mol cell proteomics 2007 pyndiah 193 206

7/22/2019 Mol Cell Proteomics 2007 Pyndiah 193 206

1/14

Two-dimensional Blue Native/SDS GelElectrophoresis of Multiprotein Complexesfrom Helicobacter pylori*

Slovenie Pyndiah, Jean Paul Lasserre, Armelle Menard, Stephane Claverol,Valerie Prouzet-Mauleon, Francis Megraud**, Frank Zerbib, and Marc Bonneu

The study of protein interactions constitutes an important

domain to understand the physiology and pathogenesis of

microorganisms. The two-dimensional blue native/SDS-

PAGE was initially reported to analyze membrane protein

complexes. In this study, both cytoplasmic and mem-

brane complexes of a bacterium, the strain J99 of the

gastric pathogen Helicobacter pylori, were analyzed by

this method. It was possible to identify 34 different pro-

teins grouped in 13 multiprotein complexes, 11 from the

cytoplasm and two from the membrane, either previously

reported partially or totally in the literature. Besides com-

plexes involved in H. pylori physiology, this method al-

lowed the description of interactions involving known

pathogenic factors such as (i) urease with the heat shock

protein GroEL or with the putative ketol-acid reductoi-

somerase IlvC and (ii) the cag pathogenicity island CagA

protein with the DNA gyrase GyrA as well as insight on the

partners of TsaA, a peroxide reductase/stress-dependent

molecular chaperone. The two-dimensional blue native/

SDS-PAGE combined with mass spectrometry is a poten-

tial tool to study the differences in complexes isolated in

various situations and also to study the interactions be-

tween bacterial and eucaryotic cell proteins. Molecular& Cellular Proteomics 6:193206, 2007.

Helicobacter pylori is a spiral, microaerophilic, Gram-neg-ative bacterium that colonizes the gastric epithelium (1) in4060% of the worlds population. Approximately 1020% ofthese infected individuals suffer from diseases, such as pepticulcer disease, or from conditions such as chronic atrophicgastritis, which can then evolve toward gastric cancer (2 4).

Identification and characterization of multiprotein com-plexes are important steps in obtaining an integrative view of

protein-protein interaction networks that determine proteinfunction and cell behavior. The availability of complete DNAsequences of two H. pyloristrains (5, 6) has led to the devel-

opment of reliable proteome-wide approaches for a betterunderstanding of the virulence mechanisms of the bacterium.One of the strategies of functional proteomics, a method usedto identify gene function at the protein level, is the compre-hensive analysis of protein-protein interactions related to thefunctional linkage among proteins and the analysis of func-tional cellular machinery to better understand the basis of theorganisms functions.

Recent progress in high throughput technologies has al-lowed the characterization of protein-protein interactionsmore directly than ever before using procedures such as thetwo-hybrid assay (7), co-purification (8, 9), or co-immunopre-cipitation (10). The workhorse of experimental proteomics hasbeen the two-hybrid screening method, although it has beencriticized for its limited accuracy of results and its labor-intensive nature (11, 12). Indeed, it is currently the most reli-able technique for large scale characterization of protein in-teractions in complete genomes (13). Protein chips mayeventually provide large scale simultaneous protein-proteininteraction data (14, 15), but technical problems (denaturation

and substrate biocompatibility) must be overcome to scale upfor high throughput analysis. To date, these technologies havegenerated large interaction networks for bacteria (16), yeast(8, 9), fruit flies (17), and nematode worms (18).

Other approaches will undoubtedly become prominent asproteomics technology continues to evolve. The limiting fac-tor for identifying protein complexes is the separation methodthat must be performed under native conditions to preventprotein dissociation. Because of these limits, the two-dimen-sional blue native (2D BN)1/SDS-PAGE method was appliedto study the H. pylorireference strain J99 complexome. Pro-tein identification was performed by using LC-MS/MS. Thishighly resolvent separation method was initially described for

the separation under native conditions of the membrane pro-tein complexes of mitochondria (19). Later on, numerous

From INSERM, U853, Bordeaux, F 33076 France and the Labo-ratoire de Bacteriologie and Pole Proteomique, PlateformeGenomique Fonctionnelle Bordeaux, Universite Victor Segalen Bor-deaux 2, Bordeaux, F 33076 France

Received, September 19, 2006Published, MCP Papers in Press, November 7, 2006, DOI 10.1074/

mcp.M600363-MCP200

1 The abbreviations used are: 2D BN, two-dimensional blue native;UreA/B, urease /subunit; GroEL, chaperone and heat shock pro-tein; AhpC, alkyl hydroperoxide reductase; TSA, thiol-specific antiox-idant; POR, pyruvate oxidoreductase; PAI, pathogenicity island; CagAor -I, cag pathogenicity island protein A or I; GyrA, DNA gyrasesubunit A; OMP, outer membrane protein; BabA/B, blood groupantigen-binding adhesin A/B.

Research

2007 by The American Society for Biochemistry and Molecular Biology, Inc. Molecular & Cellular Proteomics 6.2 193This paper is available on line at http://www.mcponline.org


2/14

studies focused on the protein complexes of the respiratorychain (2027). More recently, because this method is repro-ducible, it was successfully used to study the detection ofprotein complex deficiencies of mitochondria (2832). In thesame manner, this method was applied to the protein com-plexes of the chloroplast membranes of cyanobacteria (33)and protein complexes of the mitochondrial and chloroplastmembranes of plants (3441). Moreover a similar methodusing agarose instead of acrylamide was developed to studyprotein complexes of approximate molecular mass greaterthan 1200 kDa (42). The anionic dye used in this method(Coomassie Brilliant Blue G-250) binds to the surface of allproteins, particularly on aromatic residues and on arginines.This binding of a large number of negatively charged dyemolecules to proteins facilitates the multiprotein complex mi-gration in a first dimension native electrophoresis (BN-PAGE),and the tendency for protein aggregation is thus reducedconsiderably. Multiprotein complexes are separated accord-ing to their size and shape. Each multiprotein complex is

denatured in a second dimension electrophoresis (SDS-PAGE), and the protein alignment allows the identification ofinteractive proteins.

Recent modifications have made it possible to apply thismethod to whole protein complexes (4345). Indeed thismethod was recently used to study the Escherichia colicellenvelope complexome (44), human embryonic kidney 293cells (43), and human platelets (45). A dialysis must be per-formed on cytoplasmic extracts to eliminate salt and smallmolecules (43).

This method was applied to analyze both H. pyloricytoplas-mic and membrane complexes. Purification steps such asliquid IEF or chromatography fractionation and enrichmentwere used to improve the multiprotein complex separationfrom the cytoplasm.

EXPERIMENTAL PROCEDURES

Bacterial Growth ConditionsH. pyloristrain J99 (ATCC 700824)(6) was used for the experiments.H. pyloricells were cultured for 48 hon Wilkins Chalgren agar (Oxoid Ltd., Hampshire, UK) plates supple-mented with 10% human blood and the following antibiotics: 10mg/ml vancomycin (Lilly France S.A., Fergesheim, France), 2 mg/mlcefsulodin (Takeda France S.A., Puteaux, France), 5 g/ml Fungizone(Bristol-Myers Squibb Co.), and 5 mg/ml trimethoprim (GlaxoSmith-Kline). A bacterial suspension was grown in brucella broth (BD Bio-sciences) supplemented with the same antibiotics cited above and

with 10% fetal calf serum (Eurobio, Les Ulis, France). The plates wereincubated at 37 C under microaerobic conditions (5% O2, 10% CO

2,

85% N2), and the broth was incubated in a 1-liter loosely capped

container, with a 150 rpm agitation, in a microaerobic atmosphere at37 C for less than 72 h.

Bacteria harvested from two or three agar plates were suspendedin 250 ml of brucella broth. The resulting liquid culture showed anapproximate bacterial growth of 0.1% (w/v). For the development ofthe 2D BN/SDS-PAGE applied to H. pyloricytoplasmic extract and toobtain the final results for both the cytoplasmic and the membraneextract, a total of 10 g of H. pyloristrain J99 was frozen.

Bacterial Lysate, Cytoplasmic, and Membrane PreparationsAll of

the bacteria and sample manipulations were performed at 4 C. Bac-teria were harvested from culture by a centrifugation at 2,500 gfor10 min and washed twice in PBS buffer. Bacteria were suspended(v/v) in native extraction buffer A (750 mM6-amino-n-caproic acid, 50mMTris/HCl, pH 7.0, at 4 C) supplemented with a 1 mMfinal con-centration of PMSF and passed three times through a One Shotdisruptor at 2 kilobars. The lysate was centrifuged at 6,000 gfor 20min, the supernatant was kept, a 0.2 mg/ml final concentration of

DNase I was added, and digestion was carried out for 1 h at 25 C.Then the supernatant was centrifuged at 100,000 g for 30 min at4 C; the resulting supernatant and pellet contained the cytoplasmicprotein and the membrane protein, respectively.

For the cytoplasmic sample preparation, the supernatant was fil-tered with a Miracloth membrane (Calbiochem). and this sample wasnamed fraction I. The concentration of 125150 mg/ml protein wasobtained, and the sample appeared limpid.

For the membrane sample preparation, a bacterial lysate wascentrifuged at 6,000 gfor 20 min, and the pellet was resuspendedin buffer A and passed three times through a One Shot disruptor at 2kilobars. The resulting lysate was centrifuged at 100,000 g for 30min, and the pellet was resuspended in buffer A and centrifuged oncemore. The washing step was performed three times. The proteinextraction was then carried out by resuspending the membrane in 12

ml of buffer A supplemented with 2% -D-dodecyl-n-maltoside de-tergent (Sigma). This sample was then centrifuged at 100,000 gfor30 min, and the membrane multiprotein complexes contained in thesupernatant were separated by 2D BN/SDS-PAGE. Concerning themembrane extract, the detergent used interfered with the Bradfordprotein dosage. For this reason and to obtain sufficiently resolventgels, a preliminary experiment was carried out with various sampledilutions directly loaded onto the first dimension of the 2D BN/SDS-PAGE gel to determine the quantity of material needed for theelectrophoresis.

Purification Steps on the Cytoplasmic SampleAll of the stepswere carried out at 4 C. Purification steps using physical or chemicalproperties of the multiprotein complexes constituted an importantaspect to confirm protein-protein interactions. Therefore, liquid IEF orexclusion filtration methods were used as purification steps beforeapplying the 2D BN/SDS-PAGE.

Liquid IEF purification was used to separate the multiprotein com-plexes according to their pI in a pH range from 3.5 to 10. An aliquotof fraction I (containing 60 mg of protein) was analyzed in a Rotoforsystem (Rotofor Prep IEF Cell, Bio-Rad). The protein mixture wasprepared according to the manufacturers recommendations beforefilling the Rotofor chamber. The IEF method produced many proteinprecipitates in the most abundant protein fractions with a pI of 56.Very low protein concentrations were found in the basic fractionsalthough a concentration step with the Centricon kit (Amicon, Inc.,Beverly, MA) was used.

After the IEF of the protein sample, the resulting fractions weredesalted in buffer A using a 5-ml HiTrapTM desalting column (Amer-sham Biosciences). Multiprotein complexes were recovered in 300-l

fractions. Indeed for H. pylori cytoplasmic extracts, a preliminarydialysis is necessary to obtain highly resolvent gels. Here, dialysis wasreplaced by a desalting step, which allows the elimination of smallmolecules and salts, as was described for the purification of thehuman embryonic kidney cell line HEK293 (43).

Gel filtration purification was also tested. An aliquot of fraction I(300 l containing 60 mg of protein) was loaded on a SuperdexTM

200 column (Amersham Biosciences). Buffer A was run at a flow rateof 0.3 ml/min using the FPLC Akta (Amersham Biosciences). Multi-protein complexes were recovered in 250-l fractions. The cytoplas-mic sample was separated into five peaks of major interest: 1000,580, 340, 100, and 93 kDa. This method allowed the adaptability of

Helicobacter pyloriProteomics

194 Molecular & Cellular Proteomics 6.2


3/14

the 2D BN/SDS-PAGE acrylamide gradient according to the mass ofinterest of the complexes. The Centricon kit (Amicon, Inc.) with acutoff of 50 kDa was also used to concentrate the purified sampleswhen the sample concentration was lower than 50 mg/ml.

First Dimension: BN-PAGESample preparation and BN-PAGEwere carried out as described by Schagger and von Jagow (19) withthe following minor modifications. The gel dimension was 22 cm 16.5 cm 1 mm. Separating gels with linear 49% acrylamide

gradient gels were used. Anode and cathode buffers contained 50 mMTris, 75 mMglycine, and only the cathode buffer was supplementedwith 0.002% Serva blue G (Serva, Heidelberg, Germany). Beforeloading the sample, 1l of sample buffer (500 mM6-amino-n-caproicacid, 5% Serva blue G) was added. The gel was run overnight at 4 Cat 1 watt. Thyroglobulin (669 kDa) and bovine serum albumin (66 kDa)were used for each BN-PAGE analysis as molecular weight sizestandards (Sigma).

Different acrylamide gradients were tested for the BN-PAGE toimprove the multiprotein complex separation. We found that the bestmultiprotein complex separation was obtained with a linear 49%acrylamide gradient. A certain balance needs to be found to optimizeboth focalization and separation of complexes with a mass greaterthan 60 kDa. The 49% acrylamide gradient was performed threetimes on the same sample and two more times on a sample originat-

ing from another extraction; this experiment showed that the migra-tion distance of the multiprotein complexes was the same (data notshown). This is proof that the multiprotein complexes of H. pylorimaintain their global conformation during the electrophoresis. There-fore, a molecular mass could be attributed to the cytoplasmic com-plexes based on the two different marker proteins mentioned above.

Second Dimension: SDS-PAGEIndividual lanes from BN-PAGEwere equilibrated for 5 min in an equilibrating buffer containing 1%SDS (w/v), 0.125 mM Tris/HCl, pH 6.8, and then dipped into equili-brating buffer supplemented with 100 mMdithiothreitol (Acros Organ-ics, Morris Plains, NJ) for 15 min. Individual lanes were subsequentlysoaked in equilibrating buffer supplemented with 55 mM iodoacet-amide (Sigma) for 15 min. An ultimate washing step lasting 5 min wasperformed in the equilibrating buffer without supplement. Individuallanes were placed on a glass plate at the usual position for stackinggels. After positioning the spacers and covering with the second glassplate, the gel was brought into a vertical position. Then the 10%separating gel mixture was poured. After polymerization the stackinggel mixture was poured.

Gel StainingSilver staining was performed using a silver stainingkit (Sigma) according to the manufacturers instructions.

In-gel Protein DigestionSilver-stained proteins separated bySDS-PAGE were excised and destained using the PROTSIL2 silverstaining kit (Sigma) according to the manufacturers instructions.Spots were subsequently washed in distilled water/methanol/aceticacid (47.5:47.5:5) until complete destaining. The solvent mixture wasremoved and replaced by acetonitrile. After shrinking of the gelpieces, acetonitrile was removed, and gel pieces were dried in avacuum centrifuge. Gel pieces were rehydrated in 10 ng/l trypsin

(Sigma) and 50 mM

bicarbonate and incubated overnight at 37 C.The supernatant was removed and stored at 20 C, and the gelpieces were incubated for 15 min in 50 mM bicarbonate at roomtemperature under rotary shaking. This second supernatant waspooled with the previous one, and a distilled water/acetonitrile/aceticacid (47.5:47.5:5) solution was added to the gel pieces for 15 min.This step was repeated again twice. Supernatants were pooled andconcentrated in a vacuum centrifuge to a final volume of 30 l.Digests were finally acidified by the addition of 1.8 l of acetic acidand stored at 20 C.

On-line Capillary HPLC Nanospray Ion Trap MS/MS Analysis

Peptide mixtures were analyzed by on-line capillary HPLC (LC Pack-

ings, Amsterdam, The Netherlands) coupled to a nanospray LCQ iontrap mass spectrometer (ThermoFinnigan, San Jose, CA). Peptideswere separated on a 75-m-inner diameter 15-cm C18PepMap

TM

column (LC Packings). The flow rate was set at 200 nl/min. Peptideswere eluted using a 550% linear gradient of solvent B for 30 min(solvent A was 0.1% formic acid in 5% acetonitrile, and solvent B was0.1% formic acid in 80% acetonitrile). The mass spectrometer wasoperated in positive ion mode at a 2-kV needle voltage and a 38-V

capillary voltage. Data acquisition was performed in a data-depend-ent mode consisting of, alternatively in a single run, a full scan MSover the rangem/z300 2000 and a full scan MS/MS in an exclusiondynamic mode. MS/MS data were acquired using a 3 m/zunit ionisolation window, a 35% relative collision energy, and a 5-min dy-namic exclusion duration.

Data AnalysisData were analyzed by SEQUEST (ThermoFinni-gan, Torrance, CA) against a subset of the National Center for Bio-technology Information (NCBI) database consisting of H. pyloristrainJ99 and 26695 protein sequences. Carbamidomethylation of cys-teines (57) and oxidation of methionines (16) were considered asdifferential modifications. Only peptides with an Xcorr values greaterthan 1.5 (single charge), 2 (double charge), and 2.5 (triple charge)were retained. In all cases, C

nvalues have to be greater than 0.1.

RESULTS

Global Presentation of the ResultsMultiprotein complexesfrom the cytoplasm were named C, and those from themembrane were named M. Putative multihomooligomericprotein complexes were named H. Certain identifications(noted S1S5) could not be attributed to complexes; they arenot presented in Table I but are noted in the legends of thefigures only. Proteins that are components of the same mul-tiprotein complex co-migrate in the first dimension and arefound aligned with a similar shape in the second dimension.This is the basic condition for the identification of a multipro-tein complex. As an example, several spots were found to be

perfectly aligned (Figs. 1Aand 2), and the five proteins with asimilar shape (spots 2125) were attributed to the multiproteincomplex named C8. However, the elongated form of thesespots in Fig. 1Ahas a more rounded form in Fig. 2. In a furtherexample, spots 46 (Fig. 3) fulfilled these criteria, and thethree corresponding proteins were considered to be subunitsof a multiprotein complex named C2. Aligned spots but withdifferent shapes were considered to belong to different mul-tiprotein complexes. Obvious examples are indicated by ar-

rows (spots A1A6) in Figs. 2 and 3. The spots A1 and A2indicated by arrows (Fig. 2) are perfectly aligned; however,spot A1 has a rounded form, whereas spot A2 has a more

elongated shape. For this reason, they were attributed todifferent complexes. Other examples of aligned spots withdifferent shapes are indicated by arrows: 1) spots A3 and A4in Fig. 2 and 2) spots A5 and A6 in Fig. 3. One can also noticethat only spot A6 was identified in the crude extract (Fig. 1A).

All these spots (spots A1A6) were considered to belong todistinct complexes.

The crude cytoplasmic and membrane protein complexesseparated by 2D BN/SDS-PAGE are shown in Fig. 1, AandB,respectively. To increase the number of multiprotein com-


Molecular & Cellular Proteomics 6.2 195


4/14


5/14


6/14

plexes observed, non-denaturing purification steps were per-formed on the cytoplasmic extract before applying the 2D

BN/SDS-PAGE separation,e.g.liquid IEF (Fig. 2) or exclusionfiltration (Fig. 3). All of the multiprotein complexes identified inthis study are described in Table I. Attempts to identify pro-teins were made on a large number of spots, but becausesome LC-MS/MS identifications failed, certain complexeshave not been reported in this study.

Different types of complexes were observed. Multiheteroo-ligomeric complexes fulfilling the previously defined criteriawere clearly identified, and in certain cases, these complexeswere found several times. Complex C1 (spots 13) is an

example; it is found in Figs. 2 and 3. The aligned spots areelongated in Fig. 3 but have a more marked curve in Fig. 2.

This complex is no doubt present in Fig. 1A, but only spots 1and 2 can be seen. Because spots 1 and 2 are more intenselycolored than spot 3 (in Figs. 2 and 3) and the spots 1 and 2 arevery light in Fig. 1, a plausible explanation is that the lattercould not be visualized in Fig. 1. One can also notice that theshape of spot 3 is more difficult to interpret because the gelmigration is diffuse in this molecular mass range. Interestinglythe genes jhp0631,jhp0632, and jhp0633 that encode thesethree proteins are located on the same operon (6). ComplexC2 (spots 46) is clearly visualized in Fig. 3 but to a lesser

FIG. 1. Analysis of the crude cyto-plasmic and membrane samples of H.

pylori reference strain J99. The first

dimension gel (BN-PAGE) was per-formed with an acrylamide gradient of49% for the crude cytoplasmic sample(A) and 6 13% for the crude membranesample (B). C and M represent com-plexes isolated from the cytoplasm andfrom the membrane compartments, re-spectively. H represents putative multi-homooligomeric complexes. * repre-sents spots where different proteinswere identified (see Table I). A secondmigration of the C3 andM1 complexes isshown in Boxes 1 and 3, respectively.The migration of the C5 complex identi-fied in Fig. 3 was also performed usingthe crude cytoplasmic extract, but thequantity of protein loaded on the gel wasincreased (Box 2). A second migration ofspots S3 and S4 is shown in Box 4.Arrow A6 refers to Fig. 3. Two proteinswere identified in spot S1: AlpB (orHopB; two peptides, coverage 2%)and AlpA (or HopC; three peptides, cov-erage 2%). Spot S2 corresponds toTsaA (five peptides, coverage 31%).Spot S3 contains FrpB3 (22 peptides,coverage 30%), CagA (four peptides,coverage 2%), and ClpB (four pep-tides, coverage 2%), and spot S4 con-tains BabB (seven peptides, coverage 13%), HydB (or HyaB; 11 peptides, cov-erage 18%), and HopM (or OMP5, sixpeptides, coverage 7%). Spots D1D14 correspond to monomers of AtpA,

AtpD, GroEL, Tig, RecA, AddB, Tfs,TufA, JHP0971, JHP1356, Adk, PorC,CagL, and JHP1494, respectively.




7/14

extent than in Fig. 1Abecause spot 4 is not perfectly alignedwith spots 5 and 6 in this figure. Indeed a mixture of proteinscontaining DnaN (23 peptides, coverage 83%) and PorA

(six peptides, coverage 18%) was identified in spot 4 in thecrude extract. In fact, spot 4 probably corresponds to twodifferent spots with the spot corresponding to DnaK masking

FIG. 2. Protein complexes identifiedfrom H. pylori reference strain J99

when the cytoplasmic sample was pu-

rified using the liquid isoelectrofocal-

ization method (Rotofor system) be-fore applying 2D BN/SDS-PAGE.

Analysis of the sample fraction contain-ing protein complexes with a global pI of5 is shown. The first dimension gel (BN-PAGE) was performed with an acrylam-ide gradient of 49%. H represents pu-tative multihomooligomeric complexes. *represents spots where different pro-teins were identified (see Table I). SpotsS5S7 all correspond to TsaA (spot S5:three peptides, coverage 13%; spotS6: three peptides, coverage 16; andspot S7: five peptides, coverage 22%). Two examples of aligned spots

with different shapes are indicated byarrows A1 and A2and byarrows A3andA4, respectively.

FIG. 3. Protein complexes identifiedfrom H. pylori reference strain J99

when the cytoplasmic sample was pu-

rified using the gel filtration method

(Superdex 200 column) before apply-

ing 2D BN/SDS-PAGE. Analysis of thesample fraction containing protein com-plexes in the range of 580 kDa is shown.The first dimension gel (BN-PAGE) wasperformed with an acrylamide gradientof 49%. An example of aligned spotswith different shapes is indicated by ar-rows A5and A6.




8/14

the less intense spot corresponding to PorA. The complex C3represented by spots 710 was identified several times in thecrude extract (Fig. 1, A and Box 1). However, after the exclu-sion filtration purification step, not all of the spots comprisingthe complex were found. Only the most intense spots, i.e.spots 9 and 10, were found at a molecular mass of 500 kDa(Fig. 3). They both correspond to alkyl hydroperoxide reduc-tase TsaA (Table I). After the liquid IEF purification step, onlythree intense spots were found at a molecular mass of 500kDa, and they were also all identified as TsaA (Fig. 2, spotsS5S7). These results are in agreement with the fact that TsaAhas been described as an abundant protein in H. pylori (46).The results also strongly suggest that spots 7 and 8 in com-plex C3, which were already very light in Fig. 1A, were prob-ably not detected after the different purification steps due totheir weak intensity or due to a loss of these two subunitsduring the purification process. In addition, after the liquid IEFpurification step, all of the visible spots on the Fig. 2 gel wereanalyzed by LC-MS/MS, and the proteins corresponding to

spots 7 and 8 were not found in the 75 analyzed spots (datanot shown).

Among the multiheterooligomeric complexes, certain oneswere not identified until after the purification step. The exclusionfiltration purification of the samples enabled the identification ofcomplexes C4, C5, and C6 (Fig. 3). The analysis of the samplefraction containing protein complexes eluted in the range of 580kDa from the exclusion filtration allowed the identification ofcomplex C4, comprised of two subunits. Indeed spots 11 and12 are aligned and have the same elongated form. Similarlyspots 13 and 14 with their lengthened form belong to complexC5 and were also found in the crude cytoplasmic extract whenthe first dimension gel was performed with a preparation con-taining more proteins (Fig. 1,A,H, andBox 2). Furthermore thiscomplex was also found in other 2D BN/SDS-PAGE gels (datanot shown). Lastly, complex C6 contains three subunits repre-sented by the similar and aligned spots 1517 (Fig. 3).

Problems were encountered for certain identifications (Figs.1Aand 2). For example, spots 1820 from complex C7 werefound to be aligned with a similar shape as were spots 2125from complex C8. Spots 18 20 (UreB, GroEL, and UreA) fromcomplex C7 were also found in complexes C8 and C9, but thequantities of the corresponding proteins appeared lower incomplexes C7 and C8 compared with complex C9, althoughthis amount was estimated on a silver-stained gel considered

as not adequate to provide a quantitative measure. This maysuggest that the C7-specific subunits are present in C8 andC9 but are near or below the detection limit of the silver stain.However, these three complexes migrate at different molec-ular mass, suggesting different states of oligomerization. Con-sequently three different complexes were attributed. In addi-tion, spots 2632 that were found to be aligned and to all havethe same shape in Fig. 1Awere also identified in Fig. 2 butwith the additional spots 3335. These spots also seem to bepresent after purification by exclusion filtration (Fig. 3); unfor-

tunately their identification after this purification step was notsufficient to attribute spots 2632 and 3335 either to a singlecomplex or to two different complexes. A hydroxyapatiteaffinity chromatography purification (HA Ultrogel column) wasalso performed, but the complex corresponding to spots2632 did not show an affinity to calcium and was directlyeluted as demonstrated by the 2D BN/SDS-PAGE analysis ofthis fraction (data not shown). In fact, the analyses by 2DBN/SDS-PAGE and LC-MS/MS of the unretained fraction andthe fractions eluted with the 1300 mM linear gradient ofsodium phosphate buffer did not allow the confirmation of thepresence of all of these spots in only one complex or in twodifferent complexes because spots 2632 were never identi-fied in these different fractions (data not shown). The absenceof spots 3335 on the gel in Fig. 1Acan be explained in twoways: 1) either the proteins present in spots 2632 and 3335are all part of the same complex and spots 3335 that wereless intense were not identified in the crude extract or 2) spots2632 belong to a different complex than that of spots 3335

but with the same apparent pI (which would explain why theywere not found in the crude extract except after the liquid IEFpurification). As a conservative measure to avoid describing afalse complex, these spots were attributed to two differentcomplexes, i.e. complexes C9 and C10 containing spots2632 and 3335, respectively.

Another problem relates to the identification of a mixture ofproteins in certain spots, and as a result, the same shapecriterion was more difficult to apply. Different complexes canindeed co-migrate in the first dimension, and therefore thespots are aligned on the second dimension. Accordingly com-plex C11 is comprised of spots 36 and 37 with a proteinmixture in spot 37 (Fig. 1A). As a result, it is not possible toevaluate whether the protein present in spot 36 interacts withonly one or with two proteins present in spot 37. To thecontrary, because the proteins present in spot 37 are thesubunits A and B of succinyl-CoA transferase (ScoA andScoB) ofH. pyloriand are known to interact together (16, 47),this complex was considered to contain JHP0739, with thetwo partners ScoA and ScoB (Table I).

Furthermore spots H1H5 (Figs. 1Aand 2) that were veryintense were identified several times. The molecular massobserved during the migration in the first dimension of thecomplexes corresponding to these spots did not correspondto the molecular mass of the proteins identified in the second

dimension. No other similarly formed spot was found in align-ment with spots H1H5 either before or after different purifi-

cations. Several hypotheses could explain these incoherentmigrations in the first and second dimensions: these spotscould correspond to multihomooligomeric complexes, or theycould also belong to multiheterooligomeric complexes thatcontain weakly expressed proteins that are not visible. Be-cause these spots were identified several times after differentpurifications, it can be assumed that they are multihomooli-gomeric complexes. Furthermore these five complexes




9/14

named H1H5 all have already been described as multiho-mooligomers in other organisms (Table I), and most of theproteins involved in H1H4 complexes, e.g.TsaA, Pfr, SodB,and AroQ, have already been reported in multihomooligo-meric protein complexes in H. pylori. For each of these fiveputative multihomooligomeric protein complexes named H1H5, the putative number of subunits was estimated to be 35,31, 12, 14, and 14, respectively.

Concerning the crude membrane sample (Fig. 1B), thecomplex M1 was easily identified with the aligned spots3841 having a slightly elongated and oval form. The spots 42and 43 were aligned; however, it was difficult to compare theirform in the second dimension clearly because the molecularmass corresponding to spot 43 is low, and the gel migrationis diffuse in this molecular mass range. The co-migration inthe first dimension seems to be correct because these twospots were observed several times (data not shown). More-over, these proteins (spots 42 and 43) have been identifiedpreviously as the subunits A and B of the fumarate reductase

(FrdA and FrdB) of H. pylori(48). This complex was namedM2. We had difficulties identifying another complex. Indeedspots S3 and S4 were aligned and presented a similar, veryelongated shape, and they were identified several times in thecrude extract (Fig. 1, Band Box 4). However, the molecularmass observed during the first migration (250 kDa) waslower than the molecular mass of all the proteins identified inthe second dimension because protein mixtures were ob-served in these two spots: spot S3 contained the predictediron-regulated outer membrane protein FrpB_3 (or FrpB3,JHP1405; 97 kDa), the cag pathogenicity island protein ACagA (or Cag26, JHP0495; 129 kDa), and the predicted ATP-dependent protease binding subunit/heat shock protein ClpB(JHP0249; 96 kDa); spot S4 contained the predicted adhesinBabB (or OMP19, JHP1164; 76 kDa), the large subunit of thequinone-reactive Ni/Fe-hydrogenase HydB (or HyaB,JHP0575; 64 kDa), and the predicted outer membrane proteinHopM (or OMP5, JHP0212; 75 kDa). The difference betweenthe molecular mass of the complex observed in the firstdimension or deduced from the migration of the proteins inthe second dimension is too important for these proteins tobelong to the same complex. These incoherent migrations inthe first and second dimensions could be explained by thepresence of at least two complexes that co-migrate in the firstdimension and whose subunits have the same mass. Given

these results, no complex including these proteins, consideredto be important in the virulence of H. pylori, can be describedyet, and complementary experiments must be carried out. Thisexample clearly reflects the various difficulties of interpretationof the results obtained by the 2D BN/SDS-PAGE technique aswell as the frequent difficulty in assigning certain proteins to acomplex when a mixture of proteins is present.

A total of 13 multiprotein complexes containing 34 differentproteins (46 in total) were identified from H. pyloristrain J99.

Among these complexes, 11 were obtained from the cyto-

plasm, and two were from the membrane. Seven multiproteincomplexes identified in this study were described previously(Table I) either totally or partially, confirming the interest of thismethod to study the H. pyloricomplexome. As an example,several interactions between the different proteins from thecytoplasmic complex C9 (Figs. 1Aand 2) were partially de-scribed in the literature (Table II). These interactions includedthe interaction between the two structural subunits, UreA andUreB, of the well known urease enzyme (49). Moreover, pre-vious studies described the urease enzyme interacting withGroEL (Hsp60), a chaperone and heat shock protein (50). Onthe other hand, some protein interactions described in thecomplex C9 and reported by Hybrigenics (Rain et al. (16))involved an intermediary protein (Table II) not identified in thecurrent study, e.g.the interaction between GroEL and RpoBincluded the predicted outer membrane protein JHP0600.This cytoplasmic complex C9 that includes nine different pro-teins contained the greatest number of interacting proteinsobserved in this study. In addition to heterooligomeric com-plexes, five putative multihomooligomeric protein complexeswere identified from the cytoplasm (H1H5, Table II). Con-cerning the membrane, very few complexes are reported in

this study due to a poor resolution and/or visualization of thespots observed on the numerous gels performed, althoughthe 2D BN/SDS-PAGE was initially reported for the separationof membrane protein complexes (51). This suggests thatmembrane purifications should also improve the gel quality.

Different oligomerization states were sometimes observedin certain complexes. For example, TsaA was found in differ-ent oligomerization states and in both cytoplasmic and mem-brane samples: C3 and spots H1 and S2 (Figs. 1, AandB; 2;and 3 and Table I). These results are in agreement with those

TABLE IIUrease complex C9 isolated from H. pylori strain J99 cytoplasm

Solid and dotted brackets represent direct protein-protein interac-tions and interactions including an intermediary protein (indicated initalics), respectively. Uvra corresponds to HP0705 and JHP0644 inH. pylorireference strains 26695 and J99, respectively. *, Dunn et al.(49), , Evans et al. (50); , Hybrigenics (Rain et al. (16)); , theintermediary protein reported in this interaction is specific to H. pylorireference strain 26695, and no homolog exists in H. pylorireferencestrain J99.




10/14

of Backert et al. (46) who also identified TsaA, both in struc-ture-bound and in soluble fractions of H. pylori, in eight oli-gomerization states in their 2D gel electrophoresis and massspectrometry study. In addition, different migrations of TsaAwere observed during the separation in the second dimension(Fig. 2, spots S5 S7; and Fig. 3, spots 9 and 10) suggestingthat multiple isoforms of TsaA exist. Their occurrence can beexplained by probable post-translational modifications mod-ifying the physicochemical criteria (pI, molecular mass, andbinding affinity) of the protein, as reported previously for nu-merous proteins (52, 53), thus altering the migration in thesecond dimension. In fact, H. pyloriproteins are subject to ahigh degree of post-translational modification, and TsaA isamong several proteins present in multiple isoforms in thebacterium (54). TsaA, originally identified as a 26-kDa antigen,belongs to a family of antioxidants called AhpC/TSA (alkylhydroperoxide reductase/thiol-specific antioxidant) involvedin the defense against oxidative stress and survival of thebacterium in the host. More recently, TsaA was shown to

switch from a peroxide reductase to a stress-dependent mo-lecular chaperone function (55). H. pyloriTsaA was initiallyreported to induce an immune response in H. pylori-infectedpatients with gastric adenocarcinoma (56) and also an immuneresponse inH. pylori-infected Mongolian gerbils that is associ-ated with the emergence of gastric diseases such as chronicactive gastritis, gastric ulcer, and gastric cancer (57). TsaAbelongs to the five immunodominant H. pylori proteins (58).Pentameric arrangements of homodimers of TsaA were de-scribed inH. pyloricytoplasm (59, 60). In our study, TsaA wasfound in homooligomeric form but also in a complex includingproteins JHP1030 and PepQ. The presence of JHP1030 withTsaA is not really surprising because JHP1030 is a predictedzinc-dependent mannitol dehydrogenase that possesses an ox-idoreductase activity (inferred from electronic annotation). Onthe other hand, the presence of PepQ in this complex is moredifficult to interpret because it is a predicted proline peptidasethat has high homology with prolidases and X-prolyl dipeptidylaminopeptidases and can be involved either in protein matura-tion or in nitrogen metabolism. Other experiments should makeit possible to determine the exact role of these two partners ofTsaA.

Complexes Involved in H. pylori VirulenceBefore consid-ering virulence factors stricto sensu, complexes containingproteins involved in the energy metabolism (electron transport

pathway, tricarboxylic acid cycle, and gluconeogenesis),which is a prerequisite for virulence, will be briefly presented.Three pyruvate ferredoxin oxidoreductases, PorA, PorB, andPorC (ex-PorG), involved in electron transport were foundtogether in complex C2 (Table I and Figs. 1A and 3). Theheterotetrameric POR complex is comprised of the subunitsPorA, PorB, PorD, and PorC (61) and was reported previouslyto be essential because inactivation of porB appeared to belethal for H. pylori(62). PorD, the fourth subunit of the PORcomplex, was not found in this study probably because its

molecular mass (14.9 kDa) is near the border limit of the gelmigration. POR has been implicated in the bioreduction ofnitroimidazole drugs, particularly metronidazole used forH. pylorieradication (61, 63). To understand the mechanismsof metronidazole activation and resistance, which are cur-rently poorly characterized, a more detailed knowledge of theelectron transport pathways of the enzymes involved wouldbe helpful.

Fumarate reductase catalyzes the reduction of fumarate tosuccinate in the Krebs cycle and appears to play an importantrole in the energy metabolism of H. pylori. The two catalyticsubunits ofH. pylorifumarate reductase, FrdA and FrdB (48),were found in the membrane complex M2 (Fig. 1Band TableI). Fumarate reductase is not necessary for H. pylorisurvivalinvitrobut is essential for H. pyloricolonization of the mousestomach (64). It was shown to be strongly immunogenic insera fromH. pylori-positive patients, suggesting that this pro-tein involved in H. pylorienergy metabolism could also beused as an anti-H. pylorivaccine candidate (65).

Examples of multiprotein complexes including knownH. pylorivirulence factors are described below.

The Urease EnzymeH. pyloriproduces large amounts ofurease that catalyzes the hydrolysis of urea to yield ammoniaand carbon dioxide, neutralizing hydrogen ions before theycan lower the intracellular pH, thus enabling the bacterium tosurvive in gastric acid and to colonize the gastric mucosa (66).This urease activity is thus required for colonization in vivo(67). The native urease complex was found in both the mem-brane and the cytoplasm of the bacteria. Different oligomer-ization forms of the urease were identified in the cytoplasm(Table I). Four urease complexes named C7, C8, C9, and M1with a global pI of 5 and a molecular mass range of 9001300kDa were identified with crude and purified extracts (Figs. 1,AandB, and 2 and Table I). In fact, UreA and UreB are knownto be present in multiple isoforms in H. pylori (54) and areamong the predominant H. pylori proteins identified duringcell infection (68). H. pylori is known to produce a 550-kDaheterohexameric enzyme composed of three (UreA) andthree subunits (UreB). Four heterohexamers (2200 kDa) forma 16-nm spherical complex also produced by the bacteria(69). This kind of oligomerization may be adapted for acidresistance. The separation resolution of the 2D BN/SDS-PAGE did not allow the visualization of large complexes witha molecular mass greater than 1500 kDa. All of the urease

complexes identified in this study included the UreA/UreB/GroEL core. These three proteins were also reported to bepresent in structure-bound and in soluble fractions of H. pylori(46) and were strongly reactive to specific antibodies (58),indicating that this core is highly immunodominant inH. pylori.Under all pH conditions, the most abundant proteins ob-served were the urease structural subunit UreB and the chap-eronin GroEL (70). A close interaction between GroEL, theco-chaperone protein GroES, and the urease enzyme hadbeen suspected (50, 71), but until now no interaction between




11/14

GroEL and the urease has been confirmed. The C8, C9, andM1 complexes included additional proteins besides the coreUreA/UreB/GroEL. The C8 complex included the IlvC protein,a putative ketol-acid reductoisomerase involved in isoleucine-valine biosynthesis and the thiol peroxidase Tpx involved indetoxification; the M1 complex included the FrpB_3 protein, aputative iron-regulated outer membrane protein; and the C9complex included six other proteins (Tables I and II).

cag Pathogenicity Island (PAI) ProteinsCagA is the markerand one of the effectors of the cagPAI, translocated in gastricepithelial cells and conferring increased virulence to H. pyloristrains (72). CagA was identified both in the cytoplasm and inthe membrane: complex C5 and spot S3, respectively (Figs. 1,

Aand B, and 3 and Table I). This localization of CagA in themembrane was reported previously (46) and is probably dueto its presence in the type IV secretion system in theH. pyloricell wall (73, 74). The multiprotein complex C5 including CagAand the DNA gyrase subunit A (GyrA) was found in the cyto-plasmic sample with a molecular mass of 475 kDa (Figs. 1A

and 3) suggesting a probable oligomerization of CagA and/orGyrA in this complex. This CagA-GyrA complex was alsoobserved on other 2D BN/SDS-PAGE (data not shown). Cul-tures of CagA-negativeH. pyloristrains show a slower growththan the CagA-positive strains (72, 75). This phenomenoncould be explained by the CagA/GyrA interaction describedin this study because this interaction may have a favorableeffect on the normal DNA replication process, leading to abetter development of the bacterial cell. However, Hybri-genics (Rain et al. (16)) described an interaction betweenGyrA and the cagPAI protein I (CagI), including a smallintermediary cysteine-rich protein B (HcpB), which is a weak-lactamase.

DISCUSSION

The classical methods widely used for large scale analysisof protein interactions are the yeast two-hybrid and the tan-dem affinity protein tag methods with the main limitation ofexpressing chimerical proteins. With the yeast two-hybridmethod, mostly binary interactions are identified. Furthermorethe proteins of interest are often expressed in a heterologoussystem, the yeast, and the interactions are observed in aparticular compartment, the nucleus. Moreover the reliabilityof high throughput yeast two-hybrid assays is 50% (76). Inthe tandem affinity protein tag method, the protein complexes

are analyzedin vivo, but the organism being studied needs tobe competent for exogenous DNA. Another disadvantage ofthis method is that multihomooligomeric complexes cannotbe analyzed.

The 2D BN/SDS-PAGE used for the study of the H. pyloricomplexome also has some limitations. First, it can be difficultto easily assign each spot to independent protein complexeswhen they are located on the same vertical line in the seconddimension; complexes C8 and C9 are very representative ofthis problem as well as spots containing a mixture of proteins

such as spots S3 and S4 for which no complex could beattributed. For this reason, it is sometimes necessary not onlyto analyze the crude extract but also to carry out variousmethods of purifications based on different physicochemicalcriteria (pI-, molecular mass-, or affinity-based purification).This makes it possible to validate the identified complex andto identify complexes sometimes with a better resolutionand/or visualization. However, subunits of unstable com-plexes can be lost during these various purification steps,therefore leading to the description of incomplete complexes.Second, proteins that are weakly expressed in certain com-plexes may not be visible, again leading to the description ofincomplete complexes or to false multihomooligomeric com-plexes. In addition, mixtures of proteins can sometimes beidentified in the same spots, making it difficult to determinewhether all of the identified proteins belong to the samecomplex. This was a frequently found case for the membranesamples analyzed in this study where the outer membraneproteins often had similar molecular mass and pI. For this

reason, several complexes were not reported in this study.Third, the extraction is carried out under non-denaturing con-ditions, and therefore the complexes requiring denaturingconditions for extraction are not easily found. Examples in-clude 1)H. pylorivacuolating cytotoxin VacA, which is quicklyexported over the membrane (77, 78) but was not detectedwith this method, and 2) flagellar sheaths, anchored in themembrane and requiring denaturing extraction conditions(79), which were not detected either. However, the use ofnon-denaturing conditions is, in fact, the major advantage ofthis technique because it provides the native form of proteincomplexes and thus allows an analysis of the physiologic

state of the organism. The method was highly reproducible,and even if it is not possible to determine the totality of theH. pyloricomplexes, this technique is of great interest to com-pare the complexes of a bacterium in two different physiologicalstates. Moreover immunoblot applied to the 2D BN/SDS-PAGEwould lead to the identification of specific protein complexes,particularly those including strain-specific proteins and viru-lence factors. Therefore, the comparison of the currently avail-able results obtained with all of the different methods used toanalyze protein complexes will allow an accurate identificationof theH. pyloriprotein-protein interaction network.

AcknowledgmentWe are grateful to Corinne Asencio for techni-

cal support in bacterial cell culture.* This work was supported in part by the Conseil Regional

dAquitaine and the Association pour la Recherche sur le Cancer. Thecosts of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Recipient of a predoctoral fellowship from the Fondation pour laRecherche Medicale.

** To whom correspondence should be addressed: UniversiteVictor Segalen Bordeaux 2, Laboratoire de Bacteriologie, Bat. 2B




12/14

RDC Zone Nord, 33076 Bordeaux cedex, France. Tel.: 33-5-56-79-59-10; Fax: 33-5-56-79-60-18; E-mail: [email protected].

REFERENCES

1. Warren, J. R., and Marshall, B. (1983) Unified curved bacilli on gastricepithelium in active chronic gastritis. Lancet1, 12731275

2. Correa, P., and Chen, V. W. (1994) Gastric cancer.Cancer Surveys1920,5576

3. Blaser, M. J. (1995) The role of Helicobacter pylori in gastritis and itsprogression to peptic ulcer disease. Alimentary Pharmacology andTherapeutics 9, 2730

4. Sipponen, P., Hyvarinen, H., Seppala, K., and Blaser, M. J. (1998) Reviewarticle: pathogenesis of the transformation from gastritis to malignancy.

Aliment. Pharmacol. Ther.12, 61715. Tomb, J. F., White, O., Kerlavage, A. R., Clayton, R. A., Sutton, G. G.,

Fleischmann, R. D., Ketchum, K. A., Klenk, H. P., Gill, S., Dougherty,B. A., Nelson, K., Quackenbush, J., Zhou, L., Kirkness, E. F., Peterson,S., Loftus, B., Richardson, D., Dodson, R., Khalak, H. G., Glodek, A.,McKenney, K., Fitzegerald, L. M., Lee, N., Adams, M. D., Hickey, E. K.,Berg, D. E., Gocayne, J. D., Utterback, T. R., Peterson, J. D., Kelley,J. M., Cotton, M. D., Weidman, J. M., Fujii, C., Bowman, C., Watthey, L.,Wallin, E., Hayes, W. S., Borodovsky, M., Karp, P. D., Smith, H., O.,Fraser, C. M., and Venter, J. C. (1997) The complete genome sequenceof the gastric pathogenHelicobacter pylori. Nature388,539547

6. Alm, R. A., Ling, L. S. L., Moir, D. T., King, B. L., Brown, E. D., Doig, P. C.,Smith, D. R., Noonan, B., Guild, B. C., deJonge, B. L., Carmel, G.,Tummino, P. J., Caruso, A., UriaNickelsen, M., Mills, D. M., Ives, C.,Gibson, R., Merberg, D., Mills, S. D., Jiang, Q., Taylor, D. E., Vovis, G. F.,and Trust, T. J. (1999) Genomic-sequence comparison of two unrelatedisolates of the human gastric pathogenHelicobacter pylori. Nature397,176180

7. Fields, S., and Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340,245246

8. Uetz, P., and Hughes, R. E. (2000) Systematic and large-scale two-hybridscreens. Curr. Opin. Microbiol. 3, 303308

9. Gavin, A. C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A.,Schultz, J., Rick, J. M., Michon, A. M., Cruciat, C. M., Remor, M., Hofert,C., Schelder, M., Brajenovic, M., Ruffner, H., Merino, A., Klein, K.,Hudak, M., Dickson, D., Rudi, T., Gnau, V., Bauch, A., Bastuck, S.,Huhse, B., Leutwein, C., Heurtier, M. A., Copley, R. R., Edelmann, A.,Querfurth, E., Rybin, V., Drewes, G., Raida, M., Bouwmeester, T., Bork,

P., Seraphin, B., Kuster, B., Neubauer, G., and Superti-Furga, G. (2002)Functional organization of the yeast proteome by systematic analysis ofprotein complexes. Nature 415,141147

10. Aebersold, R., and Mann, M. (2003) Mass spectrometry-based proteom-ics. Nature 422,198207

11. Enright, A. J., Iliopoulos, I., Kyrpides, N. C., and Ouzounis, C. A. (1999)Protein interaction maps for complete genomes based on gene fusionevents. Nature402,8690

12. Ito, T., Tashiro, K., and Kuhara, T. (2001) Systematic analysis of Saccha-romyces cerevisiaegenome: gene network and protein-protein interac-tion network. Tanpakushitsu Kakusan Koso 46, 24072413

13. Legrain, P., and Selig, L. (2000) Genome-wide protein interaction mapsusing two-hybrid systems. FEBS Lett. 480,3236

14. MacBeath, G., and Schreiber, S. L. (2000) Printing proteins as microarraysfor high-throughput function determination. Science 289,17601763

15. Hynes, S. O., McGuire, J., and Wadstrom, T. (2003) Potential for pro-teomic profiling ofHelicobacter pyloriand other Helicobacterspp. using

a ProteinChiparray. FEMS Immunol. Med. Microbiol. 36, 15115816. Rain, J. C., Selig, L., De Reuse, H., Battaglia, V., Reverdy, C., Simon, S.,

Lenzen, G., Petel, F., Wojcik, J., Schachter, V., Chemana, Y., Labigne,A., and Legrain, P. (2001) The protein-protein interaction map of Heli-cobacter pylori. Nature409,211215

17. Giot, L., Bader, J. S., Brouwer, C., Chaudhuri, A., Kuang, B., Li, Y., Hao,Y. L., Ooi, C. E., Godwin, B., Vitols, E., Vijayadamodar, G., Pochart, P.,Machineni, H., Welsh, M., Kong, Y., Zerhusen, B., Malcolm, R., Varrone,Z., Collis, A., Minto, M., Burgess, S., McDaniel, L., Stimpson, E.,Spriggs, F., Williams, J., Neurath, K., Ioime, N., Agee, M., Voss, E.,Furtak, K., Renzulli, R., Aanensen, N., Carrolla, S., Bickelhaupt, E.,Lazovatsky, Y., DaSilva, A., Zhong, J., Stanyon, C. A., Finley, R. L., Jr.,White, K. P., Braverman, M., Jarvie, T., Gold, S., Leach, M., Knight, J.,

Shimkets, R. A., McKenna, M. P., Chant, J., and Rothberg, J. M. (2003)A protein interaction map of Drosophila melanogaster. Science 302,17271736

18. Li, S., Armstrong, C. M., Bertin, N., Ge, H., Milstein, S., Boxem, M.,Vidalain, P. O., Han, J. D., Chesneau, A., Hao, T., Goldberg, D. S., Li, N.,Martinez, M., Rual, J. F., Lamesch, P., Xu, L., Tewari, M., Wong, S. L.,Zhang, L. V., Berriz, G. F., Jacotot, L., Vaglio, P., Reboul, J., Hirozane-Kishikawa, T., Li, Q., Gabel, H. W., Elewa, A., Baumgartner, B., Rose,D. J., Yu, H., Bosak, S., Sequerra, R., Fraser, A., Mango, S. E., Saxton,W. M., Strome, S., Van Den Heuvel, S., Piano, F., Vandenhaute, J.,Sardet, C., Gerstein, M., Doucette-Stamm, L., Gunsalus, K. C., Harper,J. W., Cusick, M. E., Roth, F. P., Hill, D. E., and Vidal, M. (2004) A mapof the interactome network of the metazoan C. elegans. Science 303,540543

19. Schagger, H., and von Jagow, G. (1991) Blue native electrophoresis forisolation of membrane protein complexes in enzymatically active form.

Anal. Biochem. 199,22323120. Reinheckel, T., Wiswedel, I., Noack, H., Augustin, W., Genova, M. L.,

Bianchi, C., Lenaz, G., Brookes, P. S., Pinner, A., Ramachandran, A.,Coward, L., Barnes, S., Kim, H., Darley-Usmar, V. M., Nijtmans, L. G.,Henderson, N. S., Holt, I. J., Schagger, H., Pfeiffer, K., Lindsay, J. G.,Lamantea, E., Zeviani, M., Singh, P., Jansch, L., Braun, H. P., Schmitz,U. K., Coenen, M. J., van den Heuvel, L. P., Morava, E., Marquardt, I.,Girschick, H. J., Trijbels, F. J., Grivell, L. A., Smeitink, J. A., Kruft, V.,Klement, P., Van den Bogert, C., and Houstek, J. (1995) Electrophoretic

evidence for the impairment of complexes of the respiratory chainduring iron/ascorbate induced peroxidation in isolated rat liver mito-chondria. Biochim. Biophys. Acta 1239,4550

21. Klement, P., Nijtmans, L. G., Van den Bogert, C., and Houstek, J. (1995)Analysis of oxidative phosphorylation complexes in cultured humanfibroblasts and amniocytes by blue-native-electrophoresis using mito-plasts isolated with the help of digitonin. Anal. Biochem. 231,218224

22. Schagger, H. (2001) Blue-native gels to isolate protein complexes frommitochondria. Methods Cell Biol. 65, 231244

23. Schagger, H., and Pfeiffer, K. (2001) The ratio of oxidative phosphorylationcomplexes IV in bovine heart mitochondria and the composition ofrespiratory chain supercomplexes. J. Biol. Chem. 276,3786137867

24. Nijtmans, L. G., Henderson, N. S., and Holt, I. J. (2002) Blue nativeelectrophoresis to study mitochondrial and other protein complexes.Methods26, 327334

25. Brookes, P. S., Pinner, A., Ramachandran, A., Coward, L., Barnes, S.,Kim, H., and Darley-Usmar, V. M. (2002) High throughput two-dimen-

sional blue-native electrophoresis: a tool for functional proteomics ofmitochondria and signaling complexes. Proteomics 2, 969977

26. Genova, M. L., Bianchi, C., and Lenaz, G. (2003) Structural organization ofthe mitochondrial respiratory chain. Ital. J. Biochem. 52, 5861

27. Navet, R., Jarmuszkiewicz, W., Douette, P., Sluse-Goffart, C. M., andSluse, F. E. (2004) Mitochondrial respiratory chain complex patternsfrom Acanthamoeba castellaniiand Lycopersicon esculentum: compar-ative analysis by BN-PAGE and evidence of protein-protein interactionbetween alternative oxidase and complex III. J. Bioenerg. Biomembr.36,471479

28. Van Coster, R., Smet, J., George, E., De Meirleir, L., Seneca, S., Van Hove,J., Sebire, G., Verhelst, H., De Bleecker, J., Van Vlem, B., Verloo, P., andLeroy, J. (2001) Blue native polyacrylamide gel electrophoresis: a pow-erful tool in diagnosis of oxidative phosphorylation defects. Pediatr.Res. 50, 658665

29. Coenen, M. J., van den Heuvel, L. P., Nijtmans, L. G., Morava, E., Mar-

quardt, I.,Girschick, H. J.,Trijbels, F. J.,Grivell, L. A.,and Smeitink,J. A.(1999) SURFEIT-1 gene analysis and two-dimensional blue native gelelectrophoresis in cytochrome c oxidase deficiency.Biochem. Biophys.Res. Commun. 265,339344

30. Wen, J. J., and Garg, N. (2004) Oxidative modification of mitochondrialrespiratory complexes in response to the stress of Trypanosoma cruziinfection. Free Radic. Biol. Med. 37, 20722081

31. Pineau, B., Mathieu, C., Gerard-Hirne, C., De Paepe, R., and Chetrit, P.(2005) Targeting the NAD7 subunit to mitochondria restores a functionalcomplex I and a wild type phenotype in the Nicotiana sylvestrisCMS IImutant lacking nad7. J. Biol. Chem. 280,2599426001

32. Perales, M., Eubel, H., Heinemeyer, J., Colaneri, A., Zabaleta, E., andBraun, H. P. (2005) Disruption of a nuclear gene encoding a mitochon-




13/14

drial gamma carbonic anhydrase reduces complex I and supercomplexI III2 levels and alters mitochondrial physiology inArabidopsis. J. Mol.Biol. 350,263277

33. Neff, D., and Dencher, N. A. (1999) Purification of multisubunit membraneprotein complexes: isolation of chloroplast FoF1-ATP synthase, CFoand CF1 by blue native electrophoresis. Biochem. Biophys. Res. Com-

mun.259,56957534. Jansch, L., Kruft, V., Schmitz, U. K., and Braun, H. P. (1996) New insights

into the composition, molecular mass and stoichiometry of the proteincomplexes of plant mitochondria. Plant J. 9, 357368

35. Singh, P., Jansch, L., Braun, H. P., and Schmitz, U. K. (2000) Resolutionof mitochondrial and chloroplast membrane protein complexes fromgreen leaves of potato on blue-native polyacrylamide gels. Indian J. Bio-chem. Biophys. 37, 5966

36. Eubel, H., Jansch, L., and Braun, H. P. (2003) New insights into therespiratory chain of plant mitochondria. Supercomplexes and a uniquecomposition of complex II. Plant Physiol. 133,274286

37. Eubel, H., Heinemeyer, J., and Braun, H. P. (2004) Identification andcharacterization of respirasomes in potato mitochondria. Plant Physiol.134,14501459

38. Eubel, H., Heinemeyer, J., Sunderhaus, S., and Braun, H. P. (2004) Res-piratory chain supercomplexes in plant mitochondria. Plant Physiol.Biochem.42, 937942

39. Herranen, M., Battchikova, N., Zhang, P., Graf, A., Sirpio, S., Paakkarinen,V., and Aro, E. M. (2004) Towards functional proteomics of membrane

protein complexes in Synechocystissp. PCC 6803. Plant Physiol. 134,47048140. Millar, A. H., Eubel, H., Jansch, L., Kruft, V., Heazlewood, J. L., and Braun,

H. P. (2004) Mitochondrial cytochrome c oxidase and succinate dehy-drogenase complexes contain plant specific subunits. Plant Mol. Biol.56,7790

41. Eubel, H., Braun, H. P., and Millar, A. H. (2005) Blue-native PAGE in plants:a tool in analysis of protein-protein interactions. Plant Methods1, 113

42. Henderson, N. S., Nijtmans, L. G., Lindsay, J. G., Lamantea, E., Zeviani,M., and Holt, I. J. (2000) Separation of intact pyruvate dehydrogenasecomplex using blue native agarose gel electrophoresis. Electrophoresis21,29252931

43. Camacho-Carvajal, M. M., Wollscheid, B., Aebersold, R., Steimle, V., andSchamel, W. W. (2004) Two-dimensional blue native/SDS gel electro-phoresis of multi-protein complexes from whole cellular lysates: a pro-teomics approach. Mol. Cell. Proteomics 3, 176182

44. Stenberg, F., Chovanec, P., Maslen, S. L., Robinson, C. V., Ilag, L. L., von

Heijne, G., and Daley, D. O. (2005) Protein complexes of the Escherichiacolicell envelope. J. Biol. Chem. 280,3440934419

45. Claeys, D., Geering, K., and Meyer, B. J. (2005) Two-dimensional bluenative/sodium dodecyl sulfate gel electrophoresis for analysis of mul-timeric proteins in platelets. Electrophoresis26, 11891199

46. Backert, S., Kwok, T., Schmid, M., Selbach, M., Moese, S., Peek, R. M.,Jr., Konig, W., Meyer, T. F., and Jungblut, P. R. (2005) Subproteomes ofsoluble and structure-bound Helicobacter pyloriproteins analyzed bytwo-dimensional gel electrophoresis and mass spectrometry.Proteom-

ics5, 1331134547. Corthesy-Theulaz, I. E., Bergonzelli, G. E., Henry, H., Bachmann, D.,

Schorderet, D. F., Blum, A. L., and Ornston, L. N. (1997) Cloning andcharacterization ofHelicobacter pylorisuccinyl CoA:acetoacetate CoA-transferase, a novel prokaryotic member of the CoA-transferase family.

J. Biol. Chem.272,256592566748. Birkholz, S., Knipp, U., Lemma, E., Kroger, A., and Opferkuch, W. (1994)

Fumarate reductase of Helicobacter pylorian immunogenic protein.J. Med. Microbiol. 41, 566249. Dunn, B. E., Campbell, G. P., Perez-Perez, G. I., and Blaser, M. J. (1990)

Purification and characterization of urease from Helicobacter pylori.J. Biol. Chem.265,94649469

50. Evans, D. J., Jr., Evans, D. G., Engstrand, L., and Graham, D. Y. (1992)Urease-associated heat shock protein of Helicobacter pylori. Infect.Immun. 60, 21252127

51. Schagger, H., and von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in therange from 1 to 100 kDa. Anal. Biochem. 166,368379

52. LeHoux, J. G., Fleury, A., Ducharme, L., and Hales, D. B. (2004) Phos-phorylation of the hamster adrenal steroidogenic acute regulatory pro-

tein as analyzed by two-dimensional polyacrylamide gel electrophore-ses. Mol. Cell. Endocrinol. 215,127134

53. Zhang, W., Bergamaschi, D., Jin, B., and Lu, X. (2005) Posttranslationalmodifications of p27kip1 determine its binding specificity to differentcyclins and cyclin-dependent kinases in vivo. Blood105,36913698

54. Lock, R. A., Cordwell, S. J., Coombs, G. W., Walsh, B. J., and Forbes,G. M. (2001) Proteome analysis ofHelicobacter pylori: Major proteins oftype strain NCTC 11637. Pathology33, 365374

55. Chuang, M. H., Wu, M. S., Lo, W. L., Lin, J. T., Wong, C. H., and Chiou,S. H. (2006) The antioxidant protein alkylhydroperoxide reductase ofHelicobacter pyloriswitches from a peroxide reductase to a molecularchaperone function. Proc. Natl. Acad. Sci. U. S. A. 103,25522557

56. Wang, J. T., Chang, C. S., Lee, C. Z., Yang, J. C., Lin, J. T., and Wang,T. H. (1998) Antibody to aHelicobacter pylorispecies specific antigen inpatients with adenocarcinoma of the stomach. Biochem. Biophys. Res.Commun.244,360363

57. Yan, J., Kumagai, T., Ohnishi, M., Ueno, I., and Ota, H. (2001) Immuneresponse to a 26-kDa protein, alkyl hydroperoxide reductase, in Heli-cobacter pylori-infected Mongolian gerbil model. Helicobacter6, 274282

58. Shin, J. H., Nam, S. W., Kim, J. T., Yoon, J. B., Bang, W. G., and Roe, I. H.(2003) Identification of immunodominant Helicobacter pyloriproteinswith reactivity to H-pylori-specific egg-yolk immunoglobulin. J. Med.Microbiol.52, 217222

59. Krah, A., Schmidt, F., Becher, D., Schmid, M., Albrecht, D., Rack, A.,Buttner, K., and Jungblut, P. R. (2003) Analysis of automatically gener-

ated peptide mass fingerprints of cellular proteins and antigens fromHelicobacter pylori26695 separated by two-dimensional electrophore-sis. Mol. Cell. Proteomics 2, 12711283

60. Papinutto, E., Windle, H. J., Cendron, L., Battistutta, R., Kelleher, D., andZanotti, G. (2005) Crystal structure of alkyl hydroperoxide-reductase(AhpC) fromHelicobacter pylori. Biochim. Biophys. Acta1753,240246

61. Hughes, N. J., Chalk, P. A., Clayton, C. L., and Kelly, D. J. (1995) Identi-fication of carboxylation enzymes and characterization of a novel four-subunit pyruvate:flavodoxin oxidoreductase fromHelicobacter pylori. J.Bacteriol.177,39533959

62. Hughes, N. J., Clayton, C. L., Chalk, P. A., and Kelly, D. J. (1998) Helico-bacter pylori porCDABand oorDABCgenes encode distinct pyruvate:flavodoxin and 2-oxoglutarate:acceptor oxidoreductases which medi-ate electron transport to NADP. J. Bacteriol.11191128

63. Hoffman, P. S., Goodwin, A., Johnsen, J., Magee, K., and Veldhuyzen vanZanten, S. J. O. (1996) Metabolic activities of metronidazole-sensitiveand -resistant strains of Helicobacter pylori: repression of pyruvate

oxidoreductase and expression of isocitrate lyase activity correlate withresistance. J. Bacteriol. 178,48224829

64. Ge, Z. M., Feng, Y., Dangler, C. A., Xu, S. L., Taylor, N. S., and Fox, J. G.(2000) Fumarate reductase is essential forHelicobacter pyloricoloniza-tion of the mouse stomach. Microb. Pathog. 29, 279287

65. Ge, Z. (2002) Potential of fumarate reductase as a novel therapeutic targetin Helicobacter pyloriinfection. Expert Opin. Ther. Targets6, 135146

66. Marshall, B. J., Barret, L., Prakash, C., McCallum, R., and Guerrant, R.(1990) Urea protects Helicobacter(Campylobacter)pylorifrom the bac-tericidal effect of acid. Gastroenterology99, 269276

67. Karita, M., Tsuda, M., and Nakazawa, T. (1995) Essential role of urease invitroandin vivo Helicobacter pyloricolonization study using a wild-typeand isogenic urease mutant strain. J. Clin. Gastroenterol. 21, Suppl. 1,S160S163

68. Backert, S., Gressmann, H., Kwok, T., Zimny Arndt, U., Konig, W., Jung-blut, P. R., and Meyer, T. F. (2005) Gene expression and protein profiling

of AGS gastric epithelial cells upon infection with Helicobacter pylori.Proteomics5, 3902391869. Ha, N. C., Oh, S. T., Sung, J. Y., Cha, K. A., Lee, M. H., and Oh, B. H.

(2001) Supramolecular assembly and acid resistance of Helicobacterpyloriurease. Nat. Struct. Biol. 8, 505509

70. Slonczewski, J. L., and Kirkpatrick, C. (2002) Proteomic analysis of pH-dependent stress responses in Escherichia coliand Helicobacter pyloriusing two-dimensional gel electrophoresis. Methods Enzymol. 358,228242

71. Dunn, B. E.,Vakil, N. B.,Schneider,B. G.,Miller, M. M.,Zitzer, J. B.,Peutz,T., and Phadnis, S. H. (1997) Localization of Helicobacter pyloriureaseand heat shock protein in human gastric biopsies. Infect. Immun. 65,11811188




14/14

72. Censini, S., Lange, C., Xiang, Z., Crabtree, J. E., Ghiara, P., Borodovsky,M., Rappuoli, R., and Covacci, A. (1996) cag, a pathogenicity island ofHelicobacter pylori, encodes type I-specific and disease-associatedvirulence factors. Proc. Natl. Acad. Sci. U. S. A. 93, 1464814653

73. Odenbreit, S., Puls, J., Sedlmaier, B., Gerland, E., Fischer, W., and Haas,R. (2000) Translocation of Helicobacter pyloriCagA into gastric epithe-lial cells by type IV secretion. Science 287,14971500

74. Tanaka, J., Suzuki, T., Mimuro, H., and Sasakawa, C. (2003) Structuraldefinition on the surface of Helicobacter pyloritype IV secretion appa-

ratus. Cell. Microbiol.5, 39540475. van Doorn, L. J., Quint, W., Schneeberger, P., Tytgat, G. M., and de Boer,

W. A. (1997) The only good Helicobacter pyloriis a dead Helicobacterpylori. Lancet350,7172

76. Sprinzak, E., Sattath, S., and Margalit, H. (2003) How reliable are experi-mental protein-protein interaction data? J. Mol. Biol. 327,919923

77. Fischer, W., Buhrdorf, R., Gerland, E., and Haas, R. (2001) Outer mem-brane targeting of passenger proteins by the vacuolating cytotoxinautotransporter of Helicobacter pylori. Infect. Immun. 69, 6769 6775

78. Bumann, D., Aksu, S., Wendland, M., Janek, K., Zimny-Arndt, U., Sabarth,N., Meyer, T. F., and Jungblut, P. R. (2002) Proteome analysis ofsecreted proteins of the gastric pathogen Helicobacter pylori. Infect.Immun. 70, 33963403

79. Geis, G., Suerbaum, S., Forsthoff, B., Leying, H., and Opferkuch, W.(1993) Ultrastructure and biochemical studies of the flagellar sheath ofHelicobacter pylori. J. Med. Microbiol. 38, 371377

80. Sanchez, S., Abel, A., Arenas, J., Criado, M. T., and Ferreiros, C. M. (2006)Cross-linking analysis of antigenic outer membrane protein complexesof Neisseria meningitidis. Res. Microbiol.157,136142

81. Ferrero, R. L., Hazell, S. L., and Lee, A. (1988) The urease enzymes ofCampylobacter pyloriand a related bacterium. J. Med. Microbiol. 27,3340

82. Voland, P., Weeks, D. L., Marcus, E. A., Prinz, C., Sachs, G., and Scott, D.(2003) Interactions among the seven Helicobacter pyloriproteins en-coded by the urease gene cluster.Am. J. Physiol. 284,G96G106

83. Sharp, J. A., and Edwards, M. R. (1978) Purification and properties ofsuccinyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheepkidney. Biochem. J. 173,759765

84. Jacob, U., Mack, M., Clausen, T., Huber, R., Buckel, W., and Messer-schmidt, A. (1997) Glutaconate CoA-transferase from Acidaminococcusfermentans: the crystal structure reveals homology with other CoA-transferases. Structure(Lond.) 5, 415426

85. Ge, Z. M., Jiang, Q., Kalisiak, M. S., and Taylor, D. E. (1997) Cloning and

functional characterization of Helicobacter pylori fumarate reductaseoperon comprising three structural genes coding for subunits C, A andB. Gene(Amst.) 204,227234

86. Mileni, M., MacMillan, F., Tziatzios, C., Zwicker, K., Haas, A. H., Mantele,W., Simon, J., and Lancaster, C. R. (2006) Heterologous production inWolinella succinogenesand characterization of the quinol:fumarate re-ductase enzymes from Helicobacter pyloriand Campylobacter jejuni.Biochem. J. 395,191201

87. Unden, G., and Kroger, A. (1981) The function of the subunits of thefumarate reductase complex of Vibrio succinogenes. Eur. J. Biochem.120,577584

88. Lancaster, C. R., Kroger, A., Auer, M., and Michel, H. (1999) Structure offumarate reductase from Wolinella succinogenes at 2.2 resolution.Nature 402,377385

89. Kitano, K., Niimura, Y., Nishiyama, Y., and Miki, K. (1999) Stimulation ofperoxidase activity by decamerization related to ionic strength: AhpCprotein from Amphibacillus xylanus. J. Biochem.(Tokyo) 126,313319

90. Wood, Z. A., Poole, L. B., Hantgan, R. R., and Karplus, P. A. (2002) Dimersto doughnuts: redox-sensitive oligomerization of 2-cysteine peroxire-doxins. Biochemistry41, 54935504

91. Frazier, B. A., Pgeifer, J. D., Russel, D. G., Klak, P., Olsen, A. N., Hammar,M., Westblom, T. U., and Normark, S. T. (1993) Paracrystalline inclu-sions of a novel ferritin containing non-heme iron, produced by thehuman gastric pathogen Helicobacter pylori: evidence for a third classof ferritins. J. Bacteriol. 175,966972

92. Yariv, J., Kalb, A. J., Sperling, R., Bauminger, E. R., Cohen, S. G., andOfer, S. (1981) The composition and the structure of bacterioferritin ofEscherichia coli. Biochem. J. 197,171175

93. Theil, E. C. (1987) Ferritin: structure, gene regulation, and cellular functionin animals, plants, and microorganisms. Annu. Rev. Biochem. 56,289315

94. Spiegelhalder, C., Gerstenecker, B., Kersten, A., Schiltz, E., and Kist, M.(1993) Purification of Helicobacter pylori superoxide dismutase andcloning and sequencing of the gene. Infect. Immun. 61, 53155325

95. Beyer, W. F., Jr., Reynolds, J. A., and Fridovich, I. (1989) Differencesbetween the manganese- and the iron-containing superoxide dismuta-ses of Escherichia colidetected through sedimentation equilibrium,hydrodynamic, and spectroscopic studies. Biochemistry28,44034409

96. Bottomley, J. R., Clayton, C. L., Chalk, P. A., and Kleanthous, C. (1996)Cloning, sequencing, expression, purification and preliminary charac-terization of a type II dehydroquinase from Helicobacter pylori. Bio-chem. J. 319,559565

97. Lee, B. I., Kwak, J. E., and Suh, S. W. (2003) Crystal structure of the typeII 3-dehydroquinase from Helicobacter pylori. Proteins51, 616617

98. Kinghorn, J. R., Schweizer, M., Giles, N. H., and Kushner, S. R. (1981) Thecloning and analysis of thearoD gene of E. coliK-12.Gene(Amst.)14,7380

99. Kleanthous, C., Deka, R., Davis, K., Kelly, S. M., Cooper, A., Harding,S. E., Price, N. C., Hawkins, A. R., and Coggins, J. R. (1992) A com-parison of the enzymological and biophysical properties of two distinct

classes of dehydroquinase enzymes. Biochem. J. 282,687695100. Kimber, M. S., Martin, F., Lu, Y., Houston, S., Vedadi, M., Dharamsi, A.,Fiebig, K. M., Schmid, M., and Rock, C. O. (2004) The structure of(3R)-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Pseudo-

monas aeruginosa. J. Biol. Chem.279,5259352602101. Boneca, I. G., de Reuse, H., Epinat, J.-C., Pupin, M., Labigne, A., and

Moszer, I. (2003) A revised annotation and comparative analysis ofHelicobacter pylorigenomes. Nucleic Acids Res. 31, 17041714



mol cell proteomics 2007 pyndiah 193 206

Documents