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

Translational Medicine Branch and Laboratory of Biochemistry, National Heart, Lung and Blood Institute, Medical Genetics Branch, National Institutes of Health, Bethesda, MD 20892, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2009; 106(47):19796-800. DOI: 10.1073/pnas.0910633106
Source: PubMed


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.


Available from: Linda A Stevens, Feb 19, 2014
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    • "The general mechanism for its antimicrobial action relies on the permeabilization of the microbial cell membrane [19] [20] and the importance of Arg and Trp residues of HNP-1 for this membrane interaction is now clear [21] [22] [23] [24]. A " dimer pore " model for HNP-1 has been suggested on the basis of a crystal structure study [25], while a multimeric pore model was proposed on the basis of vesicle leakage and dextran permeability experiments [26]. "
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    ABSTRACT: Antimicrobial peptides are an important component of innate immunity and have generated considerable interest as a new potential class of natural antibiotics. The biological activity of antimicrobial peptides is strongly influenced by peptide-membrane interactions. Human Neutrophil Peptide 1 (HNP-1) is a 30 aminoacid peptide, belonging to the class of α-defensins. Many biophysical studies have been performed on this peptide to define its mechanism of action. Combining spectroscopic and thermodynamic analysis, insights on the interaction of the α-defensin with POPE:POPG:CL negative charged bilayers are given. The binding states of the peptide below and above the threshold concentration have been analyzed showing that the interaction with lipid bilayers is dependent by peptide concentration. These novel results that indicate how affinity and biological activities of natural antibiotics are depending by their concentration, might open new way of investigation of the antimicrobial mode of action.
    Biochimica et Biophysica Acta 11/2012; 1828(2). DOI:10.1016/j.bbamem.2012.11.011 · 4.66 Impact Factor
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    • "Moreover, it prevents the modified Arg to serve as substrate for subsequent ADPribosylation since ornithine is not recognized as substrate by ART1. BAL of patients with idiopathic pulmonary fibrosis and asthma other than mono-and di-ADP-ribosylated- HNP-1 contained HNP-1 ADP-ribosylated at Arg24 with ornithine replacing Arg in position 14 suggesting a relevance of this conversion for the innate immunity in the airway [28]. Interestingly, when 3 of the 4 Arg (not Arg5) were replaced by ornithine the bactericidal activity was decreased suggesting that the conversion of arginine with ornithine can be important in altering HNP-1 activities [24]. "
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    ABSTRACT: HNP-1 is an antimicrobial peptide that undergoes proteolytic cleavage to become a mature peptide. This process represents the mechanism commonly used by the cells to obtain a fully active antimicrobial peptide. In addition, it has been recently described that HNP-1 is recognized as substrate by the arginine-specific ADP-ribosyltransferase-1. Arginine-specific mono-ADP-ribosylation is an enzyme-catalyzed post-translational modification in which NAD(+) serves as donor of the ADP-ribose moiety, which is transferred to the guanidino group of arginines in target proteins. While the arginine carries one positive charge, the ADP-ribose is negatively charged at the phosphate moieties at physiological pH. Therefore, the attachment of one or more ADP-ribose units results in a marked change of cationicity. ADP-ribosylation of HNP-1 drastically reduces its cytotoxic and antibacterial activities. While the chemotactic activity of HNP-1 remains unaltered, its ability to induce interleukin-8 production is enhanced. The arginine 14 of HNP-1 modified by the ADP-ribose is in some cases processed into ornithine, perhaps representing a different modality in the regulation of HNP-1 activities.
    International Journal of Peptides 08/2011; 2011(1687-9767):594723. DOI:10.1155/2011/594723
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    • "A second possibility for reverting arginine ADP-ribosylation has recently been described for HNP-1, i.e. the non-enzymatic hydrolysis at the guanidino carbon, resulting in replacement of ADP-ribosylarginine by ornithine (Stevens et al. 2009). This modification also precludes re-ADP-ribosylation at this site. "
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    ABSTRACT: Arginine adenosine-5'-diphosphoribosylation (ADP-ribosylation) is an enzyme-catalyzed, potentially reversible posttranslational modification, in which the ADP-ribose moiety is transferred from NAD(+) to the guanidino moiety of arginine. At 540 Da, ADP-ribose has the size of approximately five amino acid residues. In contrast to arginine, which, at neutral pH, is positively charged, ADP-ribose carries two negatively charged phosphate moieties. Arginine ADP-ribosylation, thus, causes a notable change in size and chemical property at the ADP-ribosylation site of the target protein. Often, this causes steric interference of the interaction of the target protein with binding partners, e.g. toxin-catalyzed ADP-ribosylation of actin at R177 sterically blocks actin polymerization. In case of the nucleotide-gated P2X7 ion channel, ADP-ribosylation at R125 in the vicinity of the ligand-binding site causes channel gating. Arginine-specific ADP-ribosyltransferases (ARTs) carry a characteristic R-S-EXE motif that distinguishes these enzymes from structurally related enzymes which catalyze ADP-ribosylation of other amino acid side chains, DNA, or small molecules. Arginine-specific ADP-ribosylation can be inhibited by small molecule arginine analogues such as agmatine or meta-iodobenzylguanidine (MIBG), which themselves can serve as targets for arginine-specific ARTs. ADP-ribosylarginine specific hydrolases (ARHs) can restore target protein function by hydrolytic removal of the entire ADP-ribose moiety. In some cases, ADP-ribosylarginine is processed into secondary posttranslational modifications, e.g. phosphoribosylarginine or ornithine. This review summarizes current knowledge on arginine-specific ADP-ribosylation, focussing on the methods available for its detection, its biological consequences, and the enzymes responsible for this modification and its reversal, and discusses future perspectives for research in this field.
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