Defensins (e.g., human neutrophil peptides, or HNPs) contribute to innate immunity through diverse actions, including microbial killing; high concentrations are present in the lung in response to inflammation. Arginines are critical for HNP activity, which is decreased by their replacement with ornithine. ADP-ribosyltransferases (ARTs) catalyze transfer of ADP-ribose from NAD to an acceptor arginine in a protein substrate, whereas ADP-ribosylarginine hydrolases release ADPribose. ART1 on the surface of airway epithelial cells ADP-ribosylated HNP-1 specifically on arginines 14 and 24, with ADP-ribosylation altering biological activity. Di-and mono-ADP-ribosylated HNP-1 were isolated from bronchoalveolar lavage fluid (BALF) of patients with asthma and idiopathic pulmonary fibrosis (IPF), suggesting a role for ADP-ribosylation in disease. In the present study, we observed that ART1-catalyzed ADP-ribosylation of HNP-1 in vitro generated a product with ADP-ribose on arginine 24, and ornithine replacing arginine at position 14. We hypothesized that ADP-ribosylarginine is susceptible to a nonenzymatic hydrolytic reaction yielding ornithine. On incubation of di-or mono-ADP-ribosyl-HNP-1 at 37°C, ADP-ribosylarginine was partially replaced by ornithine, whereas ornithine was not detected by amino acid analysis and mass spectrometry of unmodified HNP-1 incubated under the same conditions. Further, ornithine was produced from the model compound, ADPribosylarginine. BALF from an IPF patient contained ADP-ribosyl-HNPornithine as well as mono-and di-ADP-ribosylated HNP-1, consistent with in vivo conversion of arginine to ornithine. Targeted ADPribosylation of specific arginines by transferases, resulting in their replacement with ornithine, is an alternative pathway for regulation of protein function through posttranslational modification.posttranslational modification ͉ NAD ͉ bacterial toxins N eutrophils, a critical component of the innate immune system, are recruited to airways in response to inflammation or infection (1). Neutrophil defensins [human neutrophil peptides (HNPs) 1-3], stored in azurophilic granules, are small cationic peptides whose main function is to defend the lung against pathogenic microorganisms (2). High levels of defensins have been found in patients with inflammatory lung diseases, such as idiopathic pulmonary fibrosis (IPF) (3) and cystic fibrosis (4). In addition to antimicrobial activities and other diverse functions (5), defensins interact with airway epithelial cells, increasing proliferation and stimulating wound repair (6). HNP1-3 are arginine rich and differ in sequence by one amino acid. The salt bridge formed by Arg 5 -Glu 13 and three disulfide bridges are conserved, but not required, for antibacterial activity in vitro (7,8). The arginines in HNP-1 are critical for maintaining activity (9). In addition, the low number of arginines in HD6 (human defensin 6 expressed in Paneth cells) may be responsible for its lack of antibacterial activity (10).Mono-ADP-ribosylation is a posttranslational ...