ADP-ribosylation of human defensin HNP-1 results
in the replacement of the modified arginine with
the noncoded amino acid ornithine
Linda A. Stevensa, Rodney L. Levineb, Bernadette R. Gochuicoc, and Joel Mossa,1
aTranslational Medicine Branch andbLaboratory of Biochemistry, National Heart, Lung and Blood Institute,cNational Human Genome Research Institute,
Medical Genetics Branch, National Institutes of Health, Bethesda, MD 20892
Communicated by M. Daniel Lane, Johns Hopkins University School of Medicine, Baltimore, MD, September 22, 2009 (received for review August 8, 2009)
Defensins (e.g., human neutrophil peptides, or HNPs) contribute to
innate immunity through diverse actions, including microbial killing;
high concentrations are present in the lung in response to inflamma-
replacement with ornithine. ADP-ribosyltransferases (ARTs) catalyze
transfer of ADP-ribose from NAD to an acceptor arginine in a protein
substrate, whereas ADP-ribosylarginine hydrolases release ADP-
ribose. ART1 on the surface of airway epithelial cells ADP-ribosylated
HNP-1 specifically on arginines 14 and 24, with ADP-ribosylation
altering biological activity. Di- and mono-ADP-ribosylated HNP-1
were isolated from bronchoalveolar lavage fluid (BALF) of patients
for ADP-ribosylation in disease. In the present study, we observed
that ART1-catalyzed ADP-ribosylation of HNP-1 in vitro generated a
product with ADP-ribose on arginine 24, and ornithine replacing
arginine at position 14. We hypothesized that ADP-ribosylarginine
is susceptible to a nonenzymatic hydrolytic reaction yielding orni-
thine. On incubation of di- or mono-ADP-ribosyl-HNP-1 at 37 °C,
ADP-ribosylarginine was partially replaced by ornithine, whereas
ornithine was not detected by amino acid analysis and mass spec-
trometry of unmodified HNP-1 incubated under the same conditions.
Further, ornithine was produced from the model compound, ADP-
ornithine as well as mono- and di-ADP-ribosylated HNP-1, consistent
with in vivo conversion of arginine to ornithine. Targeted ADP-
ribosylation of specific arginines by transferases, resulting in their
replacement with ornithine, is an alternative pathway for regulation
of protein function through posttranslational modification.
posttranslational modification ? NAD ? bacterial toxins
tion or infection (1). Neutrophil defensins [human neutrophil
peptides (HNPs) 1–3], stored in azurophilic granules, are small
cationic peptides whose main function is to defend the lung
against pathogenic microorganisms (2). High levels of defensins
have been found in patients with inflammatory lung diseases,
(4). In addition to antimicrobial activities and other diverse
functions (5), defensins interact with airway epithelial cells,
increasing proliferation and stimulating wound repair (6).
HNP1–3 are arginine rich and differ in sequence by one amino
acid. The salt bridge formed by Arg5–Glu13and three disulfide
bridges are conserved, but not required, for antibacterial activity
activity (9). In addition, the low number of arginines in HD6
(human defensin 6 expressed in Paneth cells) may be responsible
for its lack of antibacterial activity (10).
Mono-ADP-ribosylation is a posttranslational modification
of proteins in which the ADP-ribose moiety of NAD is
transferred to a specific amino acid. Several well-characterized
mono-ADP-ribosyltransferases were identified in viruses, bac-
teria, and eukaryotes. The modification can be reversed by
eutrophils, a critical component of the innate immune
system, are recruited to airways in response to inflamma-
ADP-ribosyl- acceptor hydrolases, which cleave the ADP-
ribose-acceptor bond (11, 12). Arginine-specific mono-ADP-
ribosyltransferase-1 (ART1) is present on the apical surface of
epithelial cells in human airways and is linked to the cell
surface by a glycosylphosphatidylinositol (GPI) anchor (13,
14). ART1 modifies the arginines of several substrates, in-
cluding HNP-1, thereby altering their activity (15, 16). ADP-
ribosylation of HNP-1 decreased the antimicrobial and
cytotoxic activities without affecting T-cell chemotaxis and
IL-8 release from A549 lung carcinoma cells (17). In vitro,
ART1 ADP-ribosylates HNP-1 on arginine 14 with a second-
ary site on arginine 24. Mono- and di-ADP-ribosylated HNP
were isolated from the bronchoalveolar lavage fluid 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. Sequence analysis revealed ornithine as
residue 14, with arginine 24 as the site of ADP-ribosylation.
We hypothesized that ADP-ribosylation of HNP-1 by ART1
was responsible for conversion of arginines to ornithine,
suggesting a unique function for ADP-ribosylation. Because
both modified forms of HNP-1 were found in bronchoalveolar
lavage fluid, we investigated the possibility that ADP-
ribosylation would change the primary sequence of HNP-1 in
vivo, altering its activity. To determine the mechanism of
ornithine production, we analyzed the HPLC-purified mono-
or di-ADP-ribosylated-HNP-1 after incubation at pH 7 and 9.
To verify that the amino acid sequence of HNP-1 was not
critical for conversion of arginines to ornithine and to assess
stability of the arginine-ADP-ribose bond, we examined the
effect of incubating purified ADP-ribosylarginine on ornithine
formation under the same conditions.
Results and Discussion
After HPLC separation of products of the ART1, HNP-1, and
NAD reaction, MS analysis identified HNP-1, mono-ADP-
ribosylated on arginine 14, and di-ADP-ribosylated-HNP-1,
with a second modification on arginine 24. Incubation of
ART1 (2 nmol/h activity) and HNP-1 for 24 h at 30 °C
decreased the amount of di-ADP-ribosylated-HNP-1 and in-
creased a fourth HPLC peak (Fig. 1). The purified 3,940-Da
product, identified as ADP-ribosylated-HNP-ornithine by MS
analysis, with ornithine mapped to position 14, was subjected
to acid hydrolysis. Amino acid analyses confirmed the presence
of ornithine in the modified but not in the substrate HNP-1
research; B.R.G. contributed new reagents/analytic tools; L.A.S., J.M., and R.L.L. analyzed
data; and L.A.S. and J.M. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: email@example.com.
November 24, 2009 ?
vol. 106 ?
(Fig. 2). Because arginine-14 is the site of ADP-ribosylation,
it appeared that ADP-ribosyl-arginine was the precursor of
ornithine in HNP. Amounts of modified HNP-ornithine re-
covered in the reaction mix increased with incubation time
reaching about 26% after 168 h, consistent with the nonen-
zymatic conversion of arginine to ornithine (Fig. 3). To verify
that ART1 (or an enzymatic contaminant) was not required
for ornithine formation, HPLC-purified mono- and di-
modified HNP were incubated for 24 h at 37 °C at pH 7 or 9,
and the reaction products analyzed by MS (Fig. 4 and Table 1).
Di-ADP-ribosylated-HNP converted to mono-ADP-ribosy-
lated HNP-ornithine of 3940 molecular mass at pH 7 and the
2.0 nmol/h of ART1 activity as indicated. Reactions were terminated by addi-
tion of guanidine HCl (final concentration 6 M) before HPLC and analysis at
(E), ADP-ribosyl-HNP-ornithine (*), mono-ADP-ribosylated HNP-1 (?), and
HNP-1 (F). Data are representative of 3 experiments.
RP-HPLC separation of reaction products from the incubation of
ribosyl-HNP-ornithine was purified by HPLC separation of the products from
HNP-1 (10 nmol) and 5 mM NAD incubated with ART1 (12.8 nmol/h) as in Fig.
1, and its identity confirmed by MS analysis (Fig. 2D). Preparation for amino
acid analysis was as described in Methods before OPA derivatization (Agilent
Technologies) and separation by HPLC with monitoring by fluorescence (ex-
citation 340 nm and emission 450 nm). (A) Purified ADP-ribosyl-HNP-1-
(B) HNP-1 (3 nmol) with ornithine (?) (23 pmol; Sigma) added after hydrolysis.
residues 6–14 from a digest of ADP-ribosyl-HNP-1. The b8 ion is generated
b9 ion is generated from residues 6–14 and contains either orn (D) or arg (E).
The data are representative of 2 experiments.
Amino acid analysis of purified ADP-ribosyl-HNP-ornithine. ADP-
nmol) was incubated for the indicated times at 30 °C with 5 mM NAD and
ART1 (5.8 nmol/h) in 150 ?L of 50 mM potassium phosphate (pH 7.5).
Reactions were terminated at the indicated time by addition of guanidine
HCl (final concentration 6 M) before analysis by HPLC with monitoring of
absorbance at 280 nm. ADP-ribosyl-HNP-ornithine was quantified as pico-
moles calculated from the area (mAu) under the peak identified as ADP-
ribosyl-HNP-ornithine at 280 nm. The reported percent was based on the
total number of picomoles of reaction products in the separation. Data are
means ? SD from 4 experiments.
Time course of ADP-ribosyl-HNP-ornithine production. HNP-1 (6
Stevens et al.PNAS ?
November 24, 2009 ?
vol. 106 ?
no. 47 ?
amount was greater at pH 9. About 50% of di-ADP-ribosylated
HNP-1 was converted to mono-ADP-ribosylated-HNP-1-
ornithine at pH 9; when the ADP-ribose and ornithine were
mapped to positions on the HNP-1, we observed ADP-
ribosylated arginine 14 and ornithine at position 24 and
ADP-ribosylarginine 24 and ornithine 14. Of note, with the in
vitro-modified HNP-1, we did not find any di-ADP-ribosylated-
HNP-1-ornithine. Purified mono-ADP-ribosylated HNP-1 was
converted to HNP-1-ornithine, 3,399.5 Da under the same
conditions at pH 7, but the amount was greater at pH 9. Since
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
pulmonary fibrosis as described (18). Briefly, 8 mL of BALF
were applied to LC-18 Supelclean SPE tubes (Supelco) equili-
brated in 10% isopropanol/0.1% TFA, washed and eluted with
50% isopropanol/0.1% TFA. The eluted proteins were vacuum
concentrated before separation by RF-HPLC. In four of the
seven IPF samples, a broad peak eluted at the retention time of
HNP-1. MS analysis confirmed one of the samples contained
HNP-1 and consisted of 38.8% HNP-1, 32.1% di-ADP-
ribosylated HNP-1, 20.8% ADP-ribosyl-HNP-1, and 8.3% ADP-
ribosyl-HNP-1-orninthine. These data are consistent with the in
vivo alteration of HNP-1 primary sequence. Of note, as with in
vitro ADP-ribosyl-HNP-1, in the in vivo-modified material, we
did not see di-ADP-ribosylated HNP-1-ornithine, consistent
with the fact that ornithine is not ADP-ribosylated by NAD:argi-
nine ADP-ribosyltransferases. Experimentally, arginine and ag-
matine served as substrates for ART1, ornithine did not. We did
not find HNP-1-ornithine in BALF from patients with asthma
(n ? 4); of the four patients, one had both di- and mono-ADP-
1-ornithine were not seen in all patients with IPF or asthma.
Effects of sequence and structure on the biological activity of
the ?-defensins have been studied by several investigators (19).
After ADP-ribosylation by ART1, HNP-1 had reduced antimi-
crobial and cytotoxic activities but maintained its ability to
recruit T lymphocytes and release IL-8 from A549 cells (17). To
(not arginine 5) were replaced with lysine or ornithine (9).
Bactericidal activity was decreased as arginines were replaced by
lysines or ornithines. These data suggested that in addition to
ADP-ribosylation, conversion of arginine to ornithine can alter
HNP-1 activity in vivo.
Arginase, found predominantly in liver, catalyses the hydro-
lysis of arginine to ornithine in the urea cycle. We considered the
possibility that, although the enzyme is reported to require free
arginine (20), HNP-1 could be a substrate for bovine liver
arginase. HNP-1 (1.4 nmol), mono-ADP-ribosylated-HNP-1 (1
nmol), ADP-ribosylarginine (18 ?M), and arginine (5 mM) were
incubated with manganese-activated arginase (0.7 units) at pH
9.5 (in 200 ?L, 37 °C, 10 min), conditions used for arginase
hydrolysis of arginine to ornithine (21). Identification of the
products by amino acid analysis and MS revealed ornithine only
in the reaction that contained free arginine.
Ornithine was found in acid hydrolysates of human skin
age of the patients from whom the proteins were isolated. It was
proposed that ornithine formation resulted from glycation of
arginine by sugar moieties (Advanced Glycation End-products,
9. Di-modified HNP-1 (6 nmol) and mono-modified HNP-1 (0.5 nmol) were iso-
lated from the overnight incubation (30 °C) of HNP-1 (10 nmol) and 5 mM NAD
with ART1 (12.8 nmol/h) in 50 mM potassium phosphate (pH 7.5). Identity and
purity were confirmed by MS analysis. Purified di-modified HNP-1 or mono-
from the reaction of HNP-1 and NAD with ART1 (pH 7.5) overnight at 30 °C as
time (pH 9). (C) Di-modified HNP-1 incubated at pH 7. (D) Di-modified HNP-1
representative of 2 experiments at pH 7 and 3 experiments at pH 9.
Table 1. MS analysis of mono- and di-ADPribosyl-HNP-1 incubated at pH 7 and 9
pH Time, hHNPADPR-HNP HNP-ornDi-orn-HNPADPR-HNP-ornDi-ADPR-HNPTotal, %
82 ? 13
61 ? 9
18 ? 13
39 ? 9
1 ? 1
9 ? 3
32 ? 17
55 ? 3
67 ? 18
36 ? 0
MS analysis of purified mono- and di-ADPribosyl-HNP-1 incubated at pH 7 and 9. HNP-1 (3,442 Da), ADP-ribosyl-HNP-1 (3,983 Da), HNP-ornithine (3,400 Da),
Di-HNP-ornithine (3,358 Da), ADP-ribosyl-HNP-ornithine (3,941 Da), and di-ADP-ribosyl-HNP (4,525 Da). Data are the mean ? SD of 2 experiments on different
www.pnas.org?cgi?doi?10.1073?pnas.0910633106Stevens et al.
AGEs), followed by time-dependent breakdown of the adduct to
yield ornithine. In addition, because furornithine and N?-
carboxymethyl-ornithine were also detected in the acid hydro-
lysates, ornithine formed in this manner appeared to be further
glycated. Reducing sugars, such as ribose and ADP-ribose,
produce protein glycation by reacting with a free amino group of
lysine or arginine (23, 24). After the in vitro reaction of ribose
with collagen, the hydrolysate contained ?-NFC-1[N?-(4-oxo-5-
dihydroimidazol-2-yl)-L-ornithine], a product of a modified ar-
ginine (25). 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 se-
quence, or arginine with free ADP-ribose by amino acid analysis
(Fig. 5). In contrast to ADP-ribose glycation reactions with
model conjugates, enzymatically, ADP-ribosylated arginine was
stable at pH 9 for 30 min at 37 °C. ADP-ribose is released from
the modified protein substrate chemically by incubation with hy-
droxylamine or by enzymatic cleavage by ADP-ribosylarginine-
Consistent with a nonenzymatic conversion of modified
arginine to ornithine in HNP, ADP-ribosylarginine generated
ornithine when incubated under the same conditions. Orni-
thine was observed by amino acid analysis without prior acid
hydrolysis after incubation at pH 7 and 9 (37 °C) for 24 h.
Arginine was cleaved from ADP-ribosylarginine incubated in
6N HCl at 37 °C for 24 h, but ornithine was not detected by
amino acid analysis (Fig. 5). These data suggest that the amino
acid sequence of HNP-1 is not required for conversion of
modified arginines to ornithine. In contrast to the enzymatic
cleavage of ADP-ribosylarginine by ADP-ribosyl-arginine
hydrolase-1 (ARH1) at carbon 1?? of ADP-ribose, which
releases ADP-ribose from arginine, the nonenzymatic hydro-
lysis of ADP-ribosylarginine at the guanidino carbon of argi-
nine produces ornithine.
In addition to altering the molecular charge, pKa, secondary
structure, and biological activity, the presence of ornithine at the
HNP-1 arginine site and the absence of ADP-ribose would
prevent the modified protein from interacting with ADP-ribosyl-
acceptor hydrolases or serving as a target for subsequent ADP-
ribosylation. ADP-ribosyltransferases, such as cholera toxin,
modify guanidine-containing compounds (e.g., arginine or ar-
ginine on peptides), not amino acids containing an amino group
[e.g., lysine (31, 32) and ornithine (32)]. We found the same to
be true of ART1. If ADP-ribosylated arginine is converted
proteins may deteriorate over time. Moreover, the effect of
ADP-ribosylation on signal transduction would be altered. We
had reported that HNP-1 is specifically mono-ADP-ribosylated
by ART1 on arginine 14, or di-ADP-ribosylated on arginines 14
and 24, suggesting that specific sites in HNP-1 were selected by
ADP-ribosyltransferases for conversion to ornithine. We re-
ported that the posttranslational modification of HNP-1 by
ADP-ribose regulated its function. We have shown that this
modification may be unstable, resulting in an HNP-ornithine
peptide with altered function which may be relevant to innate
immunity in the airway.
Human Subjects Protection. The clinical protocol (99-H-0068) was approved by
the National Heart, Lung, and Blood Institute Institutional Review Board.
Written informed consent was obtained from all participants.
with plasmids containing mART1 were grown in Eagle’s MEM with 10% FBS
(Invitrogen) and Geneticin (G-418) 0.5 mg/mL. 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).
and 10 mM arginine (0.5 ?Ci14C/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
0.8 mL/min), followed by a 5-min linear gradient of 0% to 100% acetonitrile.
Peaks, monitored by absorbance at 254 nm, and radioactivity identified as
ADP-ribosyl[14C]arginine, were confirmed by MS analysis. Samples (25 ?L) of
ADP-ribosyl[14C]arginine were vacuum dried, dissolved in 200 ?L 6N HCl
(Fluka), and hydrolyzed under nitrogen (155 °C, 45 min) to release arginine.
The hydrolysate was vacuum dried, dissolved in 25 ?L 0.05% TFA, and sub-
jected to OPA derivatization (Agilent Technologies) before HPLC separation
pH 7 and 9. ADP-ribosyl[14C]arginine was prepared and purified as described
in Methods. ADP-ribosyl[14C]arginine, arginine, ADP-ribose plus arginine or
ornithine (15 pmol) in 20 mM potassium phosphate (pH 7.5) adjusted to pH 9
by NaOH (measured by microelectrode) in 100 ?L were incubated at 37 °C for
24 h except where noted. The samples were vacuum dried, dissolved in 25 ?L
0.05% TFA (without acid hydrolysis) before amino acid analysis. The HPLC
separation (see Methods) was monitored by UV detection at 338 nm. (A)
ADP-ribose and arginine (F). (B) Ornithine (*) (Sigma). (C) ADP-
ribosyl[14C]arginine incubated 24 h in 6N HCl at 37 °C. (D) ADP-
ribosyl[14C]arginine (E) 0 time. (E) ADP-ribosyl[14C]arginine. Data are repre-
sentative of 3 experiments. ADP-ribosyl[14C]arginine (1.8 nmol) in 20 mM
potassium phosphate (pH 7) incubated for the indicated times at 37 °C before
amino acid analysis monitored by fluorescence (340 nm excitation/450 emis-
Amino acid analysis following incubation of ADP-ribosylarginine at
Stevens et al.PNAS ?
November 24, 2009 ?
vol. 106 ?
no. 47 ?
(see text following). The arginine peak was quantified by absorbance at 338 Download full-text
nm and fluorescence (340 excitation/450 emission), then compared with a
Amino Acid Analysis. The indicated amount HNP-1 (Bachem) was vacuum
dried, dissolved in 200 ?L of 6N HCl plus 5 ?L of 40 mM DTT before hydrolysis
under nitrogen at 155 °C for 45 min. The hydrolysate was vacuum dried and
solubilized in 25 ?L water, 0.05% TFA before OPA (Agilent Technologies)
precolumn automated derivatization. The conditions for the derivatization
reaction and the HPLC separation (with the modification following) are
described in Agilent Technologies Technical Note (publication no. 5980–
equilibrated with mobile phase A, 40 mM sodium dibasic phosphate buffer
(pH 7.8) and amino acids were eluted with a linear gradient of 0% to 40% of
phase B, acetonitrile/MeOH/water (45:45:10) for 1.9–15 min; 15–18.1 min
gradient to 57% B, 18.1–18.6 min gradient to 100% B.
HPLC on a Discovery BioWide Pore C18 column (Supelco) as described (18).
MS and Sequence Analysis. HNP-1 was reduced, cleaved by trypsin, and
analyzed by reverse-phase chromatography/mass spectrometry as described
(17, 18), except that the reverse-phase column was a Zorbax 300SB-C18, 2.1 ?
50 mm 3.5 ?m, and the mass spectrometer was an Agilent model G1969
(Agilent Technologies) with a time-of-flight detector. Mass spectra were
deconvoluted with the Agilent software, MassHunter version 2, and the
fraction of each species was calculated from the areas of the deconvoluted
Preparation of Ornithine from ADP-Ribosyl[14C]arginine at pH 9 at 30 °C and
37 °C. ADP-ribosyl[14C]arginine was prepared as described, followed by incu-
bation at pH 9. Three samples were analyzed by amino acid analysis at 30 °C
and 37 °C. The production of ornithine at 30 °C was 4.4 ? 0.03 pmol/?L, and
ACKNOWLEDGMENTS. We thank Dr. Martha Vaughan and Dr. Gustavo
Pacheco-Rodriguez (National Heart, Lung, and Blood Institute) for helpful
discussions and critical review of the manuscript. These studies were sup-
ported by the Intramural Research Program of the National Institutes of
Health, National Heart, Lung, and Blood Institute.
Ann Med 34:96–101.
2. Bals R, Hiemstra PS (2004) Innate immunity in the lung: How epithelial cells fight
against respiratory pathogens. Eur Respir J 23:327–333.
idiopathic pulmonary fibrosis. Thorax 57:623–628.
4. Soong LB, Ganz T, Ellison A, Caughey GH (1997) Purification and characterization of
defensins from cystic fibrosis sputum. Inflamm Res 46:98–102.
immune function. Crit Rev Immunol 28:185–200.
6. van Wetering S, Tjabringa GS, Hiemstra PS (2005) Interactions between neutrophil-
derived antimicrobial peptides and airway epithelial cells. J Leukoc Biol 77:444–450.
7. Wu Z, Li X, de Leeuw E, Ericksen B, Lu W (2005) Why is the Arg5-Glu13 salt bridge
conserved in mammalian alpha-defensins? J Biol Chem 280:43039–43047.
8. Lundy FT, et al. (2008) Antimicrobial activity of truncated alpha-defensin (human
neutrophil peptide (HNP)-1) analogues without disulphide bridges. Mol Immunol
9. Zou G, et al. (2007) Toward understanding the cationicity of defensins. Arg and Lys
versus their noncoded analogs. J Biol Chem 282:19653–19665.
10. Szyk A, et al. (2006) Crystal structures of human alpha-defensins HNP4, HD5, and HD6.
Protein Sci 15:2749–2760.
11. Corda D, Di Girolamo M (2003) Functional aspects of protein mono-ADP-ribosylation.
EMBO J 22:1953–1958.
ADP-ribosyltransferases and ADP-ribosylhydrolases. Front Biosci 13:6716–6729.
13. Balducci E, et al. (1999) Selective expression of RT6 superfamily in human bronchial
epithelial cells. Am J Respir cell Mol Biol 21:337–346.
14. Okazaki IJ, Moss J (1998) Glycosylphosphatidylinositol-anchored and secretory iso-
forms of mono-ADP-ribosyltransferases. J Biol Chem 273:23617–23620.
response and cell signaling. Sci STKE 2002:PE53.
16. Di Girolamo M, Dani N, Stilla A, Corda D (2005) Physiological relevance of the endog-
enous mono(ADP-ribosyl)ation of cellular proteins. FEBS J 272:4565–4575.
17. Paone G, et al. (2002) ADP ribosylation of human neutrophil peptide-1 regulates its
biological properties. Proc Natl Acad Sci USA 99:8231–8235.
18. Paone G, et al. (2006) ADP-ribosyltransferase-specific modification of human neutro-
phil peptide-1. J Biol Chem 281:17054–17060.
19. Pazgier M, Li X, Lu W, Lubkowski J (2007) Human defensins: Synthesis and structural
properties. Curr Pharm Des 13:3096–3118.
20. Jenkinson CP, Grody WW, Cederbaum SD (1996) Comparative properties of arginases.
Comp Biochem Physiol B Biochem Mol Biol 114:107–132.
21. Bachetti T, et al. (2004) Arginase pathway in human endothelial cells in pathophysi-
ological conditions. J Mol Cell Cardiol 37:515–523.
22. Sell DR, Monnier VM (2004) Conversion of arginine into ornithine by advanced glyca-
tion in senescent human collagen and lens crystallins. J Biol Chem 279:54173–54184.
23. Jacobson EL, Cervantes-Laurean D, Jacobson MK (1994) Glycation of proteins by
ADP-ribose. Mol Cell Biochem 138:207–212.
histones by ADP-ribose. J Biol Chem 271:10461–10469.
25. Paul RG, Avery NC, Slatter DA, Sims TJ, Bailey AJ (1998) Isolation and characterization
of advanced glycation end products derived from the in vitro reaction of ribose and
collagen. Biochem J 330 (Pt 3):1241–1248.
26. Shapiro R, McManus MJ, Zalut C, Bunn HF (1980) Sites of nonenzymatic glycosylation
of human hemoglobin A. J Biol Chem 255:3120–3127.
through the Maillard reaction: Effect on protein structure and gel properties. J Agric
Food Chem 52:1293–1299.
of [cysteine(ADP-ribose)]protein and [arginine(ADP-ribose)]protein linkages. J Biol
ADP-ribose: Studies of model conjugates. Biochemistry 32:1528–1534.
30. Moss J, et al. (1992) Molecular and immunological characterization of ADP-
ribosylarginine hydrolases. J Biol Chem 267:10481–10488.
31. Moss J, Vaughan M (1978) Isolation of an avian erythrocyte protein possessing ADP-
ribosyltransferase activity and capable of activating adenylate cyclase. Proc Natl Acad
Sci USA 75:3621–3624.
32. Moss J, Vaughan M (1977) Mechanism of action of choleragen. Evidence for ADP-
ribosyltransferase activity with arginine as an acceptor. J Biol Chem 252:2455–2457.
www.pnas.org?cgi?doi?10.1073?pnas.0910633106Stevens et al.