Interaction between Complement Subcomponent C1q and the ...

INFECTION AND IMMUNITY, Nov. 1996, p. 4719–4725 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 64, No. 11

Interaction between Complement Subcomponent C1q and the Klebsiella pneumoniae Porin OmpK36 ´ N ALBERTI´,1 GUILLERMO MARQUE ´ S,2 SANTIAGO HERNA ´ NDEZ-ALLE ´ S,1 SEBASTIA ´ S,3 FERNANDO VIVANCO,2 AND VICENTE J. BENEDI´1* XAVIER RUBIRES,3 JUAN M. TOMA Area de Microbiologı´a, Departamento de Biologı´a Ambiental, Universidad de las Islas Baleares, E-07071-Palma de ´n Jime´nez Dı´az and Departamento de Bioquı´mica y Mallorca,1 Departamento de Inmunologı´a de la Fundacio 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, and OmpK36, 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 classical pathway and the subsequent deposition of complement components C3b and C5b-9 on the OmpK36 porin. Scatchard analysis of the binding of [125I]C1q to the OmpK36 porin showed two binding sites with dissociation constants of 1.5 and 75 nM. The decrease of [125I]C1q binding to the OmpK36 porin in buffer with increasing salt concentrations and the pIs of the C1q subcomponent (10.3) and OmpK36 porin (4.5) suggest that charged amino acids are involved in the binding phenomenon. In inhibition assays, only the globular regions of C1q inhibited the interaction between C1q and OmpK36 porin, demonstrating that C1q binds to porin through its globular region and not through the collagen-like stalks. (5, 10, 18). Porins are more efficient activators of the CP than is the rough LPS of Klebsiella pneumoniae (1). This activation is an effective mechanism to eliminate K. pneumoniae strains sensitive to complement-mediated lysis (1) and may represent a relevant mechanism of defense against this kind of pathogen in certain individuals because (i) K. pneumoniae is an important pathogen for people with impaired immunological responses and (ii) C1q binding and CP activation by K. pneumoniae porins is antibody independent, i.e., does not require preformed specific antibodies. Interactions between C1q and porins in enterobacterial species other than K. pneumoniae have also been reported, but direct in vivo demonstration of C1q binding to porins, i.e., binding of C1q to bacterial outer membrane-bound porins during CP activation, has not been demonstrated so far. Also, many characteristics of the C1q-binding process remain unknown. For these reasons and because of the possible important implications of CP activation in the defense against Klebsiella infections, we extend here our previous work by studying the influence of different physicochemical conditions and the regions involved in the C1q-porin interaction and by demonstrating that this binding occurs in vivo.

The complement system plays an important role in the defense against bacterial infections. Activation of this system by serum-sensitive bacteria produces two major effects to the microorganism: deposition of opsonic proteins (C3b or iC3b) onto the microbial surface, and lysis of the sensitive microorganisms because of the formation of the membrane attack complex (C5b to C9 [C5b-9]). The proteins of this system are sequentially activated in the form of two enzyme-type cascades called the classical pathway (CP) and the alternative pathway. C1 is the first component of the CP and is composed of one molecule of C1q and two molecules of each C1r and C1s, which circulate in the plasma as a calcium-dependent zymogen complex (45). C1q is called the recognition protein of C1 because immunoglobulin G (IgG)- and IgM-bearing immunocomplexes, the prototypic activators of the CP, bind C1 through C1q. C1q is formed by three polypeptide chains (A, B, and C) and presents two different regions: the collagen-like fragment and the globular headpieces. The globular domain contains the binding sites for the IgG (14) and IgM immunocomplexes, and interaction between C1q and these immunocomplexes results in activation of the CP (45). A large variety of substances interact directly with C1q independently of antibodies. RNA tumor viruses (12), C-reactive protein (26), fibronectin (47), and laminin (8) are but some examples. Only some of the C1q-binding substances are able to activate the CP after C1q binding, whereas binding of C1q to other substances does not lead to CP activation. Lipopolysaccharide (LPS) and outer membrane proteins (OMP) of the porin class (porins) from some enterobacteria are examples of molecules that bind C1q and activate the CP

MATERIALS AND METHODS Strains, media, and serum. K. pneumoniae C3 (serotype O1:K66), a serumresistant strain, and its isogenic serum-sensitive mutant strain KT707 (serotype 2 O :K66) have been described previously (6). Strain KT707 presents only one porin, OmpK36, in its outer membrane (1). Bacteria were grown in Luria-Bertani broth (46) at 378C. Normal nonimmune human sera (NHS) were obtained from healthy volunteers, pooled, aliquoted, and frozen at 2708C until its use. Antisera. Polyclonal antisera against C1q and OmpK36 porin were raised in New Zealand White rabbits by immunization with 50 to 100 mg of purified proteins in complete Freund’s adjuvant (Difco). The rabbits were given intramuscular injections once a week for a 6-week period and bled 10 to 14 days after the last injection. Monoclonal antibodies specific for complement components C3b 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 address: [email protected]. 4719

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subjected to a standard method for the isolation of bacterial porin proteins (39) as previously described (1). Briefly, 2% sodium dodecyl sulfate (SDS) was added to the cell envelopes, and they were incubated at 328C for 1 h. Insoluble material was pelleted by centrifugation at 100,000 3 g for 1 h, treated again with 2% SDS, and pelleted again. After solubilization in 50 mM Tris-HCl (pH 7.7) containing 1% SDS, 0.4 M NaCl, 5 mM EDTA, and 0.05% b-mercaptoethanol, proteins were separated from LPS by Sephacryl S-200 chromatography equilibrated in the solubilization 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 containing OmpK36 porin, as assessed by SDS-polyacrylamide gel electrophoresis (SDSPAGE; see below), were pooled and dialyzed against 3 mM sodium azide, first for 1 day at room temperature and then for 7 to 9 days at 48C. They were finally treated with phenol to remove LPS contamination (21). The LPS content of the isolated 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 standard. 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 was dialyzed against 5 mM 1,3-diaminopropane (pH 8.8) (30). Precipitated material was fractionated by ion-exchange chromatography with Bio-Rex70 (200 to 400 mesh; 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 HR5/5 column (Pharmacia) equilibrated with 20 mM Tris-HCl–100 mM NaCl (pH 7.35) (35). The isolated C1q was iodinated with lactoperoxidase-glucose oxidase as described previously (48). Purified and labeled C1q was assayed by published methods (4, 48) and shown to be hemolytically active and able to interact with immunocomplexes (35). Isolation of C1q fragments. The collagen-like fragment was isolated as previously 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 378C with 0.3 mg of pepsin (Sigma, St. Louis, Mo.). The reaction was stopped by pH titration to 8.0 with 1 M Tris-HCl (pH 8). Aggregates were cleared by centrifugation, and the supernatant fluid was chromatographed on a Sephacryl S-200 HR column 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 48C against 0.1 M Tris-HCl (pH 7.4) containing 0.2 M NaCl and 20 mM CaCl2. After centrifugation to remove aggregates and incubation at 548C for 10 min, 0.3 mg of collagenase type III (Sigma) was added to the solution and the reaction mixture was removed from the water bath. Aggregates were cleared by centrifugation, and the solution was dialyzed overnight at 48C against 1 M sodium citrate (pH 7.4) containing 0.2 M NaCl. The precipitated protein was collected by centrifugation, washed, and resuspended in 0.1 M Tris-HCl (pH 7.4), containing 0.2 M NaCl. After dialysis against 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 48C with 100 ml of the OmpK36 porin diluted in phosphate-buffered saline (PBS) at 100 mg/ml. The binding of the OmpK36 porin to the microtiter wells was checked with [125I]OmpK36 porin under the conditions described above. Only 0.5 mg of the protein was fixed under these 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 378C. To determine the binding affinity of C1q for solid-phase OmpK36 porin, the wells were washed three times with Veronal-buffered saline diluted 1:1 with distilled water (VBS21/H2O). Radiolabeled C1q (1% [wt/wt] of unlabeled C1q) and increasing amounts of unlabeled C1q were diluted in VBS21/H2O containing 1% BSA (final volume, 100 ml) and incubated for 1 h at 378C. After the plates were washed six times, the radioactivity 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 (Kd, dissociation constant; Bmax, maximum density of binding sites) and the fittings of data to the appropriate binding model were performed by computer-assisted nonlinear analysis from untransformed data by using the EBDA-LIGAND programs (36, 38). All experiments were initially analyzed by assuming a one-site binding model and then assuming a two-site binding model. The selection between different models was made statistically by using 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 the NaCl concentration, all steps were done as described above, except that a constant amount of radiolabeled C1q (5 mg) diluted in VBS21/H2O containing different concentrations of NaCl were used in the binding step. In the inhibition assays, a constant concentration of [125I]C1q (0.05 nM) was diluted in VBS21/H2O containing different amounts of the inhibitor (collagen fragment 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 these experiments, plates were coated and blocked as described above. They were incubated at 378C with NHS for different times and washed, and a polyclonal antiserum against C1q, a monoclonal anti-C3b antibody, or a monoclonal anti-

INFECT. IMMUN. C5b-9 antibody, appropriately diluted in PBS–1% BSA, was used to detect the binding of these complement components to OmpK36-coated plate wells. Plates were subsequently incubated with alkaline phosphatase-labeled anti-IgG, developed with p-nitrophenyl phosphate, and read at 405 nm. Antiserum incubations were carried out at 378C, and washing steps with PBS were included between incubations. Control wells without NHS were run simultaneously. All bindingassay points were determined in triplicate, and experiments were performed at least 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 bacterial surface receptor(s). The Tris-glycine-SDS Laemmli’s electrode and sample buffers were used, and samples were incubated at 1008C for 5 min before analysis. For the separation of C1q and its fragments, OMP, and porins, SDS-PAGE was performed in 11% acrylamide–0.5% bisacrylamide–0.1% SDS gels. C1q-receptor complexes were analyzed in 10% acrylamide–0.3% bisacrylamide–0.1% SDS. Electrophoretic analysis of the LPS content of purified OmpK36 porin was carried 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), except that an SDS-containing sample buffer (15) was used to load the first-dimension gels. First-dimension gels contained 4% acrylamide, 8 M urea, 4.5% ampholyte 4/6 (Sigma) and 0.5% ampholyte 3.5/10. Gels were casted in the Mighty Small Hoefer’s tube format and were run for 12 to 14 h at 400 V followed by 2 h at 800 V. Distribution of pH in the electrophoresed tube gels was measured at room temperature as described by O’Farrell (40) or with the two-dimensional PAGE standards (Bio-Rad, Madrid, Spain). After equilibration in 62.5 mM Tris-HCl (pH 6.8) containing 3% SDS and 5.5% b-mercaptoethanol for 20 min, the electrophoresed tube gels were loaded onto 11% acrylamide gels and electrophoresed as explained above for OMP. The gels were stained with Coomassie blue. Cross-linking assays. C1q-SASD was prepared by incubating 200 mg of purified C1q with sulfosuccinimidyl-2-(p-azidosalicylamido)-1,39-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 removed by ultrafiltration through 10-kDa-pore-size Ultrafree-MC filters (Millipore, Bedford, Mass.). A final solution of C1q-SASD at 1 mg/ml in PBS was obtained and stored at 2708C 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-receptors complexes by SDS-PAGE and Western blotting (immunoblotting) with anti-OmpK36 and anti-C1q. Briefly, 4 3 109 cells of strain KT707 were washed and suspended in 100 ml of distilled water. The C1q-SASD solution prepared as described above (100 ml) was added to the bacterial suspension, and the mixture was incubated for 30 min at 378C in the dark, followed by a 45-s irradiation with long-wavelength UV light at room temperature. After three washes with 0.1 M Tris-HCl (pH 10.0) containing 0.7 M NaCl to remove noncovalently bound C1q, bacterial OMP were isolated as described previously (17). Briefly, bacterial lysates were obtained by sonication and cell envelopes were collected by centrifugation at 13,000 3 g for 30 min at 48C. OMP were pelleted by insolubilization in 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 1008C in sample buffer, the proteins were separated in a 10% polyacrylamide gel and electrophoretically transferred to nitrocellulose membranes at 1 A for 1 h (49). The nonspecific binding sites were blocked by overnight incubation with PBS containing 1% BSA. Nitrocellulose filters were then incubated in a 1:1,000 dilution of rabbit anti-OmpK36 or rabbit anti-human C1q antiserum for 1 h at 378C. After three washes with PBS and incubation with alkaline phosphataseconjugated goat anti-rabbit IgG (1:3,000 dilution), blots were developed with 5-bromo-4-chloro-3-indolylphosphate–nitroblue tetrazolium (7).

RESULTS K. pneumoniae KT707 is a mutant obtained from K. pneumoniae C3 which has only OmpK36 as porin in its outer membrane, and thus it is a suitable starting strain for the isolation of OmpK36. OmpK36 was purified, giving a single 36-kDa band in polyacrylamide gels (Fig. 1). Densitometric scanning of SDS-PAGE lanes containing purified OmpK36 porin showed that it was more than 90% pure. Moreover, LPS contamination of purified OmpK36 porin was not detected by SDS-PAGE and silver staining (data not shown). The KDO assay indicated that the protein/LPS ratio in the purified OmpK36 porin preparations was routinely in the order of 5 3 105:1 (by weight). Isolation of complement component C1q and its collagen and globular moieties is also shown in Fig. 1. Purified C1q incubated at 1008C for 5 min in SDS sample buffer containing

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FIG. 1. SDS-PAGE and Coomassie blue staining of the OMP from K. pneumoniae C3 or KT793 (lane 1), the OMP from strain KT707 (lane 2), purified OmpK36 porin from strain KT707 (lane 3), purified C1q (lane 4), a purified globular fragment of C1q generated by collagenase treatment (lane 5), and a purified 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.

b-mercaptoethanol separates into three distinct bands of about 27 to 32 kDa. The binding of C1q to OmpK36 porin was dependent on the NaCl concentration used in the assay buffer (Fig. 2). An increment of the ionic strength in the assay drastically reduced the binding of C1q to OmpK36 porin. At 1 M NaCl, the binding was only 4% of that observed at 0.01 M NaCl, which is the optimum condition assayed. However, for all the NaCl concentrations studied, the binding was greater than in the controls. The theoretical pI of the OmpK36 porin (pI 4.3) was calculated from the amino acid-derived sequence of the ompK36 gene (GenBank/EMBL accession number Z33506 [2]) by using the Isoelectric program of the Wisconsin Genetics Computer Group software package. The isoelectric point of the OMP from K. pneumoniae KT707 was also determined by two-di-

FIG. 2. Binding of [125I]C1q to the OmpK36 porin as a function of ionic strength. Wells of ELISA plates coated with OmpK36 porin were incubated with radiolabeled C1q in a buffer of variable ionic strength. Ionic strength variations in the assay were obtained by increasing the NaCl concentration in the buffer from 0.01 to 1 M. After 60 min of incubation at 378C, the amount of bound [125I]C1q was measured. Background binding to albumin-coated wells has been subtracted from the plotted values. Results are the means of triplicate determinations 6 standard deviations for three independent experiments.

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FIG. 3. Inhibition of [125I]C1q binding to OmpK36 porin by C1q fragments. Different amounts of the collagen-like fragment (open boxes) or globular fragment (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. Controls were incubated under the same conditions, but no inhibitors (C1q fragments) were added. After 1 h of incubation at 378C, the amount of bound [125I]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 6 standard deviations for three independent experiments.

mensional electrophoresis, giving a pI of approximately 4.5 for the OmpK36 porin (data not shown); i.e., it is the most acidic protein seen in the outer membrane of K. pneumoniae. Therefore, the results shown in Fig. 2 and the known pI of C1q (pI 10.3 [31]) suggest that the binding of C1q to the OmpK36 porin is of ionic nature or that an ionic component is involved in the interaction. To investigate the region of C1q involved in the binding to OmpK36 porin, the globular and collagen-like fragments were prepared by C1q digestion with collagenase and pepsin, respectively. The fragments generated were pure, as shown in Fig. 1, and were used in inhibition assays. In these assays, the binding of C1q to the OmpK36 porin was inhibited by all the concentrations of the globular fragment used, inhibiting more than 80% of the binding of [125I]C1q to the OmpK36 porin at a concentration of 0.6 nM (Fig. 3). No significant inhibition could be observed with the collagen-like fragment by using the same molar concentrations tested with the globular fragment. These results demonstrate that the interaction between C1q and OmpK36 porin is by the globular region of C1q and not by its collagen-like fragment. The binding of complement components C1q, C3b, and C5b-9 to the OmpK36 porin was determined by enzyme-linked immunosorbent assay (ELISA) experiments with OmpK36coated microplates. NHS was used instead of pure components, and bound C1q, C3b, and C5b-9 were detected by specific antibodies. The equilibrium for the binding of C1q to the OmpK36 porin was reached in 15 min. Increasing the incubation time to 60 min did not significantly increase the amount of bound C1q (Fig. 4). These results ensure that other binding assays carried out in this work with radiolabeled C1q were done under equilibrium conditions, since 1 h was the incubation time in those experiments. The binding of C3b was maximal at 15 min of incubation (Fig. 4). As for C1q binding, the deposition of C3b could be detected immediately after addition of NHS to OmpK36-coated surfaces. In both cases, more than 50% of the binding occurred in the first 10 min, but in the case of C3b we did not observe saturation. In a short time, approximately 20 min, the levels of bound C3b dropped dras-

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FIG. 4. Binding of complement components C1q, C3b, and C5b-9 to the OmpK36 porin as a function of time. Wells coated with OmpK36 were incubated with NHS. At different times, the wells were washed and bound C1q (open circles), C3b (solid circles), and C5b-9 (squares) were detected with specific antibodies in ELISA experiments. Control wells were coated with OmpK36 porin but without the NHS incubation step, and their final ELISA values were subtracted from experimental points to give the plotted values. Results are the means of triplicate determinations 6 standard deviations for three independent experiments.

tically, most probably as a result of degradation of this component into other fragments (C3dg, C3d, etc.) that were not detectable by the monoclonal anti-C3b antibody used. Finally, binding of the C5b-9 complex was detected later than was binding of other complement components studied (Fig. 4). The deposition of the C5b-9 complex was gradual, and saturation was not observed in incubations extending up to 1 h. Altogether, the patterns of deposition of C1q, C3b, and C5b-9 are as expected from an in vivo complement activation: C1q binds first and is followed by C3b and C5b-9 deposition. Also, this demonstrates that OmpK36 porin alone is able to trigger and complete the activation of the complement cascade. The results of binding of radiolabeled C1q to the OmpK36 porin by using increasing amounts of unlabeled C1q are shown in Fig. 5. This binding was a concentration-dependent saturable process, suggesting the presence of a discrete number of

FIG. 5. Direct binding curve and Scatchard plot of [125I]C1q binding to ELISA plate wells coated with OmpK36 porin (0.5 mg per well). Binding of [125I]C1q to OmpK36 porin in the presence of increasing amounts of unlabeled C1q is indicated by solid circles. Results are the mean of at least three independent experiments. Background binding was subtracted before plotting. The Scatchard plot of the C1q-OmpK36 binding (open circles) and two Kd of 1.5 and 75 nM are shown in the insert.

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FIG. 6. Western blot analysis with specific antibodies against OmpK36 porin (A) and complement C1q (B) of the complexes formed between the K. pneumoniae OMP and C1q-SASD. Lanes in both panels contain UV-activated C1qSASD (lanes 1 and 4), isolated OMP from strain KT707 previously incubated with C1q-SASD without UV irradiation (lanes 2 and 5), and isolated OMP from strain KT707 after incubation with C1q-SASD and irradiation with UV (lanes 3 and 6). Samples from lanes 1, 2, 4, and 5 were run reduced, and samples from lanes 3 and 6 were run unreduced. Arrows indicate the putative C1q-SASDOmpK36 complexes, while OmpK36 porin alone is labeled with white asterisks. Molecular mass markers are shown in kilodaltons between the two panels.

C1q-binding sites in the OmpK36 porin. The affinity of C1q for the OmpK36 porin was calculated by Scatchard analysis of the binding data. The Scatchard plot obtained is shown as an insert in Fig. 5 and suggests that there are two binding sites with Kd of 1.5 and 75 nM. Analysis of the binding of C1q to the porin high- and low-affinity sites revealed that approximately 80% of the C1q molecules bind to the low-affinity site. Taking into account that the C1q-OmpK36 porin interaction is mediated by the globular heads of C1q, the high-affinity constant (1.5 nM) would correspond to the globular head-OmpK36 porin interaction. The low-affinity interactions may also be due to binding of the globular heads, especially since this fragment inhibited almost all the sites measured in Fig. 2. Having characterized the binding process and demonstrated that this binding leads to complete activation of the complement cascade, we were interested in studying if those phenomena occurred in vivo. In vivo binding of C1q and characterization of the bacterial C1q-binding molecules were studied by incubation of bacteria with C1q labeled with the SASD crosslinker (Fig. 6). As shown in Fig. 6A, lane 3, after incubation of bacterial cells with C1q-SASD and UV activation of the crosslinker, we detected by Western blot experiments with antiOmpK36 serum two bands of 88 and 84 kDa plus the 36-kDa band corresponding to the OmpK36 porin monomer. Figure 6A, lane 1, provides proof of the specificity of the assay: C1qSASD alone was not detected by the anti-OmpK36 antiserum. Also, in lane 2, OMP from bacteria incubated with C1q-SASD but without the UV activation (cross-linking) step were analyzed; anti-OmpK36 antiserum detected just OmpK36 porin, but no other bands were visualized. These results are consistent with the conclusions that (i) high-molecular-mass (88 and 84 kDa) complexes are formed and maintained throughout the analysis only after covalent bonds are induced by UV activation of the cross-linker (as in lane 3) and (ii) C1q-SASDOmpK36 porin complexes are formed. Further confirmation of the above results was obtained in duplicate experiments with anti-C1q antiserum (Fig. 6B). Results from lane 4 disprove the hypothesis that complexes (the 88- and 84-kDa bands of Fig. 6A, lane 3) could be a result of intramolecular C1q cross-linking: C1q-SASD alone after UV activation of the cross-linker, at the concentration used, does not show any band in addition to those expected for purified C1q run reduced. Lane 5 shows again that no C1q-porin com-

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plexes are formed and maintained throughout the analysis unless the bifunctional reagent SASD is activated to form covalent bonds between C1q-SASD and its corresponding receptor in the bacterial outer membrane. Figure 6B, lane 6, confirms that C1q is a part of the 88- and 84-kDa bands seen in Fig. 6A lane 3; also, since samples of lane 6 were run unreduced, the two subunits of C1q (A-B, 52 kDa; C-C, 48 kDa) not complexed with the porin are also detected. Therefore, taking together the results in lanes 3 and 6 in both panels and the molecular masses of the two bands detected with anti-C1q and anti-OmpK36, we suggest that C1q binds to the OmpK36 porin and form complexes that, after dissociation of porin trimers into monomers, consist of (i) porin monomers (36 kDa) plus the A and B chains of C1q (52 kDa) and (ii) OmpK36 monomer plus two C chains (48 kDa) of C1q. DISCUSSION We have studied the interaction between complement C1q and OmpK36, a porin from K. pneumoniae, and demonstrated by cross-linking assays that this interaction occurs in vivo, i.e., while porins are in the outer membrane. C1q binding is sequentially followed by C3b binding and C5b-9 deposition in the manner expected for a molecule that is able to trigger complement CP activation. Binding assays revealed two affinity constants for the binding of C1q to the OmpK36 porin (see below), but the dimensions of porin and C1q preclude a situation in which more than one C1q molecule is bound to one porin molecule: porins form trimers in the outer membrane with a distance of about 35 Å (3.5 nm) between the center of each porin monomer (13), while the C1q molecule has a size of about 50 Å (5.0 nm) in its globular region and about 250 Å (25.0 nm) in the collagenic-like region (28). It is therefore conceivable that only one C1q molecule could bind to one porin trimer and impede binding of further C1q molecules to the same and neighbor porin trimers. This could explain why in the in vivo binding experiments described in Fig. 6, a relatively high proportion of porin molecules did not form complexes with C1q. Binding experiments indicate two affinity constants, one of them a relatively high-affinity constant (Kd 5 1.5 nM) that is higher than that of C1q for IgG-bearing immunocomplexes (11 nM) (14). A second binding constant of low affinity was seen (Kd 5 75 nM). The second Kd is very similar to that of other C1q-binding molecules, e.g., fibrinogen (16), and may be associated with low-level LPS contamination of the isolated porin. Rough LPS, such as the one produced by strain KT707, the source of OmpK36 porin used here, bind C1q and activate the complement CP, as has been demonstrated for K. pneumoniae (1) and in other cases (33, 37). However, at least for K. pneumoniae, the ability of rough LPS to bind C1q and activate the CP is several times lower than that of porins (1). Also, when LPS-C1q interaction was studied in detail (53), a different pattern was seen. First, LPS binds C1q through the collagenic region and not, as in the porin case reported here, through the globular heads (see below). Second, the above LPS-C1q interaction has a lower affinity (Ka 1.4 3 108 M21) than in the porin case. Alternatively, it is conceivable that C1q possesses two binding sites for OmpK36, as happens with other activators like DNA. DNA binds to both the collagen-like and globular regions of C1q, although it binds preferentially to the A chain (residues 14 to 26) in the collagen-like region (25). The situation with OmpK36 seems to be just the opposite: OmpK36 binds to the globular heads of C1q with high affinity, but no significant inhibition of C1q binding was seen with the collagen-like region.

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C1q appears to bind to the OmpK36 porin by its globular regions as suggested by results from inhibition assays carried out with the purified collagenic and globular domains of C1q. Since porins are the most important activators of the CP in the outer membrane of K. pneumoniae (1) the binding of C1q to this microorganism will probably occur via the globular headpieces too. Some authors have reported that binding of C1q to the E. coli D13m4 heptoseless LPS is mediated by the collagenlike region (5, 53). This situation is in contrast to what we have observed with porin here, as well as with the prototypic case of C1q binding to immunocomplexes (14). Both in the porin and immunocomplex cases, as well as for other activators of the CP, binding of C1q has been described to be mediated by the globular headpieces, leaving the collagen-like region available for further interactions, e.g., with the C1q receptor on the surface of phagocytic and nonphagocytic cells (19). C1 activators have been divided into “immune” and “nonimmune” activators depending on the nature of the activator. Immune activators include IgG and IgM immune aggregates, which bind C1q through its globular region. Examples of nonimmune activators are DNA (25), C-reactive protein (26), and b-amyloid peptide (24), which bind through the collagen-like region. The major binding site for these nonimmune activators was localized to residues 14 to 26 within the collagen-like region of the C1q A chain, because synthetic C1q A-chain peptide 14 to 26 effectively inhibited activation by these substances (25, 26, 52). According to this classification, OmpK36 would be considered a nonimmune activator but one that binds C1q through the globular region (such as the above immune activators). A 33-kDa cell surface glycoprotein, designated gC1q-R, that also binds C1q through the globular heads has recently been described in several cell lines (20), but its possible relationship, if any, to the porins has not been studied. OmpK36 porin binds C1q at physiological salt concentrations, and the binding decreases as the salt concentration increases. Similar results were obtained when we studied the binding of [125I]C1q to cells of K. pneumoniae (data not shown). These data are comparable to those reported with C1q and IgG immunocomplexes, in which the strongest IgG-C1q binding was observed at an ionic strength of 0.15 to 0.17 M. Higher values of ionic strength decrease or inhibit the interaction (9, 29). Our results agree with experiments by other authors showing increased C1q binding to different activators of the CP at subphysiological ionic strength (11, 22, 43). Our data also suggest that charged amino acids from both molecules, C1q and OmpK36 porin, are involved in the interaction or, alternatively, that the ionic strength modifies the conformation of these proteins. As a porin, OmpK36 is an extremely stable molecule (2). Likewise, the conformation of C1q is not affected by variations in the ionic strength (32, 42), so the latter hypothesis seems less convincing. As in the case of the immunocomplexes and other molecules (14), the first possibility is more reasonable. OmpK36 porin and enterobacterial porins in general present charged amino acids in the extracellular domains or “loops” (2, 13, 23), which are available for interaction with C1q. The acidic pI of the OmpK36 porin described here and the basic pI of the C1q molecule further support the idea that binding depends on the existence of charged amino acids on the porin molecule. The arginine residues of the globular region of human C1q involved in the interaction with IgG have recently been described, and these residues map to two exposed regions, which presumably recognize the Cg2 domain of IgG (35) and could also be involved in the interaction with the OmpK36 porin. Hence, C1q binding to OmpK36 may be primarily, but not exclusively, due to ionic interactions. The present work constitutes a step forward the understand-

4724

ALBERTI´ ET AL.

ing of the interaction between enterobacterial porins and the complement CP. In the K. pneumoniae case we are studying, we have answered here several questions that remained open in previous works (1, 2). First, we have demonstrated by crosslinking that porin OmpK36 binds C1q while in its native state in the bacterial outer membrane of complement-sensitive strains. Second, the interaction between the porin and C1q is mediated by the globular region of C1q. Third, we have shown that the binding phenomenon is dependent on the ionic strength. The existence on the porin molecule of a conserved motif of one negative and two positive side chains, amino acids K-278, K-280, and D-281 of OmpK36 porin (2), is similar to the E-318, K-320, and K-322 C1q-binding motif described for IgG (14). Given the description here that the globular region mediates the binding of C1q to porin and the above binding motifs, a model of interaction between enterobacterial porins and C1q similar to the one described for C1q and immunocomplexes is anticipated. ACKNOWLEDGMENTS This work was supported by CICYT grants PB93-0423 and PB940906 to V.J.B. and J.M.T. and grant PB92-0788 to F.V. Predoctoral fellowships from CICYT (to S.A. and X.R.), Programa 07, Salud (to S.A.), and Fundacio ´n Conchita Ra´bago (to G.M.) are also acknowledged. We thank Antoni Miralles (UIB) for helpful discussions of the binding results and the CNB node of EMBL/Net for accession to DNA databases and to the Wisconsin GCG package. Members of the UIB also thank J. Lalucat for continuous support.

INFECT. IMMUN.

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