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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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Inhibition of C5aR activity by CHIPS variants with C-terminal truncation. Fluo-3AM labeled U937/C5aR transfectants were preincubated with CHIPS variants and stimulated with C5a. Results are expressed as percentage inhibition of buffer treated cells.
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Figure 6: Inhibition of C5aR activity by CHIPS variants with C-terminal truncation. Fluo-3AM labeled U937/C5aR transfectants were preincubated with CHIPS variants and stimulated with C5a. Results are expressed as percentage inhibition of buffer treated cells.

Mentions: To investigate whether the selected new CHIPS variants with reduced antibody binding were still functional in preventing C5a induced activation of the C5aR, the 9 mutants were purified and analyzed in a calcium flux assay. Serial dilutions of the variants were preincubated with Fluo-3AM labeled U937/C5aR cells and the cells were stimulated with 1 nM C5a. Flow cytometry data showed that mutants N55K, N68A, K69T, K92E and N111K retained full C5aR blocking activity at 1 μg/ml. In contrast, Y97K completely abolished and K95S, K100A and S107N partially abolished the biological activity in comparison to wt C-terminally truncated CHIPS (Figure 6). Thus, some mutations that interfere with antibody binding are not compatible with biological function.


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)

Inhibition of C5aR activity by CHIPS variants with C-terminal truncation. Fluo-3AM labeled U937/C5aR transfectants were preincubated with CHIPS variants and stimulated with C5a. Results are expressed as percentage inhibition of buffer treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Inhibition of C5aR activity by CHIPS variants with C-terminal truncation. Fluo-3AM labeled U937/C5aR transfectants were preincubated with CHIPS variants and stimulated with C5a. Results are expressed as percentage inhibition of buffer treated cells.
Mentions: To investigate whether the selected new CHIPS variants with reduced antibody binding were still functional in preventing C5a induced activation of the C5aR, the 9 mutants were purified and analyzed in a calcium flux assay. Serial dilutions of the variants were preincubated with Fluo-3AM labeled U937/C5aR cells and the cells were stimulated with 1 nM C5a. Flow cytometry data showed that mutants N55K, N68A, K69T, K92E and N111K retained full C5aR blocking activity at 1 μg/ml. In contrast, Y97K completely abolished and K95S, K100A and S107N partially abolished the biological activity in comparison to wt C-terminally truncated CHIPS (Figure 6). Thus, some mutations that interfere with antibody binding are not compatible with biological function.

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