肝癌中四跨膜蛋白CD151分子伴侣筛选和鉴定.docVIP

  肝癌中四跨膜蛋白 CD151 分子伴侣筛选和 鉴定# * 5 10 15 20 25 30 35 40  摘要：目的：用免疫共沉淀结合质谱分析筛选肝癌 CD151 功能复合体。方法：应用 CD151 抗体从肝癌细胞株 HCCLM3 细胞蛋白中富集与 CD151 结合蛋白，通过质谱分析与多肽比对 初步确立 CD151 功能复合体，采用免疫共沉淀后蛋白质印迹验证。结果：免疫共沉淀后质 谱鉴定出 58 个包括 CD151 有定量信息的蛋白。 其中膜蛋白 11 个，包括整合素 α3、α6、β1 和 c-Met、LDLR 以及 TNFα1R 等，信号转导蛋白 6 个包括 14-3-3、p195IQGAP1、HSP70、 PKC、RuvB1 与 2、IKK 等，通过免疫共沉淀后的 westem blot 验证了整合素 α6、β1 与 CD151 形成复合物，证实了质谱分析结果的可靠性。结论：四跨膜蛋白 CD151 与整合素 α6、β1 分 子伴侣在肝癌细胞表面形成复合物。 关键词：四跨膜蛋白；CD151；整合素 β1；肝细胞癌；免疫共沉淀；质谱 中图分类号：R735.7 Screen and identification of partner of CD151 in HCC SHI Guoming, RUAN Jin, KA Aiwu, ZHOU Jian, FAN Jia (Liver Cancer Institute, Zhongshan hospital, Fudan University, ShangHai 200032) Abstract: Objective: To screen the associates of HCC invasion related CD151 functional complex using co-immnoprecipitation and Liquid chromatography coupled to mass spectrometry (LC-MS/MS). Methods: Freshly extracted proteins from HCCLM3 cell line were immunoprecipitated with CD151 antibody. LC-MS/MS was used to identify the associates of CD151 functional complex. Bioinformatics analysis and protein validation were performed. Results: Fifty 58 proteins including CD151 were identified after LC-MS/MS. Among them, 11 were membrane proteins including α3, α6, β1, c-Met, LDLR and TNFα1R etc. and 6 signal transduction proteins such as 14-3-3, p195IQGAP1, HSP70, RuvB1 and 2, PKC and IKK. The functional complex formed by CD151, integrins α6 and β1 was examined by western blot and confirmed the reliability of the LC-MS/MS result. Conclusion: Tetaspanin CD151 and integrins α6 and β1 form a functional complex in HCC cell line. Keywords: Tetraspanin; CD151; Integrin β1; HCC; immnoprecipitation; Liquid chromatography coupled to mass spectrometry 0 引言 四跨膜蛋白 CD151 在人体细胞中分布广泛，参与细胞多种生理功能和病理过程[1-3]。近 年研究发现，CD151 参与多种肿瘤的侵袭和转移[1-5]。我们前期研究发现 CD151 通过影响肝 细胞生长因子（hepatic growth factor, HGF）/肝细胞生长因子受体（c-Met）信号参与肝癌细 胞侵袭、转移，其是影响肝癌侵袭和转移的关键分子[1]。CD151 过度表达还可促进金属基质 蛋白酶 9（MMP9）的分泌和表达，参与肝癌新生血管形成、侵袭和转移[3]；而且，肝癌细 胞中整合素 α6 与内源性 CD151 形成