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

Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):19796-800. doi: 10.1073/pnas.0910633106. Epub 2009 Nov 6.

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.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Adenosine Diphosphate Ribose / metabolism*
  • Animals
  • Arginine* / chemistry
  • Arginine* / metabolism
  • Bronchoalveolar Lavage Fluid / chemistry
  • Cell Line
  • Chromatography, High Pressure Liquid
  • Humans
  • Mass Spectrometry
  • Ornithine / metabolism*
  • Rats
  • alpha-Defensins / chemistry*
  • alpha-Defensins / genetics
  • alpha-Defensins / metabolism*

Substances

  • alpha-Defensins
  • human neutrophil peptide 1
  • Adenosine Diphosphate Ribose
  • Arginine
  • Ornithine