Andersson E, Hellman L, Gullberg U, Olsson I
Department of Hematology, Research Department 2, E-blocket, University Hospital, S-221 85 Lund, Sweden.
J Biol Chem. 1998 Feb 20;273(8):4747-53. doi: 10.1074/jbc.273.8.4747.
Myeloperoxidase (MPO), stored in azurophil granules of neutrophils, is critical for an optimal oxygen-dependent microbicidal activity of these cells. Pro-MPO goes through a stepwise proteolytic trimming with elimination of an amino-terminal propeptide to yield one heavy and one light polypeptide chain. The propeptide of MPO may have a role in retention and folding of the nascent protein into its tertiary structure or in targeting of pro-MPO for processing and storage in granules. A propeptide-deleted pro-MPO mutant (MPODeltapro) was constructed to determine if deletion of the propeptide interferes with processing and targeting after transfection to the myeloid 32D cell line. Transfection of full-length cDNA for human MPO results in normal processing and targeting of MPO to cytoplasmic dense organelles. Although the efficiency of incorporation was lower for MPODeltapro, both pro-MPO and MPODeltapro showed heme incorporation indicating that the propeptide is not critical for this process. Deletion of the propeptide results in synthesis of a protein that lacks processing into mature two-chain forms but rather is degraded intracellularly or secreted. The finding of continued degradation of MPODeltapro in the presence of lysosomotrophic agents or brefeldin A rules out that the observed degradation takes place after transfer to granules. Intracellular pro-MPO has high mannose oligosaccharide side chains, whereas stored mature MPO was found to have both high mannose and complex oligosaccharide side chains as judged by only partial sensitivity to endoglycosidase H. The propeptide may normally interfere with the generation of certain complex oligosaccharide chain(s) supported by the finding of high mannose side chains in secreted pro-MPO and lack of them in MPODeltapro that contained complex oligosaccharide side chains only. In conclusion, elimination of the propeptide of pro-MPO blocks the maturation process and abolishes accumulation of the final product in granules suggesting a critical role of the propeptide for late processing of pro-MPO and targeting for storage in granules.
髓过氧化物酶（MPO）储存于中性粒细胞的嗜天青颗粒中，对这些细胞的最佳氧依赖性杀菌活性至关重要。前MPO经历逐步的蛋白水解修剪，去除氨基末端前肽，产生一条重链和一条轻链多肽。MPO的前肽可能在新生蛋白保留并折叠成三级结构中发挥作用，或在前MPO加工和储存于颗粒的靶向过程中发挥作用。构建了一个缺失前肽的前MPO突变体（MPOΔpro），以确定前肽缺失是否会在转染至髓系32D细胞系后干扰加工和靶向。转染人MPO全长cDNA可导致MPO正常加工并靶向细胞质致密细胞器。虽然MPOΔpro的掺入效率较低，但前MPO和MPOΔpro均显示有血红素掺入，表明前肽对该过程并非至关重要。前肽缺失导致合成的蛋白质缺乏加工成成熟双链形式，而是在细胞内降解或分泌。在存在溶酶体营养剂或布雷菲德菌素A的情况下，MPOΔpro持续降解，这排除了观察到的降解发生在转移至颗粒之后。细胞内前MPO具有高甘露糖寡糖侧链，而储存的成熟MPO被发现同时具有高甘露糖和复杂寡糖侧链，这是根据对内切糖苷酶H的部分敏感性判断的。前肽通常可能会干扰某些复杂寡糖链的生成，这一发现得到了支持，即分泌的前MPO中存在高甘露糖侧链，而仅含有复杂寡糖侧链的MPOΔpro中则没有。总之，去除前MPO的前肽会阻断成熟过程，并消除最终产物在颗粒中的积累，这表明前肽对前MPO的后期加工和储存于颗粒的靶向具有关键作用。
