Analysis of receptor/ligand interactions using whole-molecule randomly-mutated ligand libraries

Cain, Stuart A.; Ratcliffe, Charlotte F.; Williams, David M.; Harris, Victoria; Monk, Peter N.

doi:10.1016/s0022-1759(00)00282-9

Cited by 11 publications

(6 citation statements)

References 14 publications

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“…Receptor activation in RBL cells was measured as the release of β-hexosaminidase from intracellular granules, as described ( Cain et al, 2000 ). The percentage of β-hexosaminidase release was calculated as a percentage of the release in the absence of CHIPS 28–149 .…”

Section: Methodsmentioning

confidence: 99%

Characterisation of receptor binding by the chemotaxis inhibitory protein of Staphylococcus aureus and the effects of the host immune response

Wright

Higginbottom

Philippe

et al. 2007

Molecular Immunology

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The chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is reported to bind to the receptors for C5a and formylated peptides and has been proposed as a promising lead for the development of new anti-inflammatory compounds. Here we have examined the receptor specificity and mode of action of recombinant CHIPS28–149 and also the immune response to CHIPS28–149 in patients with S. aureus infections and in uninfected controls. Recombinant CHIPS28–149 bound with high affinity to the human C5a receptor (C5aR), but had low affinity for the second C5a receptor, C5L2, and the formyl peptide receptor, FPR. Although ligand binding to C5aR was potently inhibited, CHIPS28–149 had much weaker effects on ligand binding to C5L2 and FPR. Similarly, CHIPS28–149 potently inhibited the ligand-induced activation of C5aR but was less potent at inhibition via FPR. NMR studies showed that CHIPS28–149 bound directly to the N-terminus of C5aR but not C5L2, and CHIPS28–149 residues involved in the interaction were identified by chemical shift analysis. All human sera examined contained high titres of IgG and IgA reactivity against CHIPS28–149, and no correlation was observed between infection status at the time of serum collection and antibody titre. Individual serum samples promoted or inhibited the binding of CHIPS28–149 to C5aR, or had no effect. IgG depletion of serum samples abrogated the effects on CHIPS binding, demonstrating that these were antibody mediated. Sera from infected individuals were more likely to inhibit CHIPS28–149 binding than sera from healthy controls. However, high antibody titres correlated well with both inhibition and enhancement of CHIPS28–149 binding to C5aR; this suggests that the inhibitory effect relates to epitope specificity rather than greater antibody binding. We conclude that CHIPS is likely to be too immunogenic to be used as an anti-inflammatory treatment but that some antibodies against CHIPS may be useful in the treatment of S. aureus infections.

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Section: Methodsmentioning

confidence: 99%

Characterisation of receptor binding by the chemotaxis inhibitory protein of Staphylococcus aureus and the effects of the host immune response

Wright

Higginbottom

Philippe

et al. 2007

Molecular Immunology

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“…Cellular Activation Assays-Cellular activation was measured as the release of ␤-hexosaminidase from intracellular granules (16). The percentage of ␤-hexosaminidase release was calculated as a percentage of the maximal release (1 M C5a or C3a for CD88 and C3aR, respectively) or total cellular ␤-hexosaminidase (C5L2).…”

Section: Methodsmentioning

confidence: 99%

The Orphan Receptor C5L2 Has High Affinity Binding Sites for Complement Fragments C5a and C5a des-Arg74

Cain

Monk

2002

Journal of Biological Chemistry

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The substantial variations in the responses of cells to the anaphylatoxin C5a and its desarginated form, C5adR 74 , suggest that more than one type of cell surface receptor for these ligands might exist. However, only a single receptor for C5a and C5adR 74 , CD88, has been characterized to date. Here we report that the orphan receptor C5L2/gpr77, which shares 35% amino acid identity with CD88, binds C5a with high affinity but has a 10-fold higher affinity for C5adR 74 than CD88. C5L2 also has a moderate affinity for anaphylatoxin C3a, but cross-competition studies suggest that C3a binds to a distinct site from C5a. C4a was able to displace C3a, suggesting that C5L2, like the C3a receptor, may have a low binding affinity for this anaphylatoxin. Unlike CD88 and C3a receptor, C5L2 transfected into RBL-2H3 cells does not support degranulation or increases in intracellular [Ca 2؉ ] and is not rapidly internalized in response to ligand binding. However, ligation of C5L2 by anaphylatoxin did potentiate the degranulation response to cross-linkage of the high affinity IgE receptor by a pertussis toxin-sensitive mechanism. These results suggest that C5L2 is an anaphylatoxin-binding protein with unique ligand binding and signaling properties.

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“…Polypeptide C5a receptor antagonist C5aRA (24) was a generous gift from Joerg Kohl (Medizinische Hochschule, Hannover, Germany), and antagonist V2[Glu 68 ] was made as described (25). Measurement of Receptor Activation-Receptor activation in RBL cells was measured as the release of ␤-hexosaminidase from intracellular granules as described (26). The percentage of ␤-hexosaminidase release was calculated as a percentage of the release stimulated by a high dose of C5a (1 M).…”

Section: Construction Of Human C5armentioning

confidence: 99%

Comparative Agonist/Antagonist Responses in Mutant Human C5a Receptors Define the Ligand Binding Site

Higginbottom

Cain

Woodruff³

et al. 2005

Journal of Biological Chemistry

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Analysis of receptor/ligand interactions using whole-molecule randomly-mutated ligand libraries

Cited by 11 publications

References 14 publications

Characterisation of receptor binding by the chemotaxis inhibitory protein of Staphylococcus aureus and the effects of the host immune response

Characterisation of receptor binding by the chemotaxis inhibitory protein of Staphylococcus aureus and the effects of the host immune response

The Orphan Receptor C5L2 Has High Affinity Binding Sites for Complement Fragments C5a and C5a des-Arg74

Comparative Agonist/Antagonist Responses in Mutant Human C5a Receptors Define the Ligand Binding Site

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