Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation.
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 alpha 4, CD45, moesin, tubulin (alpha and beta), actin, G(i)alpha(2), and a multitude of other proteins. An alpha 4-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.
Wubbolts
Richard
R
Department of Cell Biology, Utrecht University Medical Centre and Institute of Biomembranes, Room G02.525, Heidelberglaan 100, 3585 CX Utrecht, The Netherlands.
Leckie
Rachel S
RS
Veenhuizen
Peter T M
PT
Schwarzmann
Guenter
G
Möbius
Wiebke
W
Hoernschemeyer
Joerg
J
Slot
Jan-Willem
JW
Geuze
Hans J
HJ
Stoorvogel
Willem
W
D001402:B-Lymphocytes / Q000378:metabolism / Q000648:ultrastructure*; D002460:Cell Line; D002784:Cholesterol / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008854:Microscopy, Electron; D015388:Organelles / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D020543:Proteome; D002784:Cholesterol