Proteomic analysis of human lysosomes: Application to monocytic and breast cancer cells
Proteomic analysis of human lysosomes: Application to monocytic and breast cancer cells
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DOI：
10.1002/1615-9861(200208)2:8
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发表时间：
2002-08
期刊：
影响因子：
3.4
通讯作者：
A. Journet;A. Chapel;S. Kieffer;F. Roux;J. Garin
中科院分区：
文献类型：
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作者：
A. Journet;A. Chapel;S. Kieffer;F. Roux;J. Garin
迄今为止，已鉴定出约 50 种溶酶体水解酶，其中大多数通过特定的甘露糖-6-磷酸 (M-6-P) 标签靶向溶酶体。随着预计会发现更多的溶酶体水解酶，我们对可溶性溶酶体蛋白进行了蛋白质组学研究。诱导人类细胞分泌 M-6-P 蛋白，该蛋白在固定化 M-6-P 受体上进行亲和纯化。纯化的蛋白质通过二维电泳解析并通过质谱分析。鉴定出 22 种蛋白质，其中 16 种是众所周知的溶酶体水解酶。其余物种分布如下：附睾特异性 α-甘露糖苷酶是一种新的甘露糖苷酶同源物，胱抑素 F 和 CREG（E1A 刺激基因的细胞阻遏物）先前被鉴定为 M-6-P 蛋白（Journet 等人，Electrophoresis 2000, 21, 3411–3419），最后三种不是水解酶，最多可达 现在被认为是非溶酶体的。此人类 U937 M-6-P 蛋白的二维参考图随后用于与从分化为巨噬细胞样细胞的 U937 或从人乳腺癌 MCF7 细胞中纯化的 M-6-P 蛋白进行比较。佛波酯诱导的 U937 细胞分化导致有限的蛋白水解酶或离散数量的水解酶的成熟。从 MCF7 样品中鉴定出另外五种溶酶体水解酶。这些结果证明了这种程序在分析各种细胞系的溶酶体含量、发现新的 M-6-P 蛋白以及指出未知的生物过程方面的有用性。
To date, about fifty lysosomal hydrolases have been identified, and most of them are targeted towards the lysosomes through a specific mannose‐6‐phosphate (M‐6‐P) tag. As more lysosomal hydrolases were expected to be discovered, we performed a proteomic study of soluble lysosomal proteins. Human cells were induced to secrete M‐6‐P proteins which were affinity purified on immobilized M‐6‐P receptor. The purified proteins were resolved by two‐dimensional electrophoresis and analyzed by mass spectrometry. Twenty‐two proteins were identified, among which 16 were well‐known lysosomal hydrolases. The remaining species distributed as follows: epididymis‐specific α‐mannosidase is a new mannosidase homolog, cystatin F and CREG (cellular repressor of E1A‐stimulated genes) were previously identified as M‐6‐P proteins (Journet et al., Electrophoresis 2000, 21, 3411–3419), and the last three, which are not hydrolases, were up to now considered as nonlysosomal. This two‐dimensional reference map of human U937 M‐6‐P proteins was afterwards used for comparison with M‐6‐P proteins purified either from U937 differentiated into macrophage‐like cells, or from human breast cancer MCF7 cells. Phorbol ester induced differentiation of U937 cells led to limited proteolytic cleavage or maturation of a discrete number of hydrolases. Five additional lysosomal hydrolases were identified from MCF7 samples. These results prove the usefulness of such a procedure to analyze the lysosomal content of various cell lines, to discover new M‐6‐P proteins, as well as to point towards unknown biological processes.