Assessments of Roles for Calreticulin in Antigen Presentation.

Abstract

MHC class I molecules are important glycoproteins of the adaptive immune response. Assembly of MHC class I molecules occurs in the endoplasmic reticulum (ER) of cells. A group of proteins termed the MHC class I peptide-loading-complex (PLC) facilitate the assembly of MHC class I molecules. Calreticulin, a member of the PLC, is a soluble ER chaperone that aids in the folding of nascent glycoproteins via a glycan-binding site contained within a globular lectin-like domain. Calreticulin also interacts with ERp57, a thiol-oxidoreductase that facilitates disulfide isomerization in calreticulin-associated glycoproteins. Although it is widely accepted that calreticulin utilizes its lectin-like domain to chaperone glycoproteins, calreticulin can also suppress aggregation of non-glycosylated substrates in vitro under conditions of ER-stress. Thus, the cellular modes of substrate binding by calreticulin remain unclear and somewhat controversial. To this end, we focus on characterizing requirements for calreticulin recruitment into the PLC and identifying substrates of calreticulin within the PLC. Although calreticulin is typically ER-localized, under conditions of cell-stress, tumorigenesis, or cell-death, calreticulin migrates to the cell-surface where it acts as a pro-phagocytic signal. Soluble extracellular calreticulin is also suggested to mediate endocytosis of associated antigen. Thus, we investigated whether calreticulin-mediated phagocytosis or endocytosis translates to enhanced presentation of exogenous antigen to CD8 T-cells. In sum, we assess mechanisms relevant to calreticulin-mediated protein folding in the ER, and impacts of extracellular calreticulin upon presentation of exogenous antigen to CD8 T-cells. We show that calreticulin recruitment into the PLC is mediated via glycan and ERp57-dependent interactions. Additionally tapasin, an assembly factor for MHC class I molecules, is a key substrate for calreticulin recruitment into the PLC, and tapasin itself is a calreticulin substrate. We also show that the pro-phagocytic/endocytic role of calreticulin per se does not impact CD8 T-cell responses against cell-associated, peptide, bead-associated, or fused antigen. We investigate factors that may have rendered calreticulin-mediated phagocytosis non-essential, including impacts of innate stimulation and alternative modes of antigen transfer. Together, these findings allow for a better understanding of the chaperone function of calreticulin in the ER and mechanisms relevant to inducing CD8 T-cell responses against soluble and cell-associated antigens.