Proteomic and Biochemical Analyses of Human B Cell-derived Exosomes

Abstract

Exosomes are 60–100-nm membrane vesicles that are secreted into the extracellular milieu as a consequence of multivesicular body fusion with the plasma membrane. Here we determined the protein and lipid compositions of highly purified human B cell-derived exosomes. Mass spectrometric analysis indicated the abundant presence of major histocompatibility complex (MHC) class I and class II, heat shock cognate 70, heat shock protein 90, integrin α4, CD45, moesin, tubulin (α and β), actin, G_iα₂, and a multitude of other proteins. An α4-integrin may direct B cell-derived exosomes to follicular dendritic cells, which were described previously as potential target cells. Clathrin, heat shock cognate 70, and heat shock protein 90 may be involved in protein sorting at multivesicular bodies. Exosomes were also enriched in cholesterol, sphingomyelin, and ganglioside GM3, lipids that are typically enriched in detergent-resistant membranes. Most exosome-associated proteins, including MHC class II and tetraspanins, were insoluble in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-containing buffers. Multivesicular body-linked MHC class II was also resistant to CHAPS whereas plasma membrane-associated MHC class II was solubilized readily. Together, these data suggest that recruitment of membrane proteins from the limiting membranes into the internal vesicles of multivesicular bodies may involve their incorporation into tetraspanin-containing detergent-resistant membrane domains.