Department of Biochemistry, Children’s Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Mol Cell Biol. 2012 Feb;32(4):774-82. doi: 10.1128/MCB.06195-11. Epub 2011 Dec 12.
Mannose 6-phosphate (Man6P) residues represent a recognition signal required for efficient receptor-dependent transport of soluble lysosomal proteins to lysosomes. Upon arrival, the proteins are rapidly dephosphorylated. We used mice deficient for the lysosomal acid phosphatase Acp2 or Acp5 or lacking both phosphatases (Acp2/Acp5(-/-)) to examine their role in dephosphorylation of Man6P-containing proteins. Two-dimensional (2D) Man6P immunoblot analyses of tyloxapol-purified lysosomal fractions revealed an important role of Acp5 acting in concert with Acp2 for complete dephosphorylation of lysosomal proteins. The most abundant lysosomal substrates of Acp2 and Acp5 were identified by Man6P affinity chromatography and mass spectrometry. Depending on the presence of Acp2 or Acp5, the isoelectric point of the lysosomal cholesterol-binding protein Npc2 ranged between 7.0 and 5.4 and may thus regulate its interaction with negatively charged lysosomal membranes at acidic pH. Correspondingly, unesterified cholesterol was found to accumulate in lysosomes of cultured hepatocytes of Acp2/Acp5(-/-) mice. The data demonstrate that dephosphorylation of Man6P-containing lysosomal proteins requires the concerted action of Acp2 and Acp5 and is needed for hydrolysis and removal of degradation products.
甘露糖 6-磷酸（Man6P）残基是溶酶体蛋白有效依赖受体运输到溶酶体所必需的识别信号。到达溶酶体后，这些蛋白会迅速去磷酸化。我们使用缺乏溶酶体酸性磷酸酶 Acp2 或 Acp5 或缺乏两种磷酸酶（Acp2/Acp5(-/-)）的小鼠，来研究它们在含 Man6P 蛋白的去磷酸化中的作用。用 tyloxapol 纯化的溶酶体级分进行的二维（2D）Man6P 免疫印迹分析表明，Acp5 与 Acp2 协同作用对于溶酶体蛋白的完全去磷酸化具有重要作用。通过 Man6P 亲和层析和质谱鉴定出 Acp2 和 Acp5 的最丰富的溶酶体底物。取决于 Acp2 或 Acp5 的存在，溶酶体胆固醇结合蛋白 Npc2 的等电点在 7.0 到 5.4 之间，因此可能调节其在酸性 pH 下与带负电荷的溶酶体膜的相互作用。相应地，在 Acp2/Acp5(-/-) 小鼠的培养肝细胞的溶酶体中发现未酯化的胆固醇积累。这些数据表明，含 Man6P 的溶酶体蛋白的去磷酸化需要 Acp2 和 Acp5 的协同作用，并且对于水解和去除降解产物是必需的。
