Nauseef W M
Department of Medicine, Veterans Administration Medical Center, Iowa City, IA.
Blood. 1987 Oct;70(4):1143-50.
Myeloperoxidase (MPO) is a lysosomal enzyme present in the azurophilic granules of human neutrophils and monocytes and is important for optimal oxygen-dependent killing of microorganisms. The native molecule is a heterodimer composed of a pair of heavy-light protomers, each containing a 59-kDa and 13.5-kDa subunit. The intracellular processing during biosynthesis of MPO was examined in the human promyelocytic cell line HL-60. Endoglycosidase H and F digestion of immunoprecipitated pro-MPO demonstrated the presence of five N-linked--high-mannose oligosaccharide side chains and no complex mannose units. Incorporation of the threonine analogue beta-hydroxynorvaline produced species approximately 2.5 kDa and approximately 5 kDa smaller than the fully glycosylated pro-MPO, suggesting that two of the glycans were in the asparagine-X-threonine tripeptide sequence. Processing of pro-MPO occurred very rapidly, within approximately five minutes, and was best identified using glucosidase inhibitors. The presence of such inhibitors resulted in synthesis of a 92-kDa glycoprotein rather than the usually identified 89-kDa peptide. Swainsonine, a Golgi mannosidase inhibitor, did not alter the size of the earliest synthesized protein, suggesting that pro-MPO exited the endoplasmic reticulum or cis-Golgi proximal to the site of mannosidases. Intracellular transport and proteolytic maturation of MPO was retarded by weak bases (NH4Cl, chloroquine) or monensin at concentrations shown to raise intralysosomal pH. However, these agents did not qualitatively alter transport nor increase secretion. Thus, although MPO biosynthesis resembled that of other lysosomal enzymes, significant differences exist, including only limited oligosaccharide processing and intracellular transport and proteolytic maturation of pro-MPO that was only retarded by alkalinizing lysosomes without affecting the products or the fraction of pro-MPO secreted. Characterization of the determinants for targeting and of the regulatory factors in processing lysosomal enzymes in myeloid cells will provide insight into the molecular mechanisms underlying common disorders such as myeloperoxidase deficiency.
髓过氧化物酶（MPO）是一种存在于人类中性粒细胞和单核细胞嗜天青颗粒中的溶酶体酶，对于微生物的最佳氧依赖性杀伤至关重要。天然分子是由一对重链-轻链原体组成的异二聚体，每个原体包含一个59 kDa和13.5 kDa的亚基。在人早幼粒细胞系HL-60中研究了MPO生物合成过程中的细胞内加工。对免疫沉淀的前MPO进行内切糖苷酶H和F消化，结果表明存在五个N-连接的高甘露糖寡糖侧链，没有复合甘露糖单元。苏氨酸类似物β-羟基正缬氨酸的掺入产生了比完全糖基化的前MPO小约2.5 kDa和约5 kDa的物种，这表明其中两个聚糖位于天冬酰胺-X-苏氨酸三肽序列中。前MPO的加工非常迅速，大约在五分钟内完成，使用葡萄糖苷酶抑制剂可以最好地识别这一过程。这些抑制剂的存在导致合成了一种92 kDa的糖蛋白，而不是通常识别的89 kDa肽。高尔基体甘露糖苷酶抑制剂苦马豆素并没有改变最早合成蛋白质的大小，这表明前MPO在甘露糖苷酶位点近端离开内质网或顺式高尔基体。MPO的细胞内运输和蛋白水解成熟受到弱碱（NH4Cl、氯喹）或莫能菌素的抑制，这些物质在所示浓度下可提高溶酶体内pH。然而，这些试剂并没有定性地改变运输，也没有增加分泌。因此，尽管MPO的生物合成与其他溶酶体酶相似，但仍存在显著差异，包括仅有限的寡糖加工以及前MPO的细胞内运输和蛋白水解成熟，其仅因溶酶体碱化而延迟，而不影响产物或分泌的前MPO比例。对髓细胞中溶酶体酶靶向决定因素和加工调节因子的表征将有助于深入了解诸如髓过氧化物酶缺乏症等常见疾病的分子机制。
