Alterations of endoplasmic reticulum (ER) homeostasis can result from mutations of chaperones or of their substrate proteins. Calreticulin is a calcium-binding ER chaperone that is important for the folding and assembly of many N-linked glycoproteins. Calreticulin is also found on the surface of macrophages and apoptotic cells, where it facilitates cellular phagocytosis. Much remains to be understood about the molecular mechanisms of calreticulin-dependent protein folding, including factors that regulate substrate binding and release in the ER. Furthermore, the extracellular functions of calreticulin are poorly understood, including the mechanisms relevant to cell-surface interactions of calreticulin, and to calreticulin-dependent phagocytosis. There is also little knowledge about the loss and gain of function of calreticulin mutants with altered calcium- binding domains that are frequently found in myleoproliferative neoplasms (MPN). Some of these gaps in knowledge will be addressed in this application. The main hypotheses are that ATP is a key regulator of calreticulin-substrate interactions and that distinct modes of protein and lipid recognition are central to the cellular functions of calreticulin. Using computational and experimental approaches, a model for the ATP binding site of calreticulin is presented. ATP binding is shown to destabilize calreticulin binding to cellular monoglucosylated major histocompatibility complex (MHC) class I molecules. The effect of ATP binding deficient mutants on the maturation of other substrates will be examined, including ?1-antitrypsin (AAT), its misfolded variant ATZ, and the low-density lipoprotein-related protein (LRP-1). The molecular mechanisms by which calreticulin induces the clearance of insoluble ATZ will be studied, examining the model that polypeptide recognition by calreticulin is relevant to this activity. The influences of substrates and ER factos upon nucleotide exchange and upon the ATPase activity of calreticulin will be studied. Preliminary data indicate that the C-terminal acidic domain of calreticulin, which contains low affinity calcium-binding sites, also contains binding sites for phosphatidylserine (PS) and apoptotic cells. Somatic calreticulin mutants that are frequently present in MPN have altered non-acidic C-termini. These mutations are predicted to not only alter calcium and PS binding, and calreticulin-dependent cellular phagocytosis, but also affect the conformation and chaperone activity of calreticulin, which will be studied. Based on the knowledge gained from these studies, we expect to develop strategies to enhance the formation of active proteins in protein misfolding disorders such as AAT deficiency, and to understand the pathogenic effects of calreticulin mutations in cancer.

Public Health Relevance

Calreticulin is one of two key ER chaperones dedicated to the folding of glycoprotein substrates. A number of protein folding diseases arise from the misfolding of glycoproteins in the ER. One example is AAT-deficiency, a genetic disorder that arises from the presence of misfolded variants of AAT, a key molecule that is important for the regulation of inflammatory responses. Calreticulin controls the secretion and solubility of AAT variants, understanding of which could allow for better strategies to increase circulating AAT levels and counter disease. Additionally, calreticulin itself is mutated in myleoproliferative neoplasms, and the mutations are predicted to affect the innate and adaptive immune functions of calreticulin. Understanding the molecular mechanism of dysfunction of mutated calreticulin could allow for the further development of new prognostic markers and anti- cancer therapies. Thus, the proposed research contributes to the mission of the NIH relating to advancement of fundamental knowledge to reduce the burden of human disease and disability.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Membrane Biology and Protein Processing Study Section (MBPP)
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Gondre-Lewis, Timothy A
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
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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