Abstract
The high specificity of antibodies for their antigen allows a fine discrimination of target conformations and post-translational modifications, making antibodies the first choice tool to interrogate the proteome. We describe here an approach based on a large-scale intracellular expression and selection of antibody fragments in eukaryotic cells, so-called intrabodies, and the subsequent identification of their natural target within living cell. Starting from a phenotypic trait, this integrated system allows the identification of new therapeutic targets together with their companion inhibitory intrabody. We applied this system in a model of allergy and inflammation. We first cloned a large and highly diverse intrabody library both in a plasmid and a retroviral eukaryotic expression vector. After transfection in the RBL-2H3 rat basophilic leukemia cell line, we performed seven rounds of selection to isolate cells displaying a defect in FcεRI-induced degranulation. We used high throughput sequencing to identify intrabody sequences enriched during the course of selection. Only one intrabody was common to both plasmid and retroviral selections, and was used to capture and identify its target from cell extracts. Mass spectrometry analysis identified protein RGD1311164 (C12orf4), with no previously described function. Our data demonstrate that RGD1311164 is a cytoplasmic protein implicated in the early signaling events following FcεRI-induced cell activation. This work illustrates the strength of the intrabody-based in-cell selection, which allowed the identification of a new player in mast cell activation together with its specific inhibitor intrabody.
Highlights
Mast cells and basophils are key effector cells in IgE-associated immediate hypersensitivity and allergic disorders
Upon FceRI crosslinking initiated by the binding of antigen-IgE complexes, cell activation results in downstream events that lead to the secretion of three classes of mediators: (a) the extracellular release of preformed mediators stored in cell cytoplasmic granules, by a process called degranulation; (b) the de novo synthesis of proinflammatory lipid mediators; and (c) the synthesis and secretion of many growth factors, cytokines, and chemokines
We performed the phenotypic selection in the rat basophilic leukemia cell line RBL-2H3, used extensively to study FceRI and the biochemical pathways for secretion in mast cells
Summary
Mast cells and basophils are key effector cells in IgE-associated immediate hypersensitivity and allergic disorders. Upon FceRI crosslinking initiated by the binding of antigen-IgE complexes, cell activation results in downstream events that lead to the secretion of three classes of mediators: (a) the extracellular release of preformed mediators stored in cell cytoplasmic granules, by a process called degranulation; (b) the de novo synthesis of proinflammatory lipid mediators; and (c) the synthesis and secretion of many growth factors, cytokines, and chemokines This IgE-dependent release of mediators begins within minutes of antigen challenge and leads to certain acute allergic reactions such as anaphylaxis and acute attacks of atopic asthma [1]. Protein therapies have permitted alternative approaches in addition to drug therapies In this respect, an important treatment for allergic conditions is the recombinant humanized IgG monoclonal antibody Omalizumab, which binds selectively to human IgE and inhibit the production and release of all mast cell mediators by antagonizing IgE action. This biologic is highly effective, it is difficult and expensive to manufacture and administer
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