title: In-cell intrabody selection from a diverse human library identifies C12orf4 protein as a new player in rodent mast cell degranulation.
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.
authors.affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France; INSERM, U896, Montpellier, France; Université Montpellier1, Montpellier, France; ICM, Institut régional du Cancer Montpellier, Montpellier, France.
authors.forename: Elsa
authors.initials: E
authors.lastname: Mazuc
authors.affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France; INSERM, U896, Montpellier, France; Université Montpellier1, Montpellier, France; ICM, Institut régional du Cancer Montpellier, Montpellier, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Guglielmi
authors.affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France; INSERM, U896, Montpellier, France; Université Montpellier1, Montpellier, France; ICM, Institut régional du Cancer Montpellier, Montpellier, France.
authors.forename: Nicole
authors.initials: N
authors.lastname: Bec
authors.affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France; INSERM, U896, Montpellier, France; Université Montpellier1, Montpellier, France; ICM, Institut régional du Cancer Montpellier, Montpellier, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Parez
authors.affiliation: Sanofi-Aventis, Bridgewater, New Jersey, United States of America.
authors.forename: Chang S
authors.initials: CS
authors.lastname: Hahn
authors.affiliation: ICM, Institut régional du Cancer Montpellier, Montpellier, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Mollevi
authors.affiliation: MGX-Montpellier GenomiX, Institut de Génomique Fonctionnelle, Montpellier, France.
authors.forename: Hugues
authors.initials: H
authors.lastname: Parrinello
authors.affiliation: MGX-Montpellier GenomiX, Institut de Génomique Fonctionnelle, Montpellier, France.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Desvignes
authors.affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France; INSERM, U896, Montpellier, France; Université Montpellier1, Montpellier, France; ICM, Institut régional du Cancer Montpellier, Montpellier, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Larroque
authors.affiliation: Sanofi-Aventis, Bridgewater, New Jersey, United States of America.
authors.forename: Ray
authors.initials: R
authors.lastname: Jupp
authors.affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France; INSERM, U896, Montpellier, France; Université Montpellier1, Montpellier, France; ICM, Institut régional du Cancer Montpellier, Montpellier, France; Université Montpellier2, Montpellier, France.
authors.forename: Piona
authors.initials: P
authors.lastname: Dariavach
authors.affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France; INSERM, U896, Montpellier, France; Université Montpellier1, Montpellier, France; ICM, Institut régional du Cancer Montpellier, Montpellier, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Martineau
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D015550:Cell Degranulation / Q000502:physiology*; D005260:Female; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000276:immunology* / Q000502:physiology*; D008407:Mast Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL
chemical_list: D000906:Antibodies; C000597204:C12orf4 protein, human; D047908:Intracellular Signaling Peptides and Proteins