; 采用Label-free非标记定量蛋白质组学技术对高浓度汞暴露组(Hg-H)牡蛎消化腺进行蛋白质鉴定与差异表达分析, 开展亚细胞定位、结构域预测、基因本体(gene ontology, GO)功能和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes, KEGG)数据库通路富集分析; 利用实时荧光定量(quantitative real-time polymerase chain reaction, qPCR)技术分析0~60 d时间内消化腺中金属硫蛋白(metallothionein, MT)和热休克蛋白70 (heat shock protein 70, Hsp70)基因的表达。结果 汞胁迫与净化实验结果显示, 牡蛎消化腺中汞、甲基汞含量与胁迫浓度成正比, 并且甲基汞含量最高峰出现延迟效应; qPCR结果显示Hsp70与MT基因表达呈时序性协同变化, Hsp70于胁迫5 d迅速上调(7.15倍), 净化40 d达峰值(32.09倍); MT表达呈双峰模式(胁迫10 d 5.81倍, 净化40 d 3.55倍), 其动态与蛋白组结果一致。蛋白质组学分析共定性蛋白5487个, 筛选得到多个时间点对比下的显著差异表达蛋白, 差异蛋白显著富集于细胞核、胞外区、细胞膜等区域, 涉及热休克蛋白(Hsp20/Hsp70)、C型凝集素、过氧化氢酶、C1q等结构域。GO和KEGG分析表明, 差异蛋白主要参与应激反应、免疫应答、抗氧化过程、氨基酸代谢和信号转导等生物过程, 且花生四烯酸代谢通路可能与汞的排出过程密切相关。结论 汞胁迫可显著影响牡蛎消化腺蛋白质表达谱, 触发包括热休克蛋白、免疫因子和代谢酶在内的多蛋白协同响应, 且与汞/甲基汞的累积、转化及排出过程密切相关。本研究通过蛋白组、qPCR与汞富集与净化的整合分析, 揭示了牡蛎应对汞胁迫的分子机制, 为贝类污染毒理研究和水产品安全监测提供了理论支撑。

英文摘要:

      Objective To investigate the mechanisms of mercury accumulation, speciation transformation and metabolic depuration in Magallana gigas, clarify the kinetics of methylmercury formation and elimination.?Methods A two-compartment kinetic model was employed to simulate the accumulation, transformation, and depuration processes of mercury in Magallana gigas. Label-free quantitative proteomics technology combined with MaxQuant software was used to identify and analyze differentially expressed proteins in the digestive gland of oysters from the high-concentration mercury exposure group (Hg-H). Analyses included subcellular localization prediction, domain prediction, gene ontology (GO) Functional enrichment analysis and kyoto encyclopedia of genes and genomes (KEGG) Database Pathway enrichment analysis. The expression dynamics of metallothionein (MT) and heat shock protein 70 (Hsp70) genes in the digestive gland over 0–60 d were detected using quantitative real-time polymerase chain reaction (qPCR). Results The results of mercury accumulation and depuration experiments indicated that the content of mercury and methylmercury in the digestive gland of oysters were positively proportional to the stress concentration, and a lag effect occurred in the peak content of methylmercury. qPCR results showed temporally coordinated changes in Hsp70 and MT gene expression: Hsp70 was rapidly up-regulated (7.15-fold) on 5 d of stress and peaked (32.09-fold) on 40 d of depuration; MT expression showed a bimodal pattern (5.81-fold on 10 d of stress and 3.55-fold on 40 d of depuration), consistent with proteomic findings. Proteomic analysis identified 5487 proteins, and differentially expressed proteins were significantly enriched in subcellular locations such as the nucleus, extracellular region, and plasma membrane, and were associated with domains including heat shock proteins (Hsp20/Hsp70), C-type lectins, catalase, and C1q. GO and KEGG analyses indicated that these proteins were primarily involved in biological processes such as stress response, immune response, antioxidant processes, amino acid metabolism and signal transduction (e.g., PI3K-Akt and MAPK pathways). The arachidonic acid metabolism pathway might be closely associated with mercury elimination. Conclusion Mercury stress significantly alters the protein expression profile in oyster digestive glands, triggering a coordinated response involving molecular chaperones, immune factors, and metabolic enzymes, which is closely related to the accumulation, transformation, and elimination of mercury/methylmercury. Through integrated analysis of proteomics, qPCR, as well as mercury bioaccumulation and depuration processes, this study systematically elucidated the molecular mechanisms by which oysters cope with mercury stress, providing a theoretical foundation for toxicological studies on shellfish contamination and safety surveillance of aquatic products.