651. In vivo identification of an HLA-G complex as ubiquitinated protein circulating in exosomes.
- Author
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Alegre E, Rebmann V, Lemaoult J, Rodriguez C, Horn PA, Díaz-Lagares A, Echeveste JI, and González A
- Subjects
- Ascites immunology, Blotting, Western, Electrophoresis, Gel, Two-Dimensional, Enzyme-Linked Immunosorbent Assay, Exosomes chemistry, Exosomes immunology, HLA-G Antigens immunology, HLA-G Antigens isolation & purification, Humans, Immunoprecipitation, Pleural Effusion immunology, Ubiquitinated Proteins immunology, Ubiquitinated Proteins isolation & purification, Ubiquitinated Proteins metabolism, Ubiquitination, Exosomes metabolism, HLA-G Antigens metabolism
- 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., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2013
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