ABSTRACT
Reticulocytes release small membrane vesicles termed exosomes during their maturation into erythrocytes. Exosomes are intraluminal vesicles of multivesicular endosomes released into the extracellular medium by fusion of these endosomal compartments with the plasma membrane. This secretion pathway contributes to reticulocyte plasma membrane remodeling by eliminating certain membrane glycoproteins. We show in this study that galectin-5, although mainly cytosolic, is also present on the cell surface of rat reticulocytes and erythrocytes. In addition, in reticulocytes, it resides in the endosomal compartment. We document galectin-5 translocation from the cytosol into the endosome lumen, leading to its secretion in association with exosomes. Galectin-5 bound onto the vesicle surface may function in sorting galactose-bearing glycoconjugates. Fittingly, we found that Lamp2, a major cellular glycoprotein presenting galectin-reactive poly-N-acetylactosamine chains, is lost during reticulocyte maturation. It is associated with released exosomes, suggestive of binding to galectin-5. Finally, we reveal that the uptake of rat reticulocyte exosomes by macrophages is dependent on temperature and the mechanoenzyme dynamin and that exosome uptake is decreased by adding galectin-5. These data imply galectin-5 functionality in the exosomal sorting pathway during rat reticulocyte maturation.
Subject(s)
Exosomes/metabolism , Galectins/metabolism , Galectins/physiology , Macrophages/metabolism , Reticulocytes/metabolism , Animals , Antigens, Surface/metabolism , Biological Transport/physiology , Lysosomal-Associated Membrane Protein 2/metabolism , Lysosomal-Associated Membrane Protein 2/physiology , Protein Binding/physiology , Protein Transport/physiology , Rats , Rats, Sprague-Dawley , Secretory Pathway/physiology , Transport Vesicles/metabolismABSTRACT
Exosomes are small membrane vesicles that are released into the extracellular compartment as a consequence of fusion of multivesicular endosomes with the plasma membrane. To unravel the molecular basis of protein sorting into exosomes, we have made a chimeric protein containing the cytosolic domain of the transmembrane subunit of the viral Env protein of BLV and the ectodomain of CD8 (CDTM-BLV-CD8). When expressed in K562 cells known to constitutively secrete exosomes, the chimera was found to be very efficiently targeted to the released vesicles. Very interestingly, the cytosolic domain of the Env protein contains peptide motifs potentially recognized by components of the ESCRT machinery that could be related to chimera sorting into the vesicles. Then, quantifying the chimera secretion, we investigated the site of exosome biogenesis in K562 cells using a pharmacological approach. We present different arguments indicating that CDTM-BLV-CD8-containing exosomes are likely formed from a recycling endosomal/TGN compartment.
Subject(s)
Exocytosis , Exosomes/metabolism , Gene Products, env/metabolism , Leukemia Virus, Bovine , Amino Acid Motifs , Amino Acid Sequence , Animals , Cattle , Cell Line, Tumor , Cell Membrane/physiology , Fluorescent Antibody Technique, Direct , Gene Products, env/drug effects , Gene Products, env/genetics , Humans , K562 Cells , Molecular Sequence Data , Protein Transport , Rats , Receptors, Antigen, T-Cell/drug effects , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/metabolism , trans-Golgi Network/metabolismABSTRACT
Reticulocytes release small membrane vesicles termed exosomes during their maturation into erythrocytes. It has been suggested that reticulocytes remodel the plasma membrane of the immature red cell during erythropoiesis by specifically eliminating various proteins. We report here that exosome release is associated with a physiologic cascade induced by the expression of a 15-lipoxygenase at the reticulocyte stage. We found that the phospholipase iPLA2 specifically associated with the endosomal and exosomal membranes could be activated by reactive oxygen species (ROSs) produced during mitochondria degeneration induced by 15-lipoxygenase. Since iPLA2 has recently been demonstrated to participate in the clearance of apoptotic cells, we investigated its role in vesicle removal. We found that exosomes isolated directly from the blood of an anemic rat or released during in vitro maturation of rat reticulocytes bind IgM antibodies on their surface, in contrast to immature and mature red cells. These natural IgM antibodies recognize lysophosphatidylcholine and are able to specifically bind to apoptotic cells. Finally, evidence of C3 deposition on the exosome surface leads us to hypothesize that this cascade may favor the clearance of exosomes by cells once released into the bloodstream, via a mechanism similar to that involved in the elimination of apoptotic cells.
Subject(s)
Immunoglobulin M/metabolism , Phospholipases A2, Calcium-Independent/metabolism , Phospholipases A2, Calcium-Independent/physiology , Reactive Oxygen Species/pharmacology , Reticulocytes/metabolism , Secretory Vesicles/metabolism , Anemia/metabolism , Anemia/pathology , Animals , Endosomes/drug effects , Endosomes/enzymology , Enzyme Activation/drug effects , Humans , Jurkat Cells , Mitochondria/pathology , Phospholipases A2, Calcium-Independent/pharmacology , Phospholipids/metabolism , Rats , Rats, Sprague-Dawley , Reticulocytes/drug effects , Secretory Vesicles/drug effectsABSTRACT
Reticulocyte maturation into erythrocytes is the final step of erythropoiesis that occurs in the blood circulation. This terminal differentiation period corresponds to a cellular remodeling phase following expulsion of the nucleus into the bone marrow. Among other events, this remodeling leads to the disappearance of intracellular organelles and acquisition of the typical cellular biconcave form. Here, we propose that exosome biogenesis and secretion, which contributes to net loss of the cell surface membrane via selective vesicular membrane secretion, is also closely interconnected with upstream (nucleus expulsion), accompanying (mitoptosis) and downstream (vesicle clearance) events.