PE
Excitation: 565nm, Emission: 578nm
Association of major histocompatibility complex (MHC) class II molecules with peptides occurs in a series of endocytic vacuoles, termed MHC class II-enriched compartments (MIICs). Morphological criteria have defined several types of MIICs, including multivesicular MIICs, which are composed of 50-60-nm vesicles surrounded by a limiting membrane. Multivesicular MIICs can fuse with the plasma membrane, thereby releasing their internal vesicles into the extracellular space. The externalized vesicles, termed exosomes, carry MHC class II and can stimulate T-cells in vitro. In this study, we show that exosomes are enriched in the co-stimulatory molecule CD86 and in several tetraspan proteins, including CD37, CD53, CD63, CD81, and CD82. Interestingly, subcellular localization of these molecules revealed that they were concentrated on the internal membranes of multivesicular MIICs. In contrast to the tetraspans, other membrane proteins of MIICs, such as HLA-DM, Lamp-1, and Lamp-2, were mainly localized to the limiting membrane and were hardly detectable on the internal membranes of MIICs nor on exosomes. Because internal vesicles of multivesicular MIICs are thought to originate from inward budding of the limiting membrane, the differential distribution of membrane proteins on the internal and limiting membranes of MIICs has to be driven by active protein sorting.
C33 Ag and M38 Ag had been identified by mAb inhibitory to HTLV-1-induced syncytium formation. The cDNA encoding C33 Ag had revealed that it belongs to the newly defined transmembrane 4 superfamily (TM4SF). M38 Ag was detected on virtually all human cell lines and fresh leukocytes except for most granulocytes. It was also expressed on a mouse hybrid cell clone containing human chromosome 11q23-pter. Immunoprecipitation and immunoblot analyses identified a monomeric 26-kDa protein. The M38 epitope was dependent on S-S bonding. These characteristics were very similar to those reported for TAPA-1 (the target of antiproliferative antibody-1), which also belongs to TM4SF as C33 Ag. We therefore cloned the cDNA of human TAPA-1 and expressed it in COS cells. M38 indeed reacted with COS cells expressing human TAPA-1. We concluded that M38 Ag was identical to TAPA-1. To further investigate the biologic functions of C33 Ag and M38 Ag (TAPA-1) and their roles in HTLV-1-induced syncytium formation, molecules associated with these Ag were examined in T cells. Immunoprecipitation from surface-iodinated cell lysates revealed that proteins co-precipitated by C33 and M38 were mostly common including each other. Sequential immunoprecipitation-immunoblot experiments confirmed that C33 Ag and M38 Ag (TAPA-1) were associated with each other. The association was further confirmed in BHK cells doubly transfected with human cDNA for C33 Ag and TAPA-1. We extended similar analyses and found that C33 Ag and M38 Ag (TAPA-1) were regularly associated with CD4 or CD8. The association of these Ag on the cell surface was further supported by co-modulation of M38 Ag (TAPA-1), CD4 and CD8 with C33 Ag. This is the first time that a physical association between the members of TM4SF is demonstrated. Furthermore, the regular association of C33 Ag and M38 Ag (TAPA-1) with CD4 or CD8 might indicate that they play a role in expression and/or function of the CD4/CD8 co-receptor complex.
