title: In vivo identification of an HLA-G complex as ubiquitinated protein circulating in exosomes.
abstract: The nonclassical human leukocyte antigen-G (HLA-G) is a tolerogenic molecule that can be released to the circulation by expressing cells. This molecule can form dimers but some other complexed HLA-G forms have been proposed to be present in vivo. Here, we further characterized these other complexed HLA-G forms in vivo. Ascitic and pleural exudates from patients were selected based on positivity for HLA-G by ELISA. Complexed HLA-G was detected in exosomes, which indicates an intracellular origin of these forms. 2D-PAGE analysis of exudates and isolated exosomes showed that these high molecular weight complexes were more heterogeneous than the HLA-G1 expressed by cell cultures. Treatment with deglycosylating enzymes did not change the molecular weight of HLA-G complexes. Immunoblot analysis of exudates and exosomes with an anti-ubiquitin antibody showed that at least some of these structures correspond to ubiquitinated HLA-G. HLA-G ubiquitination could be reproduced in vitro in HLA-G1-transfected cell lines, although with a lower modified/nonmodified protein proportion than in exudates. In summary, we demonstrate new circulating HLA-G forms in vivo that open a new perspective in the study of HLA-G function and analysis.
authors.affiliation: Department of Biochemistry, University Clinic of Navarra, Pamplona, Spain.
authors.forename: Estibaliz
authors.initials: E
authors.lastname: Alegre
authors.forename: Vera
authors.initials: V
authors.lastname: Rebmann
authors.forename: Joel
authors.initials: J
authors.lastname: Lemaoult
authors.forename: Carmen
authors.initials: C
authors.lastname: Rodriguez
authors.forename: Peter A
authors.initials: PA
authors.lastname: Horn
authors.forename: Angel
authors.initials: A
authors.lastname: Díaz-Lagares
authors.forename: José I
authors.initials: JI
authors.lastname: Echeveste
authors.forename: Alvaro
authors.initials: A
authors.lastname: González
mesh_terms: D001201:Ascites / Q000276:immunology; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D055354:Exosomes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D059951:HLA-G Antigens / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D010996:Pleural Effusion / Q000276:immunology; D057149:Ubiquitinated Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D059951:HLA-G Antigens; D057149:Ubiquitinated Proteins
keywords: Exosome; Exudates; HLA-G; Molecular immunology; Ubiquitin