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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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Affinity-purified anti-CHIPS IgG recognize non-linear epitopes. Affinity-purified anti-CHIPS1–121 IgG was tested for the ability to bind CHIPS derived 25-mer peptides or full-length CHIPS in ELISA (A). Full-length CHIPS1–121 or CHIPS with N-terminal truncation (CHIPS ΔN), or CHIPS with truncations in both the N- and C-terminal ends (CHIPS ΔN/C) were compared for reactivity with different human affinity-purified anti-full-length CHIPS1–121 IgG (B), anti-CHIPS ΔN/C IgG (C), and the CHIPS specific mouse monoclonal antibody 2G8 as control (D).
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Figure 2: Affinity-purified anti-CHIPS IgG recognize non-linear epitopes. Affinity-purified anti-CHIPS1–121 IgG was tested for the ability to bind CHIPS derived 25-mer peptides or full-length CHIPS in ELISA (A). Full-length CHIPS1–121 or CHIPS with N-terminal truncation (CHIPS ΔN), or CHIPS with truncations in both the N- and C-terminal ends (CHIPS ΔN/C) were compared for reactivity with different human affinity-purified anti-full-length CHIPS1–121 IgG (B), anti-CHIPS ΔN/C IgG (C), and the CHIPS specific mouse monoclonal antibody 2G8 as control (D).

Mentions: In order to map antibody epitopes on CHIPS, affinity purification of human anti-CHIPS IgG was initially performed on the wt CHIPS1–121 protein. Pooled human IgG (IV-IgG) was affinity-purified using a column packed with immobilized CHIPS1–121 resin. We tested the binding of affinity-purified anti-CHIPS1–121 antibodies to a set of CHIPS derived 25-mer peptides spanning the complete CHIPS1–121 sequence. As shown in Figure 2A, linear epitopes do appear to be confined to the N-terminus of CHIPS. To confirm the presence of conformational epitopes, we tested the reactivity of two different affinity-purified antibody preparations (anti-full-length CHIPS1–121 and anti-CHIPS ΔN/C) to wt CHIPS1–121, CHIPS ΔN and CHIPS ΔN/C. Figure 2B–D shows that both preparations of affinity isolated antibodies react with all three CHIPS variants. There is no significant difference in reactivity towards the different CHIPS variants between the preparations. Reactivity of the different CHIPS variants with the control antibody 2G8 (a monoclonal mouse anti-CHIPS antibody [27]) was also comparable, indicating that all CHIPS variants coated equally well. These data also imply that epitopes outside the N-terminus may be of importance for antibody binding and that some of these epitopes are conformational.


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)

Affinity-purified anti-CHIPS IgG recognize non-linear epitopes. Affinity-purified anti-CHIPS1–121 IgG was tested for the ability to bind CHIPS derived 25-mer peptides or full-length CHIPS in ELISA (A). Full-length CHIPS1–121 or CHIPS with N-terminal truncation (CHIPS ΔN), or CHIPS with truncations in both the N- and C-terminal ends (CHIPS ΔN/C) were compared for reactivity with different human affinity-purified anti-full-length CHIPS1–121 IgG (B), anti-CHIPS ΔN/C IgG (C), and the CHIPS specific mouse monoclonal antibody 2G8 as control (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Affinity-purified anti-CHIPS IgG recognize non-linear epitopes. Affinity-purified anti-CHIPS1–121 IgG was tested for the ability to bind CHIPS derived 25-mer peptides or full-length CHIPS in ELISA (A). Full-length CHIPS1–121 or CHIPS with N-terminal truncation (CHIPS ΔN), or CHIPS with truncations in both the N- and C-terminal ends (CHIPS ΔN/C) were compared for reactivity with different human affinity-purified anti-full-length CHIPS1–121 IgG (B), anti-CHIPS ΔN/C IgG (C), and the CHIPS specific mouse monoclonal antibody 2G8 as control (D).
Mentions: In order to map antibody epitopes on CHIPS, affinity purification of human anti-CHIPS IgG was initially performed on the wt CHIPS1–121 protein. Pooled human IgG (IV-IgG) was affinity-purified using a column packed with immobilized CHIPS1–121 resin. We tested the binding of affinity-purified anti-CHIPS1–121 antibodies to a set of CHIPS derived 25-mer peptides spanning the complete CHIPS1–121 sequence. As shown in Figure 2A, linear epitopes do appear to be confined to the N-terminus of CHIPS. To confirm the presence of conformational epitopes, we tested the reactivity of two different affinity-purified antibody preparations (anti-full-length CHIPS1–121 and anti-CHIPS ΔN/C) to wt CHIPS1–121, CHIPS ΔN and CHIPS ΔN/C. Figure 2B–D shows that both preparations of affinity isolated antibodies react with all three CHIPS variants. There is no significant difference in reactivity towards the different CHIPS variants between the preparations. Reactivity of the different CHIPS variants with the control antibody 2G8 (a monoclonal mouse anti-CHIPS antibody [27]) was also comparable, indicating that all CHIPS variants coated equally well. These data also imply that epitopes outside the N-terminus may be of importance for antibody binding and that some of these epitopes are conformational.

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