Immune Evasion of Enterococcus faecalis by an Extracellular Gelatinase That Cleaves C3 and iC3b

GelE, Abs, and complement components C3, C3b, iC3b, factors B, D, I, and H

GelE was purified from the Ef GM culture media in accordance with the procedures described in our previous study (19). Anti-human C3 Ab was purchased from Sigma-Aldrich (Cat. No. C7761). In the case of the anti-GelE Ab, we utilized an antiserum that had been previously generated via an injection of GelE into a rabbit (19). All human complement components used in this study were commercially obtained from three companies as follows: C3, Sigma-Aldrich C2910; C3b, Calbiochem 204860; iC3b, Calbiochem 204863; fB, Quidel 902985; fD, Sigma-Aldrich C5688; fI, Sigma-Aldrich C5938; fH, Sigma-Aldrich C5813.

Normal human serum and PMNs

Fresh normal human serum (NHS) and PMNs were prepared from blood samples that were collected from three healthy human donors after obtaining informed consent. PMNs were isolated from 15 ml of EDTA-anticoagulated blood via three-step procedures consisting of dextran sedimentation, Ficoll-Hypaque centrifugation, and hypotonic lysis of residual erythrocytes as previously described by Rakita et al. (20). They were then suspended in HBSS containing 1% BSA without Ca²⁺ and Mg²⁺ at approximately 6 × 10⁶ cells/ml.

Degradation of C3 in NHS by GelE

Purified C3 was mixed with GelE in the presence of both fH and fI or each of two factors. Alternatively, the same reaction was conducted in the absence of GelE. The reaction volume was 10 μl of PBS and the quantity of each protein used in these tests was adjusted to 1 μg. Each mixture was incubated for 30 min at room temperature and dried down completely via vacuum centrifugation. The sample was then resuspended in 10 μl of SDS-PAGE sample buffer (0.5 M Tris-HCl (pH 6.8), containing 10% (w/v) SDS and 40 mM DTT) and subjected to SDS-PAGE analysis. Duplicate SDS-PAGE gel was employed for immunoblot assay conducted with anti-human C3 Ab. In an effort to detect the proteolytic activity of GelE to C3, fH, fI, fB, or fD, each complement factor was incubated with increasing concentration of GelE under conditions identical to those listed above, then assessed via SDS-PAGE analyses. In addition, C3 (0.5 μg), fB (0.6 μg), and fD (4 ng) were incubated with GelE (0.5 μg) in 10 μl of PBS containing 5 mM MgCl₂ for 30 min at room temperature to examine whether the C3b-like molecule generated by GelE is capable of functioning as a component of soluble C3 convertase. For the control assembly of C3 convertase, commercial C3b (0.5 μg) was incubated with fB (0.6 μg) and fD (4 ng) under the same condition as above. The assembly of C3 convertase was verified on SDS-PAGE gel by monitoring a band of fB fragment (Bb) generated via proteolysis by fD. In an effort to assess the activity of soluble C3 convertase against the purified C3, C3 convertase was assembled via the 30-min incubation of C3b (2 μg) with fB (2 μg) and fD (0.4 μg) in 50 μl of PBS containing 5 mM MgCl₂ and mixed with 10 μl of PBS containing C3 (10 μg). Alternatively, 50 μl of the assembled C3 convertase was mixed with 10 μl of PBS containing C3 (10 μg), fI (2 μg), and fH (4 μg), then incubated for 30 min at room temperature. In addition, GelE (2 μg) was added to the mixture of fB (2 μg), fD (0.4 μg), fI (2 μg), fH (4 μg), and C3 (10 μg), and then incubated for 30 min at room temperature. Thereafter, 6 μl of each sample was subjected to SDS-PAGE analysis.

To examine the fD cofactor activity of C3b or C3b′, C3b (2 μg) or C3 (2 μg) preincubated for 30 min with GelE, was incubated for 10 min with fB (4 μg) at room temperature. Then, each sample was loaded onto 10% native SDS-PAGE gel. C3 (2 μg) was incubated with fB (4 μg) under the same condition as above and used for control.

Immunofluorescence microscopic observation

Using zymosan (Sigma-Aldrich, Z4025) and C3-deficient human serum (C3-def HS) (Sigma-Aldrich, C8788), we assessed the opsonization of foreign particles by the complement system. Sixty microliters of PBS containing C3 (10 μg) was mixed with 40 μl of Gelatin Veronal Buffer (GVB, Sigma-Aldrich, G6514) containing 50% (v/v) C3-def HS and 100 μg of zymosan. The mixture was then incubated for 1 h at 37°C in a rotary shaker and washed twice in PBS. After centrifugation, the sediment was resuspended in 100 μl of PBS containing 4% (v/v) FITC-labeled C3c complement Ab (GeneTex, 16262), then incubated for an additional 30 min at 37°C in a rotary shaker. The sample was then washed twice in PBS and resuspended in 100 μl of PBS. In brief, 20 μl of sample was removed and observed under the fluorescence microscopy (Leica DMBL). To assess an effect of soluble C3 convertase assembled with C3b (2 μg), fB (2 μg), and fD (0.4 μg) on C3, C3 (10 μg) was incubated in 60 μl of PBS containing C3 convertase for 30 min at room temperature. The sample was then treated sequentially with C3-def HS and zymosan in accordance with the procedure described above, and then subjected to microscopic analysis. Alternatively, C3 was incubated with C3 convertase in the presence of fH (4 μg) and fI (2 μg) under the same condition as above and the sample was subjected to the same microscopic observation procedure. To assess the effects of GelE on C3 opsonization, C3 (10 μg) was incubated for 30 min with GelE (2 μg) in the presence of fH (4 μg) and fI (2 μg) at room temperature. After further incubation in Gelatin Veronal Buffer containing zymosan and C3-def HS, the sample was subjected to microscopic analysis.

PMN-mediated bactericidal assay

One hundred microliters of PMN solution (6 × 10⁶ cells/ml) prepared in HBSS were added to 90 μl of PBS containing 10 μl of NHS. In this experiment, we utilized PBS containing 1 mM CaCl₂ and 1 mM MgCl₂. This PMN-NHS sample was mixed with log-phase Ef (6 × 10⁵ cells) in 10 μl of PBS. After 30 min of incubation at 37°C in a rotary shaker, 10 μl of the mixture was removed and added to 1.49 ml of distilled water to lyse the PMNs. Then, 10 μl of the sample was plated on tryptic soy agar (Difco). The resultant colonies were counted following overnight incubation at 37°C. For a control, 10 μl of Ef solution (6 × 10⁵ cells) was incubated in buffer mixture (100 μl of HBSS plus 90 μl of PBS) in the absence of NHS and PMNs under the same condition described above. According to the same procedure, bacteria were numerated after overnight incubation. In an effort to assess the effects of GelE on the Ef killing mediated by NHS and PMNs, 100 μl of PMN solution was added to 90 μl of PBS containing various quantities of GelE (0.5, 1, or 2 μM) and 10 μl of NHS and each sample was then subjected to the same procedure as described above. Alternatively, to evaluate the effects of GelE on the killing of pre-opsonized Ef (O-Ef) by PMN, 900 μl of Ef solution (6 × 10⁹ cells/ml) were first incubated with 100 μl of NHS for 15 min at 37°C in a rotary shaker. The reaction was stopped by the addition of 50 mM EDTA solution in PBS, after which the mixture was washed twice in PBS. After a brief centrifugation, the O-Ef sediment was resuspended in 1 ml of PBS. Then, 10 μl of O-Ef (6 × 10⁷ cells/ml) was mixed with 90 μl of PBS containing varying concentration of GelE (0.5, 1 or 2 μM) or no GelE. Following 30 min of incubation at 37°C in a rotary shaker, 100 μl of PMN solution (6 × 10⁶ cells/ml) in HBSS was added to the mixture. After 30 min of incubation, each sample was subjected to the same procedure as described above to count the recovered colonies. For a 100% survival control, O-Ef was incubated under the same conditions in HBSS containing no PMNs.

In addition, we attempted to examine whether GelE affected PMNs and damaged their phagocytic activity against O-Ef. GelE at different concentrations (0.5, 1, or 2 μM) were initially incubated for 30 min with PMNs in HBSS at 37°C. After three times washing in HBSS to remove the remaining GelE, 190 μl of GelE-treated PMN sample (6 × 10⁵ cells) was mixed with 10 μl of PBS containing O-Ef (6 × 10⁵ cells), and incubated for 30 min at 37°C. The survival colonies from each sample were numerated in accordance with the same procedure as described above.

Degradation of iC3b by GelE and microscopic observation for effect of GelE on CR3 (CD11b/CD18) on human PMNs

It has been determined that GelE is capable of degrading purified iC3b and surface-bound iC3b, both of which were generated via the activity of fI in the presence of fH. Purified C3b (2 μg) was incubated for 30 min with GelE (0.5 μg) in 10 μl of PBS containing fI (1 μg) and fH (2 μg) at 37°C. For a control, C3b was treated with an identical amount of fI and fH in PBS without GelE. Each sample was subjected to SDS-PAGE and immunobloting analyses were conducted with anti-C3 Ab. The proteolytic activity of GelE to iC3b was also evaluated using purified iC3b. One microgram of iC3b was incubated with GelE of 2-fold serially diluted concentrations from 0.8 μM for 30 min at 37°C, then subjected to SDS-PAGE analysis. To assess the effect of GelE on surface-bound iC3b, 900 μl of PBS containing 200 μg of zymosan was mixed with 100 μl of NHS and incubated for 20 min at 37°C. After two washings in PBS, zymosan was treated with GelE (2 μg) for 30 min at 37°C. The zymosan sample was then extensively washed in PBS and boiled for 5 min in 50 μl of SDS-sample buffer to elute zymosan-bound proteins. After a brief centrifugation, the supernatant was subjected to SDS-PAGE and duplicate gels were utilized for immunoblot analysis. The protein sample detached from zymosan that was incubated with NHS but not treated with GelE was utilized as a control. Furthermore, zymosan was incubated with NHS for different times, and each sample was treated with GelE. Then, the eluted proteins were also examined by immunobloting analysis. To examine the effect of GelE on CR3, 100 μl of PMNs suspension (2 × 10⁷ cells/ml) in HBSS was mixed with 100 μl of 2 μM GelE in PBS and incubated for 30 min at 37°C. After washing twice with HBSS, PMNs were treated with FITC-conjugated anti-CD11b Ab (sc-52686, Santa Cruz Biotechnology) for 30 min and observed under fluorescence microscopy.

Phagocytosis and flow cytometry analysis

Methanol-fixed Ef was first labeled with FITC (Sigma-Aldrich F7250). FITC-labeled O-Ef was treated with 2 μM GelE for 30 min at 37°C and then incubated for 30 min with PMNs at 37°C. For a control, FITC-labeled O-Ef untreated with 2 μM GelE was incubated with PMNs. According to the same procedure as described above, each sample was then visualized via fluorescence microscopy. To assess the effects of GelE on PMNs, PMN samples were treated with 2 μM GelE for 30 min at 37°C and washed twice in HBSS before incubation with FITC-labeled O-Ef. The phagocytosis of O-Ef by GelE-treated PMNs was then observed in accordance with the same procedures as above. Alternatively, the phagocytosis of FITC-labeled O-Ef by PMNs was assessed via flow cytometry using a FACS flow cytometer (BD Biosciences) with computer-assisted evaluation of data (FACScan software). In these experiments, two times more PMNs (6 × 10⁵ cells) and FITC-labeled O-Ef (1.2 × 10⁶ cells) than those employed in the microscopic experiments were used, and 20,000 PMNs of each sample were analyzed for FACS analyses. Also, before cell counting, the samples were consistently fixed with 4% paraformaldehyde.

Hemolysis assay

Complement activation in sera from human and other animals was assessed by measuring their hemolytic activity against RBC obtained from a rabbit. Human (20 μl), dog (25 μl), chicken (25 μl), mouse (55 μl), or guinea pig (45 μl) sera were mixed with PBS to a total volume of 90 μl. Each serum sample was supplemented with 10 μl of rabbit RBC solution (10⁹ cells/ml) in HBSS, then incubated for 30 min at 37°C. After 3 min centrifugation at 10,000 × g, 50 μl of the supernatant was removed and its OD was measured at 405 nm using a 96-well plate reader (Bio-Tek Instruments ELx 800). Alternatively, each serum sample was treated with 2 μM GelE for 30 min at 37°C before the addition of rabbit RBC, and the hemolysis of each sample was then examined according to the same procedure as described above.

Inactivation of C3 in human serum by GelE

Using purified components of the human complement, the effect of GelE on C3 was assessed via SDS-PAGE and immunoblotting assays. As shown in Fig. 1⇓A, the C3 α-chain vanished from the gel when it was incubated with GelE in the presence of fI and fH. However, without fI or fH in the reaction mixture, C3 was changed into a C3b-like molecule (C3b′) by GelE. In addition, C3 was not affected by fI and fH in the absence of GelE. Therefore, it was concluded that GelE acted on C3 in a manner similar to that of C3 convertase and generated a C3b-like protein which was degraded further by fI in the presence of its cofactor, fH. We also attempted to determine whether this C3b′ can behave like an original C3b as a component of C3 convertase (C3bBb). When it was incubated with fB and fD, it was confirmed that an fB fragment (Bb) was generated via the action of fD with the aid of its cofactor as in the control mixture, to which an original C3b was added (Fig. 1⇓C). This result demonstrated that C3b′ retained an fD cofactor activity for the proteolysis of fB. The proteolytic activity of the assembled C3 convertase (C3b, fB, and fD) against C3 was evaluated in the absence or presence of fI and fH (Fig. 1⇓D). As a result, the C3 α-chain was found to be intact in the mixture containing C3 convertase, fI and fH, thereby indicating that C3 convertase was inactivated by fI and fH (lane 2 in Fig. 1⇓D). In contrast, once C3 was incubated with fB, fD and GelE in the presence of fI and fH, the C3 α-chain disappeared but Bb was detected, thereby suggesting that C3b′ could exert its fD cofactor activity before degradation by fI. In addition, the fD cofactor activity of C3b′ was confirmed via an SDS-PAGE analysis under non-reducing condition (Fig. 1⇓E). When GelE-treated C3 was incubated with fB, the band for C3b′ was shifted to upper position (sixth lane) as did an original C3b incubated with fB (fourth lane), indicating C3b′ was capable of binding to fB and acting as a cofactor of fD. In parallel, it was verified that the assembled C3 convertase did not affect C3 in NHS upon its addition to NHS but that the addition of GelE to NHS resulted in the degradation of the C3 α-chain as had been observed previously (data not shown). From these results, it was concluded that GelE functioned as a soluble C3 convertase which was resistant to proteolysis by fI and fH. Using C3-deficient human serum (C3-def HS) and zymosan, we also determined the effects of GelE on the opsonization of complement on zymosan surfaces (Fig. 2⇓). When C3-def HS was supplemented with C3 that had been preincubated with the assembled C3 convertase, fluorescence resulting from opsonization by C3 fragments, such as C3b and iC3b, was not detected on zymosan surfaces (Fig. 2⇓B). However, C3-def HS supplemented with C3 pre-treated with soluble C3 convertase in the presence of fI and fH evidenced a significant opsonization effect (Fig. 2⇓C) as in the case of the control sample (Fig. 2⇓A). This result indicates that the activity of soluble C3 convertase was abolished by fI and fH, and thus an intact C3 was provided to C3-def HS. In contrast, as expected from the results shown in Fig. 1⇓, C3-def HS supplemented with GelE-treated C3 evidenced no opsonization effects on zymosan (Fig. 2⇓D).

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FIGURE 1.

Proteolytic effects of GelE on complement components in the human serum. A, Each reaction mixture was subjected to SDS-PAGE (upper panel) and immunoblotting analysis (lower panel). B, Proteolytic activity of GelE to each purified complement factor. One microgram of each complement factor was treated with GelE at the indicated concentrations for 30 min at room temperature and then subjected to SDS-PAGE. C, The assembly of fluid-phase C3 convertase with fB, fD, and C3b′ generated via GelE action. Validity of C3b′ as a component of C3 convertase was monitored by the appearance of Bb after the incubation of the mixture. Lane 1, human C3b; lane 2, human C3 treated with GelE; lane 3, mixture of C3b, fB, and fD; lane 4, mixture of C3, GelE, fB, and fD. D, Effects of fH and fI on assembled C3 convertase. Lane 1, C3 treated with assembled C3 convertase for control; lane 2, C3 treated with assembled C3 convertase in the presence of fH and fI; lane 3, mixture of C3, GelE, fB, fD, fH, and fI. E, Native SDS-PAGE analysis to examine the fD cofactor activity of C3b and C3b′. Of note, whereas the band for C3 was not changed upon incubation with fB (third lane), those for C3b and C3b′ were moved upward in the presence of fB. In A and E, symbols + and − indicate addition and no addition of corresponding protein into the sample, respectively.

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FIGURE 2.

Immunofluorescence analysis for an effect of GelE on C3 opsonization of zymosan. A, Zymosan incubated with C3-def HS supplemented with C3. B, Zymosan incubated with C3-def HS supplemented with C3 treated by C3 convertase. C, Zymosan incubated with C3-def HS supplemented with C3 treated by C3 convertase in the presence of fI and fH. D, Zymosan incubated with C3-def HS supplemented with C3 treated by GelE in the presence of fI and fH. For immunostaining, FITC-labeled anti-C3c Ab was diluted to a concentration of 1/25 in PBS.

Effect of GelE on PMN-mediated bactericidal activity

When Ef was incubated at 5% NHS containing constant quantities of human PMNs and different concentrations of GelE, the rate of Ef survival was enhanced with increasing concentration of GelE in the reaction mixture (Fig. 3⇓A). From this result, it was concluded that GelE inhibited an opsonization by NHS and substantially attenuated PMN-mediated Ef killing. In the following experiments, we also attempted to determine whether this inhibitory effect of GelE could be induced by its action against pre-opsonized Ef (O-Ef) with NHS or against PMNs. Firstly, O-Ef was incubated at varying concentrations of GelE and then the PMN-mediated bactericidal effect was assessed. As a result, the higher the concentration of GelE that was applied to O-Ef, the higher the survival rates of Ef were (Fig. 3⇓B). Secondly, before incubation with O-Ef, PMNs were treated with varying concentrations of GelE. As shown in Fig. 3⇓C, all PMN samples treated with different concentrations of GelE evidenced an equivalent antibacterial activity against O-Ef as was observed with the untreated PMN. Thus, we concluded that GelE affected O-Ef but not PMNs, and thereby damaged the PMN-mediated Ef killing process.

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FIGURE 3.

Effects of GelE on PMN-mediated bacterial killing. A, All elements (NHS, PMN, GelE, and Ef) were incubated together in the reaction mixture. B, O-Ef was treated with GelE before incubation with PMN. C, PMN was treated with GelE before incubation with O-Ef. In control sample, Ef bacteria (N-Ef) that were not pre-opsonized by NHS were incubated with PMN, after which the recovered CFU was numerated. In all tests, PMN-mediated Ef killing was expressed as the percent of bacterial survival in each sample as compared with that of the control sample, according to the following equation: [(number of colony forming units (CFU) in test sample/number of CFU in control sample) × 100]. Data were obtained from three independent experiments and expressed as the mean values ± SDs (p < 0.01).

Effects of GelE on C3 fragments bound to zymosan and CR3 on PMN surfaces

As it has been determined that iC3b is a predominant C3 fragment bound to microbial surfaces and performs a pivotal function as a major complement opsonin in phagocytosis by PMNs, we assessed the proteolytic effects of GelE on iC3b. As shown in Fig. 4⇓A, treatment of C3b with fI and fH generated two fragments (62 and 43 kDa) on SDS-PAGE gel. The appearance of two fragments is indicative of the generation of iC3b, which resulted from the removal of the C3f fragment from C3b α-chain. Two fragments of the iC3b α-chain were further degraded upon the addition of GelE into the mixture, a finding which differed from the result that the C3b α-chain was intact against GelE attack. To further verify it, we directly assessed the proteolytic activity of GelE against a purified iC3b. As shown in Fig. 4⇓B, two bands of iC3b corresponding to 62 and 43 kDa vanished from the gel as the concentration of GelE increased in the reaction mixture. In addition, we attempted to evaluate the effects of GelE on C3 fragments bound to the zymosan surface. The protein sample eluted from zymosan pre-opsonized by NHS was subjected to immunoblotting analysis with anti-C3 Ab (Fig. 4⇓C). As a result, iC3b was detected principally as C3 fragments, although some C3b was also detected. However, when opsonized zymosan was treated with GelE, only C3b was shown to have been eluted from zymosan. This result demonstrated that iC3b was stripped off from the zymosan surfaces, as two fragments of the iC3b α-chain were degraded by GelE. Alternatively, GelE was added to the incubation mixture (zymosan and NHS) at different postincubation times (0, 5, and 10 min). After 10 min of further incubation, C3 fragments eluted from the zymosan surfaces were also assessed via an immunoblotting analysis. As a result, both C3b and iC3b were detected in the zymosan samples not treated with GelE. In contrast, only C3b was detected in all samples, to which GelE was added (Fig. 4⇓D). It is worthy of note that the longer zymosan and NHS were incubated before addition of GelE, the more C3b was detected in the sample eluted from zymosan, although iC3b was not detected in any of the GelE-treated samples. According to these results, it was concluded that GelE degraded iC3b, but not C3b bound to the surfaces of foreign particles, and consequently paralyzed an opsonization induced by the complement activation. In addition, the proteolytic effect of GelE against CR3 on the surfaces of human PMNs was examined by fluorescence microscopy (data not shown). Fluorescence resulting from binding of FITC-conjugated anti-CD11b Ab to the PMN surfaces was consistently detected after treatment with GelE, suggesting that CR3 on the PMN surfaces was not affected by Ef GelE.

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FIGURE 4.

Proteolysis of human iC3b by GelE. A, Immunoblotting assay for proteolytic activity of GelE against iC3b generated from purified C3b via the action of fI and fH. B, Purified iC3b was treated with varying concentrations of GelE. The sample was then electrophoresed on 10% SDS-PAGE gel and the gel was stained with Coomassie blue. C, Immunoblotting analysis for the proteolytic activity of GelE against C3 fragments bound to the zymosan surfaces. Protein samples were subjected to 8% SDS-PAGE analysis and duplicate gels were used for immunoblotting assay. Lane 1, purified C3b; lane 2, purified iC3b; lane 3, protein sample eluted from zymosan opsonized by NHS; lane 4, proteins eluted from zymosan after the treatment of opsonized-zymosan with 2 μM GelE. D, GelE (2 μM) was added to the reaction mixture at different postincubation times of zymosan and 20% NHS, and protein samples eluted from zymosan were subjected to immunoblotting analysis with anti-C3 Ab as described by Towbin et al. (44 ). In A and D, symbols + and − indicate addition and no addition of corresponding protein into the reaction mixture, respectively.

GelE inhibits complement-mediated phagocytosis of Ef by human PMN

To further assess the functional role of GelE in protecting Ef against the PMN-mediated bactericidal effects, we conducted a series of experiments regarding the phagocytosis of Ef by PMNs under conditions similar to those in the PMN-mediated bactericidal assay. FACS analysis showed that the phagocytosis of Ef was severely disrupted upon the incubation of PMN with NHS containing GelE as compared with the control samples (Fig. 5⇓A). In addition, when O-Ef was treated with GelE and then incubated with PMN, phagocytosis was reduced significantly (Fig. 5⇓B). In contrast, GelE-treated PMN was confirmed to maintain its activity for the phagocytosis of O-Ef (Fig. 5⇓C). Collectively, these results were generally consistent with our previous data obtained from PMN-mediated bactericidal assays (Fig. 3⇑).

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FIGURE 5.

Effect of GelE on phagocytosis of Ef by human PMNs. Effects of GelE on phagocytosis of Ef by PMN under different conditions were assessed by FACScan flow cytometry. A, Phagocytosis of Ef by PMNs once FITC-labeled Ef and PMNs were coincubated in 5% NHS containing GelE or no GelE. B, Phagocytosis of O-Ef by PMNs once O-Ef was incubated with PMNs after O-Ef was treated with GelE. C, Phagocytosis by PMNs that were treated with GelE before incubation with O-Ef. In all data, the left ones show the results of the positive controls conducted without the addition of GelE into the sample, and the right ones show the results of the test samples treated with GelE.

Effect of GelE on complement activation in sera from other animals

To get further information about the actual site at which GelE cleaved C3, we tested its activity against sera of animals whose C3 has defined differences in sequence from human C3. Whereas the hemolytic activity of NHS was reduced significantly after GelE treatment, those of sera from four animals against rabbit RBC were not affected by GelE (Fig. 6⇓A). This result suggested that the C3 molecules of four animals were resistant to GelE-mediated proteolysis; consequently, the MAC was assembled successfully on rabbit RBC. Furthermore, the proteolytic effect of GelE on C3 in mouse serum was assessed via an immunoblotting assay conducted with an anti-mouse C3 mAb (CL7503AP: Cedarlane Laboratories) (Fig. 6⇓B). As a result, the band of human C3 was detected at a slightly lower position upon treatment with GelE than was an untreated C3, thereby indicating that C3a was removed from C3 via degradation by GelE. In addition, it was noted that the C3 band was moved to a much lower position when NHS was treated with GelE. This result shows that C3 was changed to C3b′ by GelE, which was subsequently cleaved into iC3b by fI and fH occurring in NHS, and iC3b was degraded further by GelE. In contrast, it was shown that the position of the C3 band in the mouse serum remained unchanged after incubation with GelE.

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FIGURE 6.

Sera from human and four other animals were tested for their hemolytic activity against rabbit RBCs and each hemolysis was compared with that of each GelE-treated serum sample (A). Experiments were iterated three times on different days, and the mean values were utilized for the preparation of data (p < 0.01). B, The proteolytic effects of GelE against human and mouse C3 were assessed via immunoblotting analyses. One microgram of purified human C3 (left panel), 10% NHS (center panel), or 10% normal mouse serum (NMS) (right panel) was treated with 2 μM GelE for 30 min at 37°C. Each sample was electrophoresed on 8% SDS-PAGE gels under non-reducing conditions and each duplicate gel was probed with anti-human C3 Ab (left and center) or anti-mouse C3 Ab (right). C, Joining regions of C3a and C3b in C3 molecules from human and other animals. Asterisks (∗) indicate the cleavage sites by C3 convertase and the action site of GelE is indicated on the sequence of human C3 by an arrow. It is worthy of note that, whereas P1 site of human C3 for action of GelE is an Asn, the putative site in other C3 proteins is an acidic amino acid, such as Asp or Glu. Sequences cited were from the database of the National Center for Biotechnology Information (human, AAA85332; chicken, NP_990736; dog, XP_533932; mouse, AAC42013; guinea pig, P12387).