Interaction between Complement Subcomponent C1q and the Klebsiella pneumoniaePorin OmpK36

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Interaction between Complement Subcomponent C1q and the Klebsiella pneumoniaePorin OmpK36
    1996, 64(11):4719. Infect. Immun. F Vivanco and V J Benedí S Albertí, G Marqués, S Hernández-Allés, X Rubires, J M Tomás,  pneumoniae porin OmpK36.subcomponent C1q and the Klebsiella Interaction between complement information and services can be found at: These include:  CONTENT ALERTS  more»cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles Information about commercial reprint orders: To subscribe to to another ASM Journal go to:  onM ar  c h 2 1  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om  onM ar  c h 2 1  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om   I NFECTION AND  I MMUNITY , Nov. 1996, p. 4719–4725 Vol. 64, No. 110019-9567/96/$04.00  0Copyright  1996, American Society for Microbiology Interaction between Complement Subcomponent C1q and the  Klebsiella pneumoniae  Porin OmpK36 SEBASTIA ´N ALBERTI´, 1 GUILLERMO MARQUE´S, 2 SANTIAGO HERNA ´NDEZ-ALLE´S, 1 XAVIER RUBIRES, 3 JUAN M. TOMA ´S, 3 FERNANDO VIVANCO, 2  AND  VICENTE J. BENEDI´ 1 *  Area de Microbiologı´a, Departamento de Biologı´a Ambiental, Universidad de las Islas Baleares, E-07071-Palma de Mallorca, 1  Departamento de Inmunologı´a de la Fundacio´n Jime´nez Dı´az and Departamento de Bioquı´mica y  Biologı´a Molecular I de la Universidad Complutense, E-28040 Madrid, 2  and Departamento de Microbiologı´a,Universidad de Barcelona, E-08071 Barcelona, 3 Spain Received 3 April 1996/Returned for modification 27 May 1996/Accepted 5 August 1996 The interaction between C1q, a subcomponent of the complement classical pathway component C1, andOmpK36, a porin protein from  Klebsiella pneumoniae , was studied in a solid-phase direct-binding assay,inhibition assays with the purified globular and collagen-like regions of C1q, and cross-linking experiments. We have shown that the binding of C1q to the OmpK36 porin of the serum-sensitive strain  K. pneumoniae KT707 occurs in an in vivo situation and that this binding leads to activation of the complement classicalpathway and the subsequent deposition of complement components C3b and C5b-9 on the OmpK36 porin.Scatchard analysis of the binding of [ 125 I]C1q to the OmpK36 porin showed two binding sites with dissociationconstants of 1.5 and 75 nM. The decrease of [ 125 I]C1q binding to the OmpK36 porin in buffer with increasingsalt concentrations and the pIs of the C1q subcomponent (10.3) and OmpK36 porin (4.5) suggest that chargedamino acids are involved in the binding phenomenon. In inhibition assays, only the globular regions of C1qinhibited the interaction between C1q and OmpK36 porin, demonstrating that C1q binds to porin through itsglobular region and not through the collagen-like stalks. The complement system plays an important role in the de-fense against bacterial infections. Activation of this system byserum-sensitive bacteria produces two major effects to the mi-croorganism: deposition of opsonic proteins (C3b or iC3b)onto the microbial surface, and lysis of the sensitive microor-ganisms because of the formation of the membrane attackcomplex (C5b to C9 [C5b-9]). The proteins of this system aresequentially activated in the form of two enzyme-type cascadescalled the classical pathway (CP) and the alternative pathway.C1 is the first component of the CP and is composed of onemolecule of C1q and two molecules of each C1r and C1s, which circulate in the plasma as a calcium-dependent zymogencomplex (45).C1q is called the recognition protein of C1 because immu-noglobulin G (IgG)- and IgM-bearing immunocomplexes, theprototypic activators of the CP, bind C1 through C1q. C1q isformed by three polypeptide chains (A, B, and C) and presentstwo different regions: the collagen-like fragment and the glob-ular headpieces. The globular domain contains the bindingsites for the IgG (14) and IgM immunocomplexes, and inter-action between C1q and these immunocomplexes results inactivation of the CP (45). A large variety of substances interactdirectly with C1q independently of antibodies. RNA tumor viruses (12), C-reactive protein (26), fibronectin (47), and lami-nin (8) are but some examples. Only some of the C1q-bindingsubstances are able to activate the CP after C1q binding, whereas binding of C1q to other substances does not lead toCP activation.Lipopolysaccharide (LPS) and outer membrane proteins(OMP) of the porin class (porins) from some enterobacteriaare examples of molecules that bind C1q and activate the CP(5, 10, 18). Porins are more efficient activators of the CP thanis the rough LPS of   Klebsiella pneumoniae  (1). This activationis an effective mechanism to eliminate  K. pneumoniae  strainssensitive to complement-mediated lysis (1) and may representa relevant mechanism of defense against this kind of pathogenin certain individuals because (i)  K. pneumoniae  is an impor-tant pathogen for people with impaired immunological re-sponses and (ii) C1q binding and CP activation by  K. pneu- moniae  porins is antibody independent, i.e., does not requirepreformed specific antibodies.Interactions between C1q and porins in enterobacterial spe-cies other than  K. pneumoniae  have also been reported, butdirect in vivo demonstration of C1q binding to porins, i.e.,binding of C1q to bacterial outer membrane-bound porinsduring CP activation, has not been demonstrated so far. Also,many characteristics of the C1q-binding process remain un-known. For these reasons and because of the possible impor-tant implications of CP activation in the defense against  Kleb- siella  infections, we extend here our previous work by studyingthe influence of different physicochemical conditions and theregions involved in the C1q-porin interaction and by demon-strating that this binding occurs in vivo. MATERIALS AND METHODSStrains, media, and serum.  K. pneumoniae  C3 (serotype O1:K66), a serum-resistant strain, and its isogenic serum-sensitive mutant strain KT707 (serotypeO  :K66) have been described previously (6). Strain KT707 presents only oneporin, OmpK36, in its outer membrane (1). Bacteria were grown in Luria-Bertanibroth (46) at 37  C.Normal nonimmune human sera (NHS) were obtained from healthy volun-teers, pooled, aliquoted, and frozen at   70  C until its use.  Antisera.  Polyclonal antisera against C1q and OmpK36 porin were raised inNew Zealand White rabbits by immunization with 50 to 100   g of purifiedproteins in complete Freund’s adjuvant (Difco). The rabbits were given intra-muscular injections once a week for a 6-week period and bled 10 to 14 days afterthe last injection. Monoclonal antibodies specific for complement componentsC3b and C5b-9 were obtained from Serva (Heidelberg, Germany). Isolation of the OmpK36 porin.  Bacterial cell envelopes from  K. pneumoniae KT707 were obtained by French press cell lysis and centrifugation. They were * Corresponding author. Mailing address: UIB-Microbiologı´a,Crtra. de Valldemosa Km. 7,5, E-07071 Palma de Mallorca, Spain.Phone: (34-71)-173335. Fax: (34-71)-173184. Electronic mail   onM ar  c h 2 1  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om   subjected to a standard method for the isolation of bacterial porin proteins (39)as previously described (1). Briefly, 2% sodium dodecyl sulfate (SDS) was addedto the cell envelopes, and they were incubated at 32  C for 1 h. Insoluble material was pelleted by centrifugation at 100,000   g   for 1 h, treated again with 2% SDS,and pelleted again. After solubilization in 50 mM Tris-HCl (pH 7.7) containing1% SDS, 0.4 M NaCl, 5 mM EDTA, and 0.05%   -mercaptoethanol, proteins were separated from LPS by Sephacryl S-200 chromatography equilibrated in thesolubilization buffer. Chromatography was carried out in a 1.5- by 90-cm column,and 2-ml fractions were monitored at 280 nm and recovered. Fractions contain-ing OmpK36 porin, as assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; see below), were pooled and dialyzed against 3 mM sodium azide, firstfor 1 day at room temperature and then for 7 to 9 days at 4  C. They were finallytreated with phenol to remove LPS contamination (21). The LPS content of theisolated OmpK36 porin was assessed by electrophoresis in polyacrylamide gels(see below) and silver staining (21, 50, 51) and by the 2-keto-3-deoxyoctonate(KDO) assay (27) with purified  Escherichia coli  O55:B5 LPS (Sigma) as a stan-dard. The protein content was determined by the method of Lowry et al. (34) with bovine serum albumin (BSA) as a standard. C1q purification and labelling.  C1q was isolated from a pool of NHS. A combination of two previously described methods was used. First, NHS wasdialyzed against 5 mM 1,3-diaminopropane (pH 8.8) (30). Precipitated material was fractionated by ion-exchange chromatography with Bio-Rex70 (200 to 400mesh; Bio-Rad) and gel filtration on Bio-Gel A-5M (100 to 200 mesh; Bio-Rad)(48). C1q was repurified by ion-exchange chromatography on a MonoS HR5/5column (Pharmacia) equilibrated with 20 mM Tris-HCl–100 mM NaCl (pH 7.35)(35). The isolated C1q was iodinated with lactoperoxidase-glucose oxidase asdescribed previously (48). Purified and labeled C1q was assayed by publishedmethods (4, 48) and shown to be hemolytically active and able to interact withimmunocomplexes (35). Isolation of C1q fragments.  The collagen-like fragment was isolated as previ-ously described (3) by the method described by Reid (44). Briefly, 10 mg of purified C1q in 0.1 M sodium acetate (pH 4.46) was digested for 20 h at 37  C with0.3 mg of pepsin (Sigma, St. Louis, Mo.). The reaction was stopped by pHtitration to 8.0 with 1 M Tris-HCl (pH 8). Aggregates were cleared by centrifu-gation, and the supernatant fluid was chromatographed on a Sephacryl S-200 HRcolumn equilibrated with 50 mM Tris-HCl (pH 7.2) containing 200 mM NaCl.Fractions were monitored at 280 nm and analyzed by SDS-PAGE.Globular regions of C1q were isolated by the method of Paques et al. (41).Briefly, 5 mg of C1q was dialyzed overnight at 4  C against 0.1 M Tris-HCl (pH7.4) containing 0.2 M NaCl and 20 mM CaCl 2 . After centrifugation to removeaggregates and incubation at 54  C for 10 min, 0.3 mg of collagenase type III(Sigma) was added to the solution and the reaction mixture was removed fromthe water bath. Aggregates were cleared by centrifugation, and the solution wasdialyzed overnight at 4  C against 1 M sodium citrate (pH 7.4) containing 0.2 MNaCl. The precipitated protein was collected by centrifugation, washed, andresuspended in 0.1 M Tris-HCl (pH 7.4), containing 0.2 M NaCl. After dialysisagainst the same buffer, globular domains were analyzed by SDS-PAGE. Solid-phase binding assays.  Microtiter plate wells (Polysorb; Nunc, Roskilde,Denmark) were incubated overnight at 4  C with 100   l of the OmpK36 porindiluted in phosphate-buffered saline (PBS) at 100   g/ml. The binding of theOmpK36 porin to the microtiter wells was checked with [ 125 I]OmpK36 porinunder the conditions described above. Only 0.5  g of the protein was fixed underthese conditions (data not shown). Unbound OmpK36 porin was removed by washing three times with PBS. To block residual binding sites in the wells, plates were incubated with PBS–1% BSA for 4 h at 37  C. To determine the bindingaffinity of C1q for solid-phase OmpK36 porin, the wells were washed three times with Veronal-buffered saline diluted 1:1 with distilled water (VBS 2   /H 2 O). Ra-diolabeled C1q (1% [wt/wt] of unlabeled C1q) and increasing amounts of unla-beled C1q were diluted in VBS 2   /H 2 O containing 1% BSA (final volume, 100  l) and incubated for 1 h at 37  C. After the plates were washed six times, theradioactivity of the wells was counted. Background binding to wells coated only with BSA was also determined for all C1q concentrations used. Analyses of saturation isotherms (  K   d , dissociation constant;  B max  , maximum density of bind-ing sites) and the fittings of data to the appropriate binding model were per-formed by computer-assisted nonlinear analysis from untransformed data byusing the EBDA-LIGAND programs (36, 38). All experiments were initiallyanalyzed by assuming a one-site binding model and then assuming a two-sitebinding model. The selection between different models was made statistically byusing the extra-sum-of-square principle (  F   test) (38). The more complex model was accepted if the  P   value resulting from the  F   test was less than 0.05.In the studies of the binding of C1q to the OmpK36 porin as a function of theNaCl concentration, all steps were done as described above, except that a con-stant amount of radiolabeled C1q (5   g) diluted in VBS 2   /H 2 O containingdifferent concentrations of NaCl were used in the binding step.In the inhibition assays, a constant concentration of [ 125 I]C1q (0.05 nM) wasdiluted in VBS 2   /H 2 O containing different amounts of the inhibitor (collagenfragment or globular fragment).Finally, experiments were also performed to determine the binding of C1q,C3b, and C5b-9 to the OmpK36 porin at different times of incubation. In theseexperiments, plates were coated and blocked as described above. They wereincubated at 37  C with NHS for different times and washed, and a polyclonalantiserum against C1q, a monoclonal anti-C3b antibody, or a monoclonal anti-C5b-9 antibody, appropriately diluted in PBS–1% BSA, was used to detect thebinding of these complement components to OmpK36-coated plate wells. Plates were subsequently incubated with alkaline phosphatase-labeled anti-IgG, devel-oped with  p -nitrophenyl phosphate, and read at 405 nm. Antiserum incubations were carried out at 37  C, and washing steps with PBS were included betweenincubations. Control wells without NHS were run simultaneously. All binding-assay points were determined in triplicate, and experiments were performed atleast three times. SDS-PAGE.  SDS-PAGE was used to analyze C1q and its fragments, OMP,porins, and LPS and to demonstrate interaction between C1q and its bacterialsurface receptor(s). The Tris-glycine-SDS Laemmli’s electrode and sample buff-ers were used, and samples were incubated at 100  C for 5 min before analysis.For the separation of C1q and its fragments, OMP, and porins, SDS-PAGE wasperformed in 11% acrylamide–0.5% bisacrylamide–0.1% SDS gels. C1q-receptorcomplexes were analyzed in 10% acrylamide–0.3% bisacrylamide–0.1% SDS.Electrophoretic analysis of the LPS content of purified OmpK36 porin wascarried out in 15% acrylamide–0.4% bisacrylamide–0.1% SDS gels.Two-dimensional gel electrophoretic analysis of the OMP of   K. pneumoniae KT707 was performed by using the O’Farrell method and buffers (40), exceptthat an SDS-containing sample buffer (15) was used to load the first-dimensiongels. First-dimension gels contained 4% acrylamide, 8 M urea, 4.5% ampholyte4/6 (Sigma) and 0.5% ampholyte 3.5/10. Gels were casted in the Mighty SmallHoefer’s tube format and were run for 12 to 14 h at 400 V followed by 2 h at 800V. Distribution of pH in the electrophoresed tube gels was measured at roomtemperature as described by O’Farrell (40) or with the two-dimensional PAGEstandards (Bio-Rad, Madrid, Spain). After equilibration in 62.5 mM Tris-HCl(pH 6.8) containing 3% SDS and 5.5%   -mercaptoethanol for 20 min, theelectrophoresed tube gels were loaded onto 11% acrylamide gels and electro-phoresed as explained above for OMP. The gels were stained with Coomassieblue. Cross-linking assays.  C1q-SASD was prepared by incubating 200   g of puri-fied C1q with sulfosuccinimidyl-2-(  p -azidosalicylamido)-1,3  -dithiopropionate(SASD; Pierce, Rockford, Ill.), both dissolved in 0.1 M sodium phosphate buffer(pH 9.0) at a molar ratio of 50:1, as suggested by the manufacturer. Incubation was done in the dark for 30 min at room temperature. Excess SASD was removedby ultrafiltration through 10-kDa-pore-size Ultrafree-MC filters (Millipore, Bed-ford, Mass.). A final solution of C1q-SASD at 1 mg/ml in PBS was obtained andstored at   70  C until its use.The bacterial cell surface receptor(s) for C1q was studied by incubation of bacterial cells with C1q-SASD, cross-linking, and analysis of C1q-SASD-recep-tors complexes by SDS-PAGE and Western blotting (immunoblotting) withanti-OmpK36 and anti-C1q. Briefly, 4    10 9 cells of strain KT707 were washedand suspended in 100   l of distilled water. The C1q-SASD solution prepared asdescribed above (100   l) was added to the bacterial suspension, and the mixture was incubated for 30 min at 37  C in the dark, followed by a 45-s irradiation withlong-wavelength UV light at room temperature. After three washes with 0.1 MTris-HCl (pH 10.0) containing 0.7 M NaCl to remove noncovalently bound C1q,bacterial OMP were isolated as described previously (17). Briefly, bacteriallysates were obtained by sonication and cell envelopes were collected by centrif-ugation at 13,000    g   for 30 min at 4  C. OMP were pelleted by insolubilizationin 0.4%  N  -lauroyl sarcosinate. Control samples were identically treated, but C1q was used without SASD. After solubilization of the OMP for 5 min at 100  C insample buffer, the proteins were separated in a 10% polyacrylamide gel andelectrophoretically transferred to nitrocellulose membranes at 1 A for 1 h (49).The nonspecific binding sites were blocked by overnight incubation with PBScontaining 1% BSA. Nitrocellulose filters were then incubated in a 1:1,000dilution of rabbit anti-OmpK36 or rabbit anti-human C1q antiserum for 1 h at37  C. After three washes with PBS and incubation with alkaline phosphatase-conjugated goat anti-rabbit IgG (1:3,000 dilution), blots were developed with5-bromo-4-chloro-3-indolylphosphate–nitroblue tetrazolium (7). RESULTS  K. pneumoniae  KT707 is a mutant obtained from  K. pneu- moniae  C3 which has only OmpK36 as porin in its outer mem-brane, and thus it is a suitable starting strain for the isolationof OmpK36. OmpK36 was purified, giving a single 36-kDaband in polyacrylamide gels (Fig. 1). Densitometric scanning of SDS-PAGE lanes containing purified OmpK36 porin showedthat it was more than 90% pure. Moreover, LPS contaminationof purified OmpK36 porin was not detected by SDS-PAGEand silver staining (data not shown). The KDO assay indicatedthat the protein/LPS ratio in the purified OmpK36 porin prep-arations was routinely in the order of 5    10 5 :1 (by weight).Isolation of complement component C1q and its collagen andglobular moieties is also shown in Fig. 1. Purified C1q incu-bated at 100  C for 5 min in SDS sample buffer containing 4720 ALBERTI´ ET AL. I NFECT . I MMUN .   onM ar  c h 2 1  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om    -mercaptoethanol separates into three distinct bands of about27 to 32 kDa.The binding of C1q to OmpK36 porin was dependent on theNaCl concentration used in the assay buffer (Fig. 2). An incre-ment of the ionic strength in the assay drastically reduced thebinding of C1q to OmpK36 porin. At 1 M NaCl, the binding was only 4% of that observed at 0.01 M NaCl, which is theoptimum condition assayed. However, for all the NaCl con-centrations studied, the binding was greater than in the con-trols.The theoretical pI of the OmpK36 porin (pI 4.3) was calcu-lated from the amino acid-derived sequence of the  ompK36 gene (GenBank/EMBL accession number Z33506 [2]) by usingthe Isoelectric program of the Wisconsin Genetics ComputerGroup software package. The isoelectric point of the OMPfrom  K. pneumoniae  KT707 was also determined by two-di-mensional electrophoresis, giving a pI of approximately 4.5 forthe OmpK36 porin (data not shown); i.e., it is the most acidicprotein seen in the outer membrane of   K. pneumoniae . There-fore, the results shown in Fig. 2 and the known pI of C1q (pI10.3 [31]) suggest that the binding of C1q to the OmpK36 porinis of ionic nature or that an ionic component is involved in theinteraction.To investigate the region of C1q involved in the binding toOmpK36 porin, the globular and collagen-like fragments wereprepared by C1q digestion with collagenase and pepsin, re-spectively. The fragments generated were pure, as shown inFig. 1, and were used in inhibition assays. In these assays, thebinding of C1q to the OmpK36 porin was inhibited by all theconcentrations of the globular fragment used, inhibiting morethan 80% of the binding of [ 125 I]C1q to the OmpK36 porin ata concentration of 0.6 nM (Fig. 3). No significant inhibitioncould be observed with the collagen-like fragment by using thesame molar concentrations tested with the globular fragment.These results demonstrate that the interaction between C1qand OmpK36 porin is by the globular region of C1q and not byits collagen-like fragment.The binding of complement components C1q, C3b, andC5b-9 to the OmpK36 porin was determined by enzyme-linkedimmunosorbent assay (ELISA) experiments with OmpK36-coated microplates. NHS was used instead of pure compo-nents, and bound C1q, C3b, and C5b-9 were detected by spe-cific antibodies. The equilibrium for the binding of C1q to theOmpK36 porin was reached in 15 min. Increasing the incuba-tion time to 60 min did not significantly increase the amount of bound C1q (Fig. 4). These results ensure that other bindingassays carried out in this work with radiolabeled C1q weredone under equilibrium conditions, since 1 h was the incuba-tion time in those experiments. The binding of C3b was max-imal at 15 min of incubation (Fig. 4). As for C1q binding, thedeposition of C3b could be detected immediately after addi-tion of NHS to OmpK36-coated surfaces. In both cases, morethan 50% of the binding occurred in the first 10 min, but in thecase of C3b we did not observe saturation. In a short time,approximately 20 min, the levels of bound C3b dropped dras- FIG. 1. SDS-PAGE and Coomassie blue staining of the OMP from  K. pneu- moniae  C3 or KT793 (lane 1), the OMP from strain KT707 (lane 2), purifiedOmpK36 porin from strain KT707 (lane 3), purified C1q (lane 4), a purifiedglobular fragment of C1q generated by collagenase treatment (lane 5), and apurified collagen-like fragment generated by limited pepsin digestion of C1q(lane 6). Molecular mass markers (in kilodaltons) are indicated on the left.Samples were run reduced.FIG. 2. Binding of [ 125 I]C1q to the OmpK36 porin as a function of ionicstrength. Wells of ELISA plates coated with OmpK36 porin were incubated withradiolabeled C1q in a buffer of variable ionic strength. Ionic strength variationsin the assay were obtained by increasing the NaCl concentration in the bufferfrom 0.01 to 1 M. After 60 min of incubation at 37  C, the amount of bound[ 125 I]C1q was measured. Background binding to albumin-coated wells has beensubtracted from the plotted values. Results are the means of triplicate determi-nations    standard deviations for three independent experiments.FIG. 3. Inhibition of [ 125 I]C1q binding to OmpK36 porin by C1q fragments.Different amounts of the collagen-like fragment (open boxes) or globular frag-ment (solid boxes) of C1q, together with a fixed amount of radiolabeled C1q(0.05 nM), were added to ELISA plate wells coated with OmpK36 porin. Con-trols were incubated under the same conditions, but no inhibitors (C1q frag-ments) were added. After 1 h of incubation at 37  C, the amount of bound[ 125 I]C1q was measured. The results are expressed as the percent inhibition of the binding of C1q to OmpK36 porin-coated plates. Results are the means of triplicate determinations    standard deviations for three independent experi-ments. V OL  . 64, 1996 C1q-PORIN INTERACTIONS 4721   onM ar  c h 2 1  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om   tically, most probably as a result of degradation of this com-ponent into other fragments (C3dg, C3d, etc.) that were notdetectable by the monoclonal anti-C3b antibody used. Finally,binding of the C5b-9 complex was detected later than wasbinding of other complement components studied (Fig. 4). Thedeposition of the C5b-9 complex was gradual, and saturation was not observed in incubations extending up to 1 h. Alto-gether, the patterns of deposition of C1q, C3b, and C5b-9 areas expected from an in vivo complement activation: C1q bindsfirst and is followed by C3b and C5b-9 deposition. Also, thisdemonstrates that OmpK36 porin alone is able to trigger andcomplete the activation of the complement cascade.The results of binding of radiolabeled C1q to the OmpK36porin by using increasing amounts of unlabeled C1q are shownin Fig. 5. This binding was a concentration-dependent satura-ble process, suggesting the presence of a discrete number of C1q-binding sites in the OmpK36 porin. The affinity of C1q forthe OmpK36 porin was calculated by Scatchard analysis of thebinding data. The Scatchard plot obtained is shown as an insertin Fig. 5 and suggests that there are two binding sites with  K   d of 1.5 and 75 nM. Analysis of the binding of C1q to the porinhigh- and low-affinity sites revealed that approximately 80% of the C1q molecules bind to the low-affinity site. Taking intoaccount that the C1q-OmpK36 porin interaction is mediatedby the globular heads of C1q, the high-affinity constant (1.5nM) would correspond to the globular head-OmpK36 porininteraction. The low-affinity interactions may also be due tobinding of the globular heads, especially since this fragmentinhibited almost all the sites measured in Fig. 2.Having characterized the binding process and demonstratedthat this binding leads to complete activation of the comple-ment cascade, we were interested in studying if those phenom-ena occurred in vivo. In vivo binding of C1q and characteriza-tion of the bacterial C1q-binding molecules were studied byincubation of bacteria with C1q labeled with the SASD cross-linker (Fig. 6). As shown in Fig. 6A, lane 3, after incubation of bacterial cells with C1q-SASD and UV activation of the cross-linker, we detected by Western blot experiments with anti-OmpK36 serum two bands of 88 and 84 kDa plus the 36-kDaband corresponding to the OmpK36 porin monomer. Figure6A, lane 1, provides proof of the specificity of the assay: C1q-SASD alone was not detected by the anti-OmpK36 antiserum. Also, in lane 2, OMP from bacteria incubated with C1q-SASDbut without the UV activation (cross-linking) step were ana-lyzed; anti-OmpK36 antiserum detected just OmpK36 porin,but no other bands were visualized. These results are consis-tent with the conclusions that (i) high-molecular-mass (88 and84 kDa) complexes are formed and maintained throughout theanalysis only after covalent bonds are induced by UV activa-tion of the cross-linker (as in lane 3) and (ii) C1q-SASD-OmpK36 porin complexes are formed.Further confirmation of the above results was obtained induplicate experiments with anti-C1q antiserum (Fig. 6B). Re-sults from lane 4 disprove the hypothesis that complexes (the88- and 84-kDa bands of Fig. 6A, lane 3) could be a result of intramolecular C1q cross-linking: C1q-SASD alone after UVactivation of the cross-linker, at the concentration used, doesnot show any band in addition to those expected for purifiedC1q run reduced. Lane 5 shows again that no C1q-porin com- FIG. 4. Binding of complement components C1q, C3b, and C5b-9 to theOmpK36 porin as a function of time. Wells coated with OmpK36 were incubated with NHS. At different times, the wells were washed and bound C1q (opencircles), C3b (solid circles), and C5b-9 (squares) were detected with specificantibodies in ELISA experiments. Control wells were coated with OmpK36 porinbut without the NHS incubation step, and their final ELISA values were sub-tracted from experimental points to give the plotted values. Results are themeans of triplicate determinations    standard deviations for three independentexperiments.FIG. 5. Direct binding curve and Scatchard plot of [ 125 I]C1q binding toELISA plate wells coated with OmpK36 porin (0.5   g per well). Binding of [ 125 I]C1q to OmpK36 porin in the presence of increasing amounts of unlabeledC1q is indicated by solid circles. Results are the mean of at least three indepen-dent experiments. Background binding was subtracted before plotting. The Scat-chard plot of the C1q-OmpK36 binding (open circles) and two  K   d  of 1.5 and 75nM are shown in the insert.FIG. 6. Western blot analysis with specific antibodies against OmpK36 porin(A) and complement C1q (B) of the complexes formed between the  K. pneu- moniae  OMP and C1q-SASD. Lanes in both panels contain UV-activated C1q-SASD (lanes 1 and 4), isolated OMP from strain KT707 previously incubated with C1q-SASD without UV irradiation (lanes 2 and 5), and isolated OMP fromstrain KT707 after incubation with C1q-SASD and irradiation with UV (lanes 3and 6). Samples from lanes 1, 2, 4, and 5 were run reduced, and samples fromlanes 3 and 6 were run unreduced. Arrows indicate the putative C1q-SASD-OmpK36 complexes, while OmpK36 porin alone is labeled with white asterisks.Molecular mass markers are shown in kilodaltons between the two panels. 4722 ALBERTI´ ET AL. I NFECT . I MMUN .   onM ar  c h 2 1  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om 
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