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Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus.

Gustafsson E, Haas PJ, Walse B, Hijnen M, Furebring C, Ohlin M, van Strijp JA, van Kessel KP - BMC Immunol. (2009)

Bottom Line: Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS.A few mutations were shown to affect this biological function as well as the antibody binding.This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG.

View Article: PubMed Central - HTML - PubMed

Affiliation: Alligator Bioscience AB, Scheelev. 19A, S-223 70 Lund, Sweden. erg@alligatorbioscience.com

ABSTRACT

Background: The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity.

Results: In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31-121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding.

Conclusion: Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG.

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Synthetic peptides can be used to purify CHIPS specific antibodies. Binding of affinity-purified anti-CHIPS ΔN/C IgG (a-CHIPS), anti-group 1 peptide (a-Gr 1 pep) and IV-IgG to immobilized CHIPS1–121 were measured in ELISA (A) and SPR (B). Black bars represent binding of the different affinity-purified antibodies. White bars show binding of antibodies that were pre-incubated with CHIPS1–121.
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Figure 4: Synthetic peptides can be used to purify CHIPS specific antibodies. Binding of affinity-purified anti-CHIPS ΔN/C IgG (a-CHIPS), anti-group 1 peptide (a-Gr 1 pep) and IV-IgG to immobilized CHIPS1–121 were measured in ELISA (A) and SPR (B). Black bars represent binding of the different affinity-purified antibodies. White bars show binding of antibodies that were pre-incubated with CHIPS1–121.

Mentions: To investigate the specificity of antibodies purified on the selected peptides, a synthetic peptide from the group 1 consensus sequence (MNKSYTI) was synthesized. As the N-terminus of CHIPS was shown to contain linear epitopes for human antibodies, a N-terminal peptide (aa 1–38) of CHIPS was used for comparison. The synthesized peptides were designed to contain a C-terminal cysteine residue that allowed immobilization by thiol coupling chemistry. They were subsequently coupled to activated Sepharose to create affinity columns, which were used to capture peptide-specific antibodies from the human polyclonal IgG pool (IV-IgG). Binding of the affinity-purified anti-peptide antibodies to the CHIPS1–121 molecule was studied by ELISA and Surface Plasmon Resonance (SPR) (Figure 4). The affinity-purified anti-group 1 peptide antibodies, the anti-CHIPS ΔN/C antibodies as well as affinity-purified anti-1–38 peptide antibodies were found to bind to the native CHIPS1–121 protein in ELISA and SPR. Preincubation with CHIPS1–121 abolished this binding. Without affinity purification, the same concentration of IV-IgG showed no detectable binding.


Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus.

Gustafsson E, Haas PJ, Walse B, Hijnen M, Furebring C, Ohlin M, van Strijp JA, van Kessel KP - BMC Immunol. (2009)

Synthetic peptides can be used to purify CHIPS specific antibodies. Binding of affinity-purified anti-CHIPS ΔN/C IgG (a-CHIPS), anti-group 1 peptide (a-Gr 1 pep) and IV-IgG to immobilized CHIPS1–121 were measured in ELISA (A) and SPR (B). Black bars represent binding of the different affinity-purified antibodies. White bars show binding of antibodies that were pre-incubated with CHIPS1–121.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662796&req=5

Figure 4: Synthetic peptides can be used to purify CHIPS specific antibodies. Binding of affinity-purified anti-CHIPS ΔN/C IgG (a-CHIPS), anti-group 1 peptide (a-Gr 1 pep) and IV-IgG to immobilized CHIPS1–121 were measured in ELISA (A) and SPR (B). Black bars represent binding of the different affinity-purified antibodies. White bars show binding of antibodies that were pre-incubated with CHIPS1–121.
Mentions: To investigate the specificity of antibodies purified on the selected peptides, a synthetic peptide from the group 1 consensus sequence (MNKSYTI) was synthesized. As the N-terminus of CHIPS was shown to contain linear epitopes for human antibodies, a N-terminal peptide (aa 1–38) of CHIPS was used for comparison. The synthesized peptides were designed to contain a C-terminal cysteine residue that allowed immobilization by thiol coupling chemistry. They were subsequently coupled to activated Sepharose to create affinity columns, which were used to capture peptide-specific antibodies from the human polyclonal IgG pool (IV-IgG). Binding of the affinity-purified anti-peptide antibodies to the CHIPS1–121 molecule was studied by ELISA and Surface Plasmon Resonance (SPR) (Figure 4). The affinity-purified anti-group 1 peptide antibodies, the anti-CHIPS ΔN/C antibodies as well as affinity-purified anti-1–38 peptide antibodies were found to bind to the native CHIPS1–121 protein in ELISA and SPR. Preincubation with CHIPS1–121 abolished this binding. Without affinity purification, the same concentration of IV-IgG showed no detectable binding.

Bottom Line: Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS.A few mutations were shown to affect this biological function as well as the antibody binding.This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG.

View Article: PubMed Central - HTML - PubMed

Affiliation: Alligator Bioscience AB, Scheelev. 19A, S-223 70 Lund, Sweden. erg@alligatorbioscience.com

ABSTRACT

Background: The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity.

Results: In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31-121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding.

Conclusion: Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG.

Show MeSH
Related in: MedlinePlus