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Complement Evasion Mediated by Enhancement of Captured Factor H: Implications for Protection of Self-Surfaces from Complement.

Herbert AP, Makou E, Chen ZA, Kerr H, Richards A, Rappsilber J, Barlow PN - J. Immunol. (2015)

Bottom Line: We found, using site-directed and truncation mutagenesis, surface plasmon resonance, nuclear magnetic resonance spectroscopy, and cross-linking and mass spectrometry, that the N-terminal domain of Streptococcus pneumoniae protein PspC (PspCN) not only binds FH extraordinarily tightly but also holds it in a previously uncharacterized conformation.This conformational change of FH doubles its affinity for C3b and increases 5-fold its ability to accelerate decay of the binary enzyme (C3bBb) responsible for converting C3 to C3b in an amplification loop.We propose that these bacterial proteins mimic molecular markers of self-surfaces, providing a compelling hypothesis for how FH prevents complement-mediated injury to host tissue while lacking efficacy on virtually all other surfaces.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Edinburgh, Edinburgh EH9 3FJ, United Kingdom;

No MeSH data available.


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Functional properties of wild-type and mutant FH with and without D39 PspCN. (A) Formation of C3bBb from C3b, FB, and FD on the chip surface (see Fig. 1A) and subsequent loss of Bb, monitored by SPR. Curves are color coded (see key). Note that 100 nM PspCN alone has no activity; 5 nM FH:(D39)PspCN complex accelerated decay better than did 10 nM FH alone. (B) Incrementally increasing FH from 10 to 80 nM correlates with greater fluid-phase FI cofactor activity, judging by stronger bands representing the C3b α′-chain cleavage products at 39 and 63 kDa, and loss of intensity of the band corresponding to intact α′-chain. PspCN lacks intrinsic cofactor activity whereas binding of PspCN to FH does not enhance its activity. (C) In a parallel experiment to that of (A), 5 nM FH(D1119G) and 5 nM wild-type FH have similar DAAs but (D39)PspCN enhances the activity of wild-type FH more than it enhances the activity of FH(D1119G).
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fig07: Functional properties of wild-type and mutant FH with and without D39 PspCN. (A) Formation of C3bBb from C3b, FB, and FD on the chip surface (see Fig. 1A) and subsequent loss of Bb, monitored by SPR. Curves are color coded (see key). Note that 100 nM PspCN alone has no activity; 5 nM FH:(D39)PspCN complex accelerated decay better than did 10 nM FH alone. (B) Incrementally increasing FH from 10 to 80 nM correlates with greater fluid-phase FI cofactor activity, judging by stronger bands representing the C3b α′-chain cleavage products at 39 and 63 kDa, and loss of intensity of the band corresponding to intact α′-chain. PspCN lacks intrinsic cofactor activity whereas binding of PspCN to FH does not enhance its activity. (C) In a parallel experiment to that of (A), 5 nM FH(D1119G) and 5 nM wild-type FH have similar DAAs but (D39)PspCN enhances the activity of wild-type FH more than it enhances the activity of FH(D1119G).

Mentions: If capture by PspCN exposes a C3d/TED-binding site in FH, does it modulate the principal role of FH, that is, inhibition of the C3b-amplification loop? We measured effects of (D39)PspCN on the convertase decay-accelerating activity (DAA) of FH (Fig. 1A). To do this, we monitored C3bBb formation on the SPR chip followed by its dissociation into C3b and Bb, before and after addition of FH or (D39)PspCN:FH (Fig. 7A). Predictably, 5 and 10 nM FH dose-dependently accelerated C3bBb decay; 100 nM (D39)PspCN alone had no effect. Strikingly, 5 nM (D39)PspCN:FH was at least twice as active as 10 nM FH (alone). We next checked whether (D39)PspCN:FH has enhanced cofactor activity (Fig. 1A) by incubating C3b and FI with either FH or (D39)PspC:FH. No difference was detected (Fig. 7B). That PspCN enhances DAA and affinity for C3b of FH, but does not increase cofactor activity, is readily explicable. For example, by increasing binding of FH to iC3b, PspCN could impede product (iC3b) release (from the FH:FI:iC3b complex) and slow down turnover. Another explanation is that FI recruitment to C3b:FH (perhaps unaffected by PspCN), rather than FH binding to C3b, limits efficacy as cofactor.


Complement Evasion Mediated by Enhancement of Captured Factor H: Implications for Protection of Self-Surfaces from Complement.

Herbert AP, Makou E, Chen ZA, Kerr H, Richards A, Rappsilber J, Barlow PN - J. Immunol. (2015)

Functional properties of wild-type and mutant FH with and without D39 PspCN. (A) Formation of C3bBb from C3b, FB, and FD on the chip surface (see Fig. 1A) and subsequent loss of Bb, monitored by SPR. Curves are color coded (see key). Note that 100 nM PspCN alone has no activity; 5 nM FH:(D39)PspCN complex accelerated decay better than did 10 nM FH alone. (B) Incrementally increasing FH from 10 to 80 nM correlates with greater fluid-phase FI cofactor activity, judging by stronger bands representing the C3b α′-chain cleavage products at 39 and 63 kDa, and loss of intensity of the band corresponding to intact α′-chain. PspCN lacks intrinsic cofactor activity whereas binding of PspCN to FH does not enhance its activity. (C) In a parallel experiment to that of (A), 5 nM FH(D1119G) and 5 nM wild-type FH have similar DAAs but (D39)PspCN enhances the activity of wild-type FH more than it enhances the activity of FH(D1119G).
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fig07: Functional properties of wild-type and mutant FH with and without D39 PspCN. (A) Formation of C3bBb from C3b, FB, and FD on the chip surface (see Fig. 1A) and subsequent loss of Bb, monitored by SPR. Curves are color coded (see key). Note that 100 nM PspCN alone has no activity; 5 nM FH:(D39)PspCN complex accelerated decay better than did 10 nM FH alone. (B) Incrementally increasing FH from 10 to 80 nM correlates with greater fluid-phase FI cofactor activity, judging by stronger bands representing the C3b α′-chain cleavage products at 39 and 63 kDa, and loss of intensity of the band corresponding to intact α′-chain. PspCN lacks intrinsic cofactor activity whereas binding of PspCN to FH does not enhance its activity. (C) In a parallel experiment to that of (A), 5 nM FH(D1119G) and 5 nM wild-type FH have similar DAAs but (D39)PspCN enhances the activity of wild-type FH more than it enhances the activity of FH(D1119G).
Mentions: If capture by PspCN exposes a C3d/TED-binding site in FH, does it modulate the principal role of FH, that is, inhibition of the C3b-amplification loop? We measured effects of (D39)PspCN on the convertase decay-accelerating activity (DAA) of FH (Fig. 1A). To do this, we monitored C3bBb formation on the SPR chip followed by its dissociation into C3b and Bb, before and after addition of FH or (D39)PspCN:FH (Fig. 7A). Predictably, 5 and 10 nM FH dose-dependently accelerated C3bBb decay; 100 nM (D39)PspCN alone had no effect. Strikingly, 5 nM (D39)PspCN:FH was at least twice as active as 10 nM FH (alone). We next checked whether (D39)PspCN:FH has enhanced cofactor activity (Fig. 1A) by incubating C3b and FI with either FH or (D39)PspC:FH. No difference was detected (Fig. 7B). That PspCN enhances DAA and affinity for C3b of FH, but does not increase cofactor activity, is readily explicable. For example, by increasing binding of FH to iC3b, PspCN could impede product (iC3b) release (from the FH:FI:iC3b complex) and slow down turnover. Another explanation is that FI recruitment to C3b:FH (perhaps unaffected by PspCN), rather than FH binding to C3b, limits efficacy as cofactor.

Bottom Line: We found, using site-directed and truncation mutagenesis, surface plasmon resonance, nuclear magnetic resonance spectroscopy, and cross-linking and mass spectrometry, that the N-terminal domain of Streptococcus pneumoniae protein PspC (PspCN) not only binds FH extraordinarily tightly but also holds it in a previously uncharacterized conformation.This conformational change of FH doubles its affinity for C3b and increases 5-fold its ability to accelerate decay of the binary enzyme (C3bBb) responsible for converting C3 to C3b in an amplification loop.We propose that these bacterial proteins mimic molecular markers of self-surfaces, providing a compelling hypothesis for how FH prevents complement-mediated injury to host tissue while lacking efficacy on virtually all other surfaces.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Edinburgh, Edinburgh EH9 3FJ, United Kingdom;

No MeSH data available.


Related in: MedlinePlus