ADP ribosylation of human neutrophil peptide-1 regulates its biological properties

Abstract

In human airways, epithelial cells lining the lumen and intraluminal cells (e.g., polymorphonuclear cells) participate in the innate immune response. These cells secrete or express on their surfaces arginine-specific ADP ribosyltransferases. Defensins, antimicrobial proteins secreted by immune cells, are arginine-rich, leading us to hypothesize that ADP ribosylation could modify their biological activities. We found that an arginine-specific ADP ribosyltransferase-1 present on airway epithelial cells modifies Arg-14 of ␣ defensin-1. ADP-ribosylated defensin-1 had decreased antimicrobial and cytotoxic activities but still stimulated T cell chemotaxis and IL-8 release from A549 cells. Further, ADP-ribosylated defensin-1 inhibited cytotoxic and antimicrobial activities of unmodified defensin-1. We identified ADP-ribosylated defensin-1 in bronchoalveolar lavage fluid from smokers but not from nonsmokers, confirming its existence in vivo. Thus, airway mono-ADP-ribosyltransferases could have an important regulatory role in the innate immune response through modification of ␣ defensin-1 and perhaps other basic molecules, with alteration of their biological properties. In the human airway, epithelial cells, in concert with intraluminal cells (e.g., macrophages), contribute to the innate immune response. Activation of airway epithelial cells releases proinflammatory mediators and chemotaxins, leading to the recruitment of inflammatory cells (e.g., neutrophils) (1, 2). Activated neutrophils, in turn, release azurophilic granules that contain human neutrophil peptides (HNP), small cationic peptides ranging in size from 29-33 aa (3). HNPs are characterized by a high arginine content and contain three pairs of disulfidelinked cysteines (4). They have a broad spectrum of antimicrobial and cytotoxic activities and play an important role in the innate immune response against both Gram-positive and -negative bacteria, fungi, and certain enveloped viruses (5-8). High HNP levels have been found in airway secretions of patients with inflammatory lung diseases (9-11).A family of eukaryotic and prokaryotic mono-ADP ribosyltransferases (ARTs) catalyze the transfer of ADP ribose from NAD ϩ to arginine residues in proteins (12). Among the monoARTs are bacterial products (e.g., cholera toxin), which are responsible for the clinical syndrome in disease (13). A family of mammalian ARTs that are secreted or localized on the cell surface through glycosylphosphatidylinositol anchors (14-19) are expressed preferentially on epithelial and inflammatory cells such as lymphocytes and neutrophils (17,20). Substrates of the five known mammalian ARTs (ART-1-5) include proteins that are involved in critical cellular events (e.g., lymphocyte activation, neutrophil chemotaxis) (21-23). Two of these transferases, ART-1 and -5, specifically modify arginine residues in proteins. The finding of ARTs on airway epithelial and inflammatory cells prompted us to evaluate potential substrates for ADP ribosylation in the airway (24). Because of the high arginine c...

Combining affinity purification by ADP-ribose-binding macro domains with mass spectrometry to define the mammalian ADP-ribosyl proteome

“…The latter are due to glycosylphosphatidylinositol-anchored or secreted enzymes that can modify either soluble or plasma-membrane-associated protein targets, including the P 2 X 7 purinergic receptor, human neutrophil protein 1, and ␣ 7 integrin (13)(14)(15). Intracellular, enzymatic mono-ADPribosylation modifies proteins with roles in cell signaling and metabolism, such as the chaperone GRP78/BiP, the ␤ subunit of the heterotrimeric G proteins, and mitochondrial glutamate dehydrogenase (GDH) (16)(17)(18)(19).…”

ADP-ribosylation of human defensin HNP-1 results in the replacement of the modified arginine with the noncoded amino acid ornithine

“…HNP-1 was reduced, cleaved by trypsin, and analyzed by reverse-phase chromatography/mass spectrometry as described (17,18), except that the reverse-phase column was a Zorbax 300SB-C18, 2.1 ϫ 50 mm 3.5 m, and the mass spectrometer was an Agilent model G1969 (Agilent Technologies) with a time-of-flight detector. Mass spectra were deconvoluted with the Agilent software, MassHunter version 2, and the fraction of each species was calculated from the areas of the deconvoluted peaks.…”

“…After ADP-ribosylation by ART1, HNP-1 had reduced antimicrobial and cytotoxic activities but maintained its ability to recruit T lymphocytes and release IL-8 from A549 cells (17). To understand the cationicity of defensins, 3 of 4 arginines in HNP-1 (not arginine 5) were replaced with lysine or ornithine (9).…”

“…ART1 modifies the arginines of several substrates, including HNP-1, thereby altering their activity (15,16). ADPribosylation of HNP-1 decreased the antimicrobial and cytotoxic activities without affecting T-cell chemotaxis and IL-8 release from A549 lung carcinoma cells (17). In vitro, ART1 ADP-ribosylates HNP-1 on arginine 14 with a secondary site on arginine 24.…”

NAD+-Consuming Enzymes in the Regulation of Lung Immune Responses

“…It has been also shown that ART1, which is constitutively expressed on the surface of cells lining the airway lumen [23], modifies defensin human neutrophil peptide-1 (HNP-1), an antimicrobial peptide released by neutrophils. Indeed, ADP-ribosylation by ART1 and not other mART decreased antimicrobial and cytotoxic activities of HNP-1 but not the ability to stimulate T cell chemotaxis and interleukin-8 (IL-8) release from lung epithelial cells [26]. Moreover ADP-ribosylated HNP-1 was identified in bronchoalveolar lavage fluid (BALF) from patients with asthma, idiopathic pulmonary fibrosis, or a history of smoking but not in normal volunteers [27].…”