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Mapping of the interleukin-10/interleukin-10 receptor combining site.

ABSTRACT: The discontinuous interleukin-10(IL-10)/interleukin-10 receptor (IL-10R) combining site was mapped using sets of overlapping peptides derived from both binding partners bound to continuous cellulose membranes. Low affinity binding of single regions of the discontinuous contact sites on IL-10 and IL-10R could be identified due to (1) high peptide density on the membrane support, (2) incubation with high protein concentrations, (3) indirect immunodetection of the ligates after electrotransfer onto polyvinylene difluoride membranes, and (4) use of highly overlapping peptide scans of different length (6-mers and 15-mers). The single binding regions identified for each protein species are separated in the protein sequences, but form continuous areas on the surface of IL-10 (X-ray structure) and IL-10R (computer model). Furthermore, four epitopes of neutralizing anti-IL-10 and anti-IL-10R antibodies were mapped and overlap with these binding regions. Soluble peptides (15- to 19-mers) each spanning one of the three identified IL-10-derived receptor binding regions displayed no significant affinity to IL-10R as expected, whereas a peptide (35-mer) comprising two of these regions had considerably higher binding activity. The data are consistent with a previously published computer model of the IL-10/IL-10R complex. This approach should be generally applicable for the mapping of non-linear protein-protein contact sites.

SUBMITTER: Reineke U

PROVIDER: S-EPMC2143979 | biostudies-other | 1998 Apr

REPOSITORIES: biostudies-other

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Mapping of the interleukin-10/interleukin-10 receptor combining site.

Reineke U U Sabat R R Volk H D HD Schneider-Mergener J J

Protein science : a publication of the Protein Society 19980401 4

The discontinuous interleukin-10(IL-10)/interleukin-10 receptor (IL-10R) combining site was mapped using sets of overlapping peptides derived from both binding partners bound to continuous cellulose membranes. Low affinity binding of single regions of the discontinuous contact sites on IL-10 and IL-10R could be identified due to (1) high peptide density on the membrane support, (2) incubation with high protein concentrations, (3) indirect immunodetection of the ligates after electrotransfer onto ...[more]

PMID: 9568901

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Project description:Coccidiosis is a major gastrointestinal disease caused by several Eimeria species in floor raised chickens. Feeding an antibody to interleukin 10 (aIL-10) ameliorates the negative symptoms of coccidiosis in broilers, i.e., lack of weight gain, decreased feed conversion, and mortality. IL-10 signals by forming a ligand-receptor complex with IL-10 Receptor 1 (IL-10 R1) and IL-10 Receptor 2 (IL-10 R2). In this study, we hypothesize oral antibodies to the IL-10 receptors will neutralize the IL-10 signaling pathway equal to or better than aIL-10 to act as an oral anti-coccidiosis immunotherapy. A total of 5 sequential feed trials, set up as a 4 (diet antibody) × 2 (Eimeria challenge) factorial design, tested oral egg yolk antibodies to a total of 6 IL-10 R1 epitopes and 3 IL-10 R2 epitopes compared to a control antibody diet. A total of 10 pens of 5 chicks/pen/diet antibody/Eimeria challenge were housed for 21 d. On day 3 of age, chicks were either infected or not infected with a 10× dose of an Eimeria vaccine containing Eimeria acervulina, Eimeria tenella, and Eimeria maxima. Pen feed consumption and mean body weights were assessed weekly (d1, d7, d14, and d21); fecal oocyst shedding was assessed on day 10. Data were analyzed using a 2-way ANOVA. No significant interaction on chick weight was observed in chicks fed IL-10 R1 antibodies compared to chicks fed the control antibody was observed. In studies evaluating aIL-10 R2 oral antibodies, infected chicks fed aIL-10 R2: epitope 1 overcame the negative effects of Eimeria infection and had similar 21-d body weight to uninfected chicks (P4 = 0.07). We hypothesized that feeding oral antibodies to the IL-10 receptors would result in equivalent anti-coccidial benefits to aIL-10. However, none of the 6 antibodies to IL-10 R1 epitopes yielded any benefits during Eimeria infection compared to controls. A total of 2 oral antibodies to IL-10 R2 showed promising results equivalent to the aIL-10 immunotherapeutic. Immunofluorescence staining shows that the IL-10R2 significantly increases in abundance in response to Eimeria infection, whereas IL-10R1 does not.

Dataset Information

Mapping of the interleukin-10/interleukin-10 receptor combining site.

Publications

Mapping of the interleukin-10/interleukin-10 receptor combining site.

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