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. 2002 Jun 11;99(12):8231-5.
doi: 10.1073/pnas.122238899.

ADP ribosylation of human neutrophil peptide-1 regulates its biological properties

Gregorino Paone  1 , Akihiro Wada, Linda A Stevens, Abul Matin, Toshiya Hirayama, Rodney L Levine, Joel Moss
Affiliations

ADP ribosylation of human neutrophil peptide-1 regulates its biological properties

Gregorino Paone et al. Proc Natl Acad Sci U S A. .

Abstract

In human airways, epithelial cells lining the lumen and intraluminal cells (e.g., polymorphonuclear cells) participate in the innate immune response. These cells secrete or express on their surfaces arginine-specific ADP ribosyltransferases. Defensins, antimicrobial proteins secreted by immune cells, are arginine-rich, leading us to hypothesize that ADP ribosylation could modify their biological activities. We found that an arginine-specific ADP ribosyltransferase-1 present on airway epithelial cells modifies Arg-14 of alpha defensin-1. ADP-ribosylated defensin-1 had decreased antimicrobial and cytotoxic activities but still stimulated T cell chemotaxis and IL-8 release from A549 cells. Further, ADP-ribosylated defensin-1 inhibited cytotoxic and antimicrobial activities of unmodified defensin-1. We identified ADP-ribosylated defensin-1 in bronchoalveolar lavage fluid from smokers but not from nonsmokers, confirming its existence in vivo. Thus, airway mono-ADP-ribosyltransferases could have an important regulatory role in the innate immune response through modification of alpha defensin-1 and perhaps other basic molecules, with alteration of their biological properties.

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Figures

Figure 1
Figure 1
Separation of products of ART-1-catalyzed ADP-ribosylation of HNP-1 by RP-HPLC. HNP were incubated with: (a) reaction buffer alone; (b) NAD+; (c) ART-1; or (d) ART-1 and NAD+, and then analyzed by RP-HPLC. Absorbance at 210 nm (normalized to 100 full scale) is shown as a function of elution time. For a and b, full scale is 750; for c and d, 650 and 250, respectively. ADP-ribosyl-HNP-1 and HNP-1 are indicated by solid and open arrows, respectively. (Inset) Absorbance spectra of ADP-ribosyl-HNP-1 (solid line) and HNP-1 (dotted line), recorded during elution by an inline array detector.
Figure 2
Figure 2
Antimicrobial and cytotoxic activities of unmodified HNP-1 and ADP-ribosyl-HNP-1. (a) Difference in zone size (corresponding to antimicrobial activity) is the diameter of the zone of E. coli growth cleared with the indicated concentration of HNP (○) or ADP-ribosyl-HNP-1 (□) minus the diameter of the central well (3 mm). Data are means ± 1/2 range of values from two experiments. (b) A549 cells were incubated with the indicated concentrations HNP (○), ADP-ribosyl-HNP-1 (□), or synthetic HNP-1 (sHNP, Δ). Percent of lysis (cytotoxic activity) was calculated as (cpmexp − cpmspont)/(cpmmax − cpmspont) × 100; exp, experimental release; max, maximal release; spont, spontaneous release. Data are means ± SEM of values from four experiments.
Figure 3
Figure 3
Effects of ADP-ribosyl-HNP-1 on HNP-1 antimicrobial and cytotoxic activity. (a) HNP-1 (100 nM) was incubated with the indicated concentration of ADP-ribosyl-HNP-1 before addition to E. coli ATCC 43827 for radial diffusion assay. Difference in zone size was calculated as in Fig. 2 (⧫). Data are means ± SEM of values from three experiments. (b) HNP-1 (12 μM) was incubated with the indicated concentration of ADP-ribosyl-HNP-1 before addition to 51Cr-labeled A549 cells (⧫). Percentage of cell lysis was determined as in Fig. 2 to compare effects of HNP-1(○) and ADP-ribosyl-HNP-1 (□). Data are means ± 1/2 range of values from two experiments. Results were similar in another experiment by using 24 μM HNP-1.
Figure 4
Figure 4
Effects of ADP-ribosyl-HNP-1 and HNP-1 on IL-8 release by A549 cells and on T cell chemotaxis. Cells were incubated for (a) 12 or (b) 24 h with the indicated concentration of HNP-1 or ADP-ribosyl-HNP-1 before analysis of medium. IL-8 concentration in medium from cells incubated without defensins has been subtracted (at 12 h, 36.4 ± 6 pg/ml; at 24 h, 85 ± 34 pg/ml). Data are means ± 1/2 range of values from two experiments, each performed in triplicate. *, P < 0.05 for difference between IL-8 release stimulated by HNP-1 and ADP-ribosyl-HNP-1. CD3+ cells were incubated with the indicated concentrations of HNP-1 or ADP-ribosyl HNP-1 (c). We used MIP-1β (5 ng/ml) in migration medium, or migration medium alone, as positive and negative controls, respectively. Chemotaxis percentage was calculated as: (number of cells migrated to the lower chamber in the experimental conditions − number of cells migrated in the negative control)/(number of cells migrated in the positive control − number of cells migrated in negative control) × 100. Data are means ± SEM values from four experiments, each performed in duplicate.
Figure 5
Figure 5
Characterization of ADP-ribosyl-HNP-1 from BAL fluid by RP-HPLC, MALDI, and enzymatic digestion. (a) Alignment of RP-HPLC chromatograms of products of ART-1-catalyzed ADP-ribosylation of HNP-1 (continuous line) and a chromatogram of a BAL sample from a smoker (dotted line). Arrows indicate elution times of ADP-ribosyl-HNP-1 and HNP-1. (b) (i) The peak with elution time compatible with ADP-ribosyl-HNP-1 (46.5 min), analyzed by MALDI-MS, shows a mass of 3,983 Da, consistent with ADP-ribosyl-HNP-1 (calculated 3,983 Da). (ii) Incubation of ADP-ribosyl-HNP-1 with pyrophosphatase and alkaline phosphatase produced ribosyl-HNP-1 (calculated 3,574 Da). (iii) ADP-ribosylarginine hydrolase cleaved the ribose-arginine linkage to release HNP-1 (calculated 3,443 Da). m/z is on the x axis.
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