Modification of

<p>β<b>-defensin by selected ADP-ribosyltransferases.</b> (<b>A</b>) HBD1 is ADP-ribosylated by CTA and LTA. HBD1 (3 µg, 38.18 µM)) was incubated with CTA (2.5 U) or LTA (8.9 U) in the presence of 10 µM biotin-NAD, in 50 mM potassium phosphate buffer, pH 7.5 at 30°C for 1 h (Toxin). Reactions were also performed in the presence of 2 mM NAD (Toxin + NAD) or 2 mM ADP-ribose (Toxin + ADP-ribose). Control reactions performed with heat-inactivated CTA or LTA (HI-Toxin) or in the absence of toxins (-Toxin) are also shown. The ADP-ribosylated peptides were separated by SDS-PAGE in a 10% NuPAGE gel and transferred to nitrocellulose. The membrane was treated as previously described, incubated with streptavidin-HRP conjugated (1∶10000 dilution) before visualization of the biotin-ADP-ribose labeled bands by chemiluminescence. (<b>B</b>) ADP-ribosylation of HBD1 by ART1. HBD1 (3 µg, 38.18 µM) was incubated with 6.8 U of ART1 (ART1) or heat-inactivated ART1 (HI-ART1) and 10 µM biotin-NAD in 50 mM potassium phosphate buffer, pH 7.5, at 30°C for 1 h. (<b>C</b>) HBD1 is ADP-ribosylated in a dose response fashion. HBD1 at the concentration shown in the Figure was incubated with CTA (2.5 U) or LTA (8.9 U) in the presence of 10 µM biotin-NAD, in 50 mM potassium phosphate buffer, pH 7.5 at 30°C for 1 h. (<b>D</b>) Time dependent ADP-ribosylation of HBD1. HBD1 (3 µg) was incubated with CTA (1.25 U) or LTA (4.45 U) using the same conditions above described. Times of incubation are indicated in the Figure. Molecular markers are on the left. Data shown are representative of two independent experiments.</p

Modification of HNP-1 by selected ADP-ribosyltransferases.

<p>(<b>A</b>) HNP-1 is ADP-ribosylated by CTA and LTA but only weakly by NarE. HNP-1 (3 µg, 43.56 µM) was incubated with CTA (2.5 U), LTA (8.9 U) or NarE (2 U) and 10 µM of biotin-NAD in 50 mM potassium phosphate buffer, pH 7.5, at 30°C for 1 h (Toxin). The same reactions were performed with heat-inactivated toxins (HI-Toxin), in the presence of 2 mM NAD (Toxin + NAD), or 2 mM ADP-ribose (Toxin + ADP-ribose). The ADP-ribosylated peptides were resolved by SDS-PAGE in a 10% NuPAGE gel, using MES as running buffer and transferred to nitrocellulose. After blocking with 5% BSA in PBS containing 0.05% Tween-20 (PBS-T) for 1 h, the blot was incubated with streptavidin-HRP conjugated (1∶10000 dilution) for 1 h at RT in the same buffer. The biotin-ADP-ribose labeled bands were visualized by chemiluminescence. (<b>B</b>) ART1 ADP-ribosylated HNP-1. HNP-1 (3 µg, 43.56 µM) was incubated with ART1 (6.8 U) and 10 µM of biotin-NAD in 50 mM potassium phosphate, pH 7.5 at 30°C for 1 h (ART1). A control reaction with heat-inactivated ART1 is also shown (HI-ART1). (<b>C</b>) SDS-PAGE analysis of the purification grade of 2 µg each of CTA, LTA and NarE. (<b>D</b>) HNP-1 is ADP-ribosylated in a dose and response dependent manner by CTA and LTA. HNP-1 at the concentration shown in the Figure was incubated with CTA (2.5 U), or LTA (8.9 U) and 10 µM of biotin-NAD in 50 mM potassium phosphate buffer, pH 7.5, at 30°C for 1 h. (<b>E</b>) HNP-1 is ADP-ribosylated in time dependent fashion. HNP-1 (3 µg) was incubated with CTA (1.25 U) or LTA (4.45 U) using the same conditions above described for the times of incubation indicated in the Figure. Molecular markers are on the left. Data shown are representative of several experiments performed in the same conditions.</p

MALDI-TOF mass spectra of HNP-1 reaction with CTA.

<p>Mass spectra analysis confirmed the mono-ADP-ribosylation of HNP-1 by CTA after incubation at 30°C for 1h in the presence of 2 mM NAD. Upper panels (left side: spectrum of <i>m/z</i> 2500 – 5300, right side: zoomed spectrum of <i>m/z</i> 3800 – 4300) show the mass of the control reaction, i.e. HNP-1 incubated only with NAD without toxin (<i>m/z</i> 3442.12). Lower panels (left side: spectrum of <i>m/z</i> 2500 – 5300, right side: zoomed spectrum of <i>m/z</i> 3800–4300) represent the unmodified HNP-1 peptide and the product of ADP-ribosylation peptide by CTA (<i>m/z</i> 3983.15). Stars (*) correspond to Sinapinic Acid adducts (+206 Da).</p

HNP-1 enhanced the auto-ADP-ribosylation of NarE.

<p>Purified NarE (0.4 µg) was auto-ADP-ribosylated with 10 µM biotin-NAD in 50 mM potassium phosphate pH 7.5 at 30°C for 3 and 6 h in the presence of the indicated concentrations of HNP-1. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. To detect biotin-ADP-ribosylated NarE, the membrane was incubated with streptavidin-HRP (upper panel). For western blotting the nitrocellulose membrane was incubated with rabbit polyclonal α-NarE (1∶10000 dilution) and with α-rabbit HRP-conjugated (lower panel). Labeled bands were detected by chemioluminescence. Data shown are representative of two independent experiments.</p

HNP-1 is ADP-ribosylated at R14.

<p>HNP-1 R14K and HNP-1 R15K protein variants (3 µg, 43.85 µM) were incubated with CTA (2.5 U), LTA (8.9 U) or ART1 (5.5 U) in the presence of 10 µM biotin-NAD, in 50 mM potassium phosphate buffer, pH 7.5 at 30°C for 1 h. The ADP-ribosylated peptides were separated by SDS-PAGE in a 10% NuPAGE gel and transferred to nitrocellulose. Membranes treated as previously described, were incubated with streptavidin-HRP conjugated (1∶10000 dilution) before visualization of the biotin-ADP-ribose labeled bands by chemiluminescence. Here shown in comparison with the modification of HNP-1 wild-type in the same reaction conditions.</p

Human defensins.

<p>Human defensins.</p