乙酰肝素酶信号肽对其活性功能的影响研究  

Impact of signal peptide on post-translation and activation process of heparanase

作　　者：李燕杰[1] 马远方[1] 李吉学[2] 汤国营[3] 彭清忠[3] 朱厚础[3] 

机构地区：[1]河南大学医学院免疫学研究所,河南开封475004 [2]河南大学天然药物与免疫工程省级重点实验室,河南开封475004 [3]军事医学科学院生物工程研究所,北京100071

出　　处：《军事医学科学院院刊》2008年第2期129-132,158,共5页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的:乙酰肝素酶在肿瘤转移过程中起关键作用,其信号肽在该酶翻译后处理过程中起重要作用。本研究旨在探索乙酰肝素酶在哺乳动物细胞中的表达特性及其信号肽对此酶功能活性的影响。方法:从人胎盘cDNA文库中扩增乙酰肝素酶全长编码基因,克隆入pGEM-T载体中,测序鉴定序列完全正确后,将此基因的全长和不含信号肽基因序列分别克隆入真核表达载体pcDNA4.1/Myc-His中构建pcDNA4.1/Myc-His full HPA和pcDNA4.1/Myc-His part HPA,转化COS-7细胞进行瞬时表达,分析这两种乙酰肝素酶蛋白的表达特性及功能活性。结果:在转染了乙酰肝素酶全基因的COS-7细胞裂解液中检测到该酶的功能活性及大小约53×103(Mr)的目的蛋白免疫印迹表达带,为切割激活后乙酰肝素酶羧基端大亚基与标签蛋白的融合体。在转染了敲除信号肽的乙酰肝素酶基因的COS-7细胞裂解液中测到未被切割激活的大小约65×103(Mr)的目的蛋白免疫印迹表达带,未能检测到该酶的功能活性。结论:在COS-7细胞中表达的完整的乙酰肝素酶蛋白和不含信号肽的乙酰肝素酶蛋白均位于细胞内,没有或很少被分泌到细胞外,提示该酶在正常状态下主要分布在细胞内。含信号肽的酶蛋白在翻译后处理过程中被细胞内的蛋白酶切割成2个亚基而被激活,具有功能活性。而不含信号肽的酶蛋白没有被切割而成为有活性的酶,提示乙酰肝素酶的信号肽对其翻译后加工、激活过程有重要的影响。Objective： To explore the role of the signal peptide in the expression regulation pathway of heparanase which is one of the key enzymes in malignant metastasis. Methods： The eukaryotic expression vector which expressed the full heparanase protein and the vector with the signal peptide knocked out were constructed, After being transfected into COS-7 cells, the c-myc epitope was fused with the C-termlnals of target proteins and detected by Western blot to analyze the heparanase expression in respective cells. Results： The full heparanase protein and the one lacking its signal peptide were detected by Western blot in the mammalian cell lysate, but not in the culture medium. Heparanase with signal peptide was processed into two subunits by proteolysis, N-terminus 8 × 10^3 and C-terminus 50× 10^3 polypeptides, which were detected by antibody against myc as it was fused with myc protein. However, the heparanase protein without signal peptide expressed by COS-7 cells was detected as 65× 10^3 polypeptides since it could not be incised and activated. Conclusion： The results suggested that heparanase be physiologically located in, but not secreted out of the cell. It should be further investigated how the enzyme is over-expressed on the surface of the malignant cells. The expressed heparanase without its signal peptide could not be processed into two subunits to become the active enzyme. This suggested that the signal peptide of heparanase has impact on its past-translation processing, activation and function.

关 键 词：乙酰肝素酶 信号肽 COS-7细胞 激活 

分 类 号：R730.2[医药卫生—肿瘤]