Identification and validation of mannose 6-phosphate glycoproteins in human plasma reveal a wide range of lysosomal and non-lysosomal proteins.
Identification and validation of mannose 6-phosphate glycoproteins in human plasma reveal a wide range of lysosomal and non-lysosomal proteins.
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人血浆中 6-磷酸甘露糖糖蛋白的鉴定和验证揭示了多种溶酶体和非溶酶体蛋白。
DOI：
10.1074/mcp.m600030-mcp200
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Lobel,Peter
中科院分区：
文献类型：
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作者：
Sleat,DavidE;Wang,Yanhong;Sohar,Istvan;Lackland,Henry;Li,Yan;Li,Hong;Zheng,Haiyan;Lobel,Peter
酸水解酶的活性通常局限于细胞内的溶酶体，这是一种膜分隔的细胞质细胞器，主要负责大分子的降解。然而，溶酶体蛋白也存在于人血浆中，其中一部分保留甘露糖6-磷酸（Man-6-P），这是一种n-链聚糖的修饰，可被Man-6-P受体（MPRs）识别，通常将这些蛋白定向到溶酶体。本研究利用固定化MPRs亲和层析技术纯化了人血浆中Man-6-P蛋白的糖型，并利用二维凝胶电泳和串联质谱技术对该亚蛋白质组进行了表征。正如预期的那样，我们鉴定了许多已知的和潜在的候选溶酶体蛋白。此外，我们还发现了许多丰富的经典血浆蛋白，即使在连续两轮亲和纯化后也能保留下来。考虑到它们在血浆中的丰度，我们最初认为这些蛋白质可能是污染物，但使用mpr纯化的糖肽对man -6磷酸化位点的质谱研究显示，这些经典血浆蛋白的某些比例含有Man-6-P修饰。我们认为这些糖蛋白被溶酶体磷酸酶在低水平上磷酸化，但它们的高丰度导致在血浆中检测到Man-6-P糖型。这些结果可能为man -6磷酸化的分子过程提供有用的见解，并突出了Man-6-P的存在可能不指示溶酶体功能的情况。此外，血浆Man-6-P糖蛋白组的表征将促进基于质谱的工具的发展，以诊断溶酶体贮积性疾病，并研究含有Man-6-P糖蛋白在更广泛的人类疾病中的作用及其作为生物标志物的潜在效用。
Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification onN-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers.