Functional Characterization of a Lysosomal Sorting Motif in the Cytoplasmic Tail of HLA-DOβ

Abstract

HLA-DO is an intracellular non-classical class II major histocompatibility complex molecule expressed in the endocytic pathway of B lymphocytes, which regulates the loading of antigenic peptides onto classical class II molecules such as HLA-DR. The activity of HLA-DO is mediated through its interaction with the peptide editor HLA-DM. Here, our results demonstrate that although HLA-DO is absolutely dependent on its association with DM to egress the endoplasmic reticulum, the cytoplasmic portion of its ␤ chain encodes a functional lysosomal sorting signal. By confocal microscopy and flow cytometry analysis, we show that reporter transmembrane molecules fused to the cytoplasmic tail of HLA-DO␤ accumulated in Lamp-1 ؉ vesicles of transfected HeLa cells. Mutagenesis of a leucine-leucine motif abrogated lysosomal accumulation and resulted in cell surface redistribution of reporter molecules. Finally, we show that mutation of the di-leucine sequence in DO␤ did not alter its lysosomal sorting when associated with DM molecules. Taken together, these results demonstrate that lysosomal expression of the DO-DM complex is mediated primarily by the tyrosine-based motif of HLA-DM and suggest that the DO␤-encoded motif is involved in the fine-tuning of the intracellular sorting. Major histocompatibility complex (MHC)1 class II molecules are heterodimeric cell surface glycoproteins that present antigens to CD4 ϩ T cells (1). The antigenic peptide-class II complexes are expressed on specialized antigen-presenting cells such as macrophages and B lymphocytes. Following their synthesis, the ␣ and ␤ subunits of the class II molecule associate in the endoplasmic reticulum (ER) together with the invariant chain (Ii) (2). The latter folds in part through the groove of the class II molecule, stabilizing the ␣␤ heterodimer and preventing the undesirable binding of ER polypeptides (3-6). Studies using mice with inactivated Ii genes suggested that Ii is necessary for efficient exit of newly synthesized class II molecules from the ER (7, 8). However, it was later demonstrated that high levels of class II molecules reach the surface of Ii Ϫ dendritic cells (9). Moreover, transfected cells express a substantial amount of class II molecules at their plasma membrane even in the absence of Ii (10). Such a phenotype probably results from the binding of endogenous peptides or polypeptides present in the ER (6, 11) and supports the notion that occupancy of the peptide-binding groove is sufficient to allow ER egress (12).Another function of Ii is to direct efficiently MHC class II molecules to the endocytic antigen-loading compartments (13-15). Two short leucine-based sequences located in the cytoplasmic tail of Ii are responsible for trafficking through the endocytic pathway (16,17). Similar motifs in many proteins are specifically recognized at the cell surface and trans-Golgi by elements of the sorting machinery (reviewed in Ref. 18).Once the class II-Ii complex reaches the endosomal compartments, the invariant chain is progressively degraded by v...

Forced Expression of HLA-DM at the Surface of Dendritic Cells Increases Loading of Synthetic Peptides on MHC Class II Molecules and Modulates T Cell Responses

“…The pBud-DMY, pcDNA3-CIITA, pREP4-CIITA, pBud-DRa+DRb, pREP4-Ii p33, and pREP4-Ii p35 plasmids have been described previously (16,17). L243, CerCLIP.1, MaP.DM1, and Mags.DO5 mAbs have been described previously (16,17). Abs for mouse CD80, CD86, CD11c, and for human CD80, CD83, and CD86 were purchased from BD Pharmingen (Mississauga, ON).…”

“…The AdDMY was constructed in pAdenoVator-CMV5 (CuO) from pBud-DMY (15). The pBud-DMY, pcDNA3-CIITA, pREP4-CIITA, pBud-DRa+DRb, pREP4-Ii p33, and pREP4-Ii p35 plasmids have been described previously (16,17). L243, CerCLIP.1, MaP.DM1, and Mags.DO5 mAbs have been described previously (16,17).…”

“…Cells, mice, and transfections HEK 293T, HEK 293T DR1, HEK 293T DR Ii, HeLa CIITA, HeLa CIITA DO, and HeLa DR1 Ii cells have been described previously (16,17). The murine fibroblast cell line DAP (ATCC CRL-1949) was transfected using Fugene6 (Roche Diagnostics, Canada).…”

“…Anti-mouse IFN-g and IL-4 were from eBioscience (San Diego, CA). Cell surface and intracellular staining were performed as described previously (16).…”

Functional Analysis of Tryptophans α62 and β120 on HLA-DM

“…Finally, those amino acids substitutions on DM did not affect its interaction with HLA-DO, an intracellular nonclassical class II molecule that binds to DM and regulates its activity. DM␤ cDNA has been described previously (25). DM␣ and DM␤ cDNAs from pBluescript were subcloned successively on the same pBudCE4-A vector (as previously described) (25).…”