ADP-ribosyltransferase-specific Modification of Human Neutrophil Peptide-1

Abstract

Epithelial cells lining human airways and cells recruited to airways participate in the innate immune response in part by releasing human neutrophil peptides (HNP). Arginine-specific ADP-ribosyltransferases (ART) on the surface of these cells can catalyze the transfer of ADP-ribose from NAD to proteins. We reported that ART1, a mammalian ADP-ribosyltransferase, present in epithelial cells lining the human airway, modified HNP-1, altering its function. ADP-ribosylated HNP-1 was identified in bronchoalveolar lavage fluid (BALF) from patients with asthma, idiopathic pulmonary fibrosis, or a history of smoking (and having two common polymorphic forms of ART1 that differ in activity), but not in normal volunteers or patients with lymphangioleiomyomatosis. Modified HNP-1 was not found in the sputum of cystic fibrosis patients or in leukocyte granules of normal volunteers. The finding of ADP-ribosyl-HNP-1 in BALF but not in leukocyte granules suggests that the modification occurred in the airway. Most of the HNP-1 in the BALF from individuals with a history of smoking was, in fact, monoor di-ADP-ribosylated. ART1 synthesized in Escherichia coli, glycosylphosphatidylinositol-anchored ART1 released with phosphatidylinositol-specific phospholipase C from transfected NMU cells, or ART1 expressed endogenously on C2C12 myotubes modified arginine 14 on HNP-1 with a secondary site on arginine 24. ADPribosylation of HNP-1 by ART1 was substantially greater than that by ART3, ART4, ART5, Pseudomonas aeruginosa exoenzyme S, or cholera toxin A subunit. Mouse ART2, which is an NAD:arginine ADP-ribosyltransferase, was able to modify HNP-1, but to a lesser extent than ART1. Although HNP-1 was not modified to a significant degree by ART5, it inhibited ART5 as well as ART1 activities. Human ␤-defensin-1 (HBD1) was a poor transferase substrate. Reduction of the cysteine-rich defensins enhanced their ability to serve as ADP-ribose acceptors. We conclude that ADP-ribosylation of HNP-1 appears to be primarily an activity of ART1 and occurs in inflammatory conditions and disease.

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

“…CTA (60 g), 30 mM DTT, 10 mM NAD, and 10 mM arginine (0.5 Ci 14 C/assay) with 30 g ovalbumin in 20 mM potassium phosphate (pH 7.5; volume 300 L) were incubated overnight at 30°C. Reaction products were separated on a strong anion exchange (SAX) column (DuPont) by gradient elution (18). Radioactive peaks were collected, vacuum concentrated, and applied to a Discovery BioWide Pore C18 RF-HPLC column (Supelco) equilibrated for 15 min with HPLC water, 0.05% TFA (flow ϭ 0.8 mL/min), followed by a 5-min linear gradient of 0% to 100% acetonitrile.…”

“…In vitro, ART1 ADP-ribosylates HNP-1 on arginine 14 with a secondary site on arginine 24. Mono-and di-ADP-ribosylated HNP were isolated from the bronchoalveolar lavage f luid of IPF and asthma patients, consistent with a role for the modified HNP-1 in disease (18). In addition to the two modified forms, a third product separated by HPLC from the reaction of HNP-1, NAD, and ART1 was identified by mass spectrometry (MS) analysis as ADP-ribosylated HNP containing ornithine, a noncoded amino acid.…”

“…Cells were purchased from American Type Culture Collection. Protein released from the cells by PI-PLC, collected in the medium for ADP-ribosyltransferase activity (nmol/h), were assayed by quantifying the transfer of ADP-ribose to agmatine in standard assays as described (18).…”

“…Nonenzymatic modification of several proteins in vitro by sugars has been reported (26,27), but ornithine in a protein primary sequence has been reported only in collagen as a result of age-related glycation. No ornithine was identified after incubation with free ADP-ribose by MS (18), and after incubation of HNP-1, which has no lysines in the native sequence, or arginine with free ADP-ribose by amino acid analysis (Fig. 5).…”

“…Since the two ADP-ribosylated HNP-1 products were isolated from the broncoalveolar lavage fluid (BALF) of patients with IPF (18), we expected that ADP-ribosylated-HNP-ornithine would be present in vivo. To look for formation of HNP-1-ornithine in vivo, we examined BALF samples from 7 patients with idiopathic pulmonary fibrosis as described (18). Briefly, 8 mL of BALF were applied to LC-18 Supelclean SPE tubes (Supelco) equilibrated in 10% isopropanol/0.1% TFA, washed and eluted with 50% isopropanol/0.1% TFA.…”