pylori extracts exhibited optimal catalytic activity with cobalt as a metal cofactor; manganese and nickel were significantly less efficient in catalyzing the hydrolysis of arginine.. coli
Trang 1Purification and characterization of Helicobacter pylori arginase, RocF: unique features among the arginase superfamily
David J McGee1, Jovanny Zabaleta2, Ryan J Viator3, Traci L Testerman1, Augusto C Ochoa2,4
and George L Mendz5
1
Department of Microbiology & Immunology, University of South Alabama, College of Medicine, Mobile, AL, USA;2Department of Pathology and Tumor Immunology Program, Stanley S Scott Cancer Center, Louisiana State University, Health Sciences Center, New Orleans, LA, USA;3Department of Biological Sciences, University of South Alabama, College of Arts & Sciences, Mobile, AL, USA;4Tumor Immunology Program, Stanley S Scott Cancer Center and Department of Pediatrics, Louisiana State University, Health Sciences Center, New Orleans, LA, USA;5School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia
The urea cycle enzyme arginase (EC 3.5.3.1) hydrolyzes
L-arginine toL-ornithine and urea Mammalian arginases
require manganese, have a highly alkaline pH optimum
and are resistant to reducing agents The gastric human
pathogen, Helicobacter pylori, also has a complete urea
cycle and contains the rocF gene encoding arginase (RocF),
which is involved in the pathogenesis of H pylori infection
Its arginase is specifically involved in acid resistance and
inhibits host nitric oxide production The rocF gene was
found to confer arginase activity to Escherichia coli;
dis-ruption of plasmid-borne rocF abolished arginase activity
A translationally fused His6–RocF was purified from
E coli under nondenaturing conditions and had catalytic
activity Remarkably, the purified enzyme had an acidic
pH optimum of 6.1 Both purified arginase and
arginase-containing H pylori extracts exhibited optimal catalytic
activity with cobalt as a metal cofactor; manganese and
nickel were significantly less efficient in catalyzing the hydrolysis of arginine Viable H pylori or E coli contain-ing rocF had significantly more arginase activity when grown with cobalt in the culture medium than when grown with manganese or no divalent metal His6–RocF arginase activity was inhibited by low concentrations of reducing agents Antibodies raised to purified His6–RocF reacted with both H pylori and E coli extracts containing argi-nase, but not with extracts from rocF mutants of H pylori
or E coli lacking the rocF gene The results indicate that
H pylori RocF is necessary and sufficient for arginase activity and has unparalleled features among the arginase superfamily, which may reflect the unique gastric ecological niche of this organism
Keywords: Helicobacter pylori; cobalt; arginase; urea; ornithine
Helicobacter pyloricauses gastritis [1], is strongly associated
with the development of peptic ulcers [2], and constitutes a
risk factor for gastric adenocarcinoma [3,4] The
mecha-nisms leading to the development of these diseases are not
well understood, but urease, which catalyzes the hydrolysis
of urea to carbon dioxide and ammonium, is critical in the
pathogenesis of H pylori infection [5,6] The role of other
H pylori nitrogen-metabolizing proteins in virulence has
only recently begun to be understood [7,8]
Together with ornithine, urea is synthesized from the catabolism of arginine by the urea cycle enzyme arginase (L-arginine ureohydrolase, EC 3.5.3.1) [9–11] Arginase, discovered 100 years ago [12], was one of the first enzymes shown to require divalent cations for catalytic activity [13] Eukaryotic arginases have a highly alkaline pH optimum (pH 9.0–11.0) and require manganese for optimal catalytic activity [14–21] However, other divalent metal cations (e.g cobalt and nickel) can activate some arginases that first have been dialyzed or treated with chelators to remove the manganese [13,18,19] Arginases generally lose significant catalytic activity during dialysis owing to the loss of the divalent metal cation [15,19] Reducing agents have little or
no effect on the activity of these enzymes [22,23] In contrast
to the well-characterized mammalian arginases, relatively few prokaryotic arginases have been purified and charac-terized Of those that have been purified and characterized, most are from the genus Bacillus; like eukaryotic arginases, these prokaryotic arginases were found to have an optimal catalytic activity with manganese and showed a highly alkaline (pH 9.0–11.0) pH optimum [17,24–27] In the presence of high concentrations of reducing agents, only modest inhibition of B brevis and B anthracis arginase was observed [26,27], whereas B licheniformis arginase was
Correspondence to D J McGee, University of South Alabama
College of Medicine, Department of Microbiology & Immunology,
307 N University Blvd, Mobile, AL 36688, USA.
Fax: + 1 251 460 7931, Tel.: + 1 251 460 7134,
E-mail: dmcgee@jaguar1.usouthal.edu
Abbreviation: CBA, Campylobacter agar containing 10% (v/v)
defibrinated sheep blood.
Enzyme: arginase (EC 3.5.3.1).
Note: A website is available at http://www.southalabama.edu/
microbiology/mcgee.html
(Received 10 February 2004, revised 16 March 2004,
accepted 23 March 2004)
Trang 2completely resistant to inhibition by 1 mM
2-mercaptoeth-anol [25]
H pylori arginase produces endogenous urea that can
be utilized by urease to generate ammonium and carbon
dioxide, with concomitant acid resistance [8] In addition to
producing endogenous urea, H pylori may also obtain
some of its urea exogenously from the host, as urea is
present in the gastric mucosa through host arginase activity
[10,28] Presently, it is unclear how much endogenous
vs exogenous urea is utilized by the abundant urease of
H pylori Exogenous urea is thought to be imported into
H pylori through the UreI transporter, under acidic
conditions [29], but this system may be inoperable in vivo
under neutral pH conditions Thus, arginase may be
important for providing endogenous urea in vivo under
conditions in which exogenous urea is limited Another
biological role of arginase is to regulate cytosolic arginine
and ornithine levels, which are required for numerous
metabolic processes, such as protein synthesis, and
produc-tion of polyamines and nitric oxide [30]
Previous studies have pointed to the role of H pylori
arginase in acid resistance [8] H pylori arginase also
inhibits host nitric oxide production, helping to escape the
toxic effects of nitric oxide, while outcompeting the host for
the limited arginine available [7] Arginase also plays a role
in inhibiting T cell proliferation by reducing expression of
CD3f (J Zabaleta, D McGee & A Ochoa, unpublished
observations) Thus, evidence is accumulating that H pylori
arginase plays important roles in pathogenesis
Previously, the rocF gene of H pylori was cloned and the
gene disrupted in three strains [8] While the gene was found
to be required for arginase activity, the study did not
establish whether expression of rocF alone in
Escheri-chia coliwas sufficient for arginase activity To characterize
this important enzyme and understand further the
mecha-nisms of arginase-mediated pathogenesis, the H pylori
RocF protein was purified and its biochemical properties
investigated This study demonstrates that RocF is
neces-sary and sufficient for arginase activity, and that the enzyme
has a number of interesting and unique features among the
arginase superfamily, including optimal enzymatic activity
with cobalt rather than manganese, inhibition by reducing
agents, and a pH optimum considerably lower than that of
previously characterized arginases
Materials and methods
Bacterial strains, growth conditions, and plasmids
E colistrain XL1-Blue MRF¢ [thi-1, gyrA96, recA1, endA1,
relA1, supE44, lac, hsdR17 (r–, m+), F¢ [proAB, lacIq Z
DM15, Tn10 [tetr]] was used for overexpressing H pylori
RocF for purification purposes Strain DH5a [F–, deoR,
thi-1, gyrA96, recA1, endA1, relA1, supE44D (lacZYA-argF)
U169, hsdR17(r–, m+), /80 dlacZ DM15, k–] was used for
standard cloning and transformation procedures E coli
strains were grown at 37C on Luria (L) agar and in L
broth plus appropriate antibiotics (100 lgÆmL)1ampicillin,
25 lgÆmL)1kanamycin, 10 lgÆmL)1tetracycline) Plasmids
pBS [pBluescript II SK(+); Stratagene], pBS-rocF [8],
pBS-rocF::aphA3 [8], pQE30 (Qiagen) and pQE30-rocF
(see below) were used in this study
H pyloristrains were cultured at 37C on Campylobac-ter agar containing 10% (v/v) defibrinated sheep blood (CBA) in a microaerobic environment for 2–3 days using the CampyPak Plus system (Becton Dickinson), or in 5% (v/v) CO2 in humidified air Kanamycin (5–10 lgÆmL)1) was added to the growth medium, as appropriate The strains employed in this study were wild type SS1 and its isogenic rocF::aphA3 mutant, and wild type ATCC 43504 and its isogenic rocF::aphA3 mutant [8]
Molecular biology techniques Plasmid DNA was isolated by the alkaline lysis method [31],
or by column chromatography (Qiagen) for sequencing-grade plasmid Restriction endonuclease digests, ligations and other enzyme reactions were conducted according to the manufacturer’s instructions (Promega) PCR reactions (50 lL) contained 10–100 ng of DNA, PCR buffer, 2.0– 2.5 mMMgCl2, dNTPs (each nucleotide at a concentration
of 0.20–0.25 mM), 50–100 pmol of each primer, and 2.5 U
of thermostable DNA polymerase E coli was transformed
by the calcium chloride method
Construction of the arginase mutant ofH pylori 43504 Plasmid pBS-rocF::aphA3 was transformed into H pylori
43504 by electroporation [8] to generate an arginase-negative mutant (rocF::aphA3) The mutant was confirmed
by PCR analysis, as described previously [8]
Cloning ofrocF into pQE30 The rocF gene, from nucleotides 1–967 of the coding region, was PCR-amplified using primers RocF-F6 (gcggatccAT GATTTTAGTAGGATTAGAAGCAGAG; BamHI site underlined; non-rocF sequence in lower case) and Roc F-R8 (gcctgcagAGTAACTCCTTGCAAAAGAGTGCT TC; PstI site underlined; non-rocF sequence in lower case) The PCR product was purified, phenol/chloroform extrac-ted, and precipitated with ethanol After digestion with BamHI and PstI, the product was cloned into pQE30 (Qiagen), predigested with the same enzymes, to generate pQE30-rocF The construct was confirmed by sequencing, restriction enzyme digestion, and mini-protein expression analyses (data not shown) The fusion protein, His6-RocF, has a predicted molecular mass of 37.8 kDa
Purification of RocF XL1-Blue MRF¢ pQE30-rocF (1.5 L) was grown to mid-log phase and induced with 5 mMisopropyl thio-b-D -galacto-side Cultures were harvested in Wash Buffer (50 mM NaH2PO4, 300 mMNaCl, 15 mMimidazole, pH 8.0), lysed
by two passages through a French Press (16 000 psi) on ice, clarified by centrifugation, and the cytosolic portion loaded onto polypropylene columns (8.5· 2.0 cm) containing nickel-nitrilotriacetic acid agarose resin ( 1 mL per
10 mg of protein) (Qiagen) The flow-through was retained
to monitor binding of arginase to the column Following six
to eight washes with 10 mL of Wash Buffer, RocF was eluted with Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 250 m imidazole, pH 8.0) in 1 mL fractions All
Trang 3manipulations were performed at 4C to minimize loss of
enzyme activity Fractions were analyzed for the presence of
the His6-RocF by SDS/PAGE (expected molecular mass:
37.8 kDa) and colorimetric arginase activity For long-term
storage, glycerol was added to a final concentration of 50%
and the enzyme stored at)20 C
Preparation of arginase-containing extracts
Bacteria were harvested in 0.9% NaCl and
ice-bath-sonicated (25% intensity, two pulses of 30 s each, with
30 s rests on ice between pulses) Following centrifugation
(12 000 g, 2 min, 4C), supernatants were retained on ice
or frozen at)20 C until measurement of arginase activity
No arginase activity was detected in the corresponding
pellets
Arginase activity assay
The colorimetric arginase assay measures the amount of
ornithine by the appearance of an orange color (read
spectrophotometrically at 515 nm) from the reaction of
ornithine with ninhydrin at low pH Equal volumes of
extract and 10 mM cobaltous chloride (CoCl2Æ6H2O, final
concentration 5 mM) were preincubated for 30 min at
50–55C to activate the enzyme (heat-activation step;
50 lL final volume) The heat activation of arginases in the
presence of metal cofactor is well documented in the
literature [16] and was first observed by Mohamed &
Greenberg [19] Next, arginase buffer [15 mMTris, pH 7.5,
or 15 mM Mes, pH 6.0, plus 10 mM L-arginine (unless
otherwise stated)] was added and incubation continued at
37C The arginine concentration could not be increased
beyond 10 mMwithout significant increase in background
(data not shown) After 1 h the reaction was stopped by the
addition of 750 lL of acetic acid, and the color developed
by the addition of 250 lL of ninhydrin (4 mgÆmL)1) at
95C for 1 h A standard curve of different ornithine
concentrations (3125–391 lM in serial twofold dilutions)
was used to generate a slope (typically 0.00045–
0.00070 lM )1) The data are presented in U, where 1 U is
defined as 1 pmol L-ornithine min)1Æmg)1 of
pro-tein (± SD) The enzyme activity was shown to be linear
under these conditions An abbreviated description of a
preliminary version of this assay has been previously
reported [11]
For experiments to determine pH optimum, buffers
(15 mM) of the appropriate pKa were employed
Homo-pipes (pH 4.0–5.0), Mes (pH 5.5–6.7), Mops (pH 6.7–7.3),
or Tris (pH 7.0–9.0), of the desired pH, were obtained by
addition of concentrated HCl or 10MNaOH to the buffer
after the addition of arginine (10 mM) Mes and Mops
buffers, at pH 6.7, resulted in identical arginase activities,
whereas Tris (pH 7.3) resulted in 20% more activity than
Mops (pH 7.3); this buffer effect was corrected to allow
comparison of activities at different pH values For
determining the temperature optimum, the
enzyme-containing samples were first activated with 5 mMCoCl2,
as described above, and then incubated at the desired
temperature for 1 h For determining metal ion optima,
5 mM CoCl2 was replaced with 5 mM MnSO4, NiCl2Æ
6HO, ZnCl, FeSOÆ7HO, CaClÆ2HO, MgSO, or
CuSO4Æ5H2O The reducing agents dithiothreitol or 2-mercaptoethanol were mixed with the enzyme in the presence of CoCl2 In control tubes, dithiothreitol or 2-mercaptoethanol was replaced with the same volume of sterile water
Kinetic analyses Proton NMR spectroscopy ([1H]NMR) was employed to measure purified arginase activity, as previously described [26] The kinetic constants, Kmand Vmax, were determined
by nonlinear regression analysis, employing the program ENZYME KINETICS (Trinity Software, Campton, NH, USA) The activity of the enzyme was measured at
pH 7.1 and 37C at arginine concentrations of 2, 5, 10,
15, 20, 30, 50, 70, and 100 mM Errors are quoted as SD values
Arginase activity in viableE coli Cultures of E coli were grown overnight at 37C with aeration (225 r.p.m.), in L broth plus ampicillin, in the presence or absence of cobalt or manganese (the concen-trations used are listed in the Figure legends) Metal concentrations higher than 500 lM could not be reliably tested owing to adverse effects on E coli growth Cultures were harvested, washed, and resuspended in ice-cold 0.9% (w/v) NaCl Viable E coli cells were added to arginase buffer (15 mMMes, pH 6.0, containing 10 mM L-arginine)
No divalent metal cation was added, nor was heat-activation conducted After 1 h at 37C, the assay was stopped and the ornithine concentration was analysed, as described above Under these experimental conditions, it
is assumed that the viable bacteria transport arginine intracellularly, where it is hydrolyzed by cytosolic holo-arginase (already contains metal cofactor) to yield ornithine
Arginase activity in viableH pylori Cultures of H pylori strain 43504 were grown at 37C with aeration (225 r.p.m.), in a microaerobic environment (Campy Pak Plus in an anaerobic jar), for 24 h in 5 mL
of Ham’s F-12 plus 1% (v/v) fetal bovine serum in the presence or absence of cobalt, manganese or nickel (1 lM) Metal concentrations greater than 1 lMcould not
be reliably tested owing to adverse effects on H pylori growth Cultures were harvested, washed and
resuspend-ed in ice-cold 0.9% (w/v) NaCl Viable H pylori cells were added to arginase buffer (50 mM potassium phos-phate, pH 7.5, plus 10 mM L-arginine) No divalent metal cation was added, nor was heat-activation conducted After 1 h at 37C, the assay was stopped and the concentration of ornithine determined, as described above This assay assumes that, under these experimental conditions, viable H pylori transport arginine into the cell where it is hydrolyzed by holo-arginase Preliminary experiments indicated that lysing the bacteria with SDS (0.4%, w/v) at the end of the assay did not affect the amount of ornithine detected, verifying that the ornithine produced was available for detection by the ninhydrin reagent
Trang 4Raising of antibodies to RocF
Polyclonal antibodies were raised by GeneMed Synthesis
Inc (San Francisco, CA) on a fee-for-service basis RocF
was emulsified with complete Freund’s adjuvant, and
injected four times intradermally along the back of two
adult New Zealand white rabbits Booster immunizations
were conducted with RocF emulsified in incomplete
Freund’s adjuvant Immune sera were collected 12 weeks
postimmunization, and the antisera titer determined (using
purified RocF) via Western blot analysis Four to five
milligrams of 95% pure protein was used for the
immunization Preimmune sera were isolated from rabbits
as a negative control The company has an Animal
Assurance number filed with USDA and an approved
animal protocol through its institutional animal care and
use committee
Protein determinations
The protein concentration was determined using the
bicinchoninic acid assay (Pierce Chemical Company),
following the manufacturer’s 30 min method BSA was
used as the standard
SDS/PAGE and Western blotting analysis
Proteins were electrophoresed through an
SDS-polyacryl-amide gel and transferred to methanol-treated
poly(viny-lidene difluoride) membrane using the Trans-blot cell
transfer system (Bio-Rad) Blots were blocked in 5% (w/
v) nonfat dry milk in Tris-buffered saline containing 0.5%
(v/v) Tween-20 (TBST-1) Primary antisera (1 : 2500, v/v)
were incubated for 2 h Following three washes with
TBST-2 [TBS containing 0.05% (v/v) Tween-TBST-20], goat anti-rabbit
IgG-conjugated alkaline phosphatase (Sigma
Immuno-chemical Co.; 1 : 5000–1 : 7500, v/v) was added and
incubation continued for 90 min The blot was washed
three times with TBST-2 and equilibrated with glycine
buffer [100 mMglycine, 1 mMZnCl2, 0.05% (w/v) sodium
azide, and 1 mMMgCl2, pH 10.4] The blot was developed
with 3-indoxyl phosphate (final concentration 10 lgÆmL)1;
adjusted to ·100 in water) and Nitro Blue tetrazolium
(100 lgÆmL)1) in glycine buffer
Results
Characterization ofH pylori arginase
In a previous study, NMR spectroscopy was employed
to measure arginase activity [8] A colorimetric assay,
described previously [11], which allows higher throughput,
was utilized and further developed in this study The
method was validated using arginase-containing extracts
from wild type H pylori First, no arginase activity was
detected when arginine was omitted from the assay mixture
(Fig 1) This indicates that H pylori extracts contained
undetectable amounts of ornithine and that no other
ornithine-generating pathways were active under the
con-ditions of the arginase assay Heat activation of the cell
extract, for 30 min at 50–55C in the presence of cobalt,
was required for optimal activity If cobalt was omitted, no
activity was observed If cobalt was present, but the heat activation temperature was lowered to 42C or 37 C, significantly reduced activities were observed compared to heat activation at 50C (Fig 1) If heat activation at 50–55C in the presence of cobalt was carried out for only
10 or 20 min, significantly lower arginase activities were obtained (data not shown) Thus, heat activation is time-, temperature-, and cobalt-dependent Finally, if the rocF gene was disrupted by a kanamycin resistance cassette, arginase activity was abolished These results demonstrated the validity of the assay used in this study
E coli containing the cloned rocF gene from H pylori exhibited arginase activity and this activity was markedly higher when heat-activated with cobalt rather than manganese
Arginase and urease are both multisubunit, metal cofac-tor-containing enzymes [11,32] E coli can express urease activity when transformed with eight H pylori genes: the ureABIEFGHgenes in the H pylori urease locus and the nixAnickel transporter [33,34] Expression of only ureAB (urease structural genes) or ureABIEFGH in E coli yields little or no urease activity [33,35,36] because nixA is required for nickel transport into the cell, and the accessory proteins UreEFGH are needed for incorpor-ation of nickel into the urease active site [34,37] It was unknown how many H pylori genes were required for arginase activity in E coli To address this, arginase activity was measured in an E coli strain containing the cloned H pylori arginase gene (rocF) on a plasmid, and its activity was compared with strains transformed with a vector control (pBS) or a plasmid with disrupted rocF (pBS-rocF::aphA3) E coli containing rocF (pBS-rocF) exhibited arginase activity, while arginase activity was
Fig 1 Characterization of Helicobacter pylori arginase using a colori-metric enzyme assay Extracts from wild type H pylori strain 43504 (wt) or the isogenic rocF::aphA3 mutant (rocF) were heat-activated for
30 min in the presence of 5 m M CoCl 2 (Co) The temperature of heat activation was 50 C (designated as 50), 42 C (designated as 42) or
37 C (designated as 37) Arginase activity was measured in 15 m M
Tris (pH 7.5) containing 10 m M L -arginine, for 1 h The values shown are the mean ± SD of one representative experiment, conducted in duplicate At least two experiments (independently prepared extracts) were conducted for each sample Lack of error bars on some values indicate that the standard deviation was too small to appear on the graph No arg, no arginine added to enzyme assay buffer; No Co, no cobalt added during the heat activation step.
Trang 5barely present in the control strain (pBS) or in the strain
carrying disrupted arginase (pBS-rocF::aphA3) (Fig 2A)
These findings indicated that, unlike urease, the H pylori
arginase gene was sufficient, by itself, to confer enzyme
activity to E coli No H pylori metal transporter appears
to be required for obtaining arginase activity in E coli
These results do not rule out the possibility that other
E coli genes, similar to those of H pylori, might also
contribute to arginase activity, or that arginase activity
might be modulated by other H pylori genes
E coli containing rocF also required heat activation in
the presence of cobalt for high level activity (28 000 U with
heat activation with cobalt vs 1800 U with no heat
activation or cobalt; Fig 2B) Heat activation with
man-ganese resulted in lower arginase activity (3100 U for
manganese; 28 000 U for cobalt) Culturing E coli
pBS-rocFin the presence of cobalt, and then heat activating the
extract with cobalt, generated the highest arginase activity (37 200 U) While culturing E coli pBS-rocF in the presence
of manganese and then heat activating the extract with cobalt yielded high arginase activity (32 400 U), culturing
E colipBS-rocF in the presence of cobalt and then heat activating the extract with manganese yielded low arginase activity (5200 U) (Fig 2B) These results suggest that arginase may lose the metal cofactor during preparation
of the extracts, requiring reintroduction of the cobalt cofactor by heat activation
TransformedE coli containing rocF had higher arginase activity thanH pylori
Routinely, E coli and H pylori are grown under different culture conditions (e.g on different media and under different concentrations of oxygen), making a direct com-parison of arginase activity in the two organisms difficult Furthermore, E coli pBS-rocF contains a very high copy-number plasmid, whereas rocF is chromosomally encoded
in H pylori Nonetheless, E coli (pBS-rocF) expressing the rocFgene and grown under similar conditions to H pylori (i.e microaerobically, on CBA plates) contained at least four times more arginase activity than wild type H pylori (Fig 1 vs Fig 2A) Arginase activities of E coli pBS-rocF grown on L agar and in L broth were similar to those of cells grown microaerobically on CBA (data not shown), indica-ting that, under these culture conditions, no major differ-ences in the regulation of H pylori arginase activity occurred in the E coli model
Purification and catalytic activity of RocF expressed
inE coli The rocF gene was translationally fused to the His6 -encoding construct, pQE30, and shown to be correct by sequence analysis and small scale whole-cell protein expres-sion experiments (data not shown) Arginase activity was detected in E coli containing pQE30-rocF, but not in the strain containing the vector control, pQE30 (Fig 3, and data not shown) SDS/PAGE analyses showed an extra protein, of 40 kDa in size, in extracts from E coli pQE30-rocF, but not in the strain containing the vector control, pQE30 (data not shown)
His6-RocF was purified under nondenaturing condi-tions, as described in the Materials and methods Binding
of the recombinant protein to the column was apparent from the loss of a protein of 40 kDa in the flow-through, compared to the whole cell lysates (Fig 3) His6 -RocF was enriched to more than 95% purity (Fig 3) Notably, arginase retained catalytic activity during puri-fication, with the specific activity increasing from
5000 U in the starting material to up to 100 000 U in the purified protein The specific activity of purified arginase varied in different elutions, owing to the partial inhibition of enzyme activity at high imidazole concen-trations (data not shown) The first elution, containing the lowest imidazole concentration, had the highest specific activity (Fig 3)
The eluted protein was diluted 1 : 2 with 100% sterile glycerol and stored at)20 C The protein was found to
be highly unstable It was determined that His-RocF lost
Fig 2 Characterization of arginase in Escherichia coli containing
var-ious plasmids (A) E coli DH5a, containing the plasmids indicated in
the figure, was grown overnight in L broth plus the appropriate
anti-biotics Extracts were prepared and arginase activity was measured
for 40 min, as described in the Materials and methods The arginine
concentration in the arginine buffer (Tris pH 7.5) was 5 m M
(B) E coli DH5a pBS-rocF was grown in L broth overnight in the
presence or absence of cobalt or manganese (100 l M ) and extracts were
prepared Arginase activity was measured for 60 min using 15 m M
Mes, pH 6.0, containing 10 m M arginine, either without heat
activa-tion, or with heat activation in the presence of cobalt (5 m M ),
man-ganese (5 m M ) or no metal.
Trang 645–65% of its activity after just 4 months of storage at
)20 C, and over 90% of its activity after 6 months (data
not shown) Storage of purified RocF at 4C instead of
)20 C resulted in > 90% loss of activity within 1 week
Dialysis of the enzyme also resulted in > 90% loss of
enzyme activity (data not shown), as observed with other
arginases [15,19] Attempts to retain catalytic activity of
the enzyme following lyophilization were also
unsuccess-ful Additionally, there was significant variation in specific
activity from one batch of freshly purified arginase to
another, which could be a result of the instability
of the enzyme during purification The instability of
arginase may be due to spontaneous degradation (see
below)
Like arginase-containing extracts from H pylori, heat
activation of the purified His6-RocF for 30 min at 50–55C
in the presence of cobalt showed catalytic activity, whereas
omission of cobalt yielded no arginase activity (data not
shown) Additionally, no arginase activity of the purified
His6-RocF was measured when arginine was omitted
from the assay mixture The kinetic parameters for the
purified arginase in 20 mM Mops, pH 7.1, were: Km¼
21.8 ± 2.3 mM and V ¼ 268 600 ± 10 600 U, using
[1H]NMR spectroscopy These kinetic findings are in agreement with those of the partially purified arginase reported previously [11] Throughout the study, the arginase assays were conducted at an arginine concentration of
10 mM(unless stated otherwise) Therefore, the enzyme is being assayed in the linear portion of the curve
Optimal temperature, pH and salt concentration for arginase activity
His6-RocF was measured at different pH values and temperatures Remarkably, the enzyme had an acidic pH optimum of 6.1, with considerable activity at pH 5.5 (Fig 4)
To our knowledge, no other arginase has a pH optimum below 9.0 The temperature optimum of His6-RocF arginase activity was 30C (data not shown) There was no repro-ducible preference for temperature optimum between 25 and
42C for H pylori extracts (data not shown) His6-RocF arginase activity was similar across a broad salt concentra-tion range of 6.25 to 200 mM (data not shown) Freshly prepared arginase-containing H pylori extracts also had an acidic pH optimum of 6.1, with considerable activity at
pH 5.5 (data not shown); its pH curve closely mirrored the curve of purified His6-RocF (Fig 4)
Activities of purified arginase andH pylori arginase-containing extracts with divalent cations
Mammalian arginases require manganese for optimal catalytic activity, although some of these arginases may be activated by cobalt or nickel, to a lesser extent [13,19] Remarkably, H pylori arginase does not have optimal catalytic activity when heat-activated with manganese Rather, both purified His6-RocF arginase and H pylori arginase-containing extracts have optimal catalytic activity when heat-activated with cobalt (Table 1) Very low activ-ities were obtained with purified His6-RocF when cobalt was replaced with manganese or nickel (Table 1) No detectable arginase activity was found using CuSO4, ZnCl2, FeSO4, CaCl2 or MgSO4 (data not shown) Similarly, significantly less arginase activity was obtained with
H pyloriarginase-containing extracts when manganese or
Fig 4 pH and temperature optima of purified His 6 -RocF Purified His 6 -RocF was heat-activated with CoCl 2 and then assayed for argi-nase activity at 37 C for 60 min in arginase buffer that varied in pH (see Materials and methods) Shown is a representative of two experiments ± SD, conducted in duplicate.
Fig 3 Purification of catalytically active Helicobacter pylori arginase
from Escherichia coli expressing His 6 -RocF E coli XL1-Blue MRF¢
containing pQE30-rocF was grown in L broth plus tetracycline and
ampicillin and induced with isopropyl thio-b- D -galactoside Bacteria
were harvested and lysed by French Press The whole cell lysate (WCL)
was separated into soluble and insoluble fractions by centrifugation.
The soluble fraction was loaded onto a nickel-nitrilotriacetic acid
column under nondenaturing conditions and the flow-through (FT)
collected The column was washed extensively, as described in the
Materials and methods Only the first wash is shown (W1) His 6 -RocF
was eluted off the column and the first four elutions (E1 to E4) are
shown Each of these fractions was assayed for arginase activity The
amount of protein loaded onto the SDS-polyacrylamide gel is shown
at the top of the gel The His 6 -RocF protein, 37.8 kDa, is labeled with
an arrow Arginase activity, assayed at 37 C for 1 h in arginase buffer
(15 m M Tris pH 7.5 with 10 m M L -arginine), is shown at the bottom of
each lane, rounded to the nearest 500 U The Coomassie Blue-stained
SDS-polyacrylamide gel was scanned.
Trang 7nickel were the metal cofactors during the heat-activation
step (Table 1) No appreciable arginase activity with other
divalent cations was detected in H pylori
arginase-contain-ing extracts (data not shown)
ViableH pylori and transformed E coli containing
theH pylori arginase gene had significantly more
arginase activity when grown with cobalt, compared
to manganese or no divalent metal
Although H pylori arginase has optimal catalytic activity
with cobalt, it was found that low reproducible activity can
be obtained if the extracts are heat-activated in the presence
of manganese or nickel (Table 1) This result raised the
possibility that in vitro heat activation with cobalt may not
necessarily reflect the metal found in arginase in vivo To
demonstrate that cobalt is the optimal metal in vivo,
arginase activity was measured in viable, intact cells
Bacteria were cultured overnight in media either lacking or
containing cobalt, manganese, or nickel Viable bacteria
were added directly to arginase buffer, in the absence of
metal ions, without a heat activation step This experiment
assessed whether live bacteria, which had preloaded the
metal ion during overnight growth, could transport
argin-ine into the cell during the assay and convert the arginargin-ine to
ornithine via arginase The results indicated that H pylori
cultured in the presence of cobalt have five- to sixfold more
arginase activity than bacteria grown with manganese or
no metal, and threefold more arginase activity than
bacteria grown with nickel (Fig 5A) Similar findings were
observed with viable E coli pBS-rocF: higher arginase
activity was measured when the culture was grown with
cobalt than with manganese or no metal (Fig 5B)
Furthermore, there was a sharp dose-dependent increase
in arginase activity when E coli (pBS-rocF) was cultured
with higher concentrations of cobalt, while only a mild
increase in arginase activity was observed with cultures
grown in the presence of higher concentrations of
man-ganese (Fig 5C) Taken together, these results
demonstra-ted that H pylori arginase activity was optimal with cobalt
in vivo, although they did not demonstrate directly that
cobalt is, in fact, the metal found in the arginase active site
in vivo
Inhibition of arginase activity by reducing agents Numerous enzymes are stabilized by the presence of reducing agents such as dithiothreitol; arginases from other
Table 1 Effect of metal ions on the arginase activity of
Helico-bacter pylori extracts or purified His 6 -RocF Arginase of H pylori
43504, or purified recombinant His 6 -RocF, was heat-activated at
50 C for 30 min in the presence of various divalent cations, and its
activity was measured as described in the Materials and methods.
Activity is presented as percentage of the control, with activity in the
presence of cobalt (the control) set at 100% At least three experiments
were conducted in duplicate; one set of representative results is shown.
Metal
concentration
H pylori RocF-containing extract (% of control)
Purified His 6 -RocF (% of control) CoCl 2 , 5 m M 100.0 100.0
MnSO 4 , 5 m M 20.9 14.6
NiCl 2 , 5 m M 29.7 6.1
Fig 5 Arginase activity of viable Helicobacter pylori or Escheri-chia coli pBS-rocF grown with or without cobalt or manganese Cells were harvested and processed to measure arginase activity without lysing the bacteria, as described in the Materials and methods No heat activation was conducted, nor was divalent cation added during the arginase assay Shown is the mean ± SD of one representative experiment (of at least three carried out), conducted in duplicate The dotted line represents the detection limit of this assay (A) H pylori strain 43504 was grown in F-12 plus 1% (v/v) fetal bovine serum [41]
in the presence or absence of cobalt, manganese or nickel (1 l M ) (B) E coli pBS-rocF was grown in L-broth, with or without cobalt (100 l M ) or manganese (100 l M ) (C) E coli pBS-rocF was grown in L-broth with various concentrations of cobalt or manganese.
Trang 8organisms retain catalytic activity in the presence of
reducing agents, suggesting that these agents may protect
H pylori arginase from losing activity Surprisingly,
dithiothreitol did not protect the enzyme activity of
purified His6-RocF Rather, dithiothreitol inhibited
argi-nase activity in a dose-dependent manner at relatively low
concentrations (Fig 6A) Similarly, another reducing
agent, 2-mercaptoethanol, inhibited arginase activity in
a dose-dependent manner (Fig 6B) The small increase in
arginase activity at 40 lM 2-mercaptoethanol was not
reproducibly obtained, nor statistically different from
arginase activity in the absence of 2-mercaptoethanol
Arginase activity was more sensitive to dithiothreitol than
2-mercaptoethanol, with 50% inhibitory concentrations
(IC50) of 25 lMand 800 lM, respectively Control
experi-ments showed that at concentrations used in Fig 6,
dithiothreitol and 2-mercaptoethanol had no effect on the
colorimetric development of ornithine, but interfered at
concentrations of > 1000 lM 2-mercaptoethanol or
200 lMdithiothreitol (data not shown)
Western blot analyses ofH pylori and E coli extracts
lacking or containing arginase activity
Preimmune serum from rabbits did not react with purified
RocF or with E coli arginase-containing extracts, but
purified RocF protein reacted in Western blots with
anti-RocF immunoglobulin (data not shown) Western blot
analyses using anti-RocF immunoglobulin showed that extracts from E coli (pBS-rocF) had high levels of arginase protein (Fig 7A) In contrast, extracts from E coli con-taining the insert-free control (pBS) or a plasmid concon-taining the disrupted rocF gene (pBS-rocF::aphA3) had no detect-able arginase protein (Fig 7A) A lower molecular weight band was observed to cross-react in extracts from E coli (pBS-rocF) (Fig 7A, arrowhead) and in the purified His6 -RocF (data not shown), but not in extracts from E coli (pBS) or E coli (pBS-rocF::aphA3), suggesting degradation
of arginase Lower amounts of the degraded product were observed in fresh batches of purified His6-RocF compared
to batches that had been stored at 4C or)20 C Arginase was detected in extracts from two wild type strains of
H pylori, but not the corresponding isogenic rocF::aphA3 mutants (Fig 7B), suggesting that the anti-RocF immuno-globulins are specific for RocF Despite using more total protein from H pylori (20 lg) than from E coli (15 lg), significantly less arginase was detected in the H pylori extracts, correlating with the reduced arginase activity observed in H pylori compared with E coli (pBS-rocF) extracts
Discussion
In this study, H pylori arginase was purified and charac-terized Five lines of evidence indicated that the rocF gene encodes arginase, namely (a) disrupting the rocF gene in
H pyloriabolishes arginase activity (Fig 1) [8], (b) trans-forming E coli with a plasmid containing rocF conferred arginase activity (Fig 2A), (c) transforming E coli with
a plasmid containing disrupted rocF abolished arginase activity (Fig 2A), (d) the purified His6-RocF expressed in
E colihad arginase activity (Figs 3, 4 and 6 and Table 1), and (e) the rocF gene and RocF protein shared homology with the arginase/agmatinase superfamily [8] These results demonstrate that rocF is necessary and sufficient for arginase activity However, the results did not exclude the possibility that other gene products modulate arginase activity in H pylori
Fig 6 Sensitivity of arginase activity to reducing agents The reducing
agents dithiothreitol (A, DTT) or 2-mercaptoethanol (B, BME), at the
final concentrations indicated, were mixed with purified His 6 -RocF
( 9 months old) in the presence of CoCl 2 and incubated at 50 C for
30 min Arginase buffer (10 m M arginine, 15 m M Tris pH 7.5) was
added and incubation continued at 37 C for 1 h In control tubes,
dithiothreitol or 2-mercaptoethanol was replaced with the same
vol-ume of sterile water The 50% inhibitory concentration (IC 50 ) was
determined from the graph.
Fig 7 Anti-RocF Western blot analysis of Helicobacter pylori and Escherichia coli Extracts from E coli (A, 15 lg per lane) or H pylori (B, 20 lg per lane) strains indicated on the figure were loaded onto an SDS/polyacrylamide gel, transferred to poly(vinylidene difluoride) membrane and probed with anti-RocF generated from purified His 6 -RocF Arrow, RocF (37 kDa); arrowhead, RocF degradation product.
Trang 9Urease and arginase are enzymes that require a metal
cofactor E coli transformed with the genes encoding the
urease structural proteins, UreA and UreB, have little or no
urease activity [33,35,36] This is because of the
require-ments for accessory proteins to incorporate the nickel metal
ion cofactor into the active site of the multimer [32], as well
as a nickel transporter [34,37] In contrast with the urease
system, E coli transformed with the arginase structural
gene, rocF, showed enzyme activity, suggesting that either
H pylori arginase does not require accessory proteins,
unlike urease, or that E coli expresses proteins similar to
those of H pylori that can serve as arginase accessory
proteins The latter possibility is not unlikely because E coli
expresses a member of the arginase superfamily,
agmatinase, which is encoded by speB [38] that shares low
amino acid identity with H pylori arginase Thus, accessory
proteins serving to incorporate metal ions into SpeB may
also be able to perform a similar function for RocF
When cultured under similar conditions, E coli
trans-formed with the rocF gene had substantially more specific
arginase activity than wild type H pylori In contrast,
E coliexpressing the H pylori urease and nickel
transpor-ter genes from multicopy to high copy plasmids exhibited
10-fold lower urease activity than that obtained with
H pylori[33] The higher arginase activity found in E coli
(pBS-rocF) compared to H pylori correlated well with the
greater amount of arginase protein detected in Western
blots of E coli extracts relative to H pylori extracts It is
possible that the difference in arginase activity between
E coli and H pylori may be a result of the high copy
number plasmid in E coli
Inhibition of arginase activity by low concentrations of
the reducing agents dithiothreitol and 2-mercaptoethanol
suggested a role for disulfide bonds in the activity of the
enzyme, perhaps through the interaction of the thiols with
metals There is no evidence in the literature to suggest the
involvement of cysteine residues in the catalytic activity of
other arginases, which are either completely resistant, or
only moderately sensitive to, very high concentrations of
reducing agents [25,27] Indeed, multisequence alignments
of the arginase family reveals no conserved cysteines [17]
Thus, the potential role of cysteinyls in H pylori arginase
activity is a novel feature of the arginase superfamily
Site-directed mutagenesis experiments are underway to
deter-mine whether any of the six cysteine residues in RocF are
required for catalytic activity
Unlike other arginases, the H pylori enzyme has
optimal catalytic activity at pH 6.1, a striking 3 pH units
below the pH optimum of all other known arginases [17]
The H pylori arginase retains activity at even lower pH
values, a condition under which all other known arginases
are catalytically inactive These characteristics suggest that
H pylori arginase evolved to operate under acidic
condi-tions encountered by the bacterium in vivo in its unique
gastric niche, supporting the ability of the organism to
tolerate acid stress [8] Survival of H pylori in vivo may
require arginase activity in situations when urea
produc-tion by the host is limited In this scenario, the H pylori
arginase could provide endogenous urea for utilization
by urease to generate acid-neutralizing ammonia, and
thus counteract a major innate defense of the stomach –
acid
Mammalian and other bacterial arginases require heat activation, in the presence of manganese, for optimal catalytic activity [16,19,22,25,27] Numerous studies have shown that manganese does not bind mammalian arginase very tightly, as dialysis of these enzymes results in a significant decrease in their activity [15,19] Heat activation
of arginase-containing extracts, or of the purified His6-RocF protein in the presence of cobalt, was required for catalytic activity, and this treatment was time-, temperature-, and cobalt-dependent The activation step may facilitate partial unfolding of arginase to allow cobalt into the active site The divalent metal ion may not be bound tightly enough and may leach out during the preparation of extracts or the purified protein, as little or no activity occurs in the absence
of cobalt The amino acid residues involved in metal binding
in H pylori arginase are currently under investigation Interestingly, it has been observed that arginases with alkaline isoelectric points bind manganese more tightly than arginases with acidic or neutral isoelectric points [16] As metal-bound arginases are more stable than apoarginases, alkaline pI arginases tend to be more stable Computer analysis of H pylori arginase predicts a pI of 6.3, suggesting that it belongs to the family of weak metal-binding and less stable arginases Indeed, the catalytic activity of purified His6-RocF was unstable, suggesting that cobalt does not bind tightly to the enzyme Instability of the H pylori arginase may, in part, be due to spontaneous degradation (Fig 7A)
We speculate that H pylori arginase evolved to have optimal activity with cobalt over that of manganese to avoid competing with host enzymes that contain manganese (manganoenzymes) Support for this hypothesis comes from the finding that only one known host protein contains cobalt, while multiple host proteins, such as arginase, Mn-superoxide dismutase, Mn-catalase, and serine/threonine protein phosphatase-1, are manganoenzymes [39] Known sources of cobalt in the host are vitamin B12 (cobalamin) and methionine aminopeptidase [40] Although H pylori does not absolutely require the addition of vitamin B12 for growth in vitro [41], it is possible that in vivo the bacterium can metabolize this vitamin so that the cobalt would be available for transport into the cell with subsequent incorporation into arginase Studies are underway to explore this possibility Cobalamin binds to the stomach glycoprotein intrinsic factor which allows for its normal absorption [42] H pylori may disrupt this delicate balance
by metabolizing the vitamin, removing the cobalt and incorporating the cobalt into arginase This could be a contributing factor to the vitamin B12 deficiencies often observed in H pylori-infected patients [43–45]
In summary, the H pylori rocF gene encodes the urea cycle enzyme arginase, and its gene product is necessary and sufficient for arginase activity The enzyme was expressed in and purified from E coli Antibodies were successfully raised to RocF, and Western blot analyses were employed
to show the expression of arginase E coli and the degra-dation of the recombinant enzyme Compared with other arginases, the H pylori enzyme has a number of unique features, including (a) optimal catalytic activity with cobalt rather than manganese, (b) stimulation of its activity by bicarbonate [11], (c) optimal catalytic activity at pH 6.1, rather than at pH 9.0–11.0, (d) inhibition by reducing
Trang 10agents at low concentrations, (e) inhibition of host nitric
oxide [7], and (f) protection of H pylori from acid These
unique features suggest that H pylori arginase has evolved
to allow the bacterium to effectively compete with the host
for available substrates (arginine, cobalt), necessary for the
organism to survive and proliferate in the seemingly
inhospitable gastric niche
Acknowledgements
This work was supported with start-up funds from the University of
South Alabama Department of Microbiology and Immunology and
the College of Medicine, by Public Health Service grant CA101931 (to
D.J.M.) from the National Institutes of Health, and the Australian
Research Council (to G.L.M.).
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