Signal sequences of human MHC class I molecules are a unique source of epitopes for newly synthesized nonclassical HLA-E molecules. Binding of such conserved peptides to HLA-E induces its cell surface expression and protects cells from NK cell attack. After cleavage from the pre-protein, we show that the liberated MHC class I signal peptide is further processed by signal peptide peptidase in the hydrophobic, membrane-spanning region. This cut is essential for the release of the HLA-E epitope-containing fragment from the lipid bilayer and its subsequent transport into the lumen of the endoplasmic reticulum via the TAP.

The nonclassical major histocompatibility complex class I molecule HLA-E acts as a ligand for CD94/NKG2 receptors on the surface of natural killer cells and a subset of T cells. HLA-E presents closely related nonameric peptide epitopes derived from the highly conserved signal sequences of classical major histocompatibility complex class I molecules as well as HLA-G. Their generation requires cleavage of the signal sequence by signal peptidase followed by the intramembrane-cleaving aspartic protease, signal peptide peptidase. In this study, we have assessed the subsequent proteolytic requirements leading to generation of the nonameric HLA-E peptide epitopes. We show that proteasome activity is required for further processing of the peptide generated by signal peptide peptidase. This constitutes the first example of capture of a naturally derived short peptide by the proteasome, producing a class I peptide ligand.Peptide presentation by major histocompatibility complex (MHC) 1 class I molecules allows the display of intracellular protein fragments to the immune system. Several parameters control this process including the ability of peptide to bind to MHC class I molecules, precursor protein expression and stability, processing efficiency, and rate of peptide transport into the endoplasmic reticulum (ER).Most peptides presented by MHC class I molecules originate from proteins fragmented in the cytosol by the ubiquitin-26 S proteasome pathway (1). Generally, the antigenic peptides produced by the proteasome are either of the correct size or extended at their N termini. Further trimming primarily by aminopeptidases occurs in the cytosol (2) or in the ER lumen (3-5). The peptides are transported into the ER lumen by the transporter associated with antigen processing (TAP) and are finally loaded onto newly synthesized MHC class I molecules, which traffic to the cell surface. In addition, a small fraction of MHC class I epitopes was reported to be generated independently of the proteasome (6 -8) and may be produced by the cytosolic subtilisin-like tripeptidyl peptidase II (9, 10).The nonclassical MHC class I molecule HLA-E is specialized in the presentation of peptides derived from MHC class I signal sequences, reporting their expression level to natural killer (NK) cells (11, 12). HLA-E interacts with CD94/NKG2A or CD94/NKG2C receptors, resulting in inhibition or activation of NK cell function, respectively (13-15). More recently, HLA-E has also been implicated in the presentation of self-peptides and bacterial antigens to T cells, although the role of these T cells in immune responses remains to be established (16 -19).We have previously demonstrated that the generation of the MHC signal peptide-derived epitopes binding to HLA-E does not follow the "conventional" antigen-processing pathway described above. During biosynthesis of the MHC class I preprotein and its translocation into the ER lumen, the signal sequence is cleaved by signal peptidase and subsequently processed by signal peptide peptidase (SPP) (20,21). Th...