When a kinase inactive form of Protein Kinase D (PKD-K618N) was expressed in HeLa cells, it localized to the trans-Golgi network (TGN) and caused extensive tubulation. Cargo that was destined for the plasma membrane was found in PKD-K618N-containing tubes but the tubes did not detach from the TGN. As a result, the transfer of cargo from TGN to the plasma membrane was inhibited. We have also demonstrated the formation and subsequent detachment of cargo-containing tubes from the TGN in cells stably expressing low levels of PKD-K618N. Our results suggest that PKD regulates the fission from the TGN of transport carriers that are en route to the cell surface.
HLA-DM catalyzes the release of MHC class II-associated invariant chain-derived peptides (CLIP) from class II molecules. Recent evidence has suggested that HLA-DO is a negative regulator of HLA-DM in B cells, but the physiological function of HLA-DO remains unclear. Analysis of antigen presentation by B cells from mice lacking H2-O (the mouse equivalent of HLA-DO), together with biochemical analysis using purified HLA-DO and HLA-DM molecules, suggests that HLA-DO/H2-O influences the peptide loading of class II molecules by limiting the pH range in which HLA-DM is active. This effect may serve to decrease the presentation of antigens internalized by fluid-phase endocytosis, thus concentrating the B cell-mediated antigen presentation to antigens internalized by membrane immunoglobulin.
HLA-DM (DM) facilitates peptide loading of major histocompatibility complex class II molecules in human cell lines. Mice lacking functional H2-M, the mouse equivalent of DM, have normal amounts of class II molecules at the cell surface, but most of these are associated with invariant chain-derived CLIP peptides. These mice contain large numbers of CD4+ T cells, which is indicative of positive selection in the thymus. Their CD4+ cells were unresponsive to self H2-M-deficient antigen-presenting cells (APCs) but were hyperreactive to wild-type APCs. H2-M-deficient APCs failed to elicit proliferative responses from wild-type T cells.
The murine MHC class II molecule H2-0 is expressed in B-cells and in thymic epithelium but the human equivalent, HLA-DO (DO), has not been detected, though the corresponding genes, HLA-DNA and HLA-DOB, are well known. Here we show DO to be a lysosomal resident in B-cells. Surprisingly, DO forms stable complexes with HLA-DM (DM), another lysosomal class 11-like molecule which is important for class IT-restricted antigen presentation. Association with DM is necessary for efficient exit of DO from the endoplasmic reticulum (ER) and thus for accumulation in lysosomes. The association is evolutionarily conserved and in mice lacking H2-M, the mouse equivalent of DM, the amount of intracellular H2-0 is decreased and only minor amounts of H2-0 appear to leave the ER. The DO-DM complexes survive in the lysosomal system suggesting that DO and DM functions may be intertwined.
Professional antigen-presenting cells (APCs) have a distinct compartment in which class II molecules are proposed to acquire antigenic peptides. Genetic evidence suggests that human leukocyte antigen (HLA)-DM, an unusual class II molecule, participates in this process. Peptide acquisition was reconstituted in nonprofessional APCs by transfection of class II, invariant chain (li), and H-2M, the murine equivalent of DM. The H-2M heterodimer appeared in an endosomal compartment, not at the cell surface, and the localization was independent of li. The data presented show that H-2M, class II, and li are the minimally required components for efficient formation of stable class II-peptide complexes, and thus for a functional class II compartment.
The nonpolymorphic human class II molecule HLA-DM (DM) has been found to play a key role in antigen presentation by MHC class II molecules. HLA-DM and its murine equivalent H2-M are located intracellularly and are absent from the cell surface. In transfected HeLa cells, H2-M was transported to an endosomal compartment in the absence of invariant chain. A tyrosine-based targeting motif in the cytoplasmic tail of H2-M beta was responsible for the endosomal location and, if this tyrosine was mutated, H2-M accumulated at the cell surface. In the presence of invariant chain the mutated H2-M was redistributed to endosomes. The targeting motif of H2-M appeared not to be crucial for efficient peptide loading of class II, but if the invariant chain targeting motif also was removed, peptide loading decreased drastically. Thus, the targeting motif of H2-M appears to be supplementary, rather than essential for class II-peptide association.
The function of major histocompatibility complex (MHC) class II molecules is to sample exogenous antigens for presentation to CD4+ T helper cells. After synthesis in the endoplasmic reticulum, class II molecules are directed into the endosomal system by association with the invariant chain (Ii), which is sequentially cleaved, generating class II dimers loaded with Ii-derived peptides (CLIP). These class II-peptide complexes are physiological substrates for H2-M/HLA-DM, a resident of the endosomal/lysosomal system which facilitates the removal of CLIP from newly synthesised class II alpha beta dimers. Exchange of CLIP for antigenic class II-binding peptides is also promoted by the action of H2-M/HLA-DM, resulting in stable peptide-class II complexes that are transported to the cell surface for presentation to CD4+ T cells. Recent evidence suggests that this H2-M/HLA-DM-mediated 'peptide editing' is influenced by another MHC class II-encoded molecule, H2-O/HLA-DO. This non-polymorphic alpha beta heterodimer is associated with H2-M/HLA-DM during intracellular transport and within the endosomal system of B cells. H2-O/HLA-DO alters the peptide exchange function of H2-M/HLA-DM in a pH-dependent manner, so that H2-M/HLA-DM activity is limited to more acidic conditions, corresponding to lysosomal compartments. Indeed, H2-O/HLA-DO may serve to limit the presentation of antigens after fluid phase uptake by B cells, while augmenting presentation of antigens internalised via membrane Ig receptors. Such a mechanism may maintain the fidelity of the B-cell-CD4+ T-cell interaction, counteracting self reactivity arising from less stringent lymphocyte activation. Here, data evaluating the role of H2-O/HLA-DO shall be reviewed and its putative function discussed.
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