Cellular and subcellular distribution of PBP72/74, a peptide-binding protein that plays a role in antigen processing

Abstract A 72/74-kDa peptide binding protein (PBP72/74) was previously described which plays a role in the processing and/or presentation of Ag, possibly by facilitating the association of processed Ag with the MHC class II molecules. PBP72/74 was recently shown to be related to the 70-kDa family of heat shock proteins (hsp70), whose members show the general characteristic of binding to denatured or inappropriately folded proteins. Here we describe the cellular and subcellular distribution of PBP72/74. By flow cytometry with PBP72/74-specific rabbit antisera, PBP72/74 is detected on the surfaces of mouse Ig+ B cells and MAC-1+ macrophages. PBP72/74 74 was not detected on the surfaces of Thy-1+ T cells or NK1.1+ NK cells. The cell surface expression of PBP72/74 does not require MHC class II expression. Indeed, the Ia- variant B cell lymphoma cell line, M12.C3, expresses PBP72/74 at levels equivalent to that of the Ia+ parent cell line, M12.4.1, from which it was derived. Furthermore, the fibroblast L cell line, DAP.3, shows no cell surface expression of PBP72/74, nor do DAP.3 lines transfected with and expressing genes encoding the alpha- and beta-chain of the I-Ad and I-Ed molecules. Moreover, treatment of B cells with either IL-4 or LPS, which increases Ia expression severalfold, does not affect PBP72/74 expression. Thus, PBP72/74 cell surface expression appears to be a property of B cells and macrophages, independent of Ia expression. In addition, the B cell surface expression of PBP72/74 is not altered by stress in the form of heat shock. Thus, PBP72/74 appears to be a constitutive noninducible member of the hsp70 family. By immunoelectron microscopy, PBP72/74 is detected in approximately 36% of early endocytic vesicles into which surface Ig is internalized after binding to anti-Ig antibodies. This compartment was previously shown to contain class II en route to the cell surface associated with invariant chain and the proteases cathepsin B and D and is suggested to be a subcellular site of antigen processing. PBP72/74 is also found associated with the plasma membrane, endoplasmic reticulum, and membranes proximal to the Golgi stacks. The cellular and subcellular distribution of PBP72/74 is consistent with its playing a role in the processing of presentation of Ag with the MHC class II molecules.