Calreticulin: not just another calcium-binding protein

In this paper we review some of the rapidly expanding information about calreticulin, a Ca²⁺-binding/storage protein of the endoplasmic reticulum. The emphasis is placed on the structure and function of calreticulin. We believe that calreticulin is a multifunctional Ca²⁺-binding protein and that distinct functional properties of the protein may be localized to each of the three structural domains of calreticulin. Most evidence indicates that calreticulin is a resident endoplasmic reticulum protein. However, it can also be found outside of the endoplasmic reticulum compartment, i.e. in the nuclear envelope, in the nucleus, in the cytotoxic granules in T-lymphocytes and in acrosomal vesicles of sperm cells. The evidence reviewed here clearly suggests that calreticulin has other functions in addition to its role as a Ca²⁺ storage protein in the endoplasmic reticulum.Abbreviations SR sarcoplasmic reticulum - ER endoplasmic reticulum     

Expression of the high capacity calcium-binding domain of calreticulin increases bioavailable calcium stores in plants

Modulation of cytosolic calcium levels in both plants and animals is achieved by a system of Ca²⁺-transport and storage pathways that include Ca²⁺ buffering proteins in the lumen of intracellular compartments. To date, most research has focused on the role of transporters in regulating cytosolic calcium. We used a reverse genetics approach to modulate calcium stores in the lumen of the endoplasmic reticulum. Our goals were two-fold: to use the low affinity, high capacity Ca²⁺ binding characteristics of the C-domain of calreticulin to selectively increase Ca²⁺ storage in the endoplasmic reticulum, and to determine if those alterations affected plant physiological responses to stress. The C-domain of calreticulin is a highly acidic region that binds 20–50 moles of Ca²⁺ per mole of protein and has been shown to be the major site of Ca²⁺ storage within the endoplasmic reticulum of plant cells. A 377-bp fragment encoding the C-domain and ER retention signal from the maize calreticulin gene was fused to a gene for the green fluorescent protein and expressed in Arabidopsis under the control of a heat shock promoter. Following induction on normal medium, the C-domain transformants showed delayed loss of chlorophyll after transfer to calcium depleted medium when compared to seedlings transformed with green fluorescent protein alone. Total calcium measurements showed a 9–35% increase for induced C-domain transformants compared to controls. The data suggest that ectopic expression of the calreticulin C-domain increases Ca²⁺ stores, and that this Ca²⁺ reserve can be used by the plant in times of stress.     

Calreticulin Induces Dilated Cardiomyopathy

Background

Calreticulin, a Ca²⁺-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.

Methodology/Principal Findings

We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca²⁺ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca²⁺-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca²⁺-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin.

Conclusions/Significance

We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca²⁺ handling genes, gap junction components and left ventricle remodeling.     

Differential Modulation of SERCA2 Isoforms by Calreticulin

In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca²⁺ oscillations in a manner consistent with inhibition of Ca²⁺ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA)2 gene display differential Ca²⁺ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.     

The function of calreticulin in plant immunity: New discoveries for an old protein

Since its initial discovery as a high affinity Ca²⁺-binding protein in the sarcoplasmic reticulum and endoplasmic reticulum (ER), calreticulin (CRT) has been documented to be a multifunctional protein in both animal and plant cells. This protein is well recognized as a Ca²⁺-binding molecular chaperone that facilitates the folding of newly synthesized glycoproteins and regulates the Ca²⁺ homeostasis in the ER lumen. However, functional relevance associated with its localization in other cellular compartments has also been reported. Recent studies suggest that both isoforms of plant CRTs (AtCRT1/2 and AtCRT3) are involved in regulating plant defense against biotrophic pathogens. Here we discuss the cellular functions of CRT and its connection to the emerging functions of AtCRTs in plant immunity.     

Effects of Ga3 and Ca2+ on barley aleurone protoplasts: a freeze-fracture study

Summary Freeze-fracture electron microscopy was used to study changes in the endomembrane system of barley (Hordeum vulgare L. cv. Himalaya) aleurone protoplasts. Protoplasts were used for this study because their response to calcium and the plant hormone gibberellic acid (Ga₃) can be monitored prior to rapid freezing of cells for electron microscopy. Protoplasts incubated in Ga₃ plus Ca²⁺ secrete elevated levels of a-amylase relative to cells incubated in Ga₃ or Ca²⁺ alone. The endoplasmic reticulum (ER) and Golgi apparatus of protoplasts incubated in Ga₃ plus Ca²⁺ undergo changes that are well correlated with the synthesis and secretion of a-amylase. The ER, which appears as short, single sheets of membrane in Ca²⁺-and Ga₃-treated protoplasts, exists as a series of long fenestrated stacks of membranes following incubation in Ga₃ plus Ca²⁺. The Golgi apparatus is also more highly developed in protoplasts treated with Ga₃ plus Ca²⁺. This organelle is larger and has more vesicles associated with its periphery in protoplasts that actively secrete a-amylase. Evidence that the Golgi apparatus participates in a-amylase secretion is also provided by experiments with the ionophore monensin, which causes pronounced swelling of Golgi cisternae and inhibits the secretion of a-amylase. We interpret these observations as showing that the ER and Golgi apparatus of barley aleurone participate in the intracellular transport and secretion of a-amylase. The plasmalemma (PF face) of barley aleurone protoplasts shows a high density of intramembranous particles (IMPs) which, in general, are evenly distributed. Occasionally, ordered arrays of IMPs are observed, possibly resulting fro m osmotic stress. after 48 hours the plasmalemma of some Ga₃-treated protoplasts show particle-free areas considered to be indications of senescence.abbreviations ER endoplasmic reticulum - Ga₃ gibberellic acid - IEF isoelectric focusing - IMP intramembranous particle - PF protoplasmic fracture - PL plasmalemma     

Calreticulin is an upstream regulator of calcineurin

Ca²⁺ is a signalling molecule involved in virtually every aspect of cell function. The endoplasmic reticulum (ER) is an important and dynamic organelle responsible for storage of the majority of intracellular Ca²⁺. Within the ER lumen are proteins that function as Ca²⁺ buffers and/or molecular chaperones including calreticulin, a multifunctional Ca²⁺-binding protein. Calreticulin-deficiency is lethal in utero due to impaired cardiac development. In the absence of calreticulin Ca²⁺ storage capacity in the ER and InsP₃ receptor mediated Ca²⁺ release from ER are compromised. Remarkably, over-expression of constitutively active calcineurin in the hearts of calreticulin deficient mice rescues them from embryonic lethality and produces live calreticulin deficient animals. These observations provide first evidence that calreticulin is a key upstream regulator of calcineurin in the Ca²⁺-signalling cascade and they highlight the importance of ER during early stages of cellular commitment and tissue development during organogenesis.     

Calcium binding proteins in the sarcoplasmic/endoplasmic reticulum of muscle and nonmuscle cells

In this paper we review some of the large quantities of information currently available concerning the identification, structure and function of Ca²⁺-binding proteins of endoplasmic and sarcoplasmic reticulum membranes. The review places particular emphasis on identification and discussion of Ca²⁺ storage proteins in these membranes. We believe that the evidence reviewed here supports the contention that the Ca²⁺-binding capacity of both calsequestrin and calreticulin favor their contribution as the major Ca²⁺-binding proteins of muscle and nonmuscle cells, respectively. Other Ca²⁺-binding proteins discovered in both endoplasmic reticulum and sarcoplasmic reticulum membranes probably contribute to the overall Ca²⁺ storage capacity of these membrane organelles, and they also play other important functional role such as posttranslational modification of newly synthesized proteins, a cytoskeletal (structural) function, or movement of Ca²⁺ within the lumen of the sarcoplasmic/endoplasmic reticulum towards the storage sites.Abbreviations SR Sarcoplasmic Reticulum - ER Endoplasmic Reticulum - InsP₃ Inositol 1,4,5-trisphosphate - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - PDI Protein Disulphide Isomerase - T₃BP Thyroid Hormone Binding Protein - Grp Glucose regulated proteins - HCP Histidine-rich Ca²⁺ binding Protein - LDL Low Density Lipoprotein     

Immunocytochemical localisation of calreticulin in<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis>

Calreticulin, a Ca²⁺ chaperone, is found in many different locations in various eukaryotic cells, including lumen of the endoplasmic reticulum, the cell surface, perinuclear areas and cytosolic granules. In the present study, a polyclonal antibody against calreticulin was used for the immunocytochemical localisation of the protein in Trypanosoma cruzi. Labelling was observed in the endoplasmic reticulum, Golgi complex, reservosomes, flagellar pocket, cell surface, cytosol, nucleus and kinetoplast. Significant differences in labelling were observed among the three evolutive forms of the protozoan. The functional role of calreticulin in T. cruzi is discussed.     

The evolutionary history of calreticulin and calnexin genes in green plants

Calreticulin and calnexin are Ca²⁺-binding chaperones localized in the endoplasmic reticulum of eukaryotes acting in glycoprotein folding quality control and Ca²⁺ homeostasis. The evolutionary histories of calreticulin and calnexin gene families were inferred by comprehensive phylogenetic analyses using 18 completed genomes and ESTs covering the major green plants groups, from green algae to angiosperms. Calreticulin and calnexin possibly share a common origin, and both proteins are present along all green plants lineages. The calreticulin founder gene within green plants duplicated in early tracheophytes leading to two possible groups of orthologs with specialized functions, followed by lineage-specific gene duplications in spermatophytes. Calnexin founder gene in land plants was inherited from basal green algae during evolution in a very conservative copy number. A comprehensive classification in possible groups of orthologs and a catalog of calreticulin and calnexin genes from green plants are provided.     

Ginkgo biloba Expresses Calreticulin,the Major Calcium-Binding Reticuloplasmin in Eukaryotic Cells

Calreticulin, the main Ca²⁺ binding protein in the endoplasmic reticulum of eukaryotic cells, was characterized in Ginkgo biloba L. pollen and seeds. Electrophoretic analysis of the partly purified extracts showed the presence of two protein bands of 57 and 50kDa apparent molecular masses, which strongly cross-reacted with antibodies against plant calreticulins. Amino acid sequence comparison with other plant and animal calreticulins revealed a much higher similarity of the N-terminus of Ginkgo calreticulins with the homologue from angiosperms rather than with that from mammals. The finding of calreticulin in Ginkgo is indicative of the conservation also in gymnosperms of Ca²⁺ homeostatic mechanisms, which seem to rely on the same molecular components as all eukaryotic cells.     

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin     

2,4,6-trinitrobenzenesulfonic acid modification of the carboxyl-terminal region (C-domain) of calreticulin

The role of the primary amino groups of lysine sidechains in Ca²⁺ binding to calreticulin was evaluated by chemical modification of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS binding to calreticulin could be described by two steps: (i) a fast reaction, with low affinity, and (ii) a slow reaction with a relatively high affinity. Inclusion of Ca²⁺ and/or Mg²⁺ decreased both the amount of TNBS bound to calreticulin and the apparent affinity constant of the slower reaction. In contrast, the properties of the faster reaction for TNBS binding were not sensitive to Ca²⁺ and/or Mg²⁺. Analysis of TNBS binding to the carboxyl-terminal (C-domain) and aminoterminal (N-domain) of calreticulin revealed that theC-domain andN-domain are responsible for the slow and fast component of the TNBS binding, respectively. In keeping with this, in the presence of Ca²⁺, TNBS binding to theC-domain was significantly reduced, whereas modification of theN-domain was unaffected. TNBS modification of calreticulin significantly decreased Ca²⁺ binding to the low affinity/high capacity Ca²⁺ binding site(s) which are localized to theC-domain but had no effect on the high affinity/low capacity Ca²⁺ binding localized to theN-domain.In theC-domain of calreticulin, which contains the low affinity/high capacity Ca²⁺ binding sites, acidic residues are interspersed at regular intervals with one or more positively charged lysine and arginine residues. Our results indicate that the aminogroups of the lysine sidechains in theC-domain of calreticulin have a role in the low affinity/high capacity Ca²⁺ binding that is characteristic of this region of the protein and which is proposed to contribute significantly to the capacity of the endoplasmic reticulum Ca²⁺ store. (Mol Cell Biochem130: 19–28, 1994)Abbreviations TNBS 2,4,6-Trinitrobenzenesulfonic Acid - GST Glutathione S-Transferase - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EDTA Ethylenediaminetetraacetic Acid - EGTA Ethylene Glycol bis(-aminoethylether)-N,N,N,N-tetraacetic Acid - MOPS 4-Morpholinepropanesulfonic Acid     

Calreticulin in the heart

Calreticulin is a Ca²⁺ binding/storage chaperone resident protein of the endoplasmic reticulum. This protein plays a key role in the calreticulin/calnexin cycle and the quality control pathways in the endoplasmic reticulum. Calreticulin deficiency is lethal due to impaired cardiac development. However, over-expression of the protein in developing and postnatal heart leads to bradycardia, complete heart block and sudden death. Ultrastructural evidence indicates that the deficiency associated with the absence of calreticulin in the heart may be due to a defect in the development of the contractile apparatus and/or a defect in development of the conductive system as well as a metabolic abnormality. Collectively, we postulate that calreticulin and endoplasmic reticulum plays an important role in cardiac development and postnatal pathologies. (Mol Cell Biochem 263: 137–142, 2004)     

Ectopic expression of a maize calreticulin mitigates calcium deficiency-like disorders in sCAX1-expressing tobacco and tomato

Deregulated expression of an Arabidopsis H⁺/Ca²⁺ antiporter (sCAX1) in agricultural crops increases total calcium (Ca²⁺) but may result in yield losses due to Ca²⁺ deficiency-like symptoms. Here we demonstrate that co-expression of a maize calreticulin (CRT, a Ca²⁺ binding protein located at endoplasmic reticulum) in sCAX1-expressing tobacco and tomato plants mitigated these adverse effects while maintaining enhanced Ca²⁺ content. Co-expression of CRT and sCAX1 could alleviate the hypersensitivity to ion imbalance in tobacco plants. Furthermore, blossom-end rot (BER) in tomato may be linked to changes in CAX activity and enhanced CRT expression mitigated BER in sCAX1 expressing lines. These findings suggest that co-expressing Ca²⁺ transporters and binding proteins at different intracellular compartments can alter the content and distribution of Ca²⁺ within the plant matrix.     

Spatial characteristics to calcium signalling; the calcium wave as a basic unit in plant cell calcium signalling

Many signals that modify plant cell growth and development initiate changes in cytoplasmic Ca²⁺. The subsequent movement of Ca²⁺ in the cytoplasm is thought to take place via waves of free Ca²⁺. These waves may be initiated at defined regions of the cell and movement requires release from a reticulated endoplasmic reticulum and the vacuole. The mechanism of wave propagation is outlined and the possible basis of repetitive reticulum wave formation, Ca²⁺ oscillations and capacitative Ca²⁺ signalling is discussed. Evidence for the presence of Ca²⁺ waves in plant cells is outlined, and from studies on raphides it is suggested that the capabilities for capacitative Ca²⁺ signalling are also present. The paper finishes with an outline of the possible interrelation between Ca²⁺ waves and organelles and describes the intercellular movement of Ca²⁺ waves and the relevance of such information communication to plant development.     

Glutathione Adducts on Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase Cys-674 Regulate Endothelial Cell Calcium Stores and Angiogenic Function as Well as Promote Ischemic Blood Flow Recovery

The sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is key to Ca²⁺ homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca²⁺ uptake activity and endothelial cell angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced S-glutathione adducts on SERCA in both whole muscle tissue and endothelial cells. To determine the role of S-glutathiolation, we used a SERCA 2 C674S heterozygote knock-in (SKI) mouse lacking half the key thiol. Following hind limb ischemia, SKI animals had decreased SERCA S-glutathione adducts and impaired blood flow recovery. We studied SKI microvascular endothelial cells in which total SERCA 2 expression was unchanged. Cultured SKI microvascular endothelial cells showed impaired migration and network formation compared with wild type (WT). Ca²⁺ studies showed decreased nitric oxide (·NO)-induced ⁴⁵Ca²⁺ uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca²⁺ stores and decreased vascular endothelial growth factor (VEGF)- and ·NO-induced Ca²⁺ influx. Adenoviral overexpression of calreticulin, an ER Ca²⁺ binding protein, increased ionomycin-releasable stores, VEGF-induced Ca²⁺ influx and endothelial cell migration. Taken together, these data indicate that the redox-sensitive Cys-674 thiol on SERCA 2 is required for normal endothelial cell Ca²⁺ homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca²⁺ stores.     

Calreticulin localizes to plant intra/extracellular peripheries of highly specialized cells involved in pollen-pistil interactions

Calcium (Ca²⁺) plays essential roles in generative reproduction of angiosperms, but the sites and mechanisms of Ca²⁺ storage and mobilization during pollen-pistil interactions have not been fully defined. Both external and internal Ca²⁺ stores are likely important during male gametophyte communication with the sporophytic and gametophytic cells within the pistil. Given that calreticulin (CRT), a Ca²⁺-buffering protein, is able to bind Ca²⁺ reversibly, it can serve as a mobile store of easily releasable Ca²⁺ (so called an exchangeable Ca²⁺) in eukaryotic cells. CRT has typical endoplasmic reticulum (ER) targeting and retention signals and resides primarily in the ER. However, localization of this protein outside the ER has also been revealed in both animal and plant cells, including Golgi/dictyosomes, nucleus, plasma membrane/cell surface, plasmodesmata, and even extracellular matrix. These findings indicate that CRT may function in a variety of different cell compartments and specialized structures. We have recently shown that CRT is highly expressed and accumulated in the ER of plant cells involved in pollen-pistil interactions in Petunia, and we proposed an essential role for CRT in intracellular Ca²⁺ storage and mobilization during the key reproductive events. Here, we demonstrate that both CRT and exchangeable Ca²⁺ are localized in the intra/extracellular peripheries of highly specialized plant cells, such as the pistil transmitting tract cells, pollen tubes, nucellus cells surrounding the embryo sac, and synergids. Based on our present results, we propose that extracellularly located CRT is also involved in Ca²⁺ storage and mobilization during sexual reproduction of angiosperms.