Nonenzymatic Conversion of ADP-Ribosylated Arginines to Ornithine Alters the Biological Activities of Human -1 This information is current as of September 27, 2021. Linda A. Stevens, Joseph T. Barbieri, Grzegorz Piszczek, Amy N. Otuonye, Rodney L. Levine, Gang Zheng and Joel Moss J Immunol published online 12 November 2014 http://www.jimmunol.org/content/early/2014/11/11/jimmun Downloaded from ol.1303068 http://www.jimmunol.org/ Why The JI? Submit online.

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 12, 2014, doi:10.4049/jimmunol.1303068 The Journal of Immunology

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

Linda A. Stevens,* Joseph T. Barbieri,† Grzegorz Piszczek,‡ Amy N. Otuonye,* Rodney L. Levine,x Gang Zheng,{ and Joel Moss*

Activated , recruited to the airway of diseased lung, release human neutrophil (HNP1–4) that are cytotoxic to airway cells as well as microbes. Airway epithelial cells express arginine-specific ADP ribosyltransferase (ART)-1, a GPI-anchored ART that transfers ADP-ribose from NAD to arginines 14 and 24 of HNP-1. We previously reported that ADP-ribosyl-arginine is converted nonenzymatically to ornithine and that ADP-ribosylated HNP-1 and ADP-ribosyl-HNP-(ornithine) were isolated from

bronchoalveolar lavage fluid of a patient with idiopathic pulmonary fibrosis, indicating that these reactions occur in vivo. To Downloaded from determine effects of HNP-ornithine on the airway, three analogs of HNP-1, HNP-(R14orn), HNP-(R24orn), and HNP-(R14,24orn), were tested for their activity against Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus; their cytotoxic effects on A549, NCI-H441, small airway epithelial-like cells, and normal human lung fibroblasts; and their ability to stimulate IL-8 and TGF-b1 release from A549 cells, and to serve as ART1 substrates. HNP and the three analogs had similar effects on IL-8 and TGF-b1 release from A549 cells and were all cytotoxic for small airway epithelial cells, NCI-H441, and normal human lung

fibroblasts. HNP-(R14,24orn), when compared with HNP-1 and HNP-1 with a single ornithine substitution for arginine 14 or 24, http://www.jimmunol.org/ exhibited reduced cytotoxicity, but it enhanced proliferation of A549 cells and had antibacterial activity. Thus, arginines 14 and 24, which can be ADP ribosylated by ART1, are critical to the regulation of the cytotoxic and antibacterial effects of HNP-1. The HNP analog, HNP-(R14,24orn), lacks the epithelial cell cytotoxicity of HNP-1, but partially retains its antibacterial activity and thus may have clinical applications in airway disease. The Journal of Immunology, 2014, 193: 000–000.

olymorphonuclear leukocytes migrate into the lung to the disease (7–9). In the bronchoalveolar lavage fluid (BALF) of healthy site of infection, inflammation, or other disease processes volunteers, concentrations of HNP1–3 are 0.2 mg/ml, but may in- in response to complex signaling events. After activation, P crease 6-fold during infection and 50-fold in inflammatory lung by guest on September 27, 2021 neutrophils release antimicrobial a- (human neutrophil diseases (10). Elevated levels of defensins were found in the BALF

peptides [HNP]1–4), cationic, amphipathic, arginine-rich peptides of patients with a1-antitrypsin deficiency (11, 12), in the sputum of with diverse biological activities (1). In addition to killing micro- patients with cystic fibrosis (13) and chronic obstructive pulmonary organisms, defensins promote proliferation (2, 3) and stimulate disease (14), and in the serum of patients with interstitial lung dis- the release of neutrophil chemotactic factor, IL-8, and TGF-b1 ease [e.g., idiopathic pulmonary fibrosis (15), sarcoidosis (16, 17)]. from lung epithelial cells and fibroblasts (4, 5). The in vitro effects Defensins at low concentrations increased proliferation and collagen on proliferation of airway epithelial cells suggested a role for synthesis in lung fibroblasts, possibly participating in the formation of defensins in wound repair. Indeed, wound closure and mucin gene the fibroproliferative lesions seen in inflammatory pulmonary diseases expression were enhanced by HNP1–3 in vitro (6). (3). Intratracheal instillation of defensins (HNP1–3) into the lungs of It has been proposed that defensins released at the site of in- mice that do not express neutrophil-derived HNP caused acute lung flammation or infections are also involved in the pathogenesis of inflammation and dysfunction (18). Transgenic mice that expressed a-defensins (HNP1, 2) in their neutrophils had more severe, acid- *Cardiovascular and Pulmonary Branch, National, Heart, Lung, and Blood Institute, induced acute lung injury than did their wild-type counterparts (19). National Institutes of Health, Bethesda, MD 20892; †Microbiology and Molecular ‡ Net charge, amphipathicity, hydrophobicity, and tertiary structure Genetics, Medical College of Wisconsin, Milwaukee, WI 53226; Biophysics Core Facility, National, Heart, Lung, and Blood Institute, National Institutes of Health, are critical for the antimicrobial activity of defensins (20). In addi- x Bethesda, MD 20892; Laboratory of Biochemistry, National, Heart, Lung, and tion to the hydrophobicity of tryptophan 26 (21), the positive charge Blood Institute, National Institutes of Health, Bethesda, MD 20892; and {Office of Biostatistics Research, National, Heart, Lung, and Blood Institute, National Institutes of three arginine residues (22) is essential for the antimicrobial activ- of Health, Bethesda, MD 20892 ity of HNP-1. ART1, an arginine-specific ADP ribosyltransferase Received for publication November 14, 2013. Accepted for publication October 1, (ART) expressed on the surface of airway epithelial cells and neu- 2014. trophils (23, 24), catalyzes the transfer of the ADP-ribose moiety of This work was supported by the Intramural Research Program of the National In- NAD to arginine 14 of HNP-1, inhibiting its antibacterial and cy- stitutes of Health, National Heart, Lung, and Blood Institute. totoxic activities (25). The primary sequence of HNP1–3 contains Address correspondence and reprint requests to Dr. Joel Moss, Room 6D05, Building 10, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1590. arginines at positions 5, 14, 15, and 24. Arginine 5 forms a salt E-mail address: [email protected] bridge with glutamic acid 13 that is required for folding stability Abbreviations used in this article: ART, ADP ribosyltransferase; BALF, bronchoal- (26). Arginines 14 and 24 in HNP-1 are ADP ribosylated by ART1, veolar lavage fluid; HNP, human neutrophil peptide; mART, mouse ART; NHLF, and ADP-ribosylated arginines can be converted nonenzymatically to normal human lung fibroblast; SAEC, small airway epithelial cell. ornithine by nucleophilic attack of water on the guanidino carbon Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 with probable release of ADP-ribose-carbamate (27, 28). Mono- and

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303068 2 ACTIVITY OF HNP-1–CONTAINING ORNITHINE di-ADP–ribosylated HNP-1 and ADP-ribosylated HNP-(ornithine) RPMI 1640, respectively, with 10% FBS. Normal human lung fibroblasts were isolated from the BALF of a patient with idiopathic pulmonary (NHLF) and normal human small airway epithelial cells (SAEC) were fibrosis, suggesting that posttranslationally modified HNP-1 is found purchased from Clonetics (Walkersville, MD) and grown, respectively, in fibroblast media or small airway epithelial cell complete medium supplied in the diseased airway. The relative ratios of native HNP and by the company. All cells were grown at 37˚C in 5% CO2. derivatives vary among patients. Airway epithelial cells play a critical role in host defense by se- Preparation of mouse ADP-ribosyltransferase-1 creting molecules (e.g., , chemokines, antimicrobial peptides) Rat mammary adenocarcinoma cells (NMU) transfected with plasmids that attract neutrophils and kill microorganisms (29, 30). IL-8, released containing the mouse ART (mART)1 gene were grown in Eagle’s MEM from epithelial cells in response to HNP-1, amplifies the inflammatory with 10% FBS and 0.5 mg/ml Geneticin (G418). Proteins released from the cells by incubation with phosphatidylinositol-specific phospholipase C, response by recruiting neutrophils that can, in turn, release additional which cleaves the GPI anchor, were collected in PBS (33), and ADP- HNP. Defensins at concentrations found in disease may be toxic to ribosyltransferase activity was assayed, as described (34). lung epithelial cells, endothelial cells, and normal lung fibroblasts, Preparation of ADP-ribosyl HNP analogs for identification of resulting in injury to the airway (31, 32). Modifications of antimi- the site of modification crobial HNP-1 that decrease its eukaryotic cytotoxicity but preserve antibacterial activity would reduce damage to the airway epithelium HNP analogs (10 or 30 mg), mART1 (9.14 nmol/h transferase activity), and while allowing the peptide to function in innate immunity. 5 mM NAD in 50 mM potassium phosphate (pH 7.5) were incubated at ADP ribosylation of specific arginines in HNP-1 by ART1 was 30˚C overnight or as indicated. Products were separated by HPLC. ADP- ribosylation sites on the purified products were mapped by mass spec- reported previously to have a role in modulating its activity, con- troscopy (28). sistent with a critical role for these arginines (25). We hypothesized Downloaded from that replacement of ADP-ribosyl-arginine with ornithine in HNP-1 Mass spectrometry and sequence analysis might also alter activity. To address this question, HNP-(R14orn), HNP analogs were reduced, trypsinized, and analyzed by reverse-phase HNP-(R24orn), and HNP-(R14,24orn) were synthesized, and their chromatography/mass spectrometry, as described (25), except that the 3 m antibacterial, cytotoxic, and other activities were compared with reverse-phase column was a Zorbax 300SB-C18, 1.0 50 mm, 3.5 m, and the mass spectrometer was an Agilent model 6520 QTOF capable of native HNP-1. The data demonstrate that replacement of specific tandem mass-spectrometric sequencing of peptides. Spectra were decon- arginines in HNP-1 with ornithine through an ADP-ribosylation– voluted with Agilent Masshunter software version 3, and spectra were http://www.jimmunol.org/ dependent reaction alters its biological activities and may protect matched to those predicted with GPMAW version 9 (Lighthouse Data, airway cells from HNP-1–induced cytotoxicity while partially pre- Odense, Denmark) (28). serving antimicrobial activity. Effects of HNP analogs and agmatine on nicotinamide release from [14C]NAD Materials and Methods HNP analogs or agmatine were incubated with mART1 (9.3 nmol/h) and Analogs 0.1 mM [nicotinamide-U-14 C]NAD (0.05 mCi) in 50 mM potassium HNP, HNP-(R14orn), HNP-(R24orn), and HNP-(R14,24orn) were syn- phosphate (pH 7.5) for 1.5 h at 30˚C. Nicotinamide release resulting from thesized by Bachem (Torrance, CA) as acetate salts. HNP-1 (Bachem), ADP-ribose transfer to an acceptor was monitored, as described (35). synthesized as a trifluroacetate salt, was included in indicated assays in by guest on September 27, 2021 addition to the acetate salt. Effects of HNP analogs on proliferation, cytotoxicity, and release of IL-8 and TGF-b1 Cell culture To measure the release of IL-8 (2 3 105 cells/ml) and TGF-b1(13 104 Human lung carcinoma epithelial cells (A549) and human lung adeno- cells/ml) from A549 cells, cells were grown in 96-well plates for 24 h in carcinoma epithelial cells (NCI-H441) were purchased from American complete media prior to treatment, washed with serum-free media, and Type Culture Collection (Manassas, VA) and grown in F-12K medium or treated for 24 h with analogs dissolved in serum-free media. Levels of

FIGURE 1. Effect of HNP-1 analogs on the viability of A549 cells and NHLF. A549 cells (1 3 105/ml) (A–C) and NHLF (4 3 104/ml) (D–F) were plated 24 h before the cells were washed with serum-free media and addition of HNP (A and D), HNP-(R14orn), HNP-(R24orn), (B and E), and HNP-(R14,24orn), (C and F) solubilized in serum-free media at the indicated concentrations for 24 h, as described in Materials and Methods. The data are the means 6 SEM of three experiments performed in triplicate. The mean cell proliferation as percentage of control of the four peptides in plots (A–C) over the range of HNP analog concentrations differs significantly from each other (p , 0.0001). The difference between the mean cell proliferation of HNP-(R14orn) and HNP- (R24orn) is not significant (p = 0.53). The mean proliferation of A549 cells increases significantly from 100% of control at concentrations ,25 mg/ml analog, for HNP (p = 0.0099), HNP-(R14orn) (p , 0.0001), HNP-(R24orn) (p = 0.0003), and HNP-(R14,24orn) (p , 0.0001). HNP and the three analogs were toxic for NHLF at concentrations ,10 mg/ml (p , 0.0001). The Journal of Immunology 3 human IL-8 and TGF-b1 in the media were quantified by immunoassay (R&D Systems, Minneapolis, MN). To determine the number of viable A549 cells (1 3 105 cells/ml), NCI-H441 (1 3 105 cells/ml), SAEC (1 3 105 cells/ml), or NHLF (4 3 104/ml) cells were grown on 96-well plates in complete media prior to treatment and treated as above. Proliferation and cytotoxicity were determined with tetrazolium-based Cell Counting Kit-8 (Dojindo Molecular Technologies, Rockville, MD), according to the manufacturer’s instructions. Antibacterial assays Escherichia coli (TG1 or ATCC 25922), Pseudomonas aeruginosa (PA103), and Staphylococcus aureus (ATCC 29213) were grown overnight in LB Broth, Miller (Luria-Bertani) (Difco: tryptone, 10 g/L; yeast extract, 5 g/L; and NaCl, 10 g/L) at 37˚C. were diluted 1/50, cultured at 37˚C, and harvested after 2 h. The bacterial OD was measured (5 3 108/ml = 1, A = 4 600 nm), and cells were diluted in 10 mM NaPO4 (pH 7.4) to 5 3 10 per ml. Bacteria (20 ml) were added to 50 ml HNP or analogs in 10 mM NaPO4 (pH 7.4). After 1 h at 37˚C, without agitation, an aliquot was plated to de- termine CFUs. Extending the incubation time to 2 h did not change the amount of cell killing. HNP or analogs and amounts used in the assay were FIGURE 2. Effect of HNP-1 analogs on the viability of A549, NHLF, HNP,1.0,0.1,and0.01mg; HNP-(R14orn), 3.0, 1.0, and 0.1 mg; HNP- SAEC, and NCI-H441 cells. Cells were plated for 24 h, washed with se- (R24orn), 3.0, 1.0, and 0.1 mg; and HNP-(R14,24orn), 3.0, 1.0, and 0.1 mg. rum-free media, and incubated with HNP and the ornithine analogs (50 mg/ml) Downloaded from Toxicity was similar in one or two incubations with HNP. in serum-free media for 24 h, as described in Materials and Methods. The data are shown with mean 6 SEM of three experiments performed in Analytical ultracentrifugation triplicate. There was no significant difference between HNP and the three Sedimentation velocity. Analytical ultracentrifugation experiments were per- analogs in NHLF and SAEC. HNP(R14,24orn) in NCI-H441 and A549 formed using the Beckman Optima XL-I analytical ultracentrifuge and a four- cells was significantly different from HNP. ****p , 0.0001, ***p , 0.001, place AN-Ti rotor. All HNP samples were dialyzed in PBS buffer (pH 7.4) and **p , 0.01, *p , 0.05. diluted to a final concentration of 60 mM. Double-sector centrifuge cells were filled with 0.4 ml sample or reference buffer. Centrifuge rotor was accelerated http://www.jimmunol.org/ to 250,000 3 g (60,000 rpm) after thermal equilibrium was reached at rest at BALF and plasma were found in idiopathic pulmonary fibrosis 20˚C. Interference and absorbance scans at 280 nm were started immediately patients compared with healthy volunteers (15, 38). HNP-1 has been after the rotor reached the set speed and collected until no further sedimen- reported to induce release of IL-8 and other chemokines and cyto- tation boundary movement was observed. The apparent sedimentation coef- kines from epithelial cells and fibroblasts (4, 5, 39, 40). To determine ficient distributions were analyzed by Lamm equation modeling using the SEDFIT software of Schuck (36). Positions of menisci and bottoms as well as the effects of HNP and the ornithine analogs on IL-8 release by frictional ratios were optimized during the fitting procedure. The final ac- A549 cells, cells were incubated with the ornithine analogs, and IL-8 cepted fits had a root mean square of deviation ,0.005. in the medium was measured by immunoassay. HNP with a single ornithine substitution at concentrations up to 20 mg/ml increased Statistical methods IL-8 release (Fig. 3A) as did HNP-(R14,24orn), up to 100 mg/ml by guest on September 27, 2021 The ANOVA and the test for trends were used to analyze the data presented (Fig. 3B), a range of concentrations not cytotoxic for A549 cells. in the figures. The effects on the mean outcome (e.g., cell proliferation) by the change in concentration levels or analogs were tested by a two-way Two HNP ornithine analogs release more TGF-b1 from A549 ANOVA. The test for trends, based on a linear regression model with cells than does HNP the peptide amounts coded with a set of equal-spaced scores, was used to test whether the mean outcome changes with concentration levels; analysis HNP-1 increased TGF-b1 release by fibroblasts, but reduced was done using SAS v9.3 software. TGF-b1 production by A549 cells (4). A549 cells were incubated with increasing amounts of HNP analogs, and TGF-b1 release was Results determined by immunoassay. HNP-(R24orn) and HNP-(R14,24orn) HNP-(R14,24orn) is cytotoxic for normal human fibroblasts, but not for A549 cells To determine the effect of the HNP analogs on eukaryotic cells, lung epithelial carcinoma cells (A549) or NHLF were incubated with increasing amounts of HNP or the three HNP-ornithine analogs for 24 h in serum-free media (Fig. 1A–F). HNP and HNP-(R14,24orn) at 10 mg/ml and HNP-(R14orn) and HNP-(R24orn) at 25 mg/ml in- creased the number of A549 cells (as measured by a tetrazolium reduction assay). HNP and the three analogs were toxic for NHLF at concentrations ,10 mg/ml (Fig. 1D–F). HNP-(R14orn) and HNP- (R24orn) were toxic for A549 cells at concentrations .40 mg/ml. In contrast, replacement of arginines with ornithines at aa 14 and 24 FIGURE 3. (A and B) Effects of HNP-1 analogs on expression of IL-8 A 3 4 abolished the cytotoxic effect of HNP on A549 cells at concen- by A549 cells. ( ) A549 cells (2 10 ) were grown for 24 h, washed with trations to 100 mg/ml (Fig. 1C). The toxicity of HNP and ornithine serum-free media, and then incubated for 24 h with HNP, HNP-(R14orn), HNP-(R24orn), or HNP-(R14,24orn) solubilized in serum-free media at analogs (50 mg/ml) was also tested on SAEC and NCI-H441 cells the indicated amounts. (B) Cells were treated with 50 and 100 ml/ml HNP- and compared with that seen with A549 and NHLF cells. The HNP (R14,24orn), as indicated. The medium was analyzed for IL-8 expression, analogs showed similar or less toxicity than HNP (Fig. 2). as described in Materials and Methods. The data are the mean 6 SEM of The three HNP ornithine analogs stimulate release of IL-8 from five experiments performed in triplicate. The mean IL-8 levels released in , A549 cells the presence of all analogs increase with increasing concentrations (p 0.0001), and the mean IL-8 level released in the presence of HNP is not IL-8, a chemoattractant, modulates the inflammatory response by significantly different from that of HNP-(R14orn), HNP-(R24orn), or recruiting neutrophils to the lung (37). Higher levels of IL-8 in HNP-(R14,24orn) (all p . 0.05). 4 ACTIVITY OF HNP-1–CONTAINING ORNITHINE

ADP ribosylation of HNP-ornithine peptides by ART1 To evaluate how well the ornithine analogs serve as substrates for ART1, nicotinamide release concurrent with ADP-ribose transfer from NAD to the acceptor was measured after incubation of the peptides with ART1 and NAD; HNP was compared with agmatine, a decarboxylated form of arginine, which is a known ADP-ribose acceptor used by ART1 (Fig. 6A). HNP and HNP-(R14orn) were better acceptors of ADP-ribose than agmatine. A small amount of nicotinamide release was observed in the presence of ART1, NAD, and HNP-(R14,24orn), indicating that, under these assay conditions, arginine 5 and arginine 15 are poor ADP-ribose acceptors in the ART1-catalyzed reaction. To identify the products of the reaction of the ornithine analogs, ART1 and NAD, the products were separated by HPLC, purified, and analyzed by mass spectroscopy (Fig. 6B). The early peaks in the HPLC separations were identified as ADP-ribosylated HNP- FIGURE 4. Effect of HNP-1 analogs on TGF-b1 release from A549 (R14orn), (Fig. 6Bb) or ADP-ribosylated-HNP-(R24orn), (Fig. 6Bc) 4 cells. A549 cells (1 3 10 /ml) were grown for 24 h and then washed with whereas the later peaks were identified as the unmodified forms. serum-free media. HNP, HNP-(R14orn), HNP-(R24orn), and HNP- In contrast, three products were separated from the reaction of Downloaded from (R14,24orn), solubilized in serum-free media, were added at the indicated HNP-1 with NAD and ART1 (Fig. 6Ba) (28). Compared with HNP- concentrations, as described in Materials and Methods. The medium was 1, (Fig. 6Bd) the unmodified analogs were found at an earlier analyzed for TGF-b1 by immunoassay. The data are the mean 6 SEM of three experiments performed in triplicate. The mean TGF-b1 levels re- HPLC elution time, suggesting that they are less hydrophobic than leased in the presence of HNP, HNP-(R24orn), HNP-(R14orn), and HNP- HNP. ADP-ribosylated HNP-(R24orn) (Fig. 6Bc) eluted at a sig- (R14, 24orn) decrease with analog concentrations [p , 0.0001 for HNP, nificantly earlier time, suggesting the product is as hydrophobic HNP-(R24orn), and HNP-(R14,24orn), and for HNP-(R14orn), p = as the dimodified HNP-1. ADP-ribosylarginine 5 or 15 HNP was http://www.jimmunol.org/ 0.0007]. At 20 mg/ml, there is no significant difference between the mean not identified in the reaction products or in HNP-(R14,24orn) TGF-b1 level observed after incubation with HNP and that of HNP- (Fig. 6Be). Increased ADP ribosylation of HNP-1 and HNP- (R14orn), p = 0.31. The amount of TGF-b1 released by HNP-(R24orn) and (R14,24orn) was observed after reduction of the disulfide bridges , HNP-(R14,24orn) differs significantly from that of HNP (p 0.0001 and with DTT (Fig. 6Bf). These data suggest that the quaternary p = 0.0006, respectively). structure of HNP and the analogs limit the accessibility of argi- nines for ADP ribosylation by ART1. b inhibited release of less TGF- 1 than did HNP and HNP-(R14orn) The three HNP ornithine analogs weakly dimerize at equivalent concentrations (Fig. 4). Previous studies showed that dimerization was important for the by guest on September 27, 2021 HNP-(R14,24orn) retains the antibacterial activity of HNP-1 antimicrobial activity of HNP-1 (41). We questioned whether the HNP1-4, released from neutrophil azurophilic granules, act as natural difference in antimicrobial activity of HNP-(R14,24orn) and HNP antibiotics, killing a variety of microorganisms (1). Toxicities of the with a single ornithine substitution could be due to differences HNP-ornithine analogs for E. coli-TG1 and P. aeruginosa were in dimerization. The degree of ornithine analog oligomerization compared with HNP (Fig. 5). After 1-h incubation, CFUs were de- in solution was investigated by analytical ultracentrifugation. termined by plate assay. HNP-(R14,24orn) was toxic for P. aerugi- The distribution of sedimentation coefficients of all HNP analogs nosa and E. coli-TG1. HNP-(R24orn) was not toxic for E. coli-TG1 showed .90% of the loaded material sedimenting as a single or P. aeruginosa. HNP-(R14orn) was toxic for P. aeruginosa at component with an s20,w value of 0.8 S (Fig. 7). The apparent 3 mg/ml, but inactive against E. coli-TG1. Surprisingly, the mono- molecular mass of this peak calculated from the sedimentation substituted analogs did not exhibit HNP-1 antibacterial activity, but velocity data is 3.8 kDa, in good agreement (,10% difference) were toxic for epithelial cells; antibacterial activity was observed, with the mass of the monomer calculated from protein sequence. however, with ornithine at positions 14 and 24. HNP (3 mg) was Only the coefficient of distributions of HNP-1 and HNP- not toxic for either E. coli (ATCC 25922) or S. aureus (ATCC (R14,24orn) showed small secondary peaks consistent with an 29213), which may possibly be due to the HNP concentration used oligomer. Despite the relatively high concentration (60 mM) of or the assay conditions (NaPO4 without added nutrients). peptide, the population of oligomer was only 8%, indicating weak

FIGURE 5. (A and B) Toxicity of HNP analogs for P. aeruginosa and E. coli. P. aeruginosa-PA103 (A) and E. coli-TG1 (B) were incubated with increasing amounts of HNP, HNP- (R14orn), HNP-(R24orn), or HNP-

(R14,24orn) in 70 ml 10 mM NaPO4 (pH 7.4) at 37˚C. After 1 h, CFU were determined by plate assay. The results shown represent an experi- ment performed with duplicates. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. (A) Effect of HNP analogs or agmatine on nicotinamide release from NAD catalyzed by ART1. HNP analogs or agmatine in the amounts indicated were incubated with ART1 (9.3 nmol/h) and 0.1 mM [nicotinamide-U-14C]NAD (0.05 mCi/reaction) in 50 mM potassium phosphate (pH 7.5) for 1.5 h at 30˚C. ADP ribosylation of HNP analogs or agmatine was measured by nicotinamide release in the standard assay described in Materials and Methods, HNP-(R14orn) (▴), HNP (n), agmatine (d), HNP-(R24orn) (;), HNP-(R14, 24orn) (♦).Thedataarethemean6 SEM of two experiments with assays run in duplicate. There is no significant difference between the mean nicotinamide release levels of HNP and HNP-(R14orn), p , 0.0001, but both were significantly different from the nicotinamide release by agmatine (p , 0.0001). Nicotinamide release in the presence of HNP-(R14,24orn) increased with analog amounts (p , 0.0001). However, HNP-(R14,24orn) was a poor ADP-ribose acceptor in the presence of ART1, compared with HNP and the mono-ornithine analogs. (B) HPLC separation of reaction products from the incubation of ornithine analogs with ART1. HNP-1 analogs (10 mg) were incubated with ART1 (4.7 nmol/h) and 5 mM NAD in 50 mM potassium phosphate (pH 7.5) for 8 h at 30˚C. HNP-1 or products from the reaction mix were separated by HPLC and monitored at 210 nm, as in Materials and Methods. The data are representative of three experiments. Peaks were identified by mass spectroscopy as (Ba). HNP-1, 48.3 min (d); ADP-ribosyl-HNP, 47.7 min; (+) ADP-ribosyl-HNP-ornithine, 46.8 min; (*), di- ADP-ribosyl-HNP, 46.1 min, (0). (Bb) HNP-(R14orn), 48.3 min (D); ADP-ribosyl-arginine 24-HNP-(R14orn), 47.6 min (▴). (Bc) HNP-(R24orn), 48.1 min (N); ADP-ribosyl-arginine 14-HNP-(R24orn), 46.4 min (n). (Bd)HNP-1,48.5min.(Be) HNP-(R14,24orn), 47.9 min. (Bf)HNP-1(3mg) or HNP-(R14,24orn) (3 mg) was incubated with ART1 (9.3 nmol/h) for 1 h in 50 mM potassium phosphate (pH 7.5), 10 mM(adenylate-[32P])NAD (1 mCi/assay), followed by 5 mM unlabeled NAD overnight, with or without 20 mM dithiodreitol at 30˚C. Proteins were trichloroacetic acid precipitated, separated on 16% tricine gels (Invitrogen), which were stained with Coomassie Blue, dried, and exposed to x-ray film (BioMax; Kodak). Data represent one of three experiments. 6 ACTIVITY OF HNP-1–CONTAINING ORNITHINE

dependent on cell type. For the four cell lines tested (Fig. 2), the ornithine analogs showed similar toxicity (i.e., for the NHLF and SAEC) or were less toxic than HNP. As observed by confocal laser microscopy, HNP-1 at high concentrations (10–20 mg/ml) rapidly entered A549 cells, colo- calized with the endoplasmic reticulum, and stimulated apoptotic cell death (44). The reduced cytotoxicity of HNP after di-ornithine substitution could result from changes to HNP folding that may affect its capacity to enter the cell or localize to the endoplasmic reticulum. Substitution of ornithines for two arginines in oncocin, a 19-aa peptide sequence derived from the insect Oncopeltus peptide 4, resulted in a peptide that was active against E. coli,but nontoxic to HeLa cells and stable in serum (46). Conversion of HNP-1 to the di-ornithine form resulted in reduction of injury to the epithelial cells in the airway and preservation of the prolif- FIGURE 7. Analysis of dimer formation in solution. Sedimentation erative response believed to be involved in wound repair. coefficient distributions [c(S)] from the analysis of the sedimentation ve- A better understanding of the essential properties of the anti- locity data obtained for HNP-1 (—), HNP-(R14, 24 orn) (– –), HNP- microbial defensins may lead to new analogs with activity against (R14orn) (– - –), HNP-(R24orn) (– --–), and HNP (acetate form) (. . .). bacteria resistant to current antibiotics. Cationic arginine residues, Downloaded from Results represent one of eight experiments. quaternary structure, and hydrophobicity are critical to HNP-1 antimicrobial activity. Bacterial lysis is thought to result from oligomerization of HNP isoforms in solution and no significant the positively charged peptide interacting with the prokaryotic difference among the analogs. In summary, ADP ribosylation of negatively charged membrane, resulting in pore formation (47). arginines at residues 14 and 24 by ART1 regulates HNP-1 bio- Surprisingly, arginines or ornithines at positions 14 and 24 in the logical activities (Fig. 8). Site-specific ADP ribosylation by ART1 HNP primary sequence are critical for antibacterial activity. Al- http://www.jimmunol.org/ abolishes HNP-1 antimicrobial and cytotoxic activities. HNP with though lysine, arginine, and ornithine have equivalent charge, the ADP-ribosylated arginines 14 and 24 nonenzymatically converted replacement of arginine by lysine affected the antimicrobial ac- to ornithines is not cytotoxic for epithelial cells, but the antibac- tivity of a-defensins (48). The virtual lethal dose for 90% of terial activity is preserved. bacteria increased when ornithine replaced arginine or lysine at residues 14, 15, and 24 (22). HNP-(R14,24orn) with arginine at Discussion residue 15, however, retained significant antibacterial activity The airway epithelium plays a critical role in host defense against against P. aeruginosa and E. coli, suggesting that ornithine 15 had infection (42). The repair process of the injured epithelium an effect other than charge (22). Moreover, in contrast to the by guest on September 27, 2021 involves cell migration and proliferation, processes that increase toxicity of replacing arginine 14 and 24 as in HNP-(R14,24orn), neutrophil defensins. As reported previously (31, 32, 43, 44), the the replacement of arginine 24 with ornithine abolished the anti- proliferative and cytotoxic effects of HNP-1 on human lung bacterial activity against P. aeruginosa and E. coli, demonstrating fibroblasts and epithelial and endothelial cells are dose dependent. that the activity of HNP-1 is affected by the number and position Low concentrations of HNP-1 have a proliferative effect on NHLF of arginine residues replaced by ornithine. (43) and A549 carcinoma epithelial cells (45). However, at con- Previous studies have reported that, in addition to charge, hy- centrations .20 mg/ml, HNP is cytotoxic for NHLF and A549 drophobicity and dimerization contribute to the antimicrobial ac- cells (31, 43). The di-ornithine form of HNP retained proliferative tivity of HNP-1 (21, 41). Analytical ultracentrifugation analysis effects, was nontoxic for A549 cells up to 100 mg/ml, but as toxic showed that HNP and the ornithine analogs sediment as a mono- as HNP-1 on NHLF, demonstrating that the biological effects were mer, indicating that there was no difference in oligomerization between the analogs and HNP. All ornithine analogs eluted earlier than HNP-1 in the HPLC separations, suggesting that they are less hydrophobic than HNP-1 and that the decrease in hydrophobicity was unrelated to the antibacterial activity of the HNP analogs. In contrast to the lack of cytotoxicity, the properties of HNP-1 that included the ability to form oligomers in solution, prolifera- tion, antibacterial activity, and the release of TGF-b1 and IL-8 from A549 cells were not significantly affected by substitution of ornithines in place of arginines 14 and 24. ART1 was unable to ADP-ribosylate arginines 5 and 15 in HNP or di-ornithine HNP. Increased ADP ribosylation required disruption of its tertiary structure with reduction of the disulfide bonds in HNP-1 and HNP- (R14,24orn). Ornithine in the primary sequence can change the stability by making the peptide bond resistant to proteolytic FIGURE 8. Schematic representation of the effects of ADP ribosylation cleavage (49). After incubation with trypsin, liquid chromatography– of HNP-1 on biological properties. Incubation of HNP-1 with ART1 and mass spectrometry analysis of the ornithine analogs did not find NAD results in ADP ribosylation of HNP-1, releasing nicotinamide (Nam) with reduction in its antimicrobial and cytotoxic activity (25). The ADP- evidence of cleavage sites C-terminal to the ornithines (data not ribosylated arginines of HNP-1 are converted nonenzymatically to orni- shown). Taken together, the data suggest that arginines 14 and 24, thine by nucleophilic attack of H2O on the guanidino carbon of arginine. targeted by ART1 for ADP ribosylation, in addition to effects on The HNP di-ornithine product retains antibacterial activity, but is no longer properties such as charge, structure, and hydrophobicity, contrib- toxic to epithelial cells. ute to the biological activities of HNP-1. The Journal of Immunology 7

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