Identification of Neisseria meningitidis Outer Membrane Vesicle Complexes Using 2-D High Resolution Clear Native/SDS-PAGE Juan Marzoa, Sandra Sa ´nchez, Carlos M. Ferreiro ´s, and Marı ´a Teresa Criado* Departamento de Microbiologı ´a y Parasitologı ´a, Facultad de Farmacia, Campus Sur, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain Received July 20, 2009 Abstract: The identification and characterization of men- ingococcal outer membrane vesicle complexes can be important for gaining an in-depth understaining of their structure and functionality. Analysis of the vesicle com- plexome by ‘traditional’ 2-D analysis, in which isoelec- trofocusing is used for separation in the first dimension, is hampered by the high hydrophobicity and extreme isoelectric points of many relevant proteins. Analysis of the meningococcal outer membrane vesicle complexome using Blue Native (nondenaturing) electrophoresis instead of isoelectrofocusing in the first dimension showed several porin complexes, but their composition could not be clearly resolved after separation by SDS-PAGE in the second dimension. In this work, using a recently described native separation technique shigh resolution Clear Native Electrophoresissand different bidimensional approaches, we were able to demonstrate the presence of relevant outer membrane complexes which could be resolved with a higher resolution than in previous analysis. The most relevant were nine porin complexes formed by different combinations of the meningococcal PorA, PorB and RmpM proteins, and comparison with the complexes formed in specific knockout mutants allowed us to infer the relevance of each porin in the formation of each complex. Keywords: Neisseria meningitidis • outer membrane pro- teome • high resolution clear native electrophoresis • outer membrane complex. Introduction The search for effective meningococcal antiserogroup B vaccines is currently focused in outer membrane (OM) proteins that can show high antigenic cross-reactivity and a low inter- strain variability. The main antigenic OM proteins, PorA and PorB 1-4 have been extensively studied, but up to date, their antigenic variability is an important drawback for the design of a widely effective vaccine based on them. Attempts to obtain vaccines using conserved minor surface antigens 5 have not resulted in a good vaccine candidate, and the search for effective antigens (or antigen combinations) still continues. 6 The detection and identification of epitopes of conformational and/or shared nature that can form in OM complexes, which could be important or even critical for immune responses, 7-10 has been hampered by the use of denaturing analytical techniques, such as 1-D SDS-PAGE or 2-D IEF/SDS-PAGE, which are the most employed methods for protein separation, previous to Western blotting, in almost all studies for antigen detection in Neisseria meningitidis outer membranes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) 11 is one of the best gel systems for the analysis of protein complexes under nondenaturing conditions. In this technique, the anionic dye Coomassie G-250 is used to confer, under physiological conditions, a negative charge to the complexes proportional to their size, avoiding the dissociation of subunits and, thus, retaining their native structure. Nevertheless, al- though we found that 1-D BN-PAGE produces a good resolu- tion of OM meningococcal complexes, 12 the high amount of Coomassie stain associated to them hinders a good transfer and detection of the antigenic proteins by Western blotting. Also, our attempts to obtain 2-D proteome maps from the BN- PAGE gels, using SDS-PAGE in the second dimension, resulted in a poor resolution of the components of some relevant complexes due to an important lateral diffusion of many protein spots after the second dimension run, particularly the porins. 12 A recent work by Wittig et al. 13 described a new technique that they named high resolution clear native electrophoresis (hrCNE) in which the good resolution of complexes attained in BN-PAGE is combined with the excellent preservation of the structure and function of complexes brought off by clear native electrophoresis (CNE), also overcoming the interferences pro- duced by the Coomassie dye in subsequent analyses, and allowing an excellent separation of membrane complexes in native conditions, making it optimal for functional proteomic analysis. In hrCNE, the Coomassie dye is omitted from the sample and substituted in the cathode buffer by a mixture of two detergents, one of them neutral and the other anionic. The mixed micelles formed by both detergents confer the charge needed for the electrophoretic migration of the complexes, which are then separated on the basis of their size by the acrylamide gradient in the gel. Using different anionic and neutral detergent mixtures allows the analysis of complexes with differing stability. So, the alternative use of n-dodecyl-- D-maltoside (DDM), Triton X-100 or digitonin could result in different complex patterns, even allowing the detection of supercomplexes in the membrane structure. The technique has * To whom correspondence should be addressed. E-mail: mteresa.criado@ usc.es. Tel. no.: +34 981 563100, ext 14946. Fax no.: +34 981 594631. 10.1021/pr9006409 2010 American Chemical Society Journal of Proteome Research 2010, 9, 611–619 611 Published on Web 11/04/2009
Transcript
Identification of Neisseria meningitidis Outer Membrane Vesicle
Complexes Using 2-D High Resolution Clear Native/SDS-PAGE
Juan Marzoa, Sandra Sanchez, Carlos M. Ferreiros, and Marıa Teresa Criado*
Departamento de Microbiologıa y Parasitologıa, Facultad de Farmacia, Campus Sur, Universidad de Santiagode Compostela, 15782 Santiago de Compostela, Spain
Received July 20, 2009
Abstract: The identification and characterization of men-ingococcal outer membrane vesicle complexes can beimportant for gaining an in-depth understaining of theirstructure and functionality. Analysis of the vesicle com-plexome by ‘traditional’ 2-D analysis, in which isoelec-trofocusing is used for separation in the first dimension,is hampered by the high hydrophobicity and extremeisoelectric points of many relevant proteins. Analysis ofthe meningococcal outer membrane vesicle complexomeusing Blue Native (nondenaturing) electrophoresis insteadof isoelectrofocusing in the first dimension showedseveral porin complexes, but their composition could notbe clearly resolved after separation by SDS-PAGE in thesecond dimension. In this work, using a recently describednative separation techniqueshigh resolution Clear NativeElectrophoresissand different bidimensional approaches,we were able to demonstrate the presence of relevantouter membrane complexes which could be resolved witha higher resolution than in previous analysis. The mostrelevant were nine porin complexes formed by differentcombinations of the meningococcal PorA, PorB andRmpM proteins, and comparison with the complexesformed in specific knockout mutants allowed us to inferthe relevance of each porin in the formation of eachcomplex.
The search for effective meningococcal antiserogroup Bvaccines is currently focused in outer membrane (OM) proteinsthat can show high antigenic cross-reactivity and a low inter-strain variability. The main antigenic OM proteins, PorA andPorB1-4 have been extensively studied, but up to date, theirantigenic variability is an important drawback for the designof a widely effective vaccine based on them. Attempts to obtainvaccines using conserved minor surface antigens5 have notresulted in a good vaccine candidate, and the search foreffective antigens (or antigen combinations) still continues.6
The detection and identification of epitopes of conformational
and/or shared nature that can form in OM complexes, whichcould be important or even critical for immune responses,7-10
has been hampered by the use of denaturing analyticaltechniques, such as 1-D SDS-PAGE or 2-D IEF/SDS-PAGE,which are the most employed methods for protein separation,previous to Western blotting, in almost all studies for antigendetection in Neisseria meningitidis outer membranes.
Blue native polyacrylamide gel electrophoresis (BN-PAGE)11
is one of the best gel systems for the analysis of proteincomplexes under nondenaturing conditions. In this technique,the anionic dye Coomassie G-250 is used to confer, underphysiological conditions, a negative charge to the complexesproportional to their size, avoiding the dissociation of subunitsand, thus, retaining their native structure. Nevertheless, al-though we found that 1-D BN-PAGE produces a good resolu-tion of OM meningococcal complexes,12 the high amount ofCoomassie stain associated to them hinders a good transferand detection of the antigenic proteins by Western blotting.Also, our attempts to obtain 2-D proteome maps from the BN-PAGE gels, using SDS-PAGE in the second dimension, resultedin a poor resolution of the components of some relevantcomplexes due to an important lateral diffusion of manyprotein spots after the second dimension run, particularly theporins.12
A recent work by Wittig et al.13 described a new techniquethat they named high resolution clear native electrophoresis(hrCNE) in which the good resolution of complexes attainedin BN-PAGE is combined with the excellent preservation of thestructure and function of complexes brought off by clear nativeelectrophoresis (CNE), also overcoming the interferences pro-duced by the Coomassie dye in subsequent analyses, andallowing an excellent separation of membrane complexes innative conditions, making it optimal for functional proteomicanalysis.
In hrCNE, the Coomassie dye is omitted from the sampleand substituted in the cathode buffer by a mixture of twodetergents, one of them neutral and the other anionic. Themixed micelles formed by both detergents confer the chargeneeded for the electrophoretic migration of the complexes,which are then separated on the basis of their size by theacrylamide gradient in the gel. Using different anionic andneutral detergent mixtures allows the analysis of complexeswith differing stability. So, the alternative use of n-dodecyl-�-D-maltoside (DDM), Triton X-100 or digitonin could result indifferent complex patterns, even allowing the detection ofsupercomplexes in the membrane structure. The technique has
* To whom correspondence should be addressed. E-mail: [email protected]. Tel. no.: +34 981 563100, ext 14946. Fax no.: +34 981 594631.
10.1021/pr9006409 2010 American Chemical Society Journal of Proteome Research 2010, 9, 611–619 611Published on Web 11/04/2009
been highly successful for the separation of physiologicallyactive mitochondrial complexes14 and we think that it couldbe also applied to obtain a high resolution analysis of the OMcomplexome of N. meningitidis, as a precursor step in the studyof conformational and/or shared epitopes that could form dueto the interaction of the components of the complexes.
The aim of this work is the analysis of meningococcal outermembrane vesicles (OMVs) using hrCNE/SDS-PAGE 2-D elec-trophoresis. An in-depth knowledge of the outer membranecomplexome will help to advance in the knowledge theirstructure and physiology, which could help to understand thepossible role of interactions between the OM proteins (the mainmeningococcal surface antigens) in the immune responseagainst this pathogen.
Material and Methods
Strains and Culture Conditions. The strain N. meningitidisH44/76 (B:15:P1.7,16:F3-3) and its homologous mutants lack-ing PorA, PorB, RmpM or FetA were kindly donated by Dr. IanFeavers (National Institute for Biological Standards and Control,Great Britain). Strain maintenance and culture were carried outas described previously.15 All cultures were done under ironrestriction in Mueller-Hinton broth with 100 µM Desferaladded (MH-Desferal), and cells were recovered for extractionof OMVs when in exponential growth phase (12 h).
Outer Membrane Vesicle Preparations. MeningococcalOMVs were obtained using a modification of a previouslydescribed protocol.15 Bacteria recovered from MH-Desferalcultures were suspended in distilled water at 250 mg/mL (wetweight) and processed three times in a French pressure cell at1.1 × 108 kPa. Cellular debris was removed by centrifugationat 10000g for 10 min at 4 °C, and OMVs were recovered at200 000g (10 min, 4 °C), suspended in distilled water and storedat -80 °C. Concentration of OMVs in samples was estimatedfrom the protein content calculated using the Bicinchoninicacid (BCA) protein assay.16
High Resolution Clear Native Polyacrylamide Gel Electro-phoresis (hrCNE). hrCNE analyses were done following amodification of a previously described protocol.17 Gradient gels(5-15% or 8-11% polyacrylamide in 50 mM Bis-Tris/500 mM6-aminohexanoic acid) were used for separation of the com-plexes in a Mini-Protean 3 Cell (BioRad Laboratories S.A.,Spain). Gels can be precast and maintained for up to 3 days at4 °C in sealed bags containing 50 mM Bis-Tris/500 mM6-aminohexanoic acid. Just before running, a 4% polyacryla-mide stacking gel was cast over the gradient gels. Anode bufferwas 50 mM Bis-Tris-HCl (pH 7.0). Cathode buffer (pH 7.0)contained 50 mM Tricine, 15 mM Bis-Tris-HCl, 0.05% (w/v)sodium deoxycholate and 0.02% (w/v) n-dodecyl-�-D-maltoside(DDM). For some experiments, 0.05% (v/v) Triton X-100 wasused instead of DDM in the cathode buffer.
For the analyses, OMV stocks were diluted to 1 mg/mL inwater; then, 100 µL of 50 mM Bis-Tris-HCl (pH 7.0)/1 M6-aminohexanoic acid and 45.5 µL of 10% (w/v) DDM wereadded to each milliliter of OMVs (final concentrations were 4.4mM Bis-Tris-HCl, 87.3 mM 6-aminohexanoic acid, 0.4% DDM,873 µg/mL OMVs; 4.6:1 DDM/protein ratio). Complexes weresolubilized by incubation at room temperature for 30 min;thereafter, insoluble material was removed by centrifugationat 100 000g for 15 min at 4 °C. Before loading the gel, 5% (v/v)glycerol and 0.01% (w/v) Ponceau red were added to eachsample. Each lane was loaded with 20 µg (24 µL) of solubilizedOMVs and electrophoretic separation was done at 4 °C. Power
was set to 50 V constant voltage for the first hour and 100 Vconstant voltage for about 6 h. Because of differences in themobility of the complexes depending on the detergent used inthe cathode buffer (DDM or Triton X-100), KaleidoscopeStandards (Bio-Rad Laboratories S.A., Spain) were used tostandardize the total running time, which was consideredcomplete when the front of the standards reached the bottomof the gel. High molecular weight markers (HMW Native;66-669 kDa; GE Healthcare Espana S.A., Spain) were used toestimate the molecular weight of the complexes. Gels were fixedwith trichloroacetic acid and stained with Coomassie blueG-250 using a standard protocol.
Bidimensional Analyses: hrCNE/SDS-PAGE and hrCNE/hCNE Diagonal Electrophoresis. Bidimensional analyses toidentify the components of membrane complexes were doneusing SDS-PAGE in the second dimension (hrCNE/SDS-PAGE).Lanes cut from hrCNE gels (first dimension) were incubatedin SDS sample buffer for 10 min at 95 °C and then placed ontop of 12% polyacrylamide gels in a Mini-Protean 3 Cell (Bio-Rad Laboratories S.A., Spain). Electrophoretic separation wascarried out at 150 V constant voltage, and gels were stainedwith Coomassie and analyzed using PDQuest software (Bio-Rad Laboratories S.A., Spain). To determine the influence ofdetergents in the separation and stability of the complexes, 2-DhrCNE/hCNE diagonal analyses were done using DDM in thecathode buffer for the first dimension run and Triton X-100for the second, and vice versa. Conditions for separation in bothdimensions was done as described above for the 1-D hrCNEanalyses.
Image Analysis and Protein Identification. The analysis ofcomplexes in 1-D separations and that of proteins in 2-D maps,and calculation of their molecular weights were carried outusing the Quantity One (1-D) and PDQuest (2-D) analysissoftware (Bio-Rad Laboratories S.A., Spain) after scanning ofstained gels at high resolution (1200 dpi). Identifications of thecomponents of the complexes resolved by 1-D hrCNE and thatof the components of the complexes resolved by 2-D diagonalhrCNE/hCNE were done at the CIC bioGUNE (Technology Parkof Bizkaia, Derio, Spain) by NanoLC/MS-MS analysis of thebands cut from the gels. Identification of the proteins associ-ated to each complex after 2-D hrCNE/SDS-PAGE was done atthe Servicio de Espectrometrıa de Masas (University of Valen-cia, Spain) by MALDI-TOF fingerprinting analysis of the spotscut from Coomassie-stained gels.
Mass spectrometry (MS) analyses were done using a ReflexIV MALDI-TOF-MS analyzer spectrometer (Bruker Daltonics,Spain). FlexControl 2.4 software (Bruker) was used for controlof the instrument and FlexAnalysis 2.4 software (Bruker) forevaluation of the spectra. After trypsin digestion, each sample(0.5 µL) was deposited on an AnchorChip MTP384-600 plate(Bruker), and consecutively covered with 0.5 µL of an R-cyano-4-hydroxycinnamic acid (HCCA) matrix. Peak lists were gener-ated from a minimum of 200 shots for acquisition of data.Signal to noise (S/N) ratio was 0.1, and the smoothing SavitzkyGolay algorithm (one cycle, width 0.2 m/z) was applied.Background threshold for subtraction of baseline was set usinga median algorithm (flatness 0.8), and the percentage peakheight used for centroiding was determined using the SNAPalgorithm (included in the FlexAnalysis software).
Mascot v. 2.2.06 (www.matrixscience.com) was used as thesearch engine. Peptide tolerance was set to 50-100 ppm andresolution for all MS modes was m/z 1619.82 > 10 000. All
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612 Journal of Proteome Research • Vol. 9, No. 1, 2010
fragmentation spectra were searched against the MSDB andSwiss-Prot databases (7 078 992 and 510 076 entries, respec-tively).
Results and Discussion
In a previous work, we analyzed the N. meningitidis outermembrane vesicle proteome using a 2-D approach in whichnative BN-PAGE was used for separation in the first dimensionand denaturing SDS-PAGE in the second to investigate thepresence and subunit composition of putative complexes.12
Several complexes were detected after native separation in thefirst dimension, but resolution of their components after thesecond dimension run was highly impaired due to a stronglateral diffusion that limited the assignation of individual spots(proteins) to each complex. Consequently, in this work, we useda recently described high resolution native technique13 insteadof BN-PAGE for separation in the first dimension run. The outermembrane vesicles used for this study were obtained fromcultures done under iron restriction to induce the expressionof some important proteins that are regulated by this metal.The vesicles were obtained from the cells using a French Press
and ultracentrifugation to eliminate cytosolic proteins. Analysesdone in previous works to compare the composition of thesevesicles with those obtained by treatment with sodium deoxy-cholate (the method employed for preparation of OMV-basedmeningococcal vaccines) demonstrated that OMVs producedusing the French Press contain substantially less cytoplasmicproteins and are similar to native OMVs released duringgrowth.12,18-21
Detection of Complexes Using High Resolution ClearNative Electrophoresis. Analysis of the meningococcal OMVsusing hrCN allowed the detection of apparently different bandsdepending on the detergent (DDM or Triton X-100) used inthe cathode buffer for electrophoresis (Figure 1). Three bandsof 740, 540, and 470 kDa (D1-D3, and T1-T3) showed similarapparent weights independently of the detergent used. Sevenrelevant bands (D4-D10 and T4-T10) with lower molecularweights seemed to be different on the basis of their apparentmobility (between 140 and 220 kDa when using DDM, andbetween 230 and 470 kDa when using Triton X-100). In bothcases, a variable number of lower molecular weight bands werealso observed. These bands were cut and analyzed by LC/MS-
Figure 1. Diagonal hrCNE/hCNE analysis of meningococcal OMVs using DDM in the cathode buffer for native separation in the firstdimension, and Triton X-100 in the second one (left panel, top; DDM/TX) and vice versa (left panel, bottom; TX/DDM). Right panelshows the Gaussian representations of the main porin complexes, obtained by image analysis. Bottom image (B) was flipped horizontallyand then turned 90° clockwise before superposition (C) over upper image (A). White ellipses show the porin spots in the DDM/TXdiagonal gel, and gray ellipses indicate those found in the TX/DDM diagonal gel. Numbers correspond to complexes Cx1-Cx9 inTable 1. D1-D10 and T1-T10 designate the main complexes detected in the 1-D hrCNE analyses using DDM or Triton X-100, respectively.
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MS to determine the composition and identify the putativecomplex components. As shown in Table 1 the first three bandswere identified as homomeric protein complexes formed bythe 60 kDa chaperonin (MSP63), the glutamine synthetase andthe ketol-acid reductoisomerase, respectively (independentlyof the detergent used in the cathode buffer). The other sevenrelevant complexes mentioned above all contained meningo-coccal porins. The major outer membrane protein PIB (PorB)was present in all of them, most also contained the major outermembrane protein PorA, and the outer membrane protein class4 (RmpM) was detected in two of them.
To determine the possible equivalence between bandsD4-D10 and T4-T10, we analyzed the OMVs in a 2-D diagonalhrCN/ hrCN electrophoresis using DDM for separation in thefirst dimension and Triton X-100 in the second one, and viceversa. The results obtained (Figure 1) showed that some of thebands are formed by mixtures (or associations) of at least twocomplexes, resulting in nine different individual complexes.They were named Cx1-Cx9 and the identification of theircomponent proteins (by LC/MS-MS) is also shown in Table 1.As can be seen, all these complexes had different mobilitiesdepending on the sequence of detergents used for the diagonalhrCN/hCN diagonal electrophoresis analyses (they appearedeither above or below the diagonal in the gels).
Image analyses and the results of the LC/MS-MS analysesof these complexes (Figure 1, right side; Table 1) allowed todemonstrate that they are exactly the same despite the differ-ences in their mobility and, consequently, in their apparentmolecular weight. In the first dimension analysis, some of theseindividual complexes are coincident in only one band, depend-ing on the detergent used. For instance, when the hrCNseparation in the first dimension is done using DDM, com-plexes Cx2 and Cx4 appear in band D9, whereas Cx3 and Cx5appear in band D8. On the contrary, when Triton X-100 is used
for the first dimension hrCN separation, complex Cx2 appearswith Cx1 in band T10, and Cx4 appears with Cx5 in band T8.Identification of the components of complexes Cx1-Cx9 by LC/MS-MS showed that all them contain the PorB protein (Cx1and Cx2 being homomeric), seven contain also the PorA protein(Cx3-Cx9), and the RmpM protein is also present in two ofthem (Cx8 and Cx9).
Notwithstanding the LC/MS-MS results did not show thepresence of other proteins associated to these complexes, wethink that this possibility should not be discarded in view ofthe results obtained in the 2-D hrCN/SDS-PAGE analyses thatwill be commented below; furthermore, LC/MS-MS identifica-tions done in replicates of the 2-D gels using different gradientsand/or variations in the concentrations of the detergents inthe cathode buffers (results not shown) suggest the associationof other proteins to some of the porin complexes, such as thetransferrin-binding protein A or the Fe-regulated protein B. Thishas not been reflected in Table 1 due to the low significanceof the scores for the corresponding peptides in the LC/MS-MSanalyses.
Analysis of Complexes Using 2-D hrCN/SDS-PAGE Electro-phoresis. The 2-D electrophoretic analysis of OMVs usingnondenaturing hrCN in the first dimension and denaturingSDS-PAGE in the second allows the attainment of proteomicmaps in which, theoretically, the spots aligned verticallycorrespond to the individual proteins forming each complex.Figure 2 shows the proteomic maps obtained using either DDMor Triton X-100 in the cathode buffer for separation undernative conditions in the first dimension. Identification byMALDI-TOF fingerprinting analysis of the main spots observedis shown in Tables 2 and 3. Table 2 lists the spots identified asproteins whose predicted location is the outer membrane, theperiplasm, and outer membrane-associated proteins, or pro-teins that have been consistently found in OMV preparations
Table 1. LC/MS-MS Identification of the Components of the Main Complexes Revealed Using 2-D Diagonal hrCNE/hCNEElectrophoresisa
a Naming of the complexes corresponds to that used in Figure 1. Sequence coverages and database IDs are those corresponding to the homologousgenes in the N. meningitidis MC58 strain. Correspondence with the bands detected in 1-D hrCNE analyses is shown in columns 2 and 3.
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in other studies18-21 Table 3 lists the spots identified as proteinsthat are described to be cytoplasmic in public databases (Swiss-Prot).
We analyzed a total of 151 spots selected on the basis of theresults obtained from five replicates and on our previous resultsobtained in BN/SDS-PAGE analyses.12 Eighty of the 151 spotswere unequivocally identified using MALDI-TOF (45 from the2-D gels obtained using DDM and 35 from the gels obtainedusing Triton X-100). Because of the coincidence of several ofthe spots in the two gels and to the presence of multiple spots(trails) for some of the proteins, a total of 46 different proteinswere distinguished. Twenty-nine of them (marked in Figure 2and listed in Table 2) are those located or associated to theouter membrane. Only 17 of them were found in both gel types,
nine only in gels obtained with DDM and three only in thosewith Triton X-100.
Some proteins that have been reported to be associatedforming complexes could not be shown as such depending onthe detergent used for separation in native conditions, whichdemonstrates the importance of a thorough search for anappropriate selection of the detergents used in the cathodebuffer for the analysis of particular complexes. One exampleis that formed by the transferrin-binding proteins A and B(TbpA and TbpB), which were detectable as a complex whenusing DDM, but not (only the TbpA could be identified) whenusing Triton X-100. The only complexes that could be invariablydetected independently of the detergent used are those thatwe named CxChap, CxGlu, CxKetol, as well as all porin
Figure 2. Bidimensional hrCNE/SDS-PAGE analysis of OMVs using DDM (top; DDM/SDS) or Triton X-100 (bottom; TX/SDS) in the cathodebuffer for native separation in the first dimension. All spots identified by MALDI-TOF and recognized as proteins associated to theouter membrane are indicated with numbers and their identification is referenced in Table 2. Proteins appearing as multiple spots areboxed.
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complexes, despite the different mobility observed for the last,already commented above, and suggests that the assignationof proteins to complexes after 2-D electrophoresis, contrary tothe commonly accepted, must not be done only under theassumption that spots of the same shape and located in thesame vertical are components of a single complex. The exist-
ence of these complexes agrees with our previous resultsobtained using BN/SDS-PAGE12 and SDS/SDS diagonal elec-trophoresis.22
The different mobility of the porin complexes when usingDDM or Triton X-100 is difficult to explain. Results obtainedby other authors already demonstrated that different detergents
Table 2. MALDI-TOF Identification of the Main Spots Detected Using hrCNE/SDS-PAGE Electrophoresisa
a Numbers correspond to those maked in Figure 2. Only spots reported to be located or associated to membranes are listed. Sequence coverage,database IDs and molecular weights are those corresponding to the homologous genes/proteins in the N. meningitidis MC58 strain. b m/u,matched/unmatched. c Missing IDs indicate no annotation for a homologous gene in strain MC58 in public databases. IDs in parentheses indicatehomologous genes in strains different from MC58 and not described in that strain.
Table 3. MALDI-TOF Identification of Spots Detected Using hrCNE/SDS-PAGE Electrophoresis That Are Not Described to BeAssociated to the Meningococcal Outer Membranea
a Sequence coverage, database IDs and molecular weights are those corresponding to the homologous genes in the N. meningitidis MC58 strain. b m/u,matched/unmatched peptides. c ID in paranthesis indicate homologous genes in strains different from MC58.
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616 Journal of Proteome Research • Vol. 9, No. 1, 2010
can affect the mobility of individual pure proteins (andconsequently their apparent molecular weight) in native elec-trophoresis.13 Although a rational explanation was not givenfor this phenomenon, it is conceivable that the association ofa protein (or a complex) with different mixtures of detergentscould produce micelles with differing sizes and/or charges.
When compared with analyses done by other authors,18-21
we found less cytoplasmic proteins in our OMV preparations(less than 30% of the total spots identified), which confirmsour previous observation that OMVs obtained using the FrenchPress results in a lower presence of cytoplasmic proteins. It isnoteworthy that only two of the 17 cytoplasmic proteinsidentified were coincident in the 2-D analyses done using DDMand Triton X-100 for separation in the first dimension (Table3), which suggests that the choice of detergents for nativeelectrophoresis also produces different effects on the presenceof cytoplasmic proteins, and these effects are more pronouncedthan on membrane proteins.
Our results also show that some of the proteins, especiallythe porins, appear in multiple locations forming spot trails.Either they are forming complexes in which the stoichiometryof the subunits must be different, or are associated with otherproteins to build different complexes. In view of the evidencethat some bands in the 1-D native analyses contain more thanone complex, assignation of spots to a particular complex aftera single 2-D analyses cannot be done only on the basis of theirlocation in the same vertical and a similar shape of the spots,as already commented. In our study, this would lead to theerroneous conclusion that band D9 is a single PorA/PorBcomplex, whereas the diagonal analyses shown in Figure 1demonstrate that band D9 contains a homomeric (PorB) anda heteromeric (PorA/PorB) complexes.
The three spots consistently observed, with the same mobil-ity, when using either DDM or Triton X-100 for native separa-tion in the first dimension (CxChap, CxGlu and CxKetol) alsobehaved similarly in the 1-D hrCN (bands D1-D3 and T1-T3)and in the 2-D diagonal hrCN/hCN analyses, and also inanalyses done using OMVs obtained from the four knockoutmutants. Complex CxChap, according to its molecular weight(740 kDa), and similar to their orthologues in some otherbacteria, could be formed by multimers of the MSP63 proteinthat aggregate forming two superimposed heptameric rings.23
Although the quaternary structure of the chaperonin complexescan be different in other species such as Escherichia coli, inwhich the Skp chaperonin associates forming trimers,24,25 itsapparent molecular mass is very close to the theoretical mass(882 kDa) of a double heptameric ring structure. Its locationin the outer membrane is consistent with the need for a correctconformation of outer membrane proteins after transport andwith the fact that sera from individuals convalescent frommeningococcal meningitis contain anti-chaperonin antibod-ies.26
Orthologues of the complex CxGlu have been also analyzedin other bacterial species, finding that the glutamine synthetasemolecules associate forming two superimposed hexamericrings27 and are cotranscribed and/or associate with integralmembrane proteins implicated in the transport of nitrogenouscompounds;28 the molecular mass calculated for our CxGlucomplexes suggests that they could have a similar quaternarystructure. Orthologues of the CxKetol complex have been alsofound in other species, being formed by associations of sixdimers that produce complexes containing 12 ketol-acid re-ductoisomerase subunits; again, this structure agrees with the
molecular mass calculated in our experiments. Although theassociation of the CxGlu and CxKetol complexes with the outermembrane is not in agreement with their reported cytoplasmiclocation and function, we found them consistently in all 1-Dand 2-D analyses, independently of the detergent used forseparation in native conditions, and in OMVs from all thehomologous mutants analyzed, as can be seen in Figure 3.It is important to remark that these three complexes arepresent in relatively high amounts and that all our OMVpreparations showed a null NADH oxidase activity, whichsuggests a minimal contamination with cytoplasmic com-ponents or inner membrane.
Some other spots identified by MALDI-TOF in the 2-Danalyses (Table 2) and known to form complexes in N.meningitidis and other species are spot 15, which correspondsto the PilS cassette, implicated in cellular adhesion, and spot8 (copper-containing nitrite reductase), which has been de-scribed as an outer membrane protein that forms homotrimersand is implicated in the growth of bacteria in oxygen-deficientenvironments. Finally, among the cytoplasmic proteins identi-fied (Table 3), it has been reported that the polyribonucleotidephosphorylase forms homotrimers. All the above data aboutformation of complexes by the different proteins mentionedhas been obtained from the ExPASy Proteomics Server (Uni-ProtKB/Swiss-Prot databases) (www.expasy.ch). It is importantto have into account that information about quaternarystructures formed by many of the proteins or their subcellularlocation in the databases is reported ‘by similarity’. Forexample, for the meningococcal polyribonucleotide nucleoti-dyltransferase (Q9K062; PNP_NEIMB), the server reports ‘Ho-motrimer. Organized into a structure containing a number of
Figure 3. Bidimensional hrCNE/SDS-PAGE analysis of OMVs fromknockout mutants lacking some of the pore components (PorA,PorB or RmpM). Analyses in left panel were done using usingDDM in the cathode buffer for native separation in the firstdimension. Those in right panel were done using Triton X-100.Only the regions containing the pore components are shown.
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RNA-processing enzymes (by similarity)’ for its structure, and‘Cytoplasmic (by similarity)’ for its location.
Analysis of Complexes in OMVs from the HomologousKnockout Mutants. The analysis of OMVs from the homolo-gous mutants lacking either PorA, PorB, or RmpM is shown inFigure 3. As can be seen, the main alterations are produced inthe porin complexes. In the mutant lacking PorA, an increasedformation of homocomplexes containing PorB can be observed,and only one of the two heterocomplexes that contained theRmpM was detectable. In OMVs from the mutant lacking PorB,the effect on the porin complexes was much more marked: thePorA-containing complexes visible in the wild-type strainshowed a much lower intensity and, in turn, two high molecularmass PorA homocomplexes (>700 kDa) were formed. Thisbehavior was also observed in a previous study in which BNEwas used for the analyses, although only one high molecularmass PorA complex could be resolved in that work.12 In themutant lacking RmpM, the two RmpM-containing complexesdetected in the wild-type strain disappeared, and an incrementof the PorB homocomplexes and PorA/PorB heterocomplexeswas observed. Also, two new high molecular mass complexeswere detectable: a PorA/PorB heterocomplex and a PorBhomoclomplex that were much better detected when TritonX-100 is used for separation in native conditions in the firstdimension. Finally, in the mutant lacking FetA, the only effectobserved in the complexome maps was a loss of the Fe-regulated protein B, with no effect on the main complexes;apart from this, the complexome map was identical to that ofthe wild-type strain.
The results obtained in this work confirm our previoushypothesis that meningococcal porins associate in multiplecomplexes, both homomeric and heteromeric, and the par-ticipation of the RmpM protein in some of them.12,22 Also, ourresults agree with our previous observations that PorB has apredominant role in the correct formation of porin complexesin the outer membrane. We were not able in this work toconsistently demonstrate the association of other proteins tothe porins, so we cannot propose an explanation to theexistence of that variety of porin complexes, and more in-depthanalysis must be done in order to find an appropriate explana-tion for that.
Conclussions
In this work, we used 1-D hrCN native electrophoresis incombination with LC-MS/MS, and 2-D diagonal hrCN/hCNand hrCN/SDS-PAGE in combination with LC-MS/MS andMALDI-TOF analysis, to identify the main outer membranevesicle meningococcal complexes. The results obtained allowedthe analysis of the complexes with a resolution much higherthan that obtained in previous studies performed using BNEfor separation in native conditions. We have been able toconfirm that the most abundant outer membrane complexesare those formed by the main porins (PorA and PorB), whichconsistently show forming different associations. The relevanceof each porin in the formation of the different complexes couldbe deduced from the modifications observed in the analysisof outer membrane vesicles from knockout mutants. We thinkthat the hrCN technique performed using different detergentsand combined with other separation or analysis techniques cansignificantly contribute to the study of membrane complexes,not only in N. meningitidis, but also in other bacterial speciesand cell types.
Acknowledgment. We wish to acknowledge CICbioGUNE (Technology Park of Bizkaia, Derio, Spain;member of CIBER-ehd and Proteored) for the LC-MS/MSand MALDI-TOF analysis. This work was supported bygrants PGIDIT05PXIB20302PR from the Xunta de Galicia,and PI050178 from the Fondo de Investigacion Sanitaria(FIS, Ministerio de Sanidad y Consumo), Spain.
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