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 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 ...

Insights into the biogenesis, function, and regulation of ADP-ribosylation

“…In humans, ARTCs are thought to MARylate arginines exclusively in protein targets, and thus exhibit specific amino acid selectivity in their targets, similar to their RSE bacterial ART ancestors 24,71 . Several ARTC family members contain a glutamate in the A-loop at the same position of Glu378 (IT numbering); therefore, it is likely that this glutamate in the A-loop of ARTCs is required for amino acid selection 39 .…”

“…Several ARTC family members can also MARylate arginines in peptide targets. For example, ARTC1 (also known as ART1) can MARylate multiple arginines in the immunemodulatory cationic peptides LL-37 and human neutrophil peptide-1 (HNP-1) 71,72 .…”

Methionine sulfoxide reductase A is a stereospecific methionine oxidase

“…sequencing by tandem mass spectrometry (40). To determine the stoichiometry of Met formation, 1 μM MsrA was incubated with different concentrations of L-methionine sulfoxide (MetO) without reducing agents in 50 mM sodium phosphate pH 7.5 with 1 mM diethylenetriaminepentaacetic acid (DTPA).…”

Nonenzymatic Conversion of ADP-Ribosylated Arginines to Ornithine Alters the Biological Activities of Human Neutrophil Peptide-1

“…Products were separated by HPLC. ADP-ribosylation sites on the purified products were mapped by MS (28). …”

“…Arginine 5 forms a salt bridge with glutamic acid 13 that is required for folding stability (26). Arginines 14 and 24 in HNP-1 are ADP-ribosylated by ART1 and ADP-ribosylated arginines can be converted non-enzymatically to ornithine by nucleophilic attack of water on the guanidino carbon with probable release of ADP-ribose-carbamate (27,28). Mono-and di-ADP-ribosylated HNP-1 and ADP-ribosylated HNP-(ornithine) were isolated from the BALF of a patient with idiopathic pulmonary fibrosis, suggesting that post-translationally modified HNP-1 is found in the diseased airway.…”