Paone Gregorino, Stevens Linda A, Levine Rodney L, Bourgeois Christelle, Steagall Wendy K, Gochuico Bernadette R, Moss Joel
Pulmonary-Critical Care Medicine Branch, National Institutes of Health, Bethesda, Maryland 20892-1590.
Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1590.
J Biol Chem. 2006 Jun 23;281(25):17054-17060. doi: 10.1074/jbc.M603042200. Epub 2006 Apr 20.
Epithelial cells lining human airways and cells recruited to airways participate in the innate immune response in part by releasing human neutrophil peptides (HNP). Arginine-specific ADP-ribosyltransferases (ART) on the surface of these cells can catalyze the transfer of ADP-ribose from NAD to proteins. We reported that ART1, a mammalian ADP-ribosyltransferase, present in epithelial cells lining the human airway, modified HNP-1, altering its function. ADP-ribosylated HNP-1 was identified in bronchoalveolar lavage fluid (BALF) from patients with asthma, idiopathic pulmonary fibrosis, or a history of smoking (and having two common polymorphic forms of ART1 that differ in activity), but not in normal volunteers or patients with lymphangioleiomyomatosis. Modified HNP-1 was not found in the sputum of cystic fibrosis patients or in leukocyte granules of normal volunteers. The finding of ADP-ribosyl-HNP-1 in BALF but not in leukocyte granules suggests that the modification occurred in the airway. Most of the HNP-1 in the BALF from individuals with a history of smoking was, in fact, mono- or di-ADP-ribosylated. ART1 synthesized in Escherichia coli, glycosylphosphatidylinositol-anchored ART1 released with phosphatidylinositol-specific phospholipase C from transfected NMU cells, or ART1 expressed endogenously on C2C12 myotubes modified arginine 14 on HNP-1 with a secondary site on arginine 24. ADP-ribosylation of HNP-1 by ART1 was substantially greater than that by ART3, ART4, ART5, Pseudomonas aeruginosa exoenzyme S, or cholera toxin A subunit. Mouse ART2, which is an NAD:arginine ADP-ribosyltransferase, was able to modify HNP-1, but to a lesser extent than ART1. Although HNP-1 was not modified to a significant degree by ART5, it inhibited ART5 as well as ART1 activities. Human beta-defensin-1 (HBD1) was a poor transferase substrate. Reduction of the cysteine-rich defensins enhanced their ability to serve as ADP-ribose acceptors. We conclude that ADP-ribosylation of HNP-1 appears to be primarily an activity of ART1 and occurs in inflammatory conditions and disease.
衬于人类气道的上皮细胞以及募集到气道的细胞，部分通过释放人中性粒细胞肽（HNP）参与固有免疫反应。这些细胞表面的精氨酸特异性ADP-核糖基转移酶（ART）可催化ADP-核糖从NAD转移至蛋白质。我们报道，存在于人类气道衬里上皮细胞中的哺乳动物ADP-核糖基转移酶ART1可修饰HNP-1，改变其功能。在哮喘、特发性肺纤维化患者或有吸烟史（且具有两种活性不同的常见ART1多态形式）患者的支气管肺泡灌洗液（BALF）中鉴定出了ADP-核糖基化的HNP-1，但在正常志愿者或淋巴管平滑肌瘤病患者中未发现。在囊性纤维化患者的痰液或正常志愿者的白细胞颗粒中未发现修饰的HNP-1。在BALF中而非白细胞颗粒中发现ADP-核糖基-HNP-1，这表明修饰发生在气道中。事实上，有吸烟史个体的BALF中的大多数HNP-1是单ADP-核糖基化或双ADP-核糖基化的。在大肠杆菌中合成的ART1、用磷脂酰肌醇特异性磷脂酶C从转染的NMU细胞中释放的糖基磷脂酰肌醇锚定的ART1，或在C2C12肌管上内源性表达的ART1可修饰HNP-1上的精氨酸14，并在精氨酸24处有一个二级位点。ART1对HNP-1的ADP-核糖基化作用明显大于ART3、ART4、ART5、铜绿假单胞菌外毒素S或霍乱毒素A亚基。小鼠ART2作为一种NAD：精氨酸ADP-核糖基转移酶，能够修饰HNP-1，但程度低于ART1。尽管HNP-1未被ART5显著修饰，但它可抑制ART5以及ART1的活性。人β-防御素-1（HBD1）是一种较差的转移酶底物。富含半胱氨酸的防御素的减少增强了它们作为ADP-核糖受体的能力。我们得出结论，HNP-1的ADP-核糖基化似乎主要是ART1的活性，且发生在炎症状态和疾病中。
