ADP-ribosyltransferase-specific Modification of Human Neutrophil Peptide-1

Gregorino Paone,Linda A Stevens,Rodney L Levine,Christelle Bourgeois,Wendy K Steagall,Bernadette R Gochuico,Joel Moss

doi:10.1074/jbc.m603042200

Abstract

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.

Highlights

Inflammatory cells, such as neutrophils, are recruited to the airway of patients with chronic lung disorders [1, 2]
In idiopathic pulmonary fibrosis and asthma bronchoalveolar lavage fluid (BALF), both mono- and di-modified human neutrophil peptides (HNP)-1 were identified suggesting that, in these patients, neutrophils were recruited to the airway where the released defensin could come in contact with epithelial cells expressing an arginine-specific ADP-ribosyltransferase
HNP was isolated from leukocyte granules, but modified forms were not identified, consistent with the modification of HNP-1 occurring after its release from neutrophils

Summary

Introduction

Inflammatory cells, such as neutrophils, are recruited to the airway of patients with chronic lung disorders [1, 2]. Defensins have 29 to 35 amino acids, are arginine-rich, and contain three disulfide bridges They are antimicrobial for Gram-negative and Gram-positive bacteria, fungi, and enveloped viruses, cytotoxic for epithelial cells, and chemotactic for T cells. Mono-ADP-ribosylation, in which the ADP-ribose moiety of NAD is transferred to a protein substrate, is catalyzed by amino acid-specific ADP-ribosyltransferases (ARTs) [10]. This post-translational modification occurs in viruses, bacterial, and eukaryotic cells. ART1, -3, and -4 appear to be glycosylphosphatidylinositol (GPI)-anchored, whereas ART5, expressed in lymphocytes, lacks the carboxyl-terminal signal sequence for addition of a GPI anchor and is predicted to be secreted [14] Both ART1 and ART5 are arginine-specific transferases. ADP-ribosylation altered its biological properties assayed in vitro, decreasing cytotoxic and antimicrobial

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