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 antiubiquitin 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.Keywords: Exosome r Exudates r HLA-G r Molecular immunology r Ubiquitin Additional supporting information may be found in the online version of this article at the publisher's web-site

The HLA-G cycle provides for both NK tolerance and immunity at the maternal–fetal interface

“…3). Some evidence of HLA-G ubiquitination has been reported, and could lead to HLA-G degradation (28).…”

Placental origins of adverse pregnancy outcomes: potential molecular targets: an Executive Workshop Summary of the Eunice Kennedy Shriver National Institute of Child Health and Human Development

“…396,397,398,399 Whereas exosomes are found in the circulation of pregnant women, little is known about trophoblastic exosomes, and many studies on extracellular vesicles in pregnancy have associated a mixture of vesicles (exosomes, MVs, ABs) with disorders of pregnancy, thus blurring some the distinct properties of these vesicles. 400,401,402,403,404 Placental exosomes have been implicated in several discrete functions, such as immune modulation, 405,406,407 where exosomal cargo proteins, such as galectin-3 408 and HLA-G 409 , may perform an immune modulatory function. 410 Other exosomal cargo molecules, such as Wnt/β-catenin, fibronectin, and prostaglandins, may play a role in supporting homeostasis throughout pregnancy.…”

Post-translational add-ons mark the path in exosomal protein sorting

“…For example, myeloid-derived suppressor cells (MDSC) secrete exosomes enriched in ubiquitinated proteins, including endosomal trafficking proteins and histones [74]. Moreover, high molecular weight complexes of non-classical human leukocyte antigen-G (HLA-G) are ubiquitinated, but not glycosylated, in exosomes obtained from ascitic and pleural exudates of patients [75]. Shedding vesicles also contain modified proteins such as arrestin domain-containing protein 1 (ARRDC1), which is ubiquitinated by the E3 ubiquitin ligase WW domain-containing protein 2 (WWP2).…”