From the SangStat Medical Corporation, Menlo Park,
California 94025 and § Department of Biochemistry,
Biophysics and Environmental Medicine, University of Rochester School
of Medicine, Rochester, New York 14642
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ABSTRACT |
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This is the first report on peptidic inhibitors
of heme oxygenase. Such peptides were originally developed from the
immunomodulatory peptide 2702.75-84 which corresponds to amino acid
residues 75 to 84 of the 1-helix of HLA-B2702
(2702.75-84) and has been shown to be immunosuppressive in
vitro and in vivo. In vitro, 2702.75-84 inhibited
cytotoxic T- and natural killer cell- mediated target cell lysis, and
in vivo peptide therapy resulted in prolongation of heart
and skin allograft survival in mice. The peptide was also shown to bind
to heat shock protein 70. However, D-enantiomers of
2702.75-84 and derivatives thereof, while still being
immunosuppressive, did not bind to heat shock protein 70. This study
was designed to identify proteins binding to peptide D2702.75-84(E
V) (rvnlrialry) consisting of D-amino acids.
Compared with 2702.75-84 (RENLRIALRY), glutamic acid residue 76 (E) was replaced with valine (V). Affinity chromatography using
immobilized D2702.75-84(E
V) and mouse and human cell extracts,
resulted in the isolation of heme oxygenase-1 (HO-1). Peptide
D2702.75-84 inhibited HO activity in vitro in a dose
dependent manner. Similar to what has been observed with other
inhibitors of HO, administration of peptide into mice resulted in an
up-regulation of HO-1 mRNA and protein, as well as enzyme activity
in liver, spleen and kidney. Other peptides derived from 2702.75-84
with similar immunomodulatory activity displayed similar effects. In
contrast, inactive derivatives of 2702.75-84 had no effect on HO
activity. Therefore, the immunosuppressive effects of the described
immunomodulatory peptides are similar to those of
cobalt-protoporphyrin, a known up-regulator of HO-1. Our results suggest that HO-1 modulation may be a novel mechanism of
immunomodulation.
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INTRODUCTION |
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Peptides derived from various regions of the HLA class I heavy
chain have been shown to exert immunomodulatory effects by influencing
T cell responses (1-4). Clayberger and co-workers (5, 6) studied the
less polymorphic region of the HLA class I 1-helix
(residues 75-84). Peptides corresponding to this region of the HLA-B7
(07.75-84) or HLA-B2702 (2702.75-84) molecule blocked the
differentiation of human CTL precursors in vitro in a
non-allele-restricted manner (5). The 2702.75-84 peptide inhibited not
only differentiation, but also T and
NK1 cell-mediated lysis of
target cells in a non-allele-restricted manner. Woo et al.
(7) subsequently demonstrated a more powerful effect when the peptides
were used in a dimeric form (2702.84-75-75-84). These peptides have
also been studied in vivo in transplantation models. In
rats, 07.75-84 induced permanent acceptance of LEW heart allografts in
ACI recipients when combined with subtherapeutic doses of cyclosporine
A (6). In congeneic LEW.1W donors and LEW.1A recipients, 07.75-84
induced long term graft survival without additional cyclosporine A
treatment (8). In a mouse heart transplant model, where C57Bl/6 mice
were used as donors and CBA mice as recipients, treatment with both
2702.75-84 and 2702.84-75-75-84 prolonged graft survival
significantly, with B2702.84-75-75-84 showing a more powerful effect
(7, 9, 10). Administration of 2702.75-84 has been shown to prolong the
survival of skin allografts significantly when the tail skin of C57Bl/6
mice was grafted on CBA mice (9).
Despite the peptide's effectiveness, its mechanism of action is not fully understood. For reasons discussed elsewhere (7, 9, 10) a direct interaction with T cell receptors or NK cell inhibitory receptors can be excluded. The recent observation that both L- and D-enantiomers of peptide 2702.75-84 prolong heart allograft survival in vivo indicated that the peptides' immunomodulatory activity is probably not based on indirect presentation by major histocompatibility class molecules (10). Similarly, the recent hypothesis that peptide 2702.75-84 modulates immune responses by binding to HSP/HSC70 can be excluded because the D-isomer of 2702.75-84 did not bind such proteins (11, 12).
Based on these observations, we looked for additional proteins that may
interact with these immunomodulatory peptides. For these studies, we
used a 2702.75-84-derived peptide (D2702.75-84(E V)) with two
modifications: (i) synthesis with D-amino acids and (ii)
substitution of glutamic acid residue at position 76 in 2702.75-84
with valine. Compared with peptide 2702.75-84, this peptide displayed
enhanced immunomodulatory activity in vitro and in
vivo (12). Affinity purification using this peptide resulted in
the isolation of heme oxygenase 1 (HO-1, HSP32). Effects of peptide
binding to this enzyme were analyzed in vitro and in
vivo.
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MATERIALS AND METHODS |
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Animals
Male, 7-8-week-old CBA/J (H-2k) and C57BL/6/J (H-2b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed according to the Animal Welfare Guidelines, Department of Health, California.
Peptide Synthesis and Immobilization
Peptides were synthesized using Fmoc chemistry, purified by HPLC and shown to be >90% homogenous by analytical reverse phase HPLC (SynPep, Dublin, CA). Peptides CSGSGrvnlrialry and CSGSGrvnlrtalry (5 mg) were dissolved in Me2SO (60%) and coupled to 2 ml of SulfoLink coupling gel according to the manufacturer's recommendations (Pierce). An additional column was coupled with cysteine-HCl alone. Affinity chromatography using all three gels was performed simultaneously.
Cell Culture and Preparation of Cell Extracts
Human T cell leukemia cell line Jurkat (clone E6-1) and a mouse
lymphoma cell line Yac-1 were obtained from ATCC and maintained in RPMI
1640 medium (Sigma) containing 10% fetal bovine serum. For
purification of 2702.75-84(E V)-binding proteins, 1 × 109 cells were washed in cold PBS and lysed in a buffer
containing 50 mM Tris-Cl (pH 7.6), 300 mM NaCl,
1% CHAPS, and 5 µg/ml each of leupeptin, aprotinin, pepstatin,
chymostatin, antipain and 0.5 mM phenylmethylsulfonyl
fluoride. Cells were lysed at 4 °C for 30 min. Insoluble material
was discarded after centrifugation at 2000 rpm for 10 min, and protein
content was estimated by the BCA assay (Pierce).
Affinity Chromatography
SulfoLink gel was washed several times with 10 mM
phosphate buffer containing 150 mM NaCl, 0.1% CHAPS, and
protease inhibitors (wash buffer) and incubated with cell lysate for 60 min at 4 °C with continuous rocking. The gel was packed in a column
and unbound cell lysate was recovered. The column was washed with 20 column volumes of wash buffer, followed by wash buffer containing 500 mM NaCl. The latter fraction was termed high salt eluate.
Bound proteins were eluted with 100 mM glycine-HCl, pH 3.0, containing 0.1% CHAPS and protease inhibitors in 1-ml fractions that
were immediately neutralized to pH 7.0 using 1 M Tris-HCl,
pH 8.2. Protein concentration was estimated by the BCA assay (Pierce), and fractions containing protein were pooled, concentrated in a
microconcentrator, and analyzed by SDS-polyacrylamide gel
electrophoresis and western blotting. All fractions were stored at
80 °C.
Gel Electrophoresis and Western Blotting
Approximately 1 µg of pH 3.0 eluate, 5 µg of the high salt eluate, and 20 µg of cell lysate were boiled in SDS sample buffer containing 2-mercaptoethanol and separated on a 12% SDS-polyacrylamide gel. Proteins were visualized by silver staining (13).
Western transfer was carried out using the blot module from Novex (San Diego, CA) on nitrocellulose (Hybond-N, Amersham Corp.) in transfer buffer (12 mM Tris, 96 mM glycine, pH 8.3) at 30 V for 45 min. Efficiency of transfer was monitored by including prestained molecular weight markers (Amersham Corp.) on the gel. Nonspecific binding sites were blocked by immersing the membrane in PBS (pH 7.5) containing 1% bovine serum albumin overnight at 4 °C. After a short wash in PBS containing 0.1% Tween-20 (PBST), the membrane was incubated in primary antibody at the specified dilution in PBST for 1 h at room temperature with continuous rocking. The primary antibodies tested were rat anti-human HSC/HSP70 (Stressgen, Victoria, BC, Canada) at a 1:10,000 dilution, rabbit anti-rat HSP32 (also called HO-1), rabbit anti-rat heme oxygenase-2 (HO-2), rabbit anti-mouse HSP27 (all from Stressgen), all diluted at 1:5000. Following extensive washing in PBST, the membrane was incubated in the appropriate secondary antibody solution at the specified dilution for 1 h at room temperature with continuous rocking. The secondary antibodies used were rabbit anti-rat at 1:10,000 or donkey anti-rabbit at 1:5000, respectively (Jackson ImmunoResearch Laboratories, West Grove, PA). All secondary antibodies were conjugated to horseradish peroxidase.
After extensive washing, bands were visualized using enhanced chemiluminescence western blotting reagents (Pierce). Exposure to film (Hyperfilm ECL, Amersham Corp.) was performed for 10-30 s.
Protein Sequencing
Amino acid sequencing of the ~30-kDa band was performed by Edman chemistry using an automated ABI 470 A system sequencer with an on-line ABI 128 HPLC and analyzed on ABI 610 system software at the Protein structure laboratory, University of California, Davis, CA. The sequence was compared with known sequences in the University of Wisconsin GCG data base.
Purification of Recombinant HO-1
A first strand HO-1 cDNA was generated with 1 µg of testis
poly(A) RNA using the cDNA cycle kit (Invitrogen), priming with oligo(dT), and used as a template for polymerase chain reaction using
the primers 5-GGGAAGCTTGGAGCGCCCACAGCTCG-3
(representing nucleotides 2 to 18 of HO-1 (17) and a HindIII restriction
site (italics) and
5
-AAGCTTATGATGATGATGATGATGCTTATCGTCATCGTCCATGGCATAAATTCCCACTG-3
which contains the reverse complements of nucleotides 848-867, an enterokinase cleavage site (underlined), histidine tag (bold), stop
codon (double underline), and HindIII site (italics). The fragment was cloned into the HindIII site of the pBS+ vector
(Stratagene, La Jolla, CA) and the resultant plasmid was transformed
into Inv F
Escherichia coli cells. Orientation of the
insert was determined by restriction analysis and confirmed by
sequencing. Fusion proteins were purified from overnight cultures
utilizing ProBondTM (Invitrogen) columns in accordance with
the manufacturer's instructions. Pooled peak fractions from the
ProBondTM column elution were buffer-exchanged into 50 mM Tris-HCl, pH 8.0, containing 10% glycerol, 1 mM CaCl2, and 0.1% Tween-20 and concentrated
to approximately 1 mg/ml for storage at
80 °C. Preparations were
judged to be >90% homogenous as assessed by SDS-polyacrylamide gel
electrophoresis followed by staining with Coomasie Brilliant Blue.
Enzyme Assays
Heme Oxygenase-- To determine HO activity in tissues, mouse spleen samples were homogenized on ice in a Tris-HCl lysis buffer (pH 7.4) containing 0.5% Triton X-100 and protease inhibitors. Samples were frozen in small aliquots until use. Spleen microsomes were prepared and used as the source of HO for activity measurements, assessed by bilirubin formation. Spleen homogenate (100 µl) was mixed with 0.8 mM NADPH, 0.8 mM glucose 6-phosphate, 1.0 unit of Glu-6-P dehydrogenase, 1 mM MgCl2, and 10 µl of purified rat liver biliverdin reductase at 4 °C. The reaction was initiated by the addition of hemin (final concentration 0.25 mM). The reaction mixture was incubated at 37 °C in the dark for 60 min. At the end of the incubation period, any insoluble material was removed by centrifugation, and supernatants were analyzed for bilirubin concentration by a modified procedure of Hillman and Beyer (Sigma Diagnostics, kit no. 552). Controls included spleen samples in the absence of the NADPH-generating system and all components of the reaction mixture in the absence of spleen homogenates. Biliverdin reductase was purified from rat liver by the method described by Kutty and Maines (14).
To determine HO activity in purified preparations of HO-1, the procedure described previously was used and the activity was determined in the presence of purified preparations of biliverdin reductase and NADPH-cytochrome P450 reductase (15).Biliverdin Reductase-- Biliverdin reductase activity was determined by measuring the rate of bilirubin formation (14). To a reaction mixture consisting of 0.1 M Tris-HCl (pH 8.7), 0.1 mM NADPH, 5 µl (approximately 200 ng of purified enzyme protein), 5.0 µM biliverdin, and peptides at a final concentration of 100 µM were added. The control reaction mixture did not contain peptides. The reaction was carried out in the dark at 37 °C for 30 min, and the bilirubin formed was measured as described above.
Lactate Dehydrogenase and Horseradish Peroxidase-- Lactate dehydrogenase enzyme activity was determined using whole cells (E6-1) as the source as described previously (16). Horseradish peroxidase enzyme activity was measured by addition of o-phenylenediamine. Following incubation at room temperature, the reaction was stopped by the addition of HCl, and the optical density was measured at 490 nm.
Northern Blotting
A full-length (1300 base pairs) HO-2 cDNA isolated from a
rat testis library (17) was used as a HO-2 hybridization probe. The
HO-1 hybridization probe was a 569-base pair HO-1 fragment corresponding to nucleotides 86-654 of HO-1 cDNA (18). Mouse -actin, HO-1, and HO-2 cDNA probes were labeled with
[32P]dCTP according to the manufacture's instructions,
using the Rediprime primer DNA labeling system (Amersham Corp.),
and further purified by spin column chromatography (19).
Total RNA was prepared from tissues by the method of Chirgwin et al. (20). Poly(A+) RNA was isolated by oligonucleotide(dT) cellulose chromatography, fractionated on a 1.2% agarose gel, and transferred to Nytran (Schleicher & Schuell). Prehybridization, hybridization to the appropriate 32P-labeled cDNA, and post-hybridization treatment of the blots were preformed as described elsewhere (21). The signals on autoradiograms were quantitated using an LKB UltroScan densitometer.
In Vivo Effects of Peptide Treatment
Normal CBA mice were treated with a single injection
(intraperitoneal) of peptide D2702.75-84(E V) at 80 mg/kg
dissolved in PBS + 10% Me2SO. Control animals were treated
with an equal volume of PBS + 10% Me2SO. At 6, 24, and
48 h post-treatment, animals were killed, and liver, spleen,
kidney, heart, and blood (serum) were collected and rapidly frozen.
Tissue samples were used for northern blotting to detect HO-1 mRNA
and western blotting to detect HO-1 protein levels. Microsomes were
prepared from spleen samples and used for HO activity measurements and
western blotting.
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RESULTS |
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Isolation of Heme Oxygenase-1--
Recently, peptide 2702.75-84
(RENLRIALRY) and 2702.75-84(E V) (RVNLRIALRY) were shown to bind
to HSC70 and HSP70 (11, 12). In contrast, D-enantiomers of
these peptides did not bind to HSC70, although these peptides inhibited
cytotoxicity and prolonged murine heart allograft survival to a similar
extent as the L-enantiomers. These findings prompted us to
explore additional peptide binding proteins that may mediate the
peptide's immunosuppressive effects. Whole cell lysates from Jurkat
cells (Fig. 1A), a human T
cell lymphoma, and YAC-1 cells (Fig. 1B), a mouse lymphoma,
were incubated with an affinity matrix consisting of peptides coupled
to agarose via an N-terminal cysteine residue. Proteins eluted from the
affinity gel at low pH were analyzed by SDS-gel electrophoresis. Using peptide D2702.75-84(E
V) (rvnlrialry) the pH 3.0 eluate contained a major band of approximately 30 kDa (Fig. 1, lane 4). This
protein was purified from both human Jurkat and murine YAC-1 cells.
Control runs using SulfoLink-agarose gel containing cysteine or peptide rvnlrtalry (negative control) did not result in the purification of the
30-kDa protein (data not shown).
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Inhibition of Heme Oxygenase Activity by D2702.75-84(E V)--
Heme oxygenase catalyzes the degradation of heme into
biliverdin which is subsequently reduced to bilirubin by biliverdin reductase. When the activity of purified recombinant HO-1 was assessed
in the presence of peptide D2702.75-84(E
V), a significant dose
dependent reduction of enzyme activity was observed (Fig. 4): half-maximal inhibition of HO-1
activity occurred at about 20 µM. The inhibition of
bilirubin formation was not due to the inhibition of biliverdin
reductase by the peptide because addition of peptide to purified
biliverdin reductase had no effect (data not shown). The specificity of
the effect was also evaluated using lactate dehydrogenase and horse
radish peroxidase. Addition of D2702.75-84(E
V) to these enzymes
had no effect on their activity (data not shown).
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Induction of HO-1 Expression in Vivo--
Expression of HO-1
in vivo can be induced by various forms of stress, as well
as enzyme inhibitors. For example, administration of
cobalt-protoporphyrin, a well characterized inhibitor of HO, was shown
to induce a long lasting up-regulation of HO gene transcription and
activity in rats (25, 27,
31).3 Similarly, northern
blot analysis of HO-1 mRNA levels in kidneys from mice treated with
a single dose of D2702.75-84(E V) (Fig. 5) showed a significant increase in HO-1
mRNA levels. A pronounced increase (>10 times) was detected at
6 h following peptide administration. At 24 h, levels were
about 2 times higher in treated tissues than controls, while at 48 h, levels had declined to control values. A more subdued but
reproducible increase (about 1.6 times) of HO-1 mRNA levels was
detected in liver 6 h after treatment (data not shown). HO-2
mRNA levels were not increased in either tissue (data not shown).
The increased production of HO-1 mRNA was reflected by increased
amounts of HO-1 protein as detected by western blotting (Fig.
6A). Changes in HO-1 levels in
spleen were monitored also by measuring enzyme activity. Increased HO-1
protein levels persisted longer than mRNA reflecting the relative
half-lives of the transcript and the protein (23). The observed
increase in HO-1 protein was also apparent when HO activity was
determined (Fig. 6B). Splenic microsomes prepared from
peptide treated animals showed an increase in HO activity of 26 and
31%, at 24 and 48 h post-treatment, respectively, when compared
with PBS/Me2SO-treated control animals.
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DISCUSSION |
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A single amino acid substitution in peptide 2702.75-84,
replacement of glutamic acid residue 75 with valine, and peptide
synthesis using D-amino acids, resulted in a peptide
(D2702.75-84 (E V)) with increased immunomodulatory activity as
analyzed by inhibition of cytotoxicity and prolongation of heart
allograft survival. This peptide did not bind to HSC70, indicating that
peptide-mediated immunomodulation is independent of binding to this
heat shock protein (12). Here we demonstrated that D2702.75-84(E
V) bound to HSP32 (HO-1) and inhibited enzyme activity in
vitro in a dose-dependent manner. All known inhibitors
of HO induce up-regulation of HO-1 transcription in cells or animals
(23, 24). Similarly, in vivo administration of
D2702.75-84(E
V) into mice resulted in rapid up-regulation of HO-1
mRNA and HO-1 protein levels with a corresponding increase in HO
activity in liver, spleen, and kidneys.
In an effort to study the specificity of peptide-mediated inhibition, various modifications of the peptide sequence were analyzed for in vitro enzyme inhibitory activity. Immunomodulatory activity correlated with HO-inhibitory activity; peptides without any immunomodulatory activity did not inhibit mouse or rat HO in vitro. In contrast, all tested peptides that prolonged allograft survival in mice or rats inhibited HO activity in vitro. Peptide 2702.75-84 was shown to prolong allograft survival in mice but not in rats (6-10, 12). This peptide inhibited mouse HO but not rat HO. In contrast, peptide 07.75-84, which prolonged allograft survival in rats but not in mice, was also a more effective inhibitor of rat HO compared with mouse HO. Based on various physicochemical and structural characteristics of peptides that demonstrated in vivo immunosuppressive activity, an "in silico screening" method was used to design a set of peptides.2 One of these rationally designed peptides, RDP1258, was shown to prolong allograft survival in both mice and rats2 and was able to inhibit both rat and mouse HO activity. Thus, our results demonstrate a strong correlation between in vitro peptide mediated inhibition of HO activity and immunomodulation.
HO-1 catalyzes the metabolic degradation of heme into biliverdin which is subsequently reduced to the potent anti-oxidant bilirubin by biliverdin reductase (25, 26). HO-1, also known as HSP32, is inducible by various forms of stress (heat, radiation, starvation, hypoxia, hyperoxia, ischemia, GSH-depletion) (26) and has been shown to be up-regulated during an inflammatory response and acute allograft rejection (27, 28). Up-regulation of HO activity has been shown to protect cells from oxidative injury (29). Apart from stress, compounds such as heme, metalloporphyrins, and heavy metals have been shown to induce expression of HO-1 in vivo (24, 26, 30, 31). Several of the investigated metalloporphyrins are known inhibitors of HO. The mechanism of feedback regulation of heme oxygenase transcription are unknown. However, one may speculate that a decrease in cellular heme oxygenase results in accumulation of heme, which subsequently induces up-regulation of HO expression.
Immunomodulatory effects similar to those mediated by D2702.75-84(E
V) were also observed following cobalt-protoporphyrin (CoPP)-induced up-regulation of HO-1.3 Both D2702.75-84(E
V) and CoPP inhibited HO activity in vitro. In contrast,
injection of D2702.75-84 (E
V) or CoPP into mice resulted in
up-regulation of HO-1 gene transcription and HO activity. HO-1
up-regulation following administration of both compounds was associated
with inhibition of lymphocyte proliferation and prolongation of heart
allograft survival. In summary, these observations suggest that the
immunomodulatory activity of CoPP and 2702.75-84-derived peptides is
due to their capability to up-regulate HO in vivo. Formal
proof of this hypothesis, however, has to await the availability of
genetically engineered animals.
Although the mechanism of immunosuppression by HO overexpression remains unclear, several possible explanations can be envisioned. Degradation of heme by HO results in the production of biliverdin, bilirubin, and carbon monoxide. Biliverdin, the intermediate compound of heme degradation, was shown to inhibit human complement in vitro (33). In this context, it is interesting to note that complement deposition has been observed in unsensitized rat allograft recipients (34). Based on this observation, one may speculate that increased production of biliverdin following overexpression of HO may protect a transplanted organ from complement mediated cell injury and prolong graft survival.
Bilirubin, one of the endproducts of heme degradation, has been shown to inhibit responses of human lymphocytes including phytohemagglutinin-induced proliferation, interleukin-2 production, and antibody-dependent and -independent cell-mediated cytotoxicity (35-38). Even though peptide therapy had no effect on serum bilirubin levels, one cannot exclude the possibility that overexpression of HO-1 resulted in substantially higher intracellular bilirubin levels. Additional studies evaluating the intracellular concentration of bilirubin in spleen cells of peptide-treated mice by flow cytometry will be necessary to clarify this issue (39).
The gaseous product of heme degradation, CO, like NO, has been shown to
stimulate the production of cGMP via activation of guanylate cyclase
(40, 41). The secondary messenger, cGMP, has been implicated in cell
growth arrest and the release of tumor necrosis factor- by activated
macrophages (42). In addition, cGMP is involved in the regulation of
various protein kinases, phosphodiesterases and ion channels (22,
43-45). Thus, one may speculate that the increased cGMP levels may
modulate several immune effector functions. Besides stimulation of
guanylate cyclase, CO may also modulate immune responses via inhibition
of inducible NO synthase. Regulation of HO-activity and NO production
are intimately linked and an increased production of CO causes
decreased NO production and vice versa (32). Whether peptide treatment
affects NO production is yet to be investigated.
It is interesting to discover peptides that inhibit HO-1. The mechanism
of inhibition could involve factors such as the interaction of peptide
with substrate and/or reductase binding sites, thus preventing
binding/activation of the substrate; alternatively, the peptide could
interact with HO-1 protein, changing its tertiary structure. The
possibility that these peptides may bind to the substrate heme, causing
inhibition of heme degradation cannot be dismissed. The latter
possibility, however, is unlikely since availability of substrate
during the in vitro enzyme assay is not limited and heme is
concentrated in much higher proportion than the peptide. Whichever may
be the case, the interaction of the peptide with HO-1 must share
similarities with HO-2, in that the peptide D2702.75-84(E V)
inhibits not only HO-1 activity but also that of HO-2, which was
recently observed.4
In conclusion, our data demonstrated binding of peptide D2702.75-84(E
V) to HO-1. The peptides and derivatives thereof inhibit HO
activity in vitro. Similar to effects observed following
administration of CoPP,3 injection of D2702.75-84(E
V)
resulted in up-regulation of HO activity in liver, spleen, and kidneys.
The mechanism of immunomodulation by HO-1 is not resolved. Our current
knowledge of the biological effects of heme degradation products
(biliverdin, bilirubin, and carbon monoxide) suggest that elevated HO
activity may affect multiple pathways of immune responses.
Identification of compounds that specifically modulate HO activity
without causing toxic side effects could lead to the development of
novel immunotherapeutics.
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FOOTNOTES |
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* This study was supported in part by National Institutes of Health Grant ES03968 (to M. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: SangStat Medical Corporation, 1505 Adams Drive, Menlo Park, CA 94025. Tel.: 415-688-2328; Fax: 415-328-8892; E-mail: Roland_Buelow{at}sangstat.com.
1 The abbreviations used are: NK, natural killer; HO, heme oxygenase; Fmoc, N-(9-fluorenyl)metoxycaronyl; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CoPP, cobalt-protoporphyrin.
2 G. Grassy, B. Calas, A. Yasri, R. Lahana, M. Kaczorek, J. Woo, S. Iyer, and R. Buelow, submitted for publication.
3 J. Woo, S. Iyer, M.-C. Cornejo, N. Mori, L. Gao, and R. Buelow, submitted for publication.
4 M. D. Maines, unpublished observation.
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REFERENCES |
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