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Identification of the high affinity binding site in the Streptococcus intermedius toxin intermedilysin for its membrane receptor, the human complement regulator CD59.

Hughes TR, Ross KS, Cowan GJ, Sivasankar B, Harris CL, Mitchell TJ, Morgan BP - Mol. Immunol. (2009)

Bottom Line: A peptide containing this sequence caused dose-dependent inhibition of intermedilysin-mediated lysis of human erythrocytes and rendered erythrocytes more susceptible to complement lysis.Surface plasmon resonance analysis of intermedilysin binding to immobilized CD59 revealed saturable fast-on, fast-off binding and a calculated affinity of 4.9 nM.The demonstration that a peptide modeled on the CD59-binding site inhibits intermedilysin-mediated haemolysis leads us to suggest that such peptides might be useful in treating infections caused by intermedilysin-producing bacteria.

View Article: PubMed Central - PubMed

Affiliation: Complement Biology Group, Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom.

ABSTRACT
The unique species specificity of the bacterial cytolysin intermedilysin is explained by its requirement for the human complement regulator CD59 as the primary receptor. Binding studies using individual domains of intermedilysin mapped the CD59-binding site to domain 4 and swap mutants between human and rabbit (non-intermedilysin-binding) CD59 implicated a short sequence (residues 42-59) in human CD59 in binding intermedilysin. We set out to map more closely the CD59 binding site in intermedilysin. We first looked for regions of homology between domain 4 in intermedilysin and the terminal complement components that bind CD59, C8 and C9. A nine amino acid sequence immediately adjacent the undecapeptide segment in intermedilysin domain 4 matched (5 of 9 identical, 3 of 9 conserved) a sequence in C9. A peptide containing this sequence caused dose-dependent inhibition of intermedilysin-mediated lysis of human erythrocytes and rendered erythrocytes more susceptible to complement lysis. Surface plasmon resonance analysis of intermedilysin binding to immobilized CD59 revealed saturable fast-on, fast-off binding and a calculated affinity of 4.9 nM. Substitution of three residues from the putative binding site caused a 5-fold reduction in lytic potency of intermedilysin and reduced affinity for immobilized CD59 by 2.5-fold. The demonstration that a peptide modeled on the CD59-binding site inhibits intermedilysin-mediated haemolysis leads us to suggest that such peptides might be useful in treating infections caused by intermedilysin-producing bacteria.

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Direct demonstration of interaction between the ILY peptide and CD59. CD59 was immobilized on a CM5-S chip surface via thiol-coupling. Interaction with the ILY-derived peptide (A) or jumbled peptide (B) was analysed at the indicated concentrations in HBS-P. Spikes at the beginning and end of the injection period in (A) are buffer artefacts that do not influence analysis of the data. (C) The affinity of the ILY peptide for CD59 was determined at steady state by flowing peptide over immobilised CD59 at a range of concentrations; the data were analysed using BIAcore T100 evaluation software to obtain the affinity of the ILY peptide for CD59. (D) ELISA plates coated with CD59 were incubated in buffer alone or with either the ILY-derived peptide (solid bars) or its jumbled control (open bars) at 1 and 100 μM final concentration. The peptides were removed and ILY (4 μM) was added for 5 min at 37 °C. After washing, binding of ILY to immobilised CD59 was detected with a polyclonal anti-ILY antiserum. Each bar is the mean of quadruplicate wells. Asterisks denote significant differences (P < 0.05).
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fig3: Direct demonstration of interaction between the ILY peptide and CD59. CD59 was immobilized on a CM5-S chip surface via thiol-coupling. Interaction with the ILY-derived peptide (A) or jumbled peptide (B) was analysed at the indicated concentrations in HBS-P. Spikes at the beginning and end of the injection period in (A) are buffer artefacts that do not influence analysis of the data. (C) The affinity of the ILY peptide for CD59 was determined at steady state by flowing peptide over immobilised CD59 at a range of concentrations; the data were analysed using BIAcore T100 evaluation software to obtain the affinity of the ILY peptide for CD59. (D) ELISA plates coated with CD59 were incubated in buffer alone or with either the ILY-derived peptide (solid bars) or its jumbled control (open bars) at 1 and 100 μM final concentration. The peptides were removed and ILY (4 μM) was added for 5 min at 37 °C. After washing, binding of ILY to immobilised CD59 was detected with a polyclonal anti-ILY antiserum. Each bar is the mean of quadruplicate wells. Asterisks denote significant differences (P < 0.05).

Mentions: To confirm that the ILY-derived peptide directly bound CD59, we examined by SPR its interaction with CD59 immobilised through its carboxy terminus to dextran on a Biacore chip. First, a concentration series of the ILY peptide and its jumbled control were flowed over the CD59 chip; the ILY peptide bound specifically but with very fast on/off rates (association/dissociation), whereas the jumbled control peptide did not bind (Fig. 3A and B). The affinity of the ILY peptide for CD59 was determined by steady state analysis of data obtained from a peptide concentration series run in duplicate over the CD59 coated chip (Fig. 3C). The measured affinity was 4.92 × 10−6 M with a Chi2 value of 0.45. By comparison, the affinity of native ILY for CD59, measured in the same way, was 4.29 × 10−9 M, a three-log higher affinity (Fig. 4C). The integrity of the CD59 immobilised on the chip surface was confirmed before and after passage of the peptides by flowing the anti-CD59 monoclonal antibody MEM43, which bound strongly and comparably before and after peptide analysis (data not shown). When interactions were tested in the reverse orientation, with peptides coupled to the chip surface through their terminal cysteine residues and flowing CD59 over the coated surface, no specific interaction was observed despite efficient coupling of peptide to the chip surface (negative data not shown). We concluded that the interaction was hindered by a combination of small peptide size and tethering to a fixed surface, impeding its ability to interact with its binding site on CD59 (data not shown). To provide further confirmation of the specificity of this interaction we examined the capacity of the ILY-derived peptide to directly inhibit the binding of ILY to immobilised CD59 in an ELISA assay. The ILY-derived peptide, but not its jumbled control, significantly inhibited the binding of ILY (Fig. 3D). Poor solubility of the peptides limited our ability to examine the effects of higher amounts of peptides in these competition assays.


Identification of the high affinity binding site in the Streptococcus intermedius toxin intermedilysin for its membrane receptor, the human complement regulator CD59.

Hughes TR, Ross KS, Cowan GJ, Sivasankar B, Harris CL, Mitchell TJ, Morgan BP - Mol. Immunol. (2009)

Direct demonstration of interaction between the ILY peptide and CD59. CD59 was immobilized on a CM5-S chip surface via thiol-coupling. Interaction with the ILY-derived peptide (A) or jumbled peptide (B) was analysed at the indicated concentrations in HBS-P. Spikes at the beginning and end of the injection period in (A) are buffer artefacts that do not influence analysis of the data. (C) The affinity of the ILY peptide for CD59 was determined at steady state by flowing peptide over immobilised CD59 at a range of concentrations; the data were analysed using BIAcore T100 evaluation software to obtain the affinity of the ILY peptide for CD59. (D) ELISA plates coated with CD59 were incubated in buffer alone or with either the ILY-derived peptide (solid bars) or its jumbled control (open bars) at 1 and 100 μM final concentration. The peptides were removed and ILY (4 μM) was added for 5 min at 37 °C. After washing, binding of ILY to immobilised CD59 was detected with a polyclonal anti-ILY antiserum. Each bar is the mean of quadruplicate wells. Asterisks denote significant differences (P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2697320&req=5

fig3: Direct demonstration of interaction between the ILY peptide and CD59. CD59 was immobilized on a CM5-S chip surface via thiol-coupling. Interaction with the ILY-derived peptide (A) or jumbled peptide (B) was analysed at the indicated concentrations in HBS-P. Spikes at the beginning and end of the injection period in (A) are buffer artefacts that do not influence analysis of the data. (C) The affinity of the ILY peptide for CD59 was determined at steady state by flowing peptide over immobilised CD59 at a range of concentrations; the data were analysed using BIAcore T100 evaluation software to obtain the affinity of the ILY peptide for CD59. (D) ELISA plates coated with CD59 were incubated in buffer alone or with either the ILY-derived peptide (solid bars) or its jumbled control (open bars) at 1 and 100 μM final concentration. The peptides were removed and ILY (4 μM) was added for 5 min at 37 °C. After washing, binding of ILY to immobilised CD59 was detected with a polyclonal anti-ILY antiserum. Each bar is the mean of quadruplicate wells. Asterisks denote significant differences (P < 0.05).
Mentions: To confirm that the ILY-derived peptide directly bound CD59, we examined by SPR its interaction with CD59 immobilised through its carboxy terminus to dextran on a Biacore chip. First, a concentration series of the ILY peptide and its jumbled control were flowed over the CD59 chip; the ILY peptide bound specifically but with very fast on/off rates (association/dissociation), whereas the jumbled control peptide did not bind (Fig. 3A and B). The affinity of the ILY peptide for CD59 was determined by steady state analysis of data obtained from a peptide concentration series run in duplicate over the CD59 coated chip (Fig. 3C). The measured affinity was 4.92 × 10−6 M with a Chi2 value of 0.45. By comparison, the affinity of native ILY for CD59, measured in the same way, was 4.29 × 10−9 M, a three-log higher affinity (Fig. 4C). The integrity of the CD59 immobilised on the chip surface was confirmed before and after passage of the peptides by flowing the anti-CD59 monoclonal antibody MEM43, which bound strongly and comparably before and after peptide analysis (data not shown). When interactions were tested in the reverse orientation, with peptides coupled to the chip surface through their terminal cysteine residues and flowing CD59 over the coated surface, no specific interaction was observed despite efficient coupling of peptide to the chip surface (negative data not shown). We concluded that the interaction was hindered by a combination of small peptide size and tethering to a fixed surface, impeding its ability to interact with its binding site on CD59 (data not shown). To provide further confirmation of the specificity of this interaction we examined the capacity of the ILY-derived peptide to directly inhibit the binding of ILY to immobilised CD59 in an ELISA assay. The ILY-derived peptide, but not its jumbled control, significantly inhibited the binding of ILY (Fig. 3D). Poor solubility of the peptides limited our ability to examine the effects of higher amounts of peptides in these competition assays.

Bottom Line: A peptide containing this sequence caused dose-dependent inhibition of intermedilysin-mediated lysis of human erythrocytes and rendered erythrocytes more susceptible to complement lysis.Surface plasmon resonance analysis of intermedilysin binding to immobilized CD59 revealed saturable fast-on, fast-off binding and a calculated affinity of 4.9 nM.The demonstration that a peptide modeled on the CD59-binding site inhibits intermedilysin-mediated haemolysis leads us to suggest that such peptides might be useful in treating infections caused by intermedilysin-producing bacteria.

View Article: PubMed Central - PubMed

Affiliation: Complement Biology Group, Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom.

ABSTRACT
The unique species specificity of the bacterial cytolysin intermedilysin is explained by its requirement for the human complement regulator CD59 as the primary receptor. Binding studies using individual domains of intermedilysin mapped the CD59-binding site to domain 4 and swap mutants between human and rabbit (non-intermedilysin-binding) CD59 implicated a short sequence (residues 42-59) in human CD59 in binding intermedilysin. We set out to map more closely the CD59 binding site in intermedilysin. We first looked for regions of homology between domain 4 in intermedilysin and the terminal complement components that bind CD59, C8 and C9. A nine amino acid sequence immediately adjacent the undecapeptide segment in intermedilysin domain 4 matched (5 of 9 identical, 3 of 9 conserved) a sequence in C9. A peptide containing this sequence caused dose-dependent inhibition of intermedilysin-mediated lysis of human erythrocytes and rendered erythrocytes more susceptible to complement lysis. Surface plasmon resonance analysis of intermedilysin binding to immobilized CD59 revealed saturable fast-on, fast-off binding and a calculated affinity of 4.9 nM. Substitution of three residues from the putative binding site caused a 5-fold reduction in lytic potency of intermedilysin and reduced affinity for immobilized CD59 by 2.5-fold. The demonstration that a peptide modeled on the CD59-binding site inhibits intermedilysin-mediated haemolysis leads us to suggest that such peptides might be useful in treating infections caused by intermedilysin-producing bacteria.

Show MeSH
Related in: MedlinePlus