ERAAP was isolated from mouse liver and spleen that had been solubilized with detergent and fractionated using ion-exchange chromatography. A single activity peak was identified, which was purified further using chromatographic techniques. Trypsin digestion of the gel-isolated protein, followed by mass spectrometry, gave a peptide fingerprint, which was used to search the databases. This search identified an aminopeptidase, which the authors named ERAAP.
ERAAP co-localizes with the ER markers BiP and gp96. The expression of ERAAP correlates with MHC class I expression in various cell types, and it is upregulated by stimulating cells with interferon-γ, which also upregulates the expression of other molecules in the antigen-processing pathway. The function of ERAAP as a peptide-trimming molecule in the ER was confirmed by knocking down ERAAP expression in antigen-presenting cells (APCs) using small interfering RNA (siRNA) and assessing the effect on the presentation of antigens to T cells. ERAAP expression was reduced by 90% by siRNA treatment, which decreased the expression of surface MHC class I molecules by 20–40% (because empty class I molecules cannot be expressed stably at the cell surface). The expression of two specific MHC class I peptides was inhibited significantly in APCs transfected with mouse siRNA for ERAAP, which confirms the importance of ERAAP for generating these peptides. So, ERAAP connects the peptides that are generated in the cytosol with the peptides that are presented at the cell surface on MHC molecules.
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