Thaysen-Andersen Morten, Venkatakrishnan Vignesh, Loke Ian, Laurini Christine, Diestel Simone, Parker Benjamin L, Packer Nicolle H
From the Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales-2109, Australia,
From the Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales-2109, Australia.
J Biol Chem. 2015 Apr 3;290(14):8789-802. doi: 10.1074/jbc.M114.631622. Epub 2015 Feb 2.
Unlike plants and invertebrates, mammals reportedly lack proteins displaying asparagine (N)-linked paucimannosylation (mannose(1-3)fucose(0-1)N-acetylglucosamine(2)Asn). Enabled by technology advancements in system-wide biomolecular characterization, we document that protein paucimannosylation is a significant host-derived molecular signature of neutrophil-rich sputum from pathogen-infected human lungs and is negligible in pathogen-free sputum. Five types of paucimannosidic N-glycans were carried by compartment-specific and inflammation-associated proteins of the azurophilic granules of human neutrophils including myeloperoxidase (MPO), azurocidin, and neutrophil elastase. The timely expressed human azurophilic granule-resident β-hexosaminidase A displayed the capacity to generate paucimannosidic N-glycans by trimming hybrid/complex type N-glycan intermediates with relative broad substrate specificity. Paucimannosidic N-glycoepitopes showed significant co-localization with β-hexosaminidase A and the azurophilic marker MPO in human neutrophils using immunocytochemistry. Furthermore, promyelocyte stage-specific expression of genes coding for paucimannosidic proteins and biosynthetic enzymes indicated a novel spatio-temporal biosynthetic route in early neutrophil maturation. The absence of bacterial exoglycosidase activities and paucimannosidic N-glycans excluded exogenous origins of paucimannosylation. Paucimannosidic proteins from isolated and sputum neutrophils were preferentially secreted upon inoculation with virulent Pseudomonas aeruginosa. Finally, paucimannosidic proteins displayed affinities to mannose-binding lectin, suggesting immune-related functions of paucimannosylation in activated human neutrophils. In conclusion, we are the first to document that human neutrophils produce, store and, upon activation, selectively secrete bioactive paucimannosidic proteins into sputum of lungs undergoing pathogen-based inflammation.
与植物和无脊椎动物不同，据报道哺乳动物缺乏显示天冬酰胺（N）连接的寡甘露糖基化（甘露糖(1-3)岩藻糖(0-1)N-乙酰葡糖胺(2)天冬酰胺）的蛋白质。借助全系统生物分子表征技术的进步，我们证明蛋白质寡甘露糖基化是来自病原体感染的人肺富含中性粒细胞痰液中一种重要的宿主衍生分子特征，而在无菌痰液中可忽略不计。人类中性粒细胞嗜天青颗粒的特定区室和炎症相关蛋白携带五种类型的寡甘露糖基化N-聚糖，包括髓过氧化物酶（MPO）、天青杀素和中性粒细胞弹性蛋白酶。及时表达的人类嗜天青颗粒驻留β-己糖胺酶A显示出通过修剪具有相对广泛底物特异性的杂合/复合型N-聚糖中间体来生成寡甘露糖基化N-聚糖的能力。使用免疫细胞化学方法，寡甘露糖基化N-糖表位在人类中性粒细胞中与β-己糖胺酶A和嗜天青标记物MPO显示出显著的共定位。此外，编码寡甘露糖基化蛋白和生物合成酶的基因在早幼粒细胞阶段特异性表达，表明在中性粒细胞早期成熟过程中有一条新的时空生物合成途径。细菌外切糖苷酶活性和寡甘露糖基化N-聚糖的缺失排除了寡甘露糖基化的外源起源。用强毒铜绿假单胞菌接种后，分离的中性粒细胞和痰液中的寡甘露糖基化蛋白优先分泌。最后，寡甘露糖基化蛋白对甘露糖结合凝集素表现出亲和力，表明寡甘露糖基化在活化的人类中性粒细胞中具有免疫相关功能。总之，我们首次证明人类中性粒细胞产生、储存并在激活后将生物活性寡甘露糖基化蛋白选择性分泌到经历基于病原体炎症的肺部痰液中。
