Abstract
Evidence suggests that most epitopes presented by MHC class I molecules are derived from those newly synthesized proteins that are defective due to errors during manufacture. We examined epitope production from model cytosolic and exocytic proteins modified in various ways. Substrates containing a degradation targeting sequence demonstrated very rapid turnover and enhanced epitope production, as was the case for substrate retargeted from endoplasmic reticulum to cytosol. For less radical alterations, including point mutation and deletion and elimination of glycosylation sites, despite detectable changes in folding, half-life was only moderately decreased and there were no significant increases in epitope production. Puromycin, which causes premature termination of protein synthesis, also had no impact upon epitope production. It appears that most defective proteins are not rapidly dispensed with and the targeting of most nascent proteins for Ag processing is not tied to quality control.
Highlights
NP from influenza virus A/PR/8/34 depleted of the twelve N-terminal amino acids that target it to the nucleus (NP13–498) was used as a model cytosolic Ag
The ␣-chain of human IL-2R (Tac Ag) was used as a model glycoprotein that would be subject to quality control within the endoplasmic reticulum (ER)
Each Ag was further engineered to contain the same two epitopes: H-2Kb-restricted OVA257–264 and H-2Kd-restricted NP147–155 (Fig. 1). These epitopes were chosen because, for all earlier constructs as well as those used here, they demonstrate strikingly different sensitivity to proteasome inhibitors; while OVA257–264 presentation is profoundly inhibited by proteasome inhibitors, NP147–155 presentation is either unchanged or increased depending upon context [16, 19, 22]
Summary
The initial goal of this project was to test the prediction that misfolding of our constructs would differentially affect the presentation of NP147–155 and OVA257–264 based upon the opposite impact that proteasome inhibitors have on their production and the prevailing models
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