ARTÍCULO

In vivo identification of an HLA-G complex as ubiquitinated protein circulating in exosomes

Autores: Alegre Martínez, Estíbaliz; Rebmann, V.; LeMaoult, J.; Rodríguez Jiménez, María del Carmen Milagros; Horn, P.A.; Díaz Lagares, Ángel; Echeveste, José Ignacio; González Hernández, Álvaro
Título de la revista: EUROPEAN JOURNAL OF IMMUNOLOGY
ISSN: 0014-2980
Volumen: 43
Número: 7
Páginas: 1933-9
Fecha de publicación: 2013
Resumen:
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