Bacterially expressed human FcγRIIb is soluble and functionally active after in vitro refolding

Kurucz, István; Hilbert, Ágnes; Kapus, Attila; Medgyesi, Dávid; Koncz, Gábor; Sármay, Gabriella; Erdei, Anna; Gergely, J.

doi:10.1016/s0165-2478(00)00281-9

Cited by 4 publications

(2 citation statements)

References 25 publications

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“…Human recombinant soluble FcRIIb was produced in our laboratory (Kurucz et al, 2000), horseradish peroxidase-labelled second antibodies, fluorescein-isothiocyanate (FITC)-conjugated Streptavidine, human IgG and other reagents were obtained from Sigma. Tumor necrosis factor (TNF) and anti-TNF were purchased form Biosource International (CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Synthesis and receptor binding of IgG1 peptides derived from the IgG Fc region

Uray

Medgyesi

Hilbert

et al. 2004

J of Molecular Recognition

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The IgG binding Fcgamma receptors (FcgammaRs) play a key role in defence against pathogens by linking humoral and cell-mediated immune responses. Impaired expression and/or function of FcgammaR may result in the development of pathological autoimmunity. Considering the functions of FcgammaRs, they are potential target molecules for drug design to aim at developing novel anti-inflammatory and immunomodulatory therapies. Previous data mostly obtained by X-ray analysis of ligand-receptor complexes indicate the profound role of the CH2 domain in binding to various FcgammaRs. Our aim was to localize linear segments, which are able to bind and also to modulate the function of the low affinity FcgammaRs, like FcgammaRIIb and FcgammaRIIIa. To this end a set of overlapping octapeptides was prepared corresponding to the 231-298 sequence of IgG1 CH2 domain and tested for binding to human recombinant soluble FcgammaRIIb. Based on these results, a second group of peptides was synthesized and their binding properties to recombinant soluble FcgammaRIIb, as well as to FcgammaRs expressed on the cell surface, was investigated. Here we report that peptide representing the Arg(255)-Ser(267) sequence of IgG1 is implicated in the binding to FcgammaRIIb. In addition we found that peptides corresponding to the Arg(255)-Ser(267), Lys(288)-Ser(298) or Pro(230)-Val(240) when presented in a multimeric form conjugated to branched chain polypeptide in uniformly oriented copies induced the release of TNFalpha, a pro-inflammatory cytokine from MonoMac monocyte cell line. These findings indicate that these conjugated peptides are able to cluster the activating FcgammaRs, and mediate FcgammaR dependent function. Peptide Arg(255)-Ser(267) can also be considered as a lead for further functional studies.

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Section: Methodsmentioning

confidence: 99%

Synthesis and receptor binding of IgG1 peptides derived from the IgG Fc region

Uray

Medgyesi

Hilbert

et al. 2004

J of Molecular Recognition

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“…Production of the extracellular part of the type II Fc + R was previously described [29]. Biotinylated anti-human IgM F(ab') 2 was purchased from Jackson ImmunoResearch (PA).…”

Section: Reagentsmentioning

confidence: 99%

Functional mapping of the FcγRII binding site on human IgG1 by synthetic peptides

et al. 2004

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Receptors specific for the Fc part of IgG (Fc + R) are expressed by several cell types and play diverse roles in immune responses. Impaired function of the activating and inhibitory Fc + R may result in autoimmunity. Thus, the modulation of IgG-Fc + R interaction can be a target for the development of treatments for some autoimmune and inflammatory diseases. This study addresses the localization and functional characterization of linear sequences in human IgG1 which bind to Fc + RII. Peptides with overlapping sequences derived from the CH2 domain of human IgG1 between P 234 and S 298 were synthesized and used in binding and functional experiments. Binding of the peptides to Fc + R was assayed in vitro and ex vivo, and peptides found to interact were functionally tested. The shortest effective peptide was T 256-P 271 complexed by avidin exhibited functional activity; they induced Fc + RIIb-mediated inhibition of the BCR-triggered Ca 2+ response of human Burkitt lymphoma cells, and inflammatory cytokine production (TNF- § and IL-6) by the human monocyte cell line MonoMac. In conclusion, our results suggest that the selected peptides functionally represent the Fc + RII-binding part of IgG1.

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