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. 2008 Dec 1;181(11):7463-7.
doi: 10.4049/jimmunol.181.11.7463.

Cutting Edge: Members of the Staphylococcus Aureus Extracellular Fibrinogen-Binding Protein Family Inhibit the Interaction of C3d With Complement Receptor 2

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Free PMC article

Cutting Edge: Members of the Staphylococcus Aureus Extracellular Fibrinogen-Binding Protein Family Inhibit the Interaction of C3d With Complement Receptor 2

Daniel Ricklin et al. J Immunol. .
Free PMC article

Abstract

Staphylococcus aureus expresses a highly diversified arsenal of immune evasion proteins, many of which target the complement system. The extracellular fibrinogen-binding protein (Efb) and the Efb homologous protein (Ehp) have previously been demonstrated to bind to C3 and inhibit complement activation and amplification. In this study we present the first evidence that Efb and Ehp are also capable of inhibiting the interaction of C3d with complement receptor 2 (CR2), which plays an important role in B cell activation and maturation. The C-terminal domain of Efb efficiently blocked this interaction both in surface plasmon resonance-based competition studies and cellular assays and prevented the CR2-mediated stimulation of B cells. Furthermore, analyses of the available structural data were consistent with a molecular mechanism that reflects both steric and electrostatic effects on the C3d-CR2 interaction. Our study therefore suggests that S. aureus may disrupt both the innate and adaptive immune responses with a single protein module.

Figures

FIGURE 1
FIGURE 1
Efb-C efficiently inhibits the C3d-CR2 interaction in two competition assays. A, CR2 bound to site-specifically immobilized C3d in a biphasic manner (blue signal). Whereas Efb-C forms a stable complex with C3d (dashed red signal) and completely inhibits binding of CR2 (solid red signal), its impaired mutant RA/NA only shows weak binding (dashed green line) and does not affect subsequent binding of CR2 (solid green line). As expected, Ehp showed the same inhibitory activity as Efb-C (inset). B, In a solution competition assay, CR2 was captured via the nonblocking mAb HB-5 and mixtures of C3d (1 μM; blue) with increasing amounts (125 nM to 8 μM) of either Efb-C wild type (red) or RA/NA (green) were injected. Again, wild-type Efb-C blocked binding of C3d at slight molar excess, whereas much higher concentrations were required in case of the mutant. Typical SPR responses for C3d alone (blue) and C3d in competition with Efb-C wild type (red) and RA/NA (green) are shown in the inset.
FIGURE 2
FIGURE 2
Efb-C blocks the binding and function of polymeric C3/C3d on CR2. A, Binding of pC3 to Raji cells (gray) caused a shift in fluorescence intensity (blue) that was only inhibited by the blocking anti-CR2 mAb OKB-7 (orange) but not by the nonblocking mAb HB-5 (cyan), indicating specific binding of pC3 to CR2. B, Although preincubation of pC3 with Efb-C (red) led to strong inhibition of the pC3-CR2 binding, an equal amount of the RA/NA mutant caused only a minor shift in intensity (green). This demonstrates specific inhibition of pC3 to Raji cell-bound CR2 by Efb-C. C, Mouse splenocytes were stimulated with mixed tetramers of anti-IgM and C3d on streptavidin (SA) as detected by calcium mobilization (blue). Excess of Efb-C inhibited the stimulation completely (red), whereas no inhibition was observed for the RA/NA mutant (green; inset).
FIGURE 3
FIGURE 3
The binding sites of Efb-C and CR2 are likely to overlap on C3d.A, Mutational analysis of the acidic cleft on C3d (10) previously identified several key residues in this area (marked blue on C3d in green). The C3d-contact residues for Efb-C (red), as derived from the cocrystal structure (Protein Data Bank code 2GOX; Ref. 7), largely overlap with this region, with residue D1156 (magenta) shared across both binding sites. B, Small-angle x-ray scattering studies of CR2 SCR1–2 in interaction with C3d (Protein Data Bank code 1W2S; Ref. 18) further support this finding, because alignment with the C3d:Efb-C cocrystal reveals that SCR1 of CR2 (blue) and Efb-C (red) bind to similar sites on C3d (green). Although the currently available cocrystal between C3d and CR2 SCR1–2 (PDB: 1GHQ; Ref. 17) only shows contacts of C3d with SCR2 (inset), subsequent studies have suggested that this does not reflect the binding in solution and that the binding of CR2 SCR1 is pivotal for correct recognition and function. Analogous studies with the structurally similar C3d: Ehp complex (Protein Data Bank code 2NOJ; Ref. 6) revealed the same results and are therefore omitted. Structural alignment and visualization was done using PyMOL (DeLano Scientific).

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