重组人CREG蛋白与溶酶体组织蛋白酶和M6P/IGFⅡR的相互作用

摘要

BACKGROUND:It has been found that cel ular repressor of E1A-stimulated genes (CREG) is a lysosomal protein binding directly to the mannose-6-phosphate (M6P)/insulin-like growth factor II receptor (IGFIIR) and depends on the interaction with M6P receptors for efficient delivery to lysosomes OBJECTIVE:To study the interactions between the exogenous CREG protein and cathepsins and M6P/IGFIIR and to confirm the effect of CREG protein on expression and distribution of M6P/IGFIIR. METHODS:Double-stained immunofluorescence and coimmunoprecipitation were applied to observe the interactions between the exogenous CREG protein and cathepsin B, cathepsin L and M6P/IGFIIR. Using gain-of-function and loss-of-function approaches, the effect of CREG on expression and distribution of M6P/IGFIIR were studied by western blot assay and immunofluorescence staining. RESULTS AND CONCLUSION:Double-stained immunofluorescence and coimmunoprecipitation analyses confirmed the direct interactions between the exogenous CREG protein and cathepsin B, cathepsin L and M6P/IGFIIR. It was verified that CREG plays a critical role not in the expression but in the distribution of M6P/IGFIIR using gain-of-function and loss-of-function approaches. These findings provide evidence that exogenous CREG protein is located in lysosomes and has interactions with cathepsins and M6P/IGFIIR, also CREG plays a critical role in the distribution of M6P/IGFIIR.%背景：以往研究证实，CREG是一种与M6P/IGFⅡR直接结合的溶酶体蛋白，并依赖于与M6P受体的相互作用有效转运至溶酶体。　　目的：分析外源性CREG蛋白与溶酶体组织蛋白酶和M6P/IGFⅡR的相互作用关系及其对M6P/IGFⅡR表达变化及细胞内定位的影响。　　方法：应用细胞免疫荧光双染和免疫共沉淀方法，观察外源性 CREG 蛋白与溶酶体组织蛋白酶和 M6P/IGFⅡR的相互作用关系，并应用gain-of-function和loss-of-function模型，通过Western blot和细胞免疫荧光双染方法，观察CREG对M6P/IGFⅡR表达变化及细胞内定位的影响。　　结果与结论：细胞免疫荧光双染和免疫共沉淀方法证实外源性 CREG 蛋白与溶酶体组织蛋白酶和 M6P/IGFⅡR有直接相互作用关系；应用gain-of-function和loss-of-function模型进一步证实，CREG对M6P/IGFⅡR表达变化无影响，而影响其在细胞内的定位。提示外源性CREG蛋白定位于溶酶体，且与溶酶体组织蛋白酶和M6P/IGFⅡR有相互作用关系，并影响M6P/IGFⅡR的细胞内定位。