Calreticulin is an endoplasmic reticulum (ER) chaperone that promotes folding and assembly of glycoproteins, including major histocompatibility complex (MHC) class I molecules. Calreticulin also has the capacity to direct exogenous antigens onto the MHC class I antigen presentation pathway, a phenomenon called cross-presentation. As a lectin, Calreticulin interacts with monoglucosylated core glycans on glycoproteins. Under certain conditions, Calreticulin is able to bind polypeptide components of substrates. Calcium depletion and heat-treatment expose calreticulin's polypeptide binding site and enhance Calreticulin binding to polypeptide substrates in vitro and in vivo cells. These treatments also induce Calreticulin dimerization and oligomerization. The formation of Calreticulin dimers is additionally induced by other types of ER stress, including virus infection and tunicamycin treatment. It is our hypothesis that these conformational transitions and polypeptide-binding properties are important for calreticulin's protein folding and cross-priming functions in cells. The first specific aim explores the role of polypeptide binding by Calreticulin during MHC class I folding and assembly in cells. We propose partial proteolysis and mass spectrometry-based approaches to identify Calreticulin sub-domains that are mobilized by calcium depletion. Conserved hydrophobic residues of Calreticulin, that are predicted to be surface-exposed, will be mutated to alanines. Mutants that display defects in interactions with polypeptide components of MHC class I heavy chains in vitro, as well as other mutants with defects in binding oligosaccharide substrates, will be expressed in calreticulin-deficient cells, and assessed for the ability to facilitate MHC class I folding and assembly. Together, these studies will allow us to refine our working model for the calreticulin-substrate interaction cycle, in which alternating interactions with oligosaccharide and polypeptide components of substrates are proposed. We will attempt to crystallize truncated versions of Calreticulin that have enhanced ability to bind polypeptide substrates, and also crystallize Calreticulin complexes with chicken IgY fragments. The second specific aim will explore mechanisms of calreticulin-mediated cross-presentation. Intracellular trafficking of Calreticulin and calreticulin-associated peptides during cross-presentation will be assessed, to investigate the hypothesis of an endosome-trans Golgi network-ER trafficking route. The requirement for Calreticulin for cross-presentation of antigens associated with apoptotic cells will also be assessed. Finally, the effects of ER stress on Calreticulin trafficking, cell surface expression, and interactions with receptors on antigen presenting cells will be assessed. Understanding the molecular mechanisms of calreticulin's functions, and elucidation of conditions that enhance calreticulin's T cell priming activities, will facilitate more effective design of vaccines.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Cellular and Molecular Immunology - B Study Section (CMIB)
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Gondre-Lewis, Timothy A
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
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Wijeyesakere, Sanjeeva Joseph; Bedi, Sukhmani Kaur; Huynh, David et al. (2016) The C-Terminal Acidic Region of Calreticulin Mediates Phosphatidylserine Binding and Apoptotic Cell Phagocytosis. J Immunol 196:3896-3909
Wijeyesakere, Sanjeeva Joseph; Gagnon, Jessica K; Arora, Karunesh et al. (2015) Regulation of calreticulin-major histocompatibility complex (MHC) class I interactions by ATP. Proc Natl Acad Sci U S A 112:E5608-17
Wijeyesakere, Sanjeeva J; Rizvi, Syed M; Raghavan, Malini (2013) Glycan-dependent and -independent interactions contribute to cellular substrate recruitment by calreticulin. J Biol Chem 288:35104-16
Raghavan, Malini; Wijeyesakere, Sanjeeva J; Peters, Larry Robert et al. (2013) Calreticulin in the immune system: ins and outs. Trends Immunol 34:13-21
Del Cid, Natasha; Shen, Lianjun; Belleisle, Janice et al. (2012) Assessment of roles for calreticulin in the cross-presentation of soluble and bead-associated antigens. PLoS One 7:e41727
Wijeyesakere, Sanjeeva J; Gafni, Ari A; Raghavan, Malini (2011) Calreticulin is a thermostable protein with distinct structural responses to different divalent cation environments. J Biol Chem 286:8771-85
Peters, Larry Robert; Raghavan, Malini (2011) Endoplasmic reticulum calcium depletion impacts chaperone secretion, innate immunity, and phagocytic uptake of cells. J Immunol 187:919-31
Rizvi, Syed Monem; Del Cid, Natasha; Lybarger, Lonnie et al. (2011) Distinct functions for the glycans of tapasin and heavy chains in the assembly of MHC class I molecules. J Immunol 186:2309-20
Jeffery, Elise; Peters, Larry Robert; Raghavan, Malini (2011) The polypeptide binding conformation of calreticulin facilitates its cell-surface expression under conditions of endoplasmic reticulum stress. J Biol Chem 286:2402-15
Del Cid, Natasha; Jeffery, Elise; Rizvi, Syed Monem et al. (2010) Modes of calreticulin recruitment to the major histocompatibility complex class I assembly pathway. J Biol Chem 285:4520-35

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