Myeloperoxidase biosynthesis by a human promyelocytic leukemia cell line: insight into myeloperoxidase deficiency.
Myeloperoxidase biosynthesis by a human promyelocytic leukemia cell line: insight into myeloperoxidase deficiency.
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DOI：
10.1182/blood.v67.4.865.865
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发表时间：
1986-04
期刊：
影响因子：
20.3
通讯作者：
W. Nauseef
中科院分区：
文献类型：
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作者：
W. Nauseef
在人早幼粒细胞白血病细胞系 HL-60 中研究了髓过氧化物酶 (MPO) 的生物合成和加工，MPO 是人多形核白细胞 (PMN) 嗜天青颗粒中存在的一种阳离子酶。 HL-60 细胞产生大量具有酶活性的 MPO，其电泳行为与从正常 PMN 中分离出的 MPO 相同。成熟的 MPO 是一种分子量约为 150,000 的糖蛋白，在还原条件下由两个重轻原聚体 (α2β2) 组成，亚基分别为 59,000 和 13,500 mol wt。从 HL-60 细胞中分离的 MPO 信使 RNA (mRNA) 的初级翻译产物是分子量为 80,000 的单一多肽。在用[35S]-蛋氨酸标记的HL-60细胞中，通过免疫沉淀分离的标记MPO的摩尔重量为89,000。用糖苷内切酶 H 处理这个 89 kDa 的物质产生了 79 kDa 的肽，表明 89 kDa 的蛋白质含有高甘露糖侧链。 89-kDa 的物种没有可检测到的过氧化物酶活性。在追踪实验期间，一些 89-kDa 肽被加工成分子量为 39,000、59,000 和 13,500 的较小种类，尽管在 100 小时的追踪后，一小部分 89-kDa 肽仍未被加工。此外，少量 89-kDa 肽出现在没有任何加工过的较小肽的培养基中。这些研究表明，MPO 生物合成中的主要翻译产物是 80 kDa 的肽，经过信号肽的共翻译切割和糖基化，产生 89 kDa pro-MPO，pro-MPO 是含有 MPO α 和 β 亚基的单一多肽，并含有内切糖苷酶 H 敏感的高甘露糖侧链，pro-MPO 的翻译后修饰导致靶向溶酶体和 pro-MPO 蛋白水解成熟为活性酶。鉴于之前观察到 MPO 缺陷和正常 PMN 含有与 MPO 免疫化学相关的 89 kDa 蛋白，这些关于 MPO 生物合成的研究间接支持了这样的假设：pro-MPO 翻译后加工缺陷可能是遗传性 MPO 缺陷的基础。
The biosynthesis and processing of myeloperoxidase (MPO), a cationic enzyme present in the azurophilic granules of human polymorphonuclear leukocytes (PMNs), were studied in the human promyelocytic leukemia cell line, HL-60. HL-60 cells produce large quantities of enzymatically active MPO that has the same electrophoretic behavior as MPO isolated from normal PMNs. Mature MPO is a glycoprotein of approximately 150,000 molecular weight (mol wt) composed of two heavy-light protomers (alpha 2 beta 2) with subunits of 59,000 and 13,500 mol wt, respectively, under reducing conditions. The primary translation product of MPO messenger RNA (mRNA) isolated from HL-60 cells was a single polypeptide of mol wt 80,000. In HL-60 cells labeled with [35S]-methionine, the labeled MPO isolated by immunoprecipitation had a mol wt of 89,000. Treatment of this 89-kilodalton (kDa) species with endoglycosidase H produced a 79-kDa peptide, suggesting that the 89-kDa protein contained high-mannose side chains. The 89-kDa species had no detectable peroxidase activity. During chase experiments some of the 89-kDa peptide was processed to smaller species of mol wt 39,000, 59,000, and 13,500, although a fraction of the 89-kDa peptide remained unprocessed after a chase of 100 hours. In addition, a small amount of the 89-kDa peptide appeared in the medium without any of the processed smaller peptides. These studies suggest that the primary translation product in MPO biosynthesis is an 80-kDa peptide that undergoes cotranslational cleavage of the signal peptide and glycosylation to produce an 89-kDa pro-MPO, that pro-MPO is a single polypeptide containing the alpha and beta subunits of MPO and contains endoglycosidase H-susceptible high-mannose side chains, and that posttranslational modification of pro-MPO results in targeting to the lysosome and proteolytic maturation of pro-MPO to active enzyme. In light of the previous observation that MPO-deficient and normal PMNs contain an 89-kDa protein immunochemically related to MPO, these studies on MPO biosynthesis indirectly support the hypothesis that defective posttranslation processing by pro-MPO may underlie hereditary MPO deficiency.