The plasma protein factor H (FH) inhibits the alternative pathway of complement activation. Previous work has shown that FH binds to group A streptococci and that the interaction does not interfere with the complement-inhibitory capacity of FH. In this work, we report a molecular analysis of this interaction. In absorption experiments with human plasma, M protein-expressing group A streptococci bound both FH and FH-like protein-1 (FHL-1), an active 42-kDa splice product of the FH-gene transcript comprising the first 7 of its 20 short consensus repeat (SCR) domains. rFHL-1 also bound to M protein-expressing streptococci, but rFH fragments containing SCR 1–5 or SCR 1–6 did not. rFHL-1 bound to purified M5 protein with an affinity that was higher than the value calculated for the interaction between FH and M5 protein. The binding of radiolabeled rFHL-1 to immobilized M5 was blocked completely by unlabeled rFHL-1, but was inhibited only partially by SCR 1–6, emphasizing the importance of SCR 7 for the interaction. In experiments with the FH-related proteins FHR-3 and FHR-4, only the former bound to M protein-expressing streptococci, again pointing to an involvement of SCR 7, since FHR-3, but not FHR-4, contains a domain that is similar to SCR 7. Finally, the interaction between rFHL-1 and purified M5 protein was inhibited by heparin, which binds FH via SCR 7. Together, these data indicate that the interaction between streptococcal M proteins and FH or FHL-1 requires SCR 7.

Factor H (FH)3 is a 150-kDa single chain plasma glycoprotein that plays a pivotal role in the regulation of the alternative pathway of complement activation (1, 2). This complement regulator prevents binding of factor B to C3b, supports the dissociation of the C3bBb complex, and acts as a cofactor for cleavage of C3b by factor I (3, 4). FH is composed of 20 so-called short consensus repeat domains, SCRs, each with approximately 60 amino acid residues. Each SCR domain contains several conserved amino acid residues, including four invariant cysteines, and folds into a compact unit with β-strands and sheets (5, 6). The plasma concentration of FH is approximately 400 mg/L. Alternative splicing of the FH gene transcript gives rise to FHL-1, a 42-kDa protein that contains the first seven SCRs of FH and occurs in plasma at a concentration of 10 to 50 mg/L (7, 8, 9, 10). Like FH, FHL-1 displays cofactor and decay-accelerating activity, indicating a role in the regulation of the complement system. The regulatory domains required for these activities of FH and FHL-1 have been mapped to the four N-terminal SCR domains (11, 12, 13).

The alternative pathway is considered to be an important part of the nonspecific defense system against infection (14). A major effect of complement activation through the alternative pathway is to enhance phagocytosis of microorganisms on which C3b has been deposited (15). To avoid this host defense system, a variety of microorganisms has developed strategies to minimize C3b deposition. For example, it is well known that group A streptococci express type-specific surface proteins called M proteins, which act as major virulence factors for the organism by protecting it from phagocytosis (16). There is evidence that one mechanism by which M proteins exert their antiphagocytic effect is through the prevention of complement activation and opsonization (17). Indeed, several strains of group A streptococci have been shown to bind FH, thereby down-regulating C3 deposition on the streptococcal surface (18). This finding suggests that FH plays an important role in the resistance of group A streptococci to phagocytosis. However, some group A streptococcal strains may not bind FH, but bind the complement regulator C4b-binding protein (C4BP), a plasma protein that preferentially inhibits complement activation via the classical pathway (19, 20, 21). Thus, available data suggest that strains of group A streptococci bind at least one important serum regulator of complement activation, FH or C4BP.

In this work, we report a study of the interaction between FH, the structurally related FHL-1, and streptococcal M protein. Previous work has shown that purified M6 protein binds to FH (18), and FH deletion mutants were used recently to map a binding site for the M6 protein to the region encompassing SCR 6–10 (22). We now present evidence that two different M proteins, M5 and M6, not only bind FH but also bind FHL-1, and that their interaction with M proteins requires SCR 7, a domain common to both complement regulatory proteins.

The type M5 group A streptococcal strain Manfredo (23) was provided by Dr. Michael Kehoe (University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, U.K.). The M6 protein-expressing group A streptococcal strain JRS4, and its derivative JRS145, in which the emm6 gene has been deleted (24), were obtained from Dr. June Scott, Emory University (Atlanta, GA). The plasmid pLM02, a derivative of pBR322 carrying the entire emm5 gene (23), was kindly provided by Dr. M. Kehoe. The plasmid pLZ12spec is an Escherichia coli-streptococcal shuttle vector carrying a spectinomycin-resistance marker (25). Streptococci were grown in Todd-Hewitt broth (Difco, Detroit, MI), in 5% CO2 at 37°C for 16 h. Streptococci transformed with pLZ12spec, or derivatives thereof, were grown in medium supplemented with spectinomycin at 100 mg/L. E. coli LE392 transformed with pLZ12spec was grown in LB medium, supplemented with spectinomycin at 20 mg/L.

For binding studies, streptococci were harvested by centrifugation, washed twice in PBSA (0.15 M NaCl, 0.03 M phosphate, 0.02% sodium azide, pH 7.2), and were finally resuspended in PBSA supplemented with 0.05% Tween-20. Dilutions were made with PBSA containing the nonbinding E. coli strain LE392 at 2 × 109 bacteria/ml. The binding of radiolabeled proteins to bacteria, at different dilutions, was measured in a total volume of 250 μl PBSA, supplemented with 0.05% Tween-20. Following incubation for 1 h at 20°C, the samples were centrifuged (4000 × g, 10 min), the supernatant was discarded, and the remaining pellet was washed in 2 ml PBSA containing 0.05% Tween-20. After centrifugation and removal of the supernatant, the radioactivity associated with the pellet was measured in a gamma counter.

Streptococci (1 × 1010 cells) were resuspended in 1.5 ml of fresh human plasma containing 0.34 M EDTA. Following incubation for 1 h at 20°C, the bacterial cells were pelleted, and washed three times with PBSA containing 0.05% Tween-20. After a final centrifugation, proteins bound to the bacteria were eluted by incubation with 0.1 M glycine-HCl, pH 2, for 15 min. The bacteria were removed by centrifugation (10,000 × g, 5 min), and the proteins in the supernatant were analyzed by SDS-PAGE and immunoblot experiments.

The plasmid pLM02 that carries the entire emm5 gene was used to construct plasmid pKEJ1, as described (20). A 2.1-kb EcoRI/SphI fragment of pKEJ1 with the emm5 gene was inserted into pLZ12spec, resulting in plasmid pLZM5. pLZM5 was transformed into E. coli LE392, and finally transformed into the emm-negative strain JRS145.

Standard rDNA techniques were used (26). Ligase and restriction enzymes were purchased from Promega (Madison, WI). Transformation of E. coli was performed by the CaCl2 method (26). Electroporation of streptococci was conducted as described (27).

The M5 protein was purified from E. coli transformed with pLM02, using whole cell lysates, as described (20). rEnn4 protein was produced and purified as described (20). The surface protein Rib was isolated from group B streptococci, as described (28). Human fibrinogen was from Sigma Chemical (St. Louis, MO). Cloning and expression of rSCR 1–7 (rFHL-1), SCR 1–6, and SCR 1–5 fragments using the baculovirus system have been described previously (12, 29). The fragments had been fitted with histidine tags, allowing their purification by Ni2+, metal affinity chromatography (12). The cloning and expression of the FHR-3 and FHR-4 proteins in the baculovirus system have been described (30, 31) (P. F. Zipfel, manuscript in preparation).

Preparation of polyclonal rabbit antisera against FH and SCR 1–4 and generation of a mAb (mAb VIG8), specific for an epitope in SCR 19–20 of FH, have been described (12, 32).4

Proteins were labeled with 125I, using the chloramine-T method (33). The homogeneity of the radiolabeled proteins was checked by autoradiography after separation by SDS-PAGE. SDS-PAGE, under reducing conditions, was performed as described (34). For immunoblot analysis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Imobilon; Millipore, Bedford, MA). The membranes were blocked with blocking buffer (PBSA containing 0.25% gelatin, 0.25% Tween-20), and incubated with antiserum diluted in blocking buffer as indicated. The membranes were then incubated with 125I-labeled protein G (2 × 105 cpm/ml corresponding to approximately 10 ng/ml), in blocking buffer, and were finally washed in blocking buffer containing 0.5 M NaCl.

For competitive binding experiments, microtiter plates (Becton Dickinson, Mountain View, CA) were used. The wells were coated overnight at 4°C with 50 μl of the M5 protein solution (1 μg/ml in PBS). The following day, the wells were washed three times with PBSA containing 0.05% Tween-20, and then incubated for 2 h at 4°C with 100 μl blocking buffer. A mixture of the radiolabeled ligand and unlabeled protein, or heparin (25,000 IU/ml; Pharmacia, Piscataway, NJ), in a total volume of 50 μl blocking buffer, was added to the wells and the plate was incubated for 6 h at 4°C. Finally, the wells were washed four times with PBSA containing 0.05% Tween-20, and the radioactivity in the wells was counted in a gamma counter. Nonspecific binding to wells was analyzed by measuring the binding of radiolabeled proteins to uncoated wells.

Association and dissociation rate constants for binding of FH and FHL-1 to purified M5 protein were determined by surface plasmon resonance using a Biacore-X equipment, following the instructions supplied by the manufacturer. The M5 protein was resuspended in 10 mM sodium acetate (pH 3) and was coupled to CM-5 sensor chips using amine-coupling chemistry. The chips were regenerated using 30 mM HCl. Association and dissociation rates were measured at 25°C with a continuous flow rate of 20 μl/min, using PBS with 0.05% Tween-20 as buffer, and with FH at concentrations varying from 125 to 500 μg/ml or FHL-1 at concentrations from 7 to 60 μg/ml. The evaluation was done separately for the association and dissociation rate constants using a 1:1 model for the association with the Biaevaluation 3.0 software.

Several strains of group A streptococci have been shown to bind purified FH. For type M6 streptococci, this interaction has been demonstrated to occur through the surface-exposed M6 protein (18, 22, 35, 36). To investigate whether FH was also capable of binding to streptococci in a physiologic setting, two group A streptococcal strains, the M5 protein-expressing strain Manfredo and the M6 protein-expressing strain JRS4, were incubated with plasma supplemented with 0.34 M EDTA to prevent complement activation. After extensive washing, absorbed proteins were eluted using 0.1 M glycine-HCl, pH 2. The eluted proteins were analyzed by Western blot experiments, using rabbit anti-FH antiserum as the probe. The antiserum reacted with a 150-kDa protein corresponding to FH, showing that M protein-expressing bacteria are able to absorb this complement regulator from plasma (Fig. 1 A, lanes 1 and 4).

FIGURE 1.

Absorption of FH and FHL-1 from human plasma by M protein-expressing streptococci. Group A streptococci were incubated with EDTA-plasma. Following repeated washings, bound proteins were eluted with 0.1 M glycine-HCl, pH 2. The eluted proteins, or plasma diluted 1/25, were separated by SDS-PAGE and electroblotted to PVDF membranes, which were then probed with anti-FH antiserum. A, M5 Manfredo, an M5 protein-expressing strain (lane 1); JRS145(pLZ12spec), an M strain carrying plasmid pLZ12spec (lane 2); JRS145(pLZM5), carrying pLZ12spec with the M5 gene (lane 3); JRS4, an M6 protein-expressing strain (lane 4); JRS145, the M derivative of JRS4 (lane 5); human plasma (lane 6). B, The strain JRS145(pLZM5) was incubated with EDTA-plasma. Bound proteins were eluted with 0.1 M glycine-HCl, pH 2. The eluted proteins (lanes 1, 2, and 3), or plasma diluted 1/25 (lanes 4, 5, and 6), were separated by SDS-PAGE and electroblotted to PVDF membranes that were probed with rabbit anti-FH (lanes 1 and 4), rabbit anti-SCR 1–4 of FH (lane 2 and 5), or mAB VIG8 that recognizes an epitope in SCR 19–20 of FH (lanes 3 and 6).

FIGURE 1.

Absorption of FH and FHL-1 from human plasma by M protein-expressing streptococci. Group A streptococci were incubated with EDTA-plasma. Following repeated washings, bound proteins were eluted with 0.1 M glycine-HCl, pH 2. The eluted proteins, or plasma diluted 1/25, were separated by SDS-PAGE and electroblotted to PVDF membranes, which were then probed with anti-FH antiserum. A, M5 Manfredo, an M5 protein-expressing strain (lane 1); JRS145(pLZ12spec), an M strain carrying plasmid pLZ12spec (lane 2); JRS145(pLZM5), carrying pLZ12spec with the M5 gene (lane 3); JRS4, an M6 protein-expressing strain (lane 4); JRS145, the M derivative of JRS4 (lane 5); human plasma (lane 6). B, The strain JRS145(pLZM5) was incubated with EDTA-plasma. Bound proteins were eluted with 0.1 M glycine-HCl, pH 2. The eluted proteins (lanes 1, 2, and 3), or plasma diluted 1/25 (lanes 4, 5, and 6), were separated by SDS-PAGE and electroblotted to PVDF membranes that were probed with rabbit anti-FH (lanes 1 and 4), rabbit anti-SCR 1–4 of FH (lane 2 and 5), or mAB VIG8 that recognizes an epitope in SCR 19–20 of FH (lanes 3 and 6).

Close modal

In addition to FH, a prominent band with lower molecular mass was detected (Fig. 1,A, lanes 1 and 4). This protein was identified as FHL-1, since it reacted with antiserum to FH and antiserum directed against the amino-terminal four SCR domains shared between FH and FHL-1 (Fig. 1,B, lanes 1 and 2; Fig. 2), but failed to react with a mAb recognizing an epitope present in SCR 19–20 (Fig. 1,B, lane 3). Neither FHL-1 nor FH was absorbed by strain JRS145, a derivative of JRS4 carrying an inactivated emm6 gene (Fig. 1,A, lanes 2 and 5). However, transcomplementation of JRS145 with the emm5 gene, located on a shuttle plasmid, restored binding of both FHL-1 and FH (Fig. 1 A, lane 3). Identical results were obtained using plasma from three different individuals (data not shown). Taken together, these data indicate that streptococci expressing the M5 or M6 protein bind FH and FHL-1 present in whole human plasma.

FIGURE 2.

Structural comparison between FH and related molecules. A, Schematic representation of FH, FHL-1, rSCR 1–6, rSCR 1–5, and the two FH-related proteins FHR-3 and FHR-4. Each SCR is represented by a circle. Domains with similar structure have been aligned vertically. Circles representing SCR 7 of FH (and its counterpart in FHR-3) are shaded. B, Alignment of the amino acid sequences of two closely related SCRs: SCR 7 of FH/FHL-1 and SCR 2 of FHR-3. The numbers refer to the amino acid positions in the whole proteins.

FIGURE 2.

Structural comparison between FH and related molecules. A, Schematic representation of FH, FHL-1, rSCR 1–6, rSCR 1–5, and the two FH-related proteins FHR-3 and FHR-4. Each SCR is represented by a circle. Domains with similar structure have been aligned vertically. Circles representing SCR 7 of FH (and its counterpart in FHR-3) are shaded. B, Alignment of the amino acid sequences of two closely related SCRs: SCR 7 of FH/FHL-1 and SCR 2 of FHR-3. The numbers refer to the amino acid positions in the whole proteins.

Close modal

Although the experiments described above were performed in EDTA-plasma, it could not be excluded that the deposition of FHL-1 (and FH) was secondary to complement activation on the streptococcal surface. To address this issue, a recombinant baculovirus-produced and functionally active form of FHL-1 was analyzed for its ability to bind to group A streptococci. Indeed, radiolabeled rFHL-1 bound to M5 protein-expressing streptococci (Fig. 3 A). Moreover, rFHL-1 did not bind to the M-negative strain JRS145, but bound to JRS145 expressing the M5 protein. Thus, the binding of rFHL-1 was mediated by M protein.

FIGURE 3.

M protein-expressing streptococci bind rFHL-1, but not rSCR 1–6 or rSCR 1–5. Binding of the indicated radiolabeled SCR-containing proteins (each at 0.2 nM) to M protein-expressing or nonexpressing streptococcal strains was measured. M5 Manfredo is an M5 protein-expressing strain, JRS145 is an M strain, and JRS145(pLZM5) carries a plasmid with the M5 gene. A, Binding of 125I-rFHL-1 as a function of bacterial number; B, binding of radiolabeled ligands to 1 × 1010 streptoccoci. The data are based on three different experiments with duplicate samples. Representative experiments are shown in A, whereas results are expressed as the mean ± SEM in B.

FIGURE 3.

M protein-expressing streptococci bind rFHL-1, but not rSCR 1–6 or rSCR 1–5. Binding of the indicated radiolabeled SCR-containing proteins (each at 0.2 nM) to M protein-expressing or nonexpressing streptococcal strains was measured. M5 Manfredo is an M5 protein-expressing strain, JRS145 is an M strain, and JRS145(pLZM5) carries a plasmid with the M5 gene. A, Binding of 125I-rFHL-1 as a function of bacterial number; B, binding of radiolabeled ligands to 1 × 1010 streptoccoci. The data are based on three different experiments with duplicate samples. Representative experiments are shown in A, whereas results are expressed as the mean ± SEM in B.

Close modal

It was shown recently that rFH fragments containing SCR 1–10 and 6–20 both bound to streptococci expressing the M6 protein (22). In contrast, a FH deletion mutant protein without SCR 6–10 lacked this binding activity, suggesting that the M protein-binding site is located in this region of the complement regulatory protein (22). On the other hand, our data indicate that FHL-1, which comprises SCR 1–7 of FH, binds to the M5 and M6 proteins. Taken together, these data suggest that a binding site for M proteins is located in the SCR 6–7 region of FH and FHL-1. To further investigate the localization of the M protein-binding site in FH and FHL-1, binding experiments were performed, in which radiolabeled rFH fragments encompassing SCR 1–5 and SCR 1–6 were analyzed for their ability to bind to whole streptococci (Fig. 3 B). These experiments demonstrated that SCR 7 is required for the interaction, since only rFHL-1 showed reactivity with M protein-expressing group A streptococci.

The interaction between rFHL-1 and FH with the M5 protein was further analyzed with purified components. In Western blot experiments, rFHL-1 bound to purified rM5 protein (Fig. 4,A). The interaction was specific, since rFHL-1 failed to bind rEnn4, another group A streptococcal surface protein belonging to the M protein family, or protein Rib, a group B streptococcal surface protein (Fig. 4,A). 125I-labeled (rFHL-1) also bound to M5 protein immobilized in the wells of microtiter plates. The binding could be blocked completely by rFHL-1, but was inhibited only partially by rSCR 1–6, although added in a 1000-fold molar excess (Fig. 4 B).

FIGURE 4.

Interaction between rFHL-1 and purified M5 protein. A, The bacterial surface proteins M5 (1 μg), Enn4 (0.5 μg), and Rib (2 μg) were separated in SDS-PAGE (STAIN) or electroblotted to a PVDF membrane, which was subsequently probed with unlabeled rFHL-1, followed by anti-FH antiserum and finally by 125I-labeled protein G (BLOT). B, The M5 protein was immobilized in microtiter wells. After blocking of unoccupied sites with 0.25% gelatin and 0.25% Tween-20, the binding of 125I-rFHL-1 (at 0.5 nM) to the M5 protein was measured in the presence of unlabeled rFHL-1 or rSCR 1–6, each at 0.85 μM. The data are based on three different experiments with duplicate samples and are expressed as the mean ± SEM.

FIGURE 4.

Interaction between rFHL-1 and purified M5 protein. A, The bacterial surface proteins M5 (1 μg), Enn4 (0.5 μg), and Rib (2 μg) were separated in SDS-PAGE (STAIN) or electroblotted to a PVDF membrane, which was subsequently probed with unlabeled rFHL-1, followed by anti-FH antiserum and finally by 125I-labeled protein G (BLOT). B, The M5 protein was immobilized in microtiter wells. After blocking of unoccupied sites with 0.25% gelatin and 0.25% Tween-20, the binding of 125I-rFHL-1 (at 0.5 nM) to the M5 protein was measured in the presence of unlabeled rFHL-1 or rSCR 1–6, each at 0.85 μM. The data are based on three different experiments with duplicate samples and are expressed as the mean ± SEM.

Close modal

In surface plasmon resonance experiments, both rFHL-1 and FH reacted with immobilized M5 protein (Table I). Quantitative analysis showed that FHL-1 binds to the M5 proteins with a higher affinity as compared with FH.

Table I.

Quantitative analysis of the interactions between factor H or FHL-1 and immobilized M5 proteina

Association Rate Constant (M−1 s−1)Dissociation Rate Constant (s−1)Equilibrium Association Constant (M−1)Equilibrium Dissociation Constant (M)
Factor H 3.39 × 104 6.22 × 10−3 5.44 × 106 1.84 × 10−7 
FHL-1 1.88 × 105 1.23 × 10−2 1.54 × 107 6.51 × 10−8 
Association Rate Constant (M−1 s−1)Dissociation Rate Constant (s−1)Equilibrium Association Constant (M−1)Equilibrium Dissociation Constant (M)
Factor H 3.39 × 104 6.22 × 10−3 5.44 × 106 1.84 × 10−7 
FHL-1 1.88 × 105 1.23 × 10−2 1.54 × 107 6.51 × 10−8 
a

Association and dissociation rate constants were obtained by surface plasmon resonance analysis. The equilibrium constants were calculated from the rate constants.

The data presented above indicated that SCR 7 is of major importance for the binding of FH and FHL-1 to streptococcal M proteins. Further support for this conclusion was obtained in binding experiments with two FHR proteins, designated FHR-3 (30) and FHR-4 (31). FHR-3 contains a domain that is highly similar to SCR 7 of FH and FHL-1, whereas FHR-4 lacks such a domain (Fig. 2,B). Both proteins also contain a domain similar to SCR 6 of FH. In these experiments, 125I-rFHR-3 bound to M protein-expressing group A streptococci as efficiently as 125I-rFHL-1, whereas 125I-rFHR-4 failed to show any binding at all (Fig. 5).

FIGURE 5.

M5 protein-expressing streptococci bind rFHR-3, but not rFHR-4. Binding of radiolabeled rFHR-3 and rFHR-4 to M protein-expressing or nonexpressing streptococcal strains was measured. M5 Manfredo is an M5 protein-expressing strain, JRS145 is an M strain, and JRS145(pLZM5) carries a plasmid with the M5 gene. A, Binding of 125I-rFHR-3 (at 0.2 nM) as a function of bacterial number; B, binding of radiolabeled ligands (at 0.2 nM) to 1 × 1010 streptoccoci. The data are based on three different experiments with duplicate samples. Representative experiments are shown in A, whereas results are expressed as the mean ± SEM in B.

FIGURE 5.

M5 protein-expressing streptococci bind rFHR-3, but not rFHR-4. Binding of radiolabeled rFHR-3 and rFHR-4 to M protein-expressing or nonexpressing streptococcal strains was measured. M5 Manfredo is an M5 protein-expressing strain, JRS145 is an M strain, and JRS145(pLZM5) carries a plasmid with the M5 gene. A, Binding of 125I-rFHR-3 (at 0.2 nM) as a function of bacterial number; B, binding of radiolabeled ligands (at 0.2 nM) to 1 × 1010 streptoccoci. The data are based on three different experiments with duplicate samples. Representative experiments are shown in A, whereas results are expressed as the mean ± SEM in B.

Close modal

FH is known to bind heparin, and a major binding site for this ligand was localized recently to SCR 7 (37). Since our data indicated that streptococcal M proteins also bind in SCR 7, it was of interest to analyze whether the heparin- and M protein-binding sites in FHL-1 overlap. Therefore, heparin was used in various concentrations to inhibit the binding of 125I-rFHL-1 to purified M5 protein immobilized in microtiter wells. Interestingly, heparin was found to inhibit the interaction between FHL-1 and the M5 protein (Fig. 6). This interaction was not unspecific since heparin did not inhibit the interaction between fibrinogen and the M5 protein (Fig. 6). These data provide further evidence for the involvement of SCR 7 in the interaction between FH and FHL-1.

FIGURE 6.

Streptococcal M5 protein and heparin compete for the same binding region(s) in FHL-1. The M5 protein was immobilized in microtiter wells, and the binding of 125I-rFHL-1 (at 0.5 nM) or 125I-labeled fibrinogen (at 0.2 nM) was measured in the presence of the indicated concentrations of heparin. The experiments were performed in PBS containing 0.25% gelatin and 0.25% Tween-20. The data are representative of two different experiments with triplicate samples.

FIGURE 6.

Streptococcal M5 protein and heparin compete for the same binding region(s) in FHL-1. The M5 protein was immobilized in microtiter wells, and the binding of 125I-rFHL-1 (at 0.5 nM) or 125I-labeled fibrinogen (at 0.2 nM) was measured in the presence of the indicated concentrations of heparin. The experiments were performed in PBS containing 0.25% gelatin and 0.25% Tween-20. The data are representative of two different experiments with triplicate samples.

Close modal

Streptococcal M proteins are major virulence factors of group A streptococci. An important role of these proteins is to prevent phagocytosis by granulocytes (16). The mechanism by which M proteins exert their antiphagocytic property remains unknown, but prevention of opsonization by minimizing C3b deposition on the streptococcal surface could be an important function of the M proteins (17). Indeed, many group A streptococcal strains have been shown to bind one of the plasma complement inhibitors, FH or C4BP, the function of which could be to prevent opsonization (18, 19). In the present investigation, we identify a novel M protein ligand that can provide protection against complement deposition and activation at the streptococcal surface, namely FHL-1.

Although FHL-1 is an alternative splice product of the FH gene, the description “novel” for the interaction with M protein appears justified since accumulated evidence suggests that FH and FHL-1 have different properties. Thus, although these two proteins have similar cofactor activity, FHL-1 has a lower decay-accelerating activity (13). Moreover, FHL-1 is capable of promoting the spreading and attachment of eukaryotic cells as a consequence of its cell-binding activity, a property that is not displayed by FH (38). The finding that FHL-1 was absorbed from plasma by M protein-expressing group A streptococci as efficiently as FH, although circulating in a 10- to 50-fold lower concentration, also supports the conclusion that FHL-1 and FH have different properties. A possible explanation for this situation is provided by structural analyses of FH showing that SCRs may interact with one another and that FH can fold back on itself, thereby changing the functional properties of the molecule (39). However, there is no evidence that FH and FHL-1 do not use the same SCR(s) to interact with M protein.

Apart from identifying FHL-1 as a new M protein ligand with complement regulatory activity, the results presented in this work extend recent findings by Sharma and Pangburn (22), who showed that SCR 6–10 of FH are required for M protein binding. Since FHL-1 encompasses the first seven SCRs of FH, the region required for binding of M protein could be narrowed down to SCR 6–7. The finding that rSCR 1–6 did not bind to M protein demonstrated that SCR 7 is required for the binding, but did not exclude that SCR 6 is also required. However, two observations indicate that SCR 7 is more important than SCR 6 for the binding to M protein. First, the binding of radiolabeled FHL-1 to the M protein was inhibited almost completely by unlabeled FHL-1, while unlabeled rSCR 1–6 only caused a very limited inhibition. Second, the results obtained with rFHR-3 and rFHR-4, two other members of the FH family, also indicate that SCR 7 is crucial for the interaction. Indeed, binding to M5 protein-expressing streptococci was observed for FHR-3, which has an SCR 7-like domain, while FHR-4, which lacks such a domain, did not bind.

A major heparin-binding site has previously been localized to SCR 7 of FH (37). The inhibition tests reported in this work indicate that this heparin-binding site overlaps with, or is identical to, the M protein-binding site in SCR 7. Taken together, these data indicate that SCR 7 plays a key role in the function of FHL-1 (and FH), since this SCR domain may promote binding to surfaces coated with heparin or M protein. Since the complement regulatory activity of FH and FHL-1 resides in SCR 1–4 (11, 12, 13), it seems likely that FH and FHL-1 that has bound via SCR 7 retain the ability to down-regulate the alternative pathway. Thus, an interaction with FH or FHL-1, mediated through SCR 7, may provide surfaces, such as those provided by M protein-expressing streptococci, with protection against complement attack.

The finding that heparin and M proteins compete for the same binding site in FHL-1 may have consequences for the interpretation of data obtained in the phagocytosis assay widely used for assessing the function of M proteins (16). Since this assay is conducted routinely in heparin-containing blood, results obtained in this system may be influenced by the presence of heparin (40). Therefore, other methods to achieve anticoagulation should be considered, particularly when the phagocytosis assay is used to analyze the role of SCR-containing ligands. Indeed, a recent study indicates that the procedure used to prevent coagulation in phagocytosis experiments can influence the results (41).

In summary, we have presented evidence that the binding of FH and FHL-1 to streptococcal M proteins requires SCR 7. Further studies of this interaction are of obvious interest from the point of view of streptococcal pathogenesis. In addition, studies of this interaction may help in dissecting the various functions displayed by different SCR regions in FH and FHL-1.

1

This work was supported by grants from Deutsche Forschungsgemeinschaft (Zi432/1-3), Swedish Medical Research Council, (Grants 9926 and 9490), Ax:son Johnson Trust, Magnus Bergvall Trust, Crafoord Trust, Johan and Greta Kock Trust, O.E. and Edla Johansson Trust, Royal Physiographic Society in Lund, Swedish Society for Medical Research, Wiberg Trust, and Alfred Österlund Trust. This work is part of a doctoral thesis of J.H. at the Faculty of Biology, University of Hamburg.

3

Abbreviations used in this paper: FH, complement factor H; C4BP, complement factor 4b-binding protein; FHL-1, factor H-like protein-1; FHR, factor H-related protein; 125I-FHL-1, 125I-labeled factor H-like protein-1; 125I-FHR, 125I-labeled factor H-related protein; PVDF, polyvinylidene difluoride; SCR, short consensus repeat.

4

W. M. Prodinger, J. Hellwage, M. Spruth, M. P. Dierich, and P. F. Zipfel. The C-terminus of factor H: monoclonal antibodies inhibit heparin binding and identify epitopes common to factor H and factor H-related proteins. Submitted for publication.

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