of March 26, 2018.This information is current as

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

MossAmy N. Otuonye, Rodney L. Levine, Gang Zheng and Joel Linda A. Stevens, Joseph T. Barbieri, Grzegorz Piszczek,

http://www.jimmunol.org/content/193/12/6144doi: 10.4049/jimmunol.1303068November 2014;

2014; 193:6144-6151; Prepublished online 12J Immunol 

Referenceshttp://www.jimmunol.org/content/193/12/6144.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2014 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Nonenzymatic Conversion of ADP-Ribosylated Arginines toOrnithine Alters the Biological Activities of HumanNeutrophil Peptide-1

Linda A. Stevens,* Joseph T. Barbieri,† Grzegorz Piszczek,‡ Amy N. Otuonye,*

Rodney L. Levine,x Gang Zheng,{ and Joel Moss*

Activated neutrophils, recruited to the airway of diseased lung, release human neutrophil peptides (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

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,

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: 6144–6151.

Polymorphonuclear leukocytes migrate into the lung to thesite of infection, inflammation, or other disease processesin response to complex signaling events. After activation,

neutrophils release antimicrobial a-defensins (human neutrophilpeptides [HNP]1–4), cationic, amphipathic, arginine-rich peptideswith diverse biological activities (1). In addition to killing micro-organisms, defensins promote proliferation (2, 3) and stimulatethe release of neutrophil chemotactic factor, IL-8, and TGF-b1from lung epithelial cells and fibroblasts (4, 5). The in vitro effectson proliferation of airway epithelial cells suggested a role fordefensins in wound repair. Indeed, wound closure and mucin geneexpression were enhanced by HNP1–3 in vitro (6).It has been proposed that defensins released at the site of in-

flammation or infections are also involved in the pathogenesis of

disease (7–9). In the bronchoalveolar lavage fluid (BALF) of healthyvolunteers, concentrations of HNP1–3 are 0.2 mg/ml, but may in-

crease 6-fold during infection and 50-fold in inflammatory lung

diseases (10). Elevated levels of defensins were found in the BALF

of patients with a1-antitrypsin deficiency (11, 12), in the sputum of

patients with cystic fibrosis (13) and chronic obstructive pulmonary

disease (14), and in the serum of patients with interstitial lung dis-

ease [e.g., idiopathic pulmonary fibrosis (15), sarcoidosis (16, 17)].

Defensins at low concentrations increased proliferation and collagen

synthesis in lung fibroblasts, possibly participating in the formation of

the fibroproliferative lesions seen in inflammatory pulmonary diseases

(3). Intratracheal instillation of defensins (HNP1–3) into the lungs of

mice that do not express neutrophil-derived HNP caused acute lung

inflammation and dysfunction (18). Transgenic mice that expressed

a-defensins (HNP1, 2) in their neutrophils had more severe, acid-

induced acute lung injury than did their wild-type counterparts (19).Net charge, amphipathicity, hydrophobicity, and tertiary structure

are critical for the antimicrobial activity of defensins (20). In addi-

tion to the hydrophobicity of tryptophan 26 (21), the positive charge

of three arginine residues (22) is essential for the antimicrobial activ-

ity of HNP-1. ART1, an arginine-specific ADP ribosyltransferase

(ART) expressed on the surface of airway epithelial cells and neu-

trophils (23, 24), catalyzes the transfer of the ADP-ribose moiety of

NAD to arginine 14 of HNP-1, inhibiting its antibacterial and cy-

totoxic activities (25). The primary sequence of HNP1–3 contains

arginines at positions 5, 14, 15, and 24. Arginine 5 forms a salt

bridge with glutamic acid 13 that is required for folding stability

(26). Arginines 14 and 24 in HNP-1 are ADP ribosylated by ART1,

and ADP-ribosylated arginines can be converted nonenzymatically to

ornithine by nucleophilic attack of water on the guanidino carbon

with probable release of ADP-ribose-carbamate (27, 28). Mono- and

*Cardiovascular and Pulmonary Branch, National, Heart, Lung, and Blood Institute,National Institutes of Health, Bethesda, MD 20892; †Microbiology and MolecularGenetics, Medical College of Wisconsin, Milwaukee, WI 53226; ‡Biophysics CoreFacility, National, Heart, Lung, and Blood Institute, National Institutes of Health,Bethesda, MD 20892; xLaboratory of Biochemistry, National, Heart, Lung, andBlood Institute, National Institutes of Health, Bethesda, MD 20892; and {Office ofBiostatistics Research, National, Heart, Lung, and Blood Institute, National Institutesof Health, Bethesda, MD 20892

Received for publication November 14, 2013. Accepted for publication October 1,2014.

This work was supported by the Intramural Research Program of the National In-stitutes of Health, National Heart, Lung, and Blood Institute.

Address correspondence and reprint requests to Dr. Joel Moss, Room 6D05, Building10, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1590.E-mail address: mossj@nhlbi.nih.gov

Abbreviations used in this article: ART, ADP ribosyltransferase; BALF, bronchoal-veolar lavage fluid; HNP, human neutrophil peptide; mART, mouse ART; NHLF,normal human lung fibroblast; SAEC, small airway epithelial cell.

Copyright� 2014 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/14/$16.00

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di-ADP–ribosylated HNP-1 and ADP-ribosylated HNP-(ornithine)were isolated from the BALF of a patient with idiopathic pulmonaryfibrosis, suggesting that posttranslationally modified HNP-1 is foundin the diseased airway. The relative ratios of native HNP andderivatives vary among patients.Airway epithelial cells play a critical role in host defense by se-

cretingmolecules (e.g., cytokines, chemokines, antimicrobial peptides)that attract neutrophils and kill microorganisms (29, 30). IL-8, releasedfrom epithelial cells in response to HNP-1, amplifies the inflammatoryresponse by recruiting neutrophils that can, in turn, release additionalHNP. Defensins at concentrations found in disease may be toxic tolung epithelial cells, endothelial cells, and normal lung fibroblasts,resulting in injury to the airway (31, 32). Modifications of antimi-crobial HNP-1 that decrease its eukaryotic cytotoxicity but preserveantibacterial activity would reduce damage to the airway epitheliumwhile allowing the peptide to function in innate immunity.ADP ribosylation of specific arginines in HNP-1 by ART1 was

reported previously to have a role in modulating its activity, con-sistent with a critical role for these arginines (25). We hypothesizedthat replacement of ADP-ribosyl-arginine with ornithine in HNP-1might also alter activity. To address this question, HNP-(R14orn),HNP-(R24orn), and HNP-(R14,24orn) were synthesized, and theirantibacterial, cytotoxic, and other activities were compared withnative HNP-1. The data demonstrate that replacement of specificarginines in HNP-1 with ornithine through an ADP-ribosylation–dependent reaction alters its biological activities and may protectairway cells from HNP-1–induced cytotoxicity while partially pre-serving antimicrobial activity.

Materials and MethodsAnalogs

HNP, HNP-(R14orn), HNP-(R24orn), and HNP-(R14,24orn) were syn-thesized by Bachem (Torrance, CA) as acetate salts. HNP-1 (Bachem),synthesized as a trifluroacetate salt, was included in indicated assays inaddition to the acetate salt.

Cell culture

Human lung carcinoma epithelial cells (A549) and human lung adeno-carcinoma epithelial cells (NCI-H441) were purchased from AmericanType Culture Collection (Manassas, VA) and grown in F-12K medium or

RPMI 1640, respectively, with 10% FBS. Normal human lung fibroblasts(NHLF) and normal human small airway epithelial cells (SAEC) werepurchased from Clonetics (Walkersville, MD) and grown, respectively, infibroblast media or small airway epithelial cell complete medium suppliedby the company. All cells were grown at 37˚C in 5% CO2.

Preparation of mouse ADP-ribosyltransferase-1

Rat mammary adenocarcinoma cells (NMU) transfected with plasmidscontaining the mouse ART (mART)1 gene were grown in Eagle’s MEMwith 10% FBS and 0.5 mg/ml Geneticin (G418). Proteins released from thecells by incubation with phosphatidylinositol-specific phospholipase C,which cleaves the GPI anchor, were collected in PBS (33), and ADP-ribosyltransferase activity was assayed, as described (34).

Preparation of ADP-ribosyl HNP analogs for identification ofthe site of modification

HNP analogs (10 or 30 mg), mART1 (9.14 nmol/h transferase activity), and5 mM NAD in 50 mM potassium phosphate (pH 7.5) were incubated at30˚C overnight or as indicated. Products were separated by HPLC. ADP-ribosylation sites on the purified products were mapped by mass spec-troscopy (28).

Mass spectrometry and sequence analysis

HNP analogs were reduced, trypsinized, and analyzed by reverse-phasechromatography/mass spectrometry, as described (25), except that thereverse-phase column was a Zorbax 300SB-C18, 1.0 3 50 mm, 3.5 mm,and the mass spectrometer was an Agilent model 6520 QTOF capable oftandem mass-spectrometric sequencing of peptides. Spectra were decon-voluted with Agilent Masshunter software version 3, and spectra werematched to those predicted with GPMAW version 9 (Lighthouse Data,Odense, Denmark) (28).

Effects of HNP analogs and agmatine on nicotinamide releasefrom [14C]NAD

HNP analogs or agmatine were incubated with mART1 (9.3 nmol/h) and0.1 mM [nicotinamide-U-14 C]NAD (0.05 mCi) in 50 mM potassiumphosphate (pH 7.5) for 1.5 h at 30˚C. Nicotinamide release resulting fromADP-ribose transfer to an acceptor was monitored, as described (35).

Effects of HNP analogs on proliferation, cytotoxicity, andrelease of IL-8 and TGF-b1

To measure the release of IL-8 (2 3 105 cells/ml) and TGF-b1 (1 3 104

cells/ml) from A549 cells, cells were grown in 96-well plates for 24 h incomplete media prior to treatment, washed with serum-free media, andtreated 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 (13 105/ml) (A–C) and NHLF (43 104/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).

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human IL-8 and TGF-b1 in the media were quantified by immunoassay(R&D Systems, Minneapolis, MN). To determine the number of viableA549 cells (1 3 105 cells/ml), NCI-H441 (1 3 105 cells/ml), SAEC (1 3105 cells/ml), or NHLF (43 104/ml) cells were grown on 96-well plates incomplete media prior to treatment and treated as above. Proliferation andcytotoxicity were determined with tetrazolium-based Cell Counting Kit-8(Dojindo Molecular Technologies, Rockville, MD), according to themanufacturer’s instructions.

Antibacterial assays

Escherichia coli (TG1 or ATCC 25922), Pseudomonas aeruginosa (PA103),and Staphylococcus aureus (ATCC 29213) were grown overnight in LBBroth, Miller (Luria-Bertani) (Difco: tryptone, 10 g/L; yeast extract, 5 g/L;and NaCl, 10 g/L) at 37˚C. Bacteria were diluted 1/50, cultured at 37˚C, andharvested after 2 h. The bacterial OD was measured (5 3 108/ml = 1, A =600 nm), and cells were diluted in 10 mM NaPO4 (pH 7.4) to 5 3 104 perml. 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 theamount of cell killing. HNP or analogs and amounts used in the assay wereHNP, 1.0, 0.1, and 0.01 mg; HNP-(R14orn), 3.0, 1.0, and 0.1 mg; HNP-(R24orn), 3.0, 1.0, and 0.1 mg; and HNP-(R14,24orn), 3.0, 1.0, and 0.1 mg.Toxicity was similar in one or two incubations with HNP.

Analytical ultracentrifugation

Sedimentation velocity. Analytical ultracentrifugation experiments were per-formed using the Beckman Optima XL-I analytical ultracentrifuge and a four-place AN-Ti rotor. All HNP samples were dialyzed in PBS buffer (pH 7.4) anddiluted to a final concentration of 60 mM. Double-sector centrifuge cells werefilled with 0.4 ml sample or reference buffer. Centrifuge rotor was acceleratedto 250,000 3 g (60,000 rpm) after thermal equilibrium was reached at rest at20˚C. Interference and absorbance scans at 280 nm were started immediatelyafter the rotor reached the set speed and collected until no further sedimen-tation boundary movement was observed. The apparent sedimentation coef-ficient distributions were analyzed by Lamm equation modeling using theSEDFIT software of Schuck (36). Positions of menisci and bottoms as well asfrictional ratios were optimized during the fitting procedure. The final ac-cepted fits had a root mean square of deviation ,0.005.

Statistical methods

The ANOVA and the test for trends were used to analyze the data presentedin the figures. The effects on the mean outcome (e.g., cell proliferation) bythe change in concentration levels or analogs were tested by a two-wayANOVA. The test for trends, based on a linear regression model withthe peptide amounts coded with a set of equal-spaced scores, was used totest whether the mean outcome changes with concentration levels; analysiswas done using SAS v9.3 software.

ResultsHNP-(R14,24orn) is cytotoxic for normal human fibroblasts,but not for A549 cells

To determine the effect of the HNP analogs on eukaryotic cells, lungepithelial carcinoma cells (A549) or NHLF were incubated withincreasing amounts of HNP or the three HNP-ornithine analogs for24 h in serum-free media (Fig. 1A–F). HNP and HNP-(R14,24orn) at10 mg/ml and HNP-(R14orn) and HNP-(R24orn) at 25 mg/ml in-creased the number of A549 cells (as measured by a tetrazoliumreduction assay). HNP and the three analogs were toxic for NHLF atconcentrations ,10 mg/ml (Fig. 1D–F). HNP-(R14orn) and HNP-(R24orn) were toxic for A549 cells at concentrations.40 mg/ml. Incontrast, replacement of arginines with ornithines at aa 14 and 24abolished the cytotoxic effect of HNP on A549 cells at concen-trations to 100 mg/ml (Fig. 1C). The toxicity of HNP and ornithineanalogs (50 mg/ml) was also tested on SAEC and NCI-H441 cellsand compared with that seen with A549 and NHLF cells. The HNPanalogs showed similar or less toxicity than HNP (Fig. 2).

The three HNP ornithine analogs stimulate release of IL-8 fromA549 cells

IL-8, a chemoattractant, modulates the inflammatory response byrecruiting neutrophils to the lung (37). Higher levels of IL-8 in

BALF and plasma were found in idiopathic pulmonary fibrosispatients compared with healthy volunteers (15, 38). HNP-1 has beenreported to induce release of IL-8 and other chemokines and cyto-kines from epithelial cells and fibroblasts (4, 5, 39, 40). To determinethe effects of HNP and the ornithine analogs on IL-8 release byA549 cells, cells were incubated with the ornithine analogs, and IL-8in the medium was measured by immunoassay. HNP with a singleornithine substitution at concentrations up to 20 mg/ml increasedIL-8 release (Fig. 3A) as did HNP-(R14,24orn), up to 100 mg/ml(Fig. 3B), a range of concentrations not cytotoxic for A549 cells.

Two HNP ornithine analogs release more TGF-b1 from A549cells than does HNP

HNP-1 increased TGF-b1 release by fibroblasts, but reducedTGF-b1 production by A549 cells (4). A549 cells were incubatedwith increasing amounts of HNP analogs, and TGF-b1 release wasdetermined by immunoassay. HNP-(R24orn) and HNP-(R14,24orn)

FIGURE 2. Effect of HNP-1 analogs on the viability of A549, NHLF,

SAEC, and NCI-H441 cells. Cells were plated for 24 h, washed with se-

rum-free media, and incubated with HNP and the ornithine analogs (50 mg/ml)

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

triplicate. There was no significant difference between HNP and the three

analogs in NHLF and SAEC. HNP(R14,24orn) in NCI-H441 and A549

cells was significantly different from HNP. ****p , 0.0001, ***p , 0.001,

**p , 0.01, *p , 0.05.

FIGURE 3. (A and B) Effects of HNP-1 analogs on expression of IL-8

by A549 cells. (A) A549 cells (2 3 104) were grown for 24 h, washed with

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

the indicated amounts. (B) Cells were treated with 50 and 100 ml/ml HNP-

(R14,24orn), as indicated. The medium was analyzed for IL-8 expression,

as described in Materials and Methods. The data are the mean 6 SEM of

five experiments performed in triplicate. The mean IL-8 levels released in

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

significantly different from that of HNP-(R14orn), HNP-(R24orn), or

HNP-(R14,24orn) (all p . 0.05).

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inhibited release of less TGF-b1 than did HNP and HNP-(R14orn)at equivalent concentrations (Fig. 4).

HNP-(R14,24orn) retains the antibacterial activity of HNP-1

HNP1-4, released from neutrophil azurophilic granules, act as naturalantibiotics, killing a variety of microorganisms (1). Toxicities of theHNP-ornithine analogs for E. coli-TG1 and P. aeruginosa werecompared with HNP (Fig. 5). After 1-h incubation, CFUs were de-termined by plate assay. HNP-(R14,24orn) was toxic for P. aerugi-nosa and E. coli-TG1. HNP-(R24orn) was not toxic for E. coli-TG1or P. aeruginosa. HNP-(R14orn) was toxic for P. aeruginosa at3 mg/ml, but inactive against E. coli-TG1. Surprisingly, the mono-substituted analogs did not exhibit HNP-1 antibacterial activity, butwere toxic for epithelial cells; antibacterial activity was observed,however, with ornithine at positions 14 and 24. HNP (3 mg) wasnot toxic for either E. coli (ATCC 25922) or S. aureus (ATCC29213), which may possibly be due to the HNP concentration usedor the assay conditions (NaPO4 without added nutrients).

ADP ribosylation of HNP-ornithine peptides by ART1

To evaluate how well the ornithine analogs serve as substrates forART1, nicotinamide release concurrent with ADP-ribose transferfrom NAD to the acceptor was measured after incubation of thepeptides with ART1 and NAD; HNP was compared with agmatine,a decarboxylated form of arginine, which is a known ADP-riboseacceptor used by ART1 (Fig. 6A). HNP and HNP-(R14orn) werebetter acceptors of ADP-ribose than agmatine. A small amount ofnicotinamide release was observed in the presence of ART1,NAD, and HNP-(R14,24orn), indicating that, under these assayconditions, arginine 5 and arginine 15 are poor ADP-riboseacceptors 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 inthe HPLC separations were identified as ADP-ribosylated HNP-(R14orn), (Fig. 6Bb) or ADP-ribosylated-HNP-(R24orn), (Fig. 6Bc)whereas the later peaks were identified as the unmodified forms.In contrast, three products were separated from the reaction ofHNP-1 with NAD and ART1 (Fig. 6Ba) (28). Compared with HNP-1, (Fig. 6Bd) the unmodified analogs were found at an earlierHPLC elution time, suggesting that they are less hydrophobic thanHNP. ADP-ribosylated HNP-(R24orn) (Fig. 6Bc) eluted at a sig-nificantly earlier time, suggesting the product is as hydrophobicas the dimodified HNP-1. ADP-ribosylarginine 5 or 15 HNP wasnot identified in the reaction products or in HNP-(R14,24orn)(Fig. 6Be). Increased ADP ribosylation of HNP-1 and HNP-(R14,24orn) was observed after reduction of the disulfide bridgeswith DTT (Fig. 6Bf). These data suggest that the quaternarystructure of HNP and the analogs limit the accessibility of argi-nines for ADP ribosylation by ART1.

The three HNP ornithine analogs weakly dimerize

Previous studies showed that dimerization was important for theantimicrobial activity of HNP-1 (41). We questioned whether thedifference in antimicrobial activity of HNP-(R14,24orn) and HNPwith a single ornithine substitution could be due to differencesin dimerization. The degree of ornithine analog oligomerizationin solution was investigated by analytical ultracentrifugation.The distribution of sedimentation coefficients of all HNP analogsshowed .90% of the loaded material sedimenting as a singlecomponent with an s20,w value of 0.8 S (Fig. 7). The apparentmolecular mass of this peak calculated from the sedimentationvelocity data is 3.8 kDa, in good agreement (,10% difference)with the mass of the monomer calculated from protein sequence.Only the coefficient of distributions of HNP-1 and HNP-(R14,24orn) showed small secondary peaks consistent with anoligomer. Despite the relatively high concentration (60 mM) ofpeptide, the population of oligomer was only 8%, indicating weak

FIGURE 4. Effect of HNP-1 analogs on TGF-b1 release from A549

cells. A549 cells (1 3 104/ml) were grown for 24 h and then washed with

serum-free media. HNP, HNP-(R14orn), HNP-(R24orn), and HNP-

(R14,24orn), solubilized in serum-free media, were added at the indicated

concentrations, as described in Materials and Methods. The medium was

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-

leased in the presence of HNP, HNP-(R24orn), HNP-(R14orn), and HNP-

(R14, 24orn) decrease with analog concentrations [p , 0.0001 for HNP,

HNP-(R24orn), and HNP-(R14,24orn), and for HNP-(R14orn), p =

0.0007]. At 20 mg/ml, there is no significant difference between the mean

TGF-b1 level observed after incubation with HNP and that of HNP-

(R14orn), p = 0.31. The amount of TGF-b1 released by HNP-(R24orn) and

HNP-(R14,24orn) differs significantly from that of HNP (p , 0.0001 and

p = 0.0006, respectively).

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.

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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) (♦). The data are the mean 6 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, 48.5 min. (Be) HNP-(R14,24orn), 47.9 min. (Bf) HNP-1 (3 mg) 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.

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oligomerization of HNP isoforms in solution and no significantdifference among the analogs. In summary, ADP ribosylation ofarginines at residues 14 and 24 by ART1 regulates HNP-1 bio-logical activities (Fig. 8). Site-specific ADP ribosylation by ART1abolishes HNP-1 antimicrobial and cytotoxic activities. HNP withADP-ribosylated arginines 14 and 24 nonenzymatically convertedto ornithines is not cytotoxic for epithelial cells, but the antibac-terial activity is preserved.

DiscussionThe airway epithelium plays a critical role in host defense againstinfection (42). The repair process of the injured epitheliuminvolves cell migration and proliferation, processes that increaseneutrophil defensins. As reported previously (31, 32, 43, 44), theproliferative and cytotoxic effects of HNP-1 on human lungfibroblasts and epithelial and endothelial cells are dose dependent.Low concentrations of HNP-1 have a proliferative effect on NHLF(43) and A549 carcinoma epithelial cells (45). However, at con-centrations .20 mg/ml, HNP is cytotoxic for NHLF and A549cells (31, 43). The di-ornithine form of HNP retained proliferativeeffects, was nontoxic for A549 cells up to 100 mg/ml, but as toxicas HNP-1 on NHLF, demonstrating that the biological effects were

dependent on cell type. For the four cell lines tested (Fig. 2), theornithine analogs showed similar toxicity (i.e., for the NHLF andSAEC) 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 apoptoticcell death (44). The reduced cytotoxicity of HNP after di-ornithinesubstitution could result from changes to HNP folding that mayaffect its capacity to enter the cell or localize to the endoplasmicreticulum. Substitution of ornithines for two arginines in oncocin,a 19-aa peptide sequence derived from the insect Oncopeltuspeptide 4, resulted in a peptide that was active against E. coli, butnontoxic to HeLa cells and stable in serum (46). Conversion ofHNP-1 to the di-ornithine form resulted in reduction of injury tothe epithelial cells in the airway and preservation of the prolif-erative response believed to be involved in wound repair.A better understanding of the essential properties of the anti-

microbial defensins may lead to new analogs with activity againstbacteria resistant to current antibiotics. Cationic arginine residues,quaternary structure, and hydrophobicity are critical to HNP-1antimicrobial activity. Bacterial lysis is thought to result fromthe positively charged peptide interacting with the prokaryoticnegatively charged membrane, resulting in pore formation (47).Surprisingly, arginines or ornithines at positions 14 and 24 in theHNP primary sequence are critical for antibacterial activity. Al-though lysine, arginine, and ornithine have equivalent charge, thereplacement of arginine by lysine affected the antimicrobial ac-tivity of a-defensins (48). The virtual lethal dose for 90% ofbacteria increased when ornithine replaced arginine or lysine atresidues 14, 15, and 24 (22). HNP-(R14,24orn) with arginine atresidue 15, however, retained significant antibacterial activityagainst P. aeruginosa and E. coli, suggesting that ornithine 15 hadan effect other than charge (22). Moreover, in contrast to thetoxicity of replacing arginine 14 and 24 as in HNP-(R14,24orn),the replacement of arginine 24 with ornithine abolished the anti-bacterial activity against P. aeruginosa and E. coli, demonstratingthat the activity of HNP-1 is affected by the number and positionof arginine residues replaced by ornithine.Previous studies have reported that, in addition to charge, hy-

drophobicity and dimerization contribute to the antimicrobial ac-tivity of HNP-1 (21, 41). Analytical ultracentrifugation analysisshowed that HNP and the ornithine analogs sediment as a mono-mer, indicating that there was no difference in oligomerizationbetween the analogs and HNP. All ornithine analogs eluted earlierthan HNP-1 in the HPLC separations, suggesting that they are lesshydrophobic than HNP-1 and that the decrease in hydrophobicitywas 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-8from A549 cells were not significantly affected by substitution ofornithines in place of arginines 14 and 24. ART1 was unable toADP-ribosylate arginines 5 and 15 in HNP or di-ornithine HNP.Increased ADP ribosylation required disruption of its tertiarystructure with reduction of the disulfide bonds in HNP-1 and HNP-(R14,24orn). Ornithine in the primary sequence can change thestability by making the peptide bond resistant to proteolyticcleavage (49). After incubation with trypsin, liquid chromatography–mass spectrometry analysis of the ornithine analogs did not findevidence of cleavage sites C-terminal to the ornithines (data notshown). Taken together, the data suggest that arginines 14 and 24,targeted by ART1 for ADP ribosylation, in addition to effects onproperties such as charge, structure, and hydrophobicity, contrib-ute to the biological activities of HNP-1.

FIGURE 7. Analysis of dimer formation in solution. Sedimentation

coefficient distributions [c(S)] from the analysis of the sedimentation ve-

locity data obtained for HNP-1 (—), HNP-(R14, 24 orn) (– –), HNP-

(R14orn) (– - –), HNP-(R24orn) (– - - –), and HNP (acetate form) (. . .).

Results represent one of eight experiments.

FIGURE 8. Schematic representation of the effects of ADP ribosylation

of HNP-1 on biological properties. Incubation of HNP-1 with ART1 and

NAD results in ADP ribosylation of HNP-1, releasing nicotinamide (Nam)

with reduction in its antimicrobial and cytotoxic activity (25). The ADP-

ribosylated arginines of HNP-1 are converted nonenzymatically to orni-

thine by nucleophilic attack of H2O on the guanidino carbon of arginine.

The HNP di-ornithine product retains antibacterial activity, but is no longer

toxic to epithelial cells.

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Antimicrobial peptides are strong candidates in the search foralternative antibiotics. Mouse neutrophils lack defensins and thuscannot be used as a model for these studies (50). Our data show thatHNP analogs generated in disease exhibit altered activities. Studiesof the diseased human airway are necessary, to understand both theposttranslational processing of HNP-1 and the effects of the ana-logs of HNP-1 that can be important for healing the damagedlung. These results may help identify potential therapeutic agents.However, the use of the analogs in a clinical setting is complicateddue to the fact that they are structurally complex (e.g., disulfidebridges) and increase release of IL-8 and TGF-b from cells, asobserved with the A549 cells, and thus may be proinflammatory.HNP-(R14,24 orn) is a promising novel candidate: a stable peptide

antibiotic that shows diminished toxicity for the epithelial cells thatline the airway.

AcknowledgmentsWe thank Dr. Martha Vaughan and Dr. Gustavo Pacheco-Rodriguez

(National, Heart, Lung, and Blood Institute, National Institutes of Health)

for helpful discussions and for assistance with preparation of the manu-

script.

DisclosuresL.S., R.L., and J.M have patents and patents pending related to the modi-

fication of defensins, in particular HNP-1.

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