# Identification of Proteins Related to Nickel Homeostasis inHelicobater pylori by Immobilized Metal Affinity Chromatography and Two-Dimensional  Gel Electrophoresis

Authors: Xuesong Sun; Ruiguang Ge; Jen-Fu Chiu; Hongzhe Sun; Qing-Yu He
Journal: Metal-Based Drugs
 (2008)

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## Abstract

Helicobacter pylori (H. pylori) is a widespread human pathogen causing peptic ulcers and chronic gastritis. Maintaining nickel homeostasis is crucial for the establishment of
H. pylori infection in humans. We used immobilized-nickel affinity chromatography to isolate Ni-related proteins from H. pylori cell extracts. Two-dimensional gel electrophoresis and mass spectrometry were employed to separate and identify twenty two Ni-interacting proteins in H. pylori. These Ni-interacting proteins can be classified into several general functional categories, including cellular processes (HspA, HspB, TsaA, and NapA), enzymes (Urease, Fumarase, GuaB, Cad, PPase, and DmpI), membrane-associated proteins (OM jhp1427 and HpaA), iron storage protein (Pfr), and hypothetical proteins (HP0271, HP jhp0216, HP jhp0301, HP0721, HP0614, and HP jhp0118). The implication of these proteins in nickel homeostasis is discussed.


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## Body

## 1. Introduction

The human gastric
pathogenHelicobacter pylori (H. pylori) is a microaerophilic,
spiral-shaped, Gram-negative bacterium responsible for the majority of peptic
ulcer diseases in humans [1]. H. pylori has been
shown to be the causative agent of type B gastritis and peptic ulcerations [2, 3]. Infection with H. pylori increases the risks of
developing gastric carcinoma and mucosa-associated lymphoid tissue (MALT)
lymphoma [4, 5]. Continuously exposed to
acidic pH during the process of colonization in the gastric mucus layer, H.
pylori requires mechanisms to survive acid shocks and to enable growth in
such acidic conditions. This bacterium expresses two nickel-containing enzymes:
urease and hydrogenase, both of which is important for its colonization. The H. pylori urease
consists of 12 UreA and 12 UreB, and activation of this apoenzyme requires the
insertion of 24 nickel ions [6]. Urease hydrolyzes urea into
carbon dioxide and ammonia, thereby neutralizing the nearby environment [7–9]. [Ni-Fe] hydrogen-uptake
hydrogenase contains a heterobimetallic center in the large subunit with nickel
coordinating to four cysteines, and permits respiratory-based energy production
for the bacteria in the mucosa [10, 11]. Therefore, H. pylori needs
significant amounts of nickel to satisfy its requirement for the maturation of
the nickel-containing enzymes [12].Two kinds of high-affinity, nickel-specific
uptake systems are found inH. pylori: ATP-binding cassette (ABC)-type
transporter (abc ABCD) and nickel-cobalt permease (NixA) [13, 14]. However, when excess nickel
ions accumulate, they exhibit toxic effects and thus inhibit bacterial growth [15–17]. H. pylori has developed a system to maintain nickel homeostasis,
keeping a balance between the import, intracellular storage, and export of
nickel ions. Analysis of the H. pylori genome sequence has discovered
several putative ion binders and membrane transporters involved in metal
homeostasis [18]. However, the related
nickel-interacting proteins have not been fully identified so far.Immobilized-metal affinity chromatography
(IMAC) is a separation technique commonly used for fractionating proteins based on their different binding affinities of the
surface-exposed amino acids towards immobilized metal ions [19, 20]. Ni2+ belongs to the
group of intermediate metal ions, preferring coordination to nitrogen, oxygen,
and sulfur, and especially favors binding to the proteins with two exposed
vicinal histidines [21]. Elution of the target
proteins is achieved by lowering the pH or by adding a competing reagent such
as imidazole [22]. Two-dimensional gel
electrophoresis (2-DE) is a common choice for separating cellular proteins first
by their isoelectric point (pI) and then by molecular weight (MW).
However, low-abundance proteins may not be detected in
2-DE due to its limited separation capacity. IMAC
may compensate this, in some way, by specifically serving to enrich the
low-abundance metal-binding proteins and to reduce protein complexity.In this study, proteomic technology was employed
for the first time to isolate and identify candidate proteins involved in
nickel transport, storage, and utilization inH. pylori, by integrating
the powerful tools of Ni-IMAC, 2-DE, and matrix-assisted laser desorption
time-of-flight mass spectrometry (MALDI-TOF MS). Those proteins with surface active coordinating residues for
binding nickel (and maybe other metals with similar coordinating features) were
retained, separated, and analyzed. The information obtained from the
identification and functional analysis of these nickel-related proteins may
improve our understanding of nickel homeostasis in bacteria.


## 2. Materials and Methods

### 2.1. Bacterial Culture Conditions

H. pylori strain 11637 was kindly supplied by Dr. H. H-X.
Xia (Department of Medicine, The University of Hong Kong), and cultured in the
basal medium, Brucella broth (Difco) with 5% (v/v) fetal bovine serum (FBS;
GIBCO/BRL Life Technologies), for 72 hours with orbital shaking (100 rpm) at 37°C in an anaerobic jar with a
microaerobic gas-generating kit (Oxoid). Solutions used in this study were
prepared with ultra-pure water (18.2 MΩ; Millipore).


### 2.2. Immobilized-Nickel Affinity

H. pylori 11637 cells grown in 100 mL liquid media to
mid-log phase were pelleted at 8000×g for 5 minutes at 4°C, and washed three times with 10 mL
ice-cold phosphate buffered saline (PBS). Cell pellets were resuspended in 10
mL of ice-cold Buffer A (20 mM sodium phosphate buffer, 500 mM NaCl, 10 mM imidazole, 1 mM PMSF,
pH 7.4). Bacteria were ruptured with sonication in the presence of 1% v/v Triton
X-100. Proteins were recovered (10,000×g, 30 minutes), and the
supernatant was filtered through a 0.45 µm
cellulose acetate syringe filter (Iwaki Glass, Japan) before being loaded onto
a house-made, Buffer A-equilibrated Ni-NTA Agarose column (0.5 mL, Qiagen). After
washing with 10 column volumes of Buffer A, proteins were eluted with Buffer B
(20 mM sodium phosphate buffer, 500 mM NaCl, 500 mM imidazole, pH 7.4). The
fractions were concentrated with Centricon YM-3
(Millipore), and buffer-exchanged into rehydration solutions (8 M urea, 4% CHAPS, 1 mM PMSF, 20 mM DTT, 2% pharmalyte pH 3–11) with PlusOne 2-D Clean-up kit (Amersham Biosciences,
Buckinghamshire,
UK). Protein concentrations were determined by BCA assay (Bio-Rad, Calif,
USA) using BSA as the standard. Cell extractswere used immediately
or frozen in aliquots at –80°C.


### 2.3. 2-DE

2-DE was carried out with an IPGphor II Isoelectric Focusing
(IEF) unit and Hoefer SE 600 Ruby electrophoresis unit (Amersham Biosciences)
according to the method detailed elsewhere [23]. Briefly, 100 µg of H.
pylori protein samples were diluted in rehydration solutions containing
traces of bromophenol blue. IEF was carried out with precast 13-cm Immobiline
DryStrip (IPG strips; Amersham Biosciences) to generate a nonlinear pH gradient
of 3–10. Following IEF, strips were immediately used for the second dimensional
SDS-PAGE (10). Proteins
were visualized with silver stain, and protein molecular weight (MW) was
calibrated with prestained SDS-PAGE marker (Broad range; Bio-Rad). To minimize
gel to gel variations, two-dimensional gels were run three times for each
sample.


### 2.4. Peptide Mass Fingerprinting (PMF)

The silver-stained gels were scanned (Image Scanner; Amersham
Biosciences) and analyzed with ImageMaster 2D Elite software (Amersham
Biosciences) [23]. The normalized intensity
(NI) for each protein spot was calculated as the ratio of the spot intensity versus
the sum of intensities of the spots present in the whole gel. Protein spots
were cut out from the silver-stained gels and enzymatically digested overnight
with sequence grade porcine trypsin (Promega) at 37°C. The masses of
the digested peptides were determined with a Voyager-DE STR Biospectrometry
Workstation (Applied Biosystems, Calif, USA). Protein identification was
performed by searching the NCBInr protein database using Protein Prospector (http://prospector.ucsf.edu) (3), with
pyroglutamic acid modification of N-terminal glutamine, oxidation of methionine,
and protein N-terminal acetylation as permissible modifications. The criteria
for searching were set at 50 ppm or better mass accuracy, at least four
matching peptide masses, as well as theoretical MW and isoelectric point (pI)
matching to the estimated values from gels.


## 2.1. Bacterial Culture Conditions

H. pylori strain 11637 was kindly supplied by Dr. H. H-X.
Xia (Department of Medicine, The University of Hong Kong), and cultured in the
basal medium, Brucella broth (Difco) with 5% (v/v) fetal bovine serum (FBS;
GIBCO/BRL Life Technologies), for 72 hours with orbital shaking (100 rpm) at 37°C in an anaerobic jar with a
microaerobic gas-generating kit (Oxoid). Solutions used in this study were
prepared with ultra-pure water (18.2 MΩ; Millipore).


## 2.2. Immobilized-Nickel Affinity

H. pylori 11637 cells grown in 100 mL liquid media to
mid-log phase were pelleted at 8000×g for 5 minutes at 4°C, and washed three times with 10 mL
ice-cold phosphate buffered saline (PBS). Cell pellets were resuspended in 10
mL of ice-cold Buffer A (20 mM sodium phosphate buffer, 500 mM NaCl, 10 mM imidazole, 1 mM PMSF,
pH 7.4). Bacteria were ruptured with sonication in the presence of 1% v/v Triton
X-100. Proteins were recovered (10,000×g, 30 minutes), and the
supernatant was filtered through a 0.45 µm
cellulose acetate syringe filter (Iwaki Glass, Japan) before being loaded onto
a house-made, Buffer A-equilibrated Ni-NTA Agarose column (0.5 mL, Qiagen). After
washing with 10 column volumes of Buffer A, proteins were eluted with Buffer B
(20 mM sodium phosphate buffer, 500 mM NaCl, 500 mM imidazole, pH 7.4). The
fractions were concentrated with Centricon YM-3
(Millipore), and buffer-exchanged into rehydration solutions (8 M urea, 4% CHAPS, 1 mM PMSF, 20 mM DTT, 2% pharmalyte pH 3–11) with PlusOne 2-D Clean-up kit (Amersham Biosciences,
Buckinghamshire,
UK). Protein concentrations were determined by BCA assay (Bio-Rad, Calif,
USA) using BSA as the standard. Cell extractswere used immediately
or frozen in aliquots at –80°C.


## 2.3. 2-DE

2-DE was carried out with an IPGphor II Isoelectric Focusing
(IEF) unit and Hoefer SE 600 Ruby electrophoresis unit (Amersham Biosciences)
according to the method detailed elsewhere [23]. Briefly, 100 µg of H.
pylori protein samples were diluted in rehydration solutions containing
traces of bromophenol blue. IEF was carried out with precast 13-cm Immobiline
DryStrip (IPG strips; Amersham Biosciences) to generate a nonlinear pH gradient
of 3–10. Following IEF, strips were immediately used for the second dimensional
SDS-PAGE (10). Proteins
were visualized with silver stain, and protein molecular weight (MW) was
calibrated with prestained SDS-PAGE marker (Broad range; Bio-Rad). To minimize
gel to gel variations, two-dimensional gels were run three times for each
sample.


## 2.4. Peptide Mass Fingerprinting (PMF)

The silver-stained gels were scanned (Image Scanner; Amersham
Biosciences) and analyzed with ImageMaster 2D Elite software (Amersham
Biosciences) [23]. The normalized intensity
(NI) for each protein spot was calculated as the ratio of the spot intensity versus
the sum of intensities of the spots present in the whole gel. Protein spots
were cut out from the silver-stained gels and enzymatically digested overnight
with sequence grade porcine trypsin (Promega) at 37°C. The masses of
the digested peptides were determined with a Voyager-DE STR Biospectrometry
Workstation (Applied Biosystems, Calif, USA). Protein identification was
performed by searching the NCBInr protein database using Protein Prospector (http://prospector.ucsf.edu) (3), with
pyroglutamic acid modification of N-terminal glutamine, oxidation of methionine,
and protein N-terminal acetylation as permissible modifications. The criteria
for searching were set at 50 ppm or better mass accuracy, at least four
matching peptide masses, as well as theoretical MW and isoelectric point (pI)
matching to the estimated values from gels.


## 3. Results and Discussion

To investigate nickel
binding proteins inH. pylori under
physiologically relevant conditions, a relatively mild and nondenaturing lysis method was applied prior to Nickel-IMAC
(Ni-IMAC) loading. Our previous experiments have shown 1-D gels for the total extract of H. pylori 11637 cells and extract fractions
eluted from a Ni-IMAC column [24], displaying many protein bands for both cases. We have now further
separated and identified nickel-interacting proteins in H. pylori using 2-DE.The resulting 2-DE images
were visualized by silver staining. The gel image of whole cell extracts
of H. pylori 11637 (see Figure 1(a)) has a protein distribution very similar
to the standard proteome pattern of H. pylori 26695 [25]. More than 800 protein spots were separated,
with molecular weights ranging from 6 to 200 kDa and pI values from 3 to 10. Figure 1(a) shows a representative gel
image of Ni-related proteins in H. pylori 11637. The ratio
between the number of Ni-binding proteins and total extracted proteins of whole
cell lysates was 1:57, indicating that nickel specifically binds to a
restricted number of proteins. Qualitative comparison of 2-D gels between the Ni-enriched
eluates (see Figure 1(a)) and the total extracts (see
Figure 1(b)) revealed remarkably different spot patterns.Nickel-interacting proteins inH. pylori 11637 analyzed by 2-DE. (a)
2-DE patterns of total Ni-binding proteins in H. pylori 11637; (b) 2-DE patterns of H. pylori total proteins.
(a)(b)Ni-binding protein spots were excised,
trypsin-digested, and subjected to MALDI-TOF MS sequencing. Mass spectra of tryptic peptides were used to identify
proteins by matching to the spectra of the protein sequence databases, and the
identification was further verified by comparing to the standard proteomic map
ofH. pylori [25]. In total, twenty two proteins were identified as candidate
Ni-interacting proteins, as
marked in Figure 1(a) and summarized in Table 1. Some of
the protein spots could not be annotated due to no substantial MS signals or inadequate
peptide coverage for confident identification. Interestingly, five of the
proteins identified,including
UreA, HspB, fumarase, Pfr, and hypothetical protein jhp0301, show
more than one spot
in the 2-DE map, indicating the presence of posttranslational modifications.
Protein isoforms typically present themselves as a series of spots that differ
slightly in their molecular masses and pI, that is, numerous closely
spaced spots in 2-DE profiles [26–28]. Five proteins (HspA, HspB,
fumarase, TsaA, and NapA) were also identified in the Bi-interacting profile [24], indicating that there was
some correlation between Bi3+ and Ni2+ interactions. The Ni-interacting
proteins can be classified into five general functional categories:
cellular processes (HspA, HspB, TsaA, and NapA), enzymes (Urease, Fumarase,
GuaB, Cad, PPase, and DmpI), membrane-associated proteins (OM jhp1427 and
HpaA), iron storage protein (Pfr), and hypothetical proteins (HP0271, HP
jhp0216, HP jhp0301, HP0721, HP0614, and HP jhp0118).Table 1
Summary of nickel-interactingH. pylori proteins identified by peptide
mass fingerprinting.
Protein no.Protein nameIdentified peptides% Sequence coverageMOWSE scoreMW (kDa)/pITheor.Obs.1*UreB21421.595e+1061.4/5.565.5/5.82*60 kDa chaperonin (HspB)65751.199e+1558.2/5.559.3/5.3360 kDa chaperonin (HspB)19379.875e+0458.2/5.556.7/5.54Inosine-5′-monophosphate
dehydrogenase (GuaB)24512.559e+0951.8/7.756.2/8.05Fumarase22511.097e+1151.0/6.552.6/7.46Fumarase20451.800e+1051.0/6.552.6/7.07Putative aminotransferase26552.466e+1242.4/5.840.4/6.28Hypothetical protein HP0271 (fragment)41298038.6/5.021.1/4.89Cinnnamyl-alcohol dehydrogenase
ELI3-2 (Cad)15361.912e+0938.6/7.040.7/7.810Cell binding factor 221485.030e+0634.0/9.331.9/9.811Outer membrane protein jhp147211326.291e+0430.2/9.126.7/9.312Hypothetical protein jhp021615577.869e+0529.5/9.127.4/9.413Hypothetical protein jhp03017241.027e+0428.6/7.827.1/8.014Hypothetical protein jhp030119612.461e+0528.6/7.827.1/8.115Putative neuraminyllactose-binding hemagglutinin homolog
(HpaA)13502.381e+0528.3/7.927.0/7.216Urease alpha subunit (UreA)14681.280e+0726.5/8.527.2/9.017Urease alpha subunit (UreA)4881.846e+1126.5/8.527.2/8.518Putative alkyl hydroperoxide reductase (TsaA)12541.297e+0721.9/5.719.9/6.219Inorganic pyrophosphatase (PPase)42357514.7/5.020.7/4.720Non-heme iron containing ferritin (Pfr)22336919.3/5.414.4/5.221Non-heme rion containing ferretin (Pfr)8562.190e+0719.3/5.414.4/5.522Hypothetical protein HP072113441.692e+0417.6/8.914.4/8.523Neutrophil activating protein (NapA)6481.570e+0516.8/5.710.2/6.224Hypothetical protein jhp01184321.620e+0316.6/9.312.2/9.825Chaperonin groES (HspA)64414613.0/6.110.2/6.426Hepothetical protein HP061413561.557e+0513.0/7.07.7/8.0274-oxalocrotonate tautomerase (DmpI)10853.420e+057.5/6.05.5/6.4*Confirmed with 2-DE images of H.
pylori 26695 (http://www.mpiib-berlin.mpg.de/2D-PAGE/EBP-PAGE/index.html).Nickel homeostasis
is required for the establishment ofH. pylori infection in animals [7], necessitating a balance in the nickel import, storage,
and delivery for the synthesis and activity of nickel-dependent metalloenzymes.
Proteins involved in nickel homeostasis are potential drug targets. Consequently,
an analysis to identify the nickel-interacting proteins in H. pylori may be able to reveal candidate proteins for further
characterization and validation to provide drug targets.In order
to live in the acidic gastric environment,H. pylori continuously synthesize urease catalyzing the hydrolysis of urea to
ammonia and carbamate to elevate pH. Urease is an oligomeric Ni-containing
heterodimer of UreA and UreB.Both subunits of urease were observed
in the 2-DE gel after Ni-NTA enrichment, suggesting each of them can bind nickel
ions (see Figure 1(a)). Expression of active urease is essential for H. pylori infection in all animal models
and for acid survival in vitro [29]. Urease activity is
significantly enhanced in the presence of nickel, additional incorporation of
Ni2+ into the apoenzyme is thus the major regulating event upon
higher Ni2+ availability.Heat shock proteins are a group of highly
conserved, abundantly expressed proteins with protective advantage through
their functions as molecular chaperones, assisting proper folding of a
number of substrate proteins that are otherwise destined to aggregation [30, 31]. Both Chaperones HspA and
HspB were isolated by Ni-IMAC. HspA and HspB are also involved in urease
activation and protection. Urease activity was increased four folds in E. coli when coexpressed with HspA,
suggesting that HspA possibly plays an important role in the activation of
urease, probably by means of its Ni-binding domain in the C-terminus [32]. HspA has two distinct Ni2+-binding
sites: a high-affinity site (Kd≈2.8 µM) and
a lower affinity site (Kd≈30 µM).
The H. pylori GroEL homologue HspB belongs to the HSP60 family [33], and has been shown to increase the risk of
gastric carcinoma, possibly by directly inducing the hyperproliferation
of gastric cells [34]. HspB also was suggested to
be responsible for the protection and regulation of urease activity [33].Many identified Ni-interacting proteins inH. pylori are involved in antioxidant,
antitoxic, and antiinflammation machinery. H. pylori TsaA is a major
component of the thioredoxin-dependent thiol-specific antioxidant (TSA) system
that catalyzes the reduction of hydroperoxides [35] and peroxynitrite [36]. The cleavage of TsaA
suppresses the protecting response of H. pylori cells against oxidative
stress in two possible ways: directly by the way of nucleophilic attacking the
peptide bond through metal-bound hydroxide ions, and/or indirectly by way of stimulating
the activities of specific proteases. NapA
is also directly involved in cell defense in response to oxidative stress.It
was named because of its ability to mediate neutrophil adhesion to endothelial
cells [37], and to bind to mucin and
neutrophil glycosphingolipids [38]. NapA was identified as a 150 kDa
DNA-binding dodecamer that protects cells from DNA damage caused by free
radicals under oxidative stress [39, 40]. NapA was found to be positively
regulated by iron, repressed by ferric uptake regulator (Fur) [40], and unaffected by copper, nickel,
or zinc [41]. An excess of iron is
potentially harmful as it catalyzes the formation of toxic reactive oxygen
species (ROS) via Fenton chemistry. Ferritin protein Pfr, the major iron
storage protein of H. pylori, is also
regulated by iron, nickel, zinc, and copper. The accumulation of this protein
under iron-rich conditions allows H. pylori to maximize the iron storage
capacity in response to an increase in iron availability [44]. It is essential for the
bacteria to adapt to low-iron conditions [42–45]. Obviously, Ni-IMAC is also able to trap Pfr with its cation binding
affinity.


## 4. Conclusion

Immobilized-nickel affinity chromatography and proteomic analysis
were integrated to separate and identify the Ni-interacting proteins inH. pylori. The results suggest that
Ni-interacting proteins are mainly involved in cellular processes, oxidative
stress, and metabolism of the bacteria. This study demonstrated that metalloproteomic
technique can be utilized to efficiently identify metal-related proteins that
may play crucial roles in metal homeostasis. These proteins may be potential targets
for designing and constructing drugs to suppress the bacterial infection.



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*Source: 289490-2007-12-05.xml*