Calreticulin was discovered in the early 1970s as a high affinity calcium-binding protein (HACBP) of the sarcoplasmic reticulum (Ostwald and MacLennan 1974). Later, based on N-terminal amino acid sequence analysis and molecular cloning of cDNA encoding calreticulin, it was realized that the protein was identical to calregulin, CRP55, CaBP3, ERp60, and calsequestrin-like protein (Michalak et al. 2009). The name “calreticulin” is now universally accepted to reflect the protein’s Ca2+ binding capacity and localization in the sarcoplasmic/endoplasmic reticulum (ER). Calreticulin is a ubiquitous protein that is present in all eukaryotic cells except for erythrocytes, as these cells lack ER. There is reduced abundance of calreticulin in differentiated tissues (heart and brain), while highly differentiated cells have an increased level of the protein (Michalak et al. 2009). There is a high abundance of calreticulin in various types of cancer, including pancreatic cancer, adrenocortical carcinomas, and breast cancer. Additionally, increased levels of calreticulin may contribute to metastasis in gastric, pancreatic, prostate, and ovarian cancers. Under stress conditions (e.g., environmental, impaired Ca2+ homeostasis, hypoxia), calreticulin is upregulated, as it attempts to support recovery of homeostasis (Michalak et al. 2009).
Calreticulin has a highly conserved amino acid sequence and it has been found in many organisms. In humans, this protein is encoded by the calreticulin (CALR) genes located on chromosome 19p13.2. Two calreticulin genes have been identified. CALR1 encodes the 46-kDa protein; however, the exact function of the CALR2 gene is not known, as it does not appear to be transcribed. In humans, CALR1 spans 3.6 kb of genomic DNA and is composed of nine exons and eight introns. The promoter of CALR1 contains several putative regulatory sites, including those for activator protein (AP)-1 and AP-2, guanine-cytosine (GC)-rich areas, which include a Sp1 site, an H4TF-1 site, and also four CCAAT sequences. Both H4TF-1 and AP-2 recognition sequences have been found in genes that are active in cellular proliferation. Several poly(G) sequences, including GGGNNGGG motifs, are also found in the promoter regions of CALR1. These motifs may play a role in the regulation of expression of calreticulin and in ER stress-dependent activation of the CALR1 (Michalak et al. 2009). Important modulators of calreticulin expression include NKx2.5 (NK2 transcription factor related, locus 5), MEF2C (myocyte enhancer factor 2C), COUP-TF1 (chicken ovalbumin upstream promoter-transcription factor 1), GATA6 (GATA-binding protein 6), Evi-1 (ecotropic viral integration site 1), and PPAR (peroxisome-proliferator-activated receptor) γ factors (Qiu and Michalak 2009). These modulators play important roles in the regulation of expression of calreticulin during cardiogenesis and have been identified as important transcription factors regulating CALR1 in general (Qiu and Michalak 2009). In addition, Ca2+ depletion and unfolded protein response (UPR, an ER stress coping response) were shown to be important activators of CALR1 transcription. Also, nerve growth factor (NGF) was shown to upregulate calreticulin expression (Lu et al. 2015).
Calreticulin is a 46-kDa ER-resident protein involved in Ca2+ binding and is a well-known molecular lectin chaperone. Calreticulin interacts with newly synthesized glycoproteins by binding to Glc1Man9GlcNAc2 oligosaccharides as well as to the polypeptide chain. Calreticulin has an N-terminal cleavable signal sequence, a C-terminal KDEL ER retention sequence, and is composed of distinct structural and functional domains: the N-globular domain, the P-arm domain, and the C-domain (Michalak et al. 2009).
Based on the secondary structure analysis, the N-terminal domain (residues 1–177) is predicted to be comprised of eight antiparallel β-strands. The N-terminal domain of calreticulin contains the oligosaccharide and polypeptide binding site. Although little is known about the molecular features of substrate binding to calreticulin, a significant portion of the oligosaccharide domain has been mapped. Mutations to residues Tyr109 and Asp135 were identified to abolish interactions between calreticulin and oligosaccharides. Additionally, other amino acids were identified as responsible for sugar binding: Lys111, Tyr128, and Asp127. Trp302 and His153 in the N-domain have been shown to be critical for chaperone function, and they also affect the structure of calreticulin. Disruption of the disulfide bridge between Cys88 and Cys120 only partially affected the structure of calreticulin. A high-resolution crystal structure of the N-domain in complex with its tetrasaccharide substrate (Glc1Man9GlcNAc2) suggests (Chouquet et al. 2011) that the shape of the sugar-binding pocket is formed by concave β-sheets with two residues, Gly124 and Lys111, responsible for the selectivity and specificity of calreticulin’s binding to monoglycosylated oligosaccharides. These two residues can form direct hydrogen bonds with the oxygen of the glucose. By mutating hydrophobic surface patches, the polypeptide-binding site responsible for the in vitro aggregation suppression function was identified. Mutations in P19K/I21E or Y22K/F84E patches impaired the ability of calreticulin to suppress the thermally induced aggregation of nonglycosylated substrate. These findings may indicate that the predominant contributor to the chaperone functions of calreticulin within the ER is the lectin-based mode of substrate interaction. Several amino acid residues within the globular domain of calreticulin, Lys7, Glu8, Asp12, Arg19, and Lys63, in proximity to the high-affinity Ca2+-binding site are important for high-affinity ATP binding and for ATPase activity. The study showed that ATP binding influences the interactions of calreticulin with cellular substrates, and that ATP and Ca2+ binding to calreticulin influences the stability of the interaction of calreticulin with cellular substrates. The N-domain also binds Zn2+, and this ion binding may have structural effects on the whole protein, and thus affect protein function. A proteolysis stable N-domain core is formed in the presence of Ca2+ and development of this stable core may have specific pathophysiological implications.
The P-domain is located in the middle of calreticulin’s amino acid sequence (residues 178–288) and forms an extended flexible arm. The P-domain is a proline-rich region similar to the one found in calnexin and is composed of three copies of two repeat amino acid sequences (motif 1: IxDPxA/DxKPEDWDx and motif 2: GxWxPPxIxNPxYx). The repeat sequences are arranged in an 111,222 pattern and are thought to be involved in oligosaccharide binding, together with N-domain. Based on NMR studies, it is known that the structure of the P-domain of calreticulin contains an extended region stabilized by three antiparallel β-sheets (Ellgaard et al. 2001). Small-angle X-ray scattering (SAXS) analysis showed that calreticulin, with the N- and C-terminal forming an extended globular structure, and the P-loop protruding from the globular part has a high degree of plasticity. Oxidoreductase PDIA3 (ERp57) can dock onto the tip of the P-domain, and mutational analysis showed that amino acids Glu239, Asp241, Asp243, and Trp244 are involved in this interaction (Coe et al. 2010). The P-domain has also been found to bind Ca2+ with a high affinity (Kd = 1 μM) and low capacity (1 mol of Ca2+ per 1 mol of protein). The binding site appears to possess a potential EF-hand-like helix-loop-helix motif. The P-loop is able to interact with the base of the acidic C-terminal of calreticulin in both the presence and absence of Ca2+. However, a higher occupancy close to the N-terminus lectin site and the peptide-binding site occurs in the presence of Ca2+.
The C-domain of calreticulin (residues 289–400) is mainly composed of negatively charged residues interrupted, at regular intervals, with one or more basic Lys or Arg amino acid residues. Disruption of these basic residues results in decreased Ca2+ binding capacity, and the binding can be directly attributed to the lysine amino group side chains. These acidic residues are suggested to be responsible for the Ca2+ buffering function and binding of Ca2+. The C-terminal part of calreticulin is predicted to be intrinsically disordered. Circular dichroism spectrometry measurements do not show a secondary structure, either in the presence or absence of Ca2+. It is known that the C-domain is responsible for binding 50% of the ER Ca2+ with low affinity (Kd = 2 mM) and high capacity (25 mol of Ca2+ per mol of protein), opposite to Ca2+ binding by the P-domain.
Calreticulin is typically retained in the ER but has also been identified in the cytosol and on the cell surface (Gold et al. 2010). However, the source of cell surface calreticulin and mechanisms of its cell surface translocation remain to be established (Eggleton et al. 2016). Posttranslational arginylation of calreticulin has been reported for stress granules but not ER-associated protein (Lopez Sambrooks et al. 2012). Cell surface arginylated calreticulin may influence cell survival, as exogenously applied arginylated calreticulin increases cellular apoptosis (Lopez Sambrooks et al. 2012). Arginylation of calreticulin may also increase the half-life of the protein and promote its dimerization. Citrullinated calreticulin may modulate immune function in rheumatoid arthritis patients (Ling et al. 2013).
Many diverse functions have been described for calreticulin and most have been explained by the protein’s role in Ca2+ homeostasis and/or protein folding (Michalak et al. 2009; Gold et al. 2010). Calreticulin has structural homology with calnexin, another ER chaperone, and both proteins function in the “calnexin/calreticulin cycle,” an N-glycan-dependent quality control process that ensures correct glycoprotein folding and/or degradation and prevents protein aggregation. Calreticulin is much more efficient at suppressing aggregation of glycosylated substrates than nonglycosylated substrates, and mutations of glycan-binding site residues specifically impair its ability to suppress aggregation of glycosylated substrates.
Calreticulin, as a component of the peptide-loading complex, is a key player in the MHC class I assembly pathway that induces MHC class I cell surface expression. Peptide-loading complex is a large complex with MHC class I molecules, composed of MHC class I-dedicated assembly factors, transporter associated with antigen processing (TAP) and tapasin, as well as the ER-folding factors PDIA3 and calreticulin. TAP provides a major source of peptides for MHC class I molecules, whereas tapasin, PDIA3, and calreticulin facilitate assembly of MHC class I molecules with peptides (Peaper and Cresswell 2008). Moreover, calreticulin-deficient cells express reduced cell surface MHC class I molecules. Calreticulin is required for the loading of optimal antigenic peptides in the MHC-I complex and is thereby critical for the generation of effective cytotoxic T-cell responses. Calreticulin also affects the immune system via binding to complement C1q and low-density lipoprotein receptor-related protein 1 (LRP-1/CD91). Extracellular calreticulin impacts the prophagocytic role of CD47 antibodies (Chao et al. 2010). Calreticulin may also be involved in necroptosis (Kaczmarek et al. 2013).
Mammalian calreticulin has been known for a decade to enhance wound healing (Gold et al. 2006), and when applied topically, enhances epithelial migration and granulation tissue formation. Trypanosoma cruzi calreticulin also has the ability to affect wounds (Ignacio Arias et al. 2015). Furthermore, T. cruzi, by virtue of its ability to bind C1q, plays an important physiological role in trypomastigotes infectivity. Calreticulin interacts with components of the extracellular matrix, including thrombospondin-1 (Goicoechea et al. 2002), collagens, and laminins, and constituents required for remodeling such as metalloproteases to support wound healing.
The discovery of a mutation in exon 9 of the calreticulin gene in the majority case of myeloproliferative neoplasms (Klampfl et al. 2013; Nangalia et al. 2013) holds yet another promise for diagnostic and potential therapeutic application for this full of surprises molecule. Calreticulin may also be an important component of the damage-associated molecular pattern (DAMP) and potentially a prognostic or predictor for cancer patients. The protein may also be a critical inducer of immunogenic cell death (ICD) during nonimmunogenic cell death modalities, in addition to binding to phosphatidylserine (Gardai et al. 2005; Obeid et al. 2007).
The domain structure of the protein provides a unique feature, enabling calreticulin to perform several functions in the ER lumen, while responding to continuous fluctuations of ER luminal Ca2+. Many functions of calreticulin are related to the protein’s ability to bind Ca2+ with high affinity via the C-domain. Thus, it is not surprising that calreticulin is known as a major ER Ca2+ buffer and functions as a molecular chaperone. Calreticulin is a major ER luminal Ca2+ buffering protein critical for maintenance of ER luminal Ca2+ homeostasis. Calreticulin-deficient cells have up to a 50% reduction in calcium storage capacity, impaired ER Ca2+ signaling, and delayed store-operated Ca2+ entry (SOCE). Increased abundance of calreticulin leads to increase ER Ca2+, reduced ER-mitochondria Ca2+ transport, and delayed SOCE.
Being able to bind Ca2+ with high capacity and low affinity, calreticulin is classified as a Class I Ca2+ binding protein. In order to understand the Ca2+ binding and buffering properties of calreticulin, it is important to describe the cellular and animal models with calreticulin deficiency and overexpression. Calreticulin-deficient cells have reduced Ca2+ storage capacity in the ER, while overexpression of calreticulin leads to an increased amount of Ca2+ in the cell’s intracellular stores. For example, in calreticulin-deficient mouse embryonic fibroblasts (MEFs), a significant decrease in Ca2+ storage was observed (Nakamura et al. 2001). However, when the P+C-domain involved in Ca2+ binding was expressed in the calreticulin-deficient cells, there was a full recovery of the ER Ca2+ storage capacity (Nakamura et al. 2001). Expression of the N+P-domains of calreticulin did not recover Ca2+storage capacity in the ER of calreticulin-deficient cells (Nakamura et al. 2001). Taken together, these results both suggest that the main role of the C-domain is in Ca2+ binding.
Calreticulin has also been found outside the ER lumen, in the nucleus, cytoplasm, and on the cell surface, where it may be involved in other biological functions within the cell. Calreticulin’s nuclear and cell surface localization, as well as its translocation to the cytoplasm, remains controversial. Cell surface calreticulin may be involved in the immune system where it functions in antigen presentation and complement activation. Calreticulin may translocate to the cell surface with PDIA3, allowing for presentation to T-cells, initiation of the immune response, and apoptosis of the target cell. It is also an important protein for focal adhesion assembly and impacts phagocytosis and the proinflammatory response. Moreover, calreticulin induces the migration and motility of cells involved in wound healing such as keratinocytes, fibroblasts, monocytes, and macrophages.
Calreticulin has been implicated in the cellular response to apoptosis. Modulation of ER Ca2+ stores impacts apoptosis as ER Ca2+ release is required for activation of transcriptional cascades. An increased level of calreticulin results in increased sensitivity to apoptosis, while calreticulin-deficient cells are resistant to apoptosis. Further studies showed that calreticulin, intraluminal Ca2+, and disruption in Ca2+ regulation, influences apoptosis events in cardiomyocytes. Cell-surface calreticulin seems to also play a role in apoptosis. In RK3 cells, cell surface calreticulin mediates apoptosis through activation of tumor necrosis factor receptor type 1 (TNFR1). Many forms of cancer display cell surface calreticulin, which invites immune cells to destroy them. Moreover, calreticulin was identified as a prophagocytic signal highly expressed on the surface of several human cancers but minimally expressed on most normal cells. Increased calreticulin expression is an adverse prognostic factor in diverse tumors, including neuroblastoma, bladder cancer, and non-Hodgkin’s lymphoma. Preapoptotic translocation of calreticulin to the cell surface occurs with PDIA3, and allows presentation to T-cells, triggering the initiation of the immune response and subsequent apoptosis of the immunogenic cell, preventing organism damage. Disruption of the interaction of calreticulin with PDIA3, as well as disruption of calreticulin, not only prevents surface exposure of calreticulin but also renders the cell resistant to T-cell attack. Exogenous application of calreticulin is able to overcome this resistance. It was also shown that anthracyclins induce the rapid preapoptotic translocation of calreticulin to the cell surface. Blockade or knockdown of calreticulin suppresses the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolishes their immunogenicity in mice (Obeid et al. 2007).
Calreticulin deficiency in mice is embryonic lethal (Mesaeli et al. 1999). During embryonic development, calreticulin is expressed at a high level in central nervous system, liver, and heart, and embryonic lethality of calreticulin-deficient mice is due to impaired cardiac development, specifically a marked decrease in ventricular wall thickness (Mesaeli et al. 1999). Calreticulin-deficient cells show a significant decrease in ER Ca2+ capacity, but free ER Ca2+ remains unchanged (Nakamura et al. 2001). Further studies on the embryonic stem cell demonstrated how critical the Ca2+ buffering function of calreticulin is to cardiac development, as well as for mice survivability. Molecular studies indicate that calreticulin deficiency leads to impaired myofibrillogenesis. Moreover, deficiency of calreticulin leads to deficient intercalated disc formation in the heart, which are adherens-type junctions of cardiac muscle containing vinculin, N-cadherin, and catenins.
Studies examining the molecular level and functional consequences of upregulation of calreticulin have demonstrated a significant increase in Ca2+ capacity of the ER. Transgenic mice overexpressing calreticulin in the heart display bradycardia, complete heart block, and sudden death. Tissue-specific activation of CALR1 leading to increased abundance of calreticulin in the adult heart results in dilated cardiomyopathy, cardiac fibrosis, and heart failure stems from impairment of ER homeostasis, transient activation of the UPR pathway, and stimulation of the TGFβ1/Smad2/3 signaling pathway (Groenendyk et al. 2016).
In Caenorhabditis elegans, calreticulin is expressed in the intestine, pharynx, body-wall muscles, head neurons, coelomocytes, and sperm. In males, calreticulin exhibits reduced mating efficiency and defects late in sperm development, in addition to defects in oocyte development and/or somatic gonad function in hermaphrodites. A crt-1 null mutant does not result in embryonic lethality but shows temperature-dependent reproduction defects. Moreover, the crt-1 transcript level is elevated under stress conditions, suggesting that calreticulin may be important for the stress-induced chaperoning function in C. elegans. (Park et al. 2001). In Zebrafish, there are three genes encoding calreticulin and the protein is differentially expressed in many Zebrafish tissues. Calreticulin is highly expressed in the hatching gland and in the floor plate. During Zebrafish gastrulation, increased levels of calreticulin transcript are found in the dorsal mesoderm, indicating an important role for calreticulin during Zebrafish development (Rubinstein et al. 2000).
Calreticulin has been also identified as an antigen in sera from patients suffering from several autoimmune diseases, including SLE (systemic lupus erythematosus), coeliac disease, rheumatic disease, and various parasitic diseases, which implies a pathological role for calreticulin in autoimmune diseases. The protein is found on the cell surface to interact with C1q, the first component of complement, thereby activating the classical complement pathway (Eggleton and Michalak 2013). Parasite calreticulin also affects parasite infectivity by modulating the host’s complement system to help the parasite to evade the immune response of the host.
Studies using cerebrospinal fluid indicate that calreticulin interacts with β-amyloid peptides, the major components of the plaques observed in the brains of patients with Alzheimer’s disease. Recently, it was shown that the serum levels of calreticulin mRNA and protein were lower in Alzheimer disease patients than those from a healthy group and negatively associated with the progression of Alzheimer disease. Calreticulin may serve as a negative biomarker for the diagnosis of Alzheimer disease patients (Lin et al. 2014).
Mutations within the calreticulin promoter co-occur with major psychiatric disorders and do not exist in the control pool, offering a prime model for the pathogenesis of these disorders. Two of the mutations identified in the promoter sequence of the calreticulin gene also increase gene expression activity in neuronal cell lines. One of the mutations reverts the human promoter sequence to the ancestral type observed in chimpanzee, mouse, and several other species, implying that the genomic block harboring nucleotide -220 may be involved in the evolution of human-specific higher-order functions of the brain (e.g., language, conceptual thinking, and judgment), which are ubiquitously impaired in psychoses. Calreticulin seems to be not only a promising candidate in the spectrum of psychoses but also a gene that may be important in the human-unique brain processes.
The correlation between calreticulin expression levels and tumorigenesis has been extensively studied in various cancers, and most reports have indicated that tumor tissues express significantly higher levels of the protein compared to normal tissues. Abnormal calreticulin levels are also correlated with pathological outcomes in different types of cancers. Calreticulin participates in a variety of cellular functions, both inside and outside of the ER lumen. One of the important calreticulin-mediated mechanisms that regulate cancer cell adhesion is through interaction with integrins. It still remains unclear how calreticulin levels are stimulated in different cancers. Future studies are required to delineate the possible upstream signal of calreticulin-related cancer progression, and these results will decipher the roles of calreticulin in cancer biology.
Very recently, mutations in the calreticulin gene were detected in myeloproliferative neoplasms (MPN). Most patients with MPN, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), were found to have mutations in the Janus kinase 2 gene (JAK2). In 2013, studies carried out by two independent groups discovered somatic recurrent insertions/deletions, which exclusively affected exon 9 in the calreticulin gene in 70–84% of wild-type JAK2, MPL primary myelofibrosis (PMF), and essential thrombocythaemia (ET) (Klampfl et al. 2013; Nangalia et al. 2013). These calreticulin mutations include a 52 bp deletion and 5 bp insertion, which leads to frameshift mutations. Proteins encoded by a mutated calreticulin gene lack the C-terminal KDEL. Calreticulin mutations are associated with specific phenotypic features in MPN and now are part of the diagnostic criteria in MPNs.
Calreticulin was first discovered over 40 years ago. The protein has been implicated in many biological processes, including protein folding, regulation of Ca2+ homeostasis, modulation of transcriptional pathways, cellular proliferation, mobility and adhesion, apoptosis, embryonic development and postnatal development, cancer pathology, and cardiovascular disease, to name a few. Most recently, cell surface calreticulin received considerable attention as potential inducer of immunogenic cell death.
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