Journal
EUROPEAN JOURNAL OF BIOCHEMISTRY
Volume 267, Issue 2, Pages 583-590Publisher
BLACKWELL SCIENCE LTD
DOI: 10.1046/j.1432-1327.2000.01036.x
Keywords
exosomes; reticulocyte; integrin alpha 4 beta 1; fibronectin
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Exosomes are vesicles formed in the endosomal compartment and released in the extracellular medium during reticulocyte maturation into erythrocytes. They have a clearing function because of their enrichment with some proteins known to decrease or disappear from the cell surface during maturation, e.g. acetylcholinesterase and transferrin receptor. We show here that integrin alpha 4 beta 1, present on the surface of erythroid precursors but absent from the mature red cell membrane, is at least partly cleared from the reticulocyte plasma membrane by the exosomal pathway. Using flow cytometry, we found that the alpha 4 subunit disappears from the reticulocyte surface during in vitro maturation. Two different monoclonal antibodies (B-5G10 and HP 2/1) were used to demonstrate the presence of the alpha 4 chain on the exosome surface. Moreover, membrane acetylcholinesterase and lumenal peroxidase-like (i.e. hemoglobin) enzymatic activities were assayed to demonstrate exosome binding to plates coated with increasing fibronectin (FN) concentrations. This interaction was dependent on divalent cations (MnCl2 > MgCl2 > CaCl2). Similarly, vesicles bound to plates coated with the chymotryptic 40 K fragment (FN-40) containing the heparin-binding region of FN. This binding was inhibited by exosome preincubation with fibronectin CS1 peptide and with a monoclonal antibody (HP 2/1) against the integrin alpha 4-chain, confirming an alpha 4 beta 1-induced interaction. The importance of the exosome clearance function is highlighted here, since the presence of VLA-4 on reticulocytes often leads to blood circulation complications in some diseases. Moreover, the presence of alpha 4 beta 1 on the exosome surface, by allowing binding to endothelial cells through vascular cell adhesion molecule 1 (VCAM-1), might confer another physiological function to the secreted vesicles.
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