title: ADP-ribosylation of human defensin HNP-1 results in the replacement of the modified arginine with the noncoded amino acid ornithine.
abstract: 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 ADP-ribose. 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 degrees 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, ADP-ribosylarginine. BALF from an IPF patient contained ADP-ribosyl-HNP-ornithine as well as mono- and di-ADP-ribosylated HNP-1, consistent with in vivo conversion of arginine to ornithine. Targeted ADP-ribosylation of specific arginines by transferases, resulting in their replacement with ornithine, is an alternative pathway for regulation of protein function through posttranslational modification.
authors.affiliation: Translational Medicine Branch and Laboratory of Biochemistry, National Heart, Lung and Blood Institute, Medical Genetics Branch, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Linda A
authors.initials: LA
authors.lastname: Stevens
authors.forename: Rodney L
authors.initials: RL
authors.lastname: Levine
authors.forename: Bernadette R
authors.initials: BR
authors.lastname: Gochuico
authors.forename: Joel
authors.initials: J
authors.lastname: Moss
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D013058:Mass Spectrometry; D009952:Ornithine / Q000378:metabolism*; D051381:Rats; D023084:alpha-Defensins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D023084:alpha-Defensins; C406413:human neutrophil peptide 1; D000246:Adenosine Diphosphate Ribose; D001120:Arginine; D009952:Ornithine