Molecular cloning and characterization of sea bass (Dicentrarchus labrax,L.) calreticulin

Mammalian calreticulin (CRT) is a key molecular chaperone and regulator of Ca²⁺ homeostasis in endoplasmic reticulum (ER), also being implicated in a variety of physiological/pathological processes outside the ER. Importantly, it is involved in assembly of MHC class I molecules. In this work, sea bass (Dicentrarchus labrax) CRT (Dila-CRT) gene and cDNA have been isolated and characterized. The mature protein retains two conserved motifs, three structural/functional domains (N, P and C), three type 1 and 2 motifs repeated in tandem, a conserved pair of cysteines and ER-retention motif. It is a single-copy gene composed of 9 exons. Dila-CRT three-dimensional homology models are consistent with the structural features described for mammalian molecules. Together, these results are supportive of a highly conserved structure of CRT through evolution. Moreover, the present data provides information that will allow further studies on sea bass CRT involvement in immunity and in particular class I antigen presentation.     

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.     

A conserved basic residue cluster is essential for the protein quality control function of the Arabidopsis calreticulin 3

Calreticulin (CRT) is a highly conserved chaperone-like lectin that regulates Ca²⁺ homeostasis and participates in protein quality control in the endoplasmic reticulum (ER). Most of our CRT knowledge came from mammalian studies, but our understanding of plant CRTs is limited. Many plants contain more than two CRTs that form two distinct groups: CRT1/CRT2 and CRT3. Previous studies on plant CRTs were focused on their Ca²⁺-binding function, but recent studies revealed a crucial role for the Arabidopsis CRT3 in ER retention of a mutant brassinosteroid receptor, brassinosteroid-insensitive 1-9 (bri1-9) and in complete folding of a plant immunity receptor EF-Tu Receptor (EFR). However, little is known about the molecular basis of the functional specification of the CRTs. We have recently shown that the C-terminal domain of CRT3, which is rich in basic residues, is essential for retaining bri1-9 in the ER; however, its role in assisting EFR folding has not been studied. Here, we used an insertional mutant of CRT3, ebs2-8 (EMS mutagenized bri1 suppressor 2-8), in the bri1-9 background as a genetic system to investigate the functional importance of two basic residue clusters in the CRT3′s C-terminal domain. Complementation experiments of ebs2-8 bri1-9 with mutant CRT3^M transgenes showed that a highly conserved basic tetrapeptide Arg³⁹²Arg³⁹³Arg³⁹⁴Lys³⁹⁵ is essential but a less conserved basic tetrapeptide Arg⁴⁰¹Arg⁴⁰²Arg⁴⁰³Arg⁴⁰⁴ is dispensable for the quality control function of CRT3 that retains bri1-9 in the ER and facilitates the complete folding of EFR.     

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.     

Calreticulin positively regulates the expression and function of epithelial sodium channel

Epithelial sodium channel (ENaC) is a heteromultimeric Na⁺ channel at the apical membrane in the kidney, colon, and lung. Because ENaC plays a crucial role in regulating Na⁺ absorption and extracellular fluid volume, its dysregulation causes severe phenotypes including hypertension, hypokalemia, and airway obstruction. Despite the importance of ENaC, its protein quality control mechanism remains less established. Here we firstly show the role of calreticulin (CRT), a lectin-like molecular chaperone in the endoplasmic reticulum (ER), on the regulation of ENaC. Overexpression and knockdown analyses clearly indicated that CRT positively affects the expression of each ENaC subunit (α, β and γ). CRT overexpression also up-regulated the cell surface expression of α-, β- and γ-ENaC. Moreover, we found that CRT directly interacts with each ENaC subunit. Although CRT knockdown did not affect the de novo synthesis of ENaC subunits, CRT overexpression decreased α-, β- and γ-ENaC expression in the detergent (RIPA)-insoluble fraction, suggesting that CRT enhanced the solubility of ENaC subunits. Consistent with the increased intracellular and cell surface expression of ENaC subunits, increased channel activity of ENaC was also observed upon overexpression of CRT. Our study thus identifies CRT as an ER chaperone that regulates ENaC expression and function.     

Lysyl tRNA synthetase is required for the translocation of calreticulin to the cell surface in immunogenic death

In response to immunogenic cell death inducers, calreticulin (CRT) translocates from its orthotopic localization in the lumen of the endoplasmic reticulum (ER) to the surface of the plasma membrane where it serves as an engulfment signal for antigen-presenting cells.¹ Here, we report that yet another ER protein, the lysyl-tRNA synthetase (KARS), was exposed on the surface of stressed cells, on which KARS co-localized with CRT in lipid rafts. Depletion of KARS with small interfering RNAs suppressed CRT exposure induced by anthracyclines or UVC light. In contrast to CRT, KARS was also found in the supernatant of stressed cells. Recombinant KARS protein was unable to influence the binding of recombinant CRT to the cell surface. Moreover, recombinant KARS protein was unable to stimulate macrophages in vitro. These results underscore the contribution of KARS to the emission of (one of) the principal signal(s) of immunogenic cell death, CRT exposure.     

Kinetics of interactions of sendai virus envelope glycoproteins, F and HN, with endoplasmic reticulum-resident molecular chaperones, BiP, calnexin, and calreticulin

Sendai virus envelope glycoproteins, F and HN, mature during their transport through the endoplasmic reticulum (ER) and Golgi complex. To better understand their maturation processes in the ER, we investigated the time course of their interactions with three ER- resident molecular chaperones, BiP, calnexin (CNX), and calreticulin (CRT), in Sendai virus-infected HeLa cells. Pulse-chase and immunoprecipitation analyses using antibodies against each virus glycoprotein or ER chaperone revealed that F precursor interacted with CNX transiently (t(1/2)=8 min), while HN protein displayed longer and sequential interactions with BiP (t(1/2)=8 min), CNX (t(1/2)=15 min), and CRT (t(1/2)=20 min). HN interacted with the three ER chaperones not only as a monomer but also as a tetramer for several hours, suggesting mechanism(s) to undergo chaperone-mediated quality control of an assembled HN oligomer in the ER. The kinetics of dissociation of the HN-chaperone complexes exhibited a marked delay in the presence of proteasome inhibitors, suggesting that a part of HN associated with BiP, CNX, and CRT is destined to be degraded in the proteasome-dependent pathway. Further, the associations between virus glycoproteins and CNX or CRT were impaired by castanospermine, an inhibitor of ER glucosidase I and II, confirming that these interactions require monoglucosylated oligosaccharide on F(0) and HN peptides. These findings together suggest that newly synthesized F protein undergoes rapid maturation in the ER through a transient interaction with CNX, whereas HN protein requires more complex processes involving prolonged association with BiP, CNX, and CRT for its quality control in the ER.     

Delayed Activation of the Store-operated Calcium Current Induced by Calreticulin Overexpression in RBL-1 Cells

Calreticulin (CRT) is a high-capacity, low-affinity Ca²⁺-binding protein located in the lumen of the endoplasmic reticulum (ER) of all eukaryotic cells investigated so far. Its high level of conservation among different species suggests that it serves functions fundamental to cell survival. The role originally proposed for CRT, i.e., the main Ca²⁺ buffer of the ER, has been obscured or even casted by its implication in processes as diverse as gene expression, protein folding, and cell adhesion. In this work we seek the role of CRT in Ca²⁺ storing and signaling by evaluating its effects on the kinetics and amplitude of the store-operated Ca²⁺ current (I_CRAC). We show that, in the rat basophilic leukemia cell line RBL-1, overexpression of CRT, but not of its mutant lacking the high-capacity Ca²⁺-binding domain, markedly retards the I_CRAC development, however, only when store depletion is slower than the rate of current activation. On the contrary, when store depletion is rapid and complete, overexpression of CRT has no effect. The present results are compatible with a major Ca²⁺-buffering role of CRT within the ER but exclude a direct, or indirect, role of this protein on the mechanism of I_CRAC activation.     

A Mechanism of Release of Calreticulin from Cells During Apoptosis

Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone responsible for glycoprotein folding and Ca²⁺ homeostasis. CRT also has extracellular functions, e.g. tumor and apoptotic cell recognition and wound healing, but the mechanism of CRT extracellular release is unknown. Cytosolic localization of CRT is determined by signal peptide and subsequent retrotranslocation of CRT into the cytoplasm. Here, we show that under apoptotic stress conditions, the cytosolic concentration of CRT increases and associates with phosphatidylserine (PS) in a Ca²⁺-dependent manner. PS distribution is regulated by aminophospholipid translocase (APLT), which maintains PS on the cytosolic side of the cell membrane. APLT is sensitive to redox modifications of its SH groups by reactive nitrogen species. During apoptosis, both CRT expression and the concentration of nitric oxide (NO) increase. By using S-nitroso-l-cysteine-ethyl-ester, an intracellular NO donor and inhibitor of APLT, we showed that PS and CRT externalization occurred together in an S-nitrosothiol-dependent and caspase-independent manner. Furthermore, the CRT and PS are relocated as punctate clusters on the cell surface. Thus, CRT induced nitrosylation and its externalization with PS could explain how CRT acts as a bridging molecule during apoptotic cell clearance.     

ERP57 membrane translocation dictates the immunogenicity of tumor cell death by controlling the membrane translocation of calreticulin

Several pieces of experimental evidence indicate the following: 1) the most efficient antitumor treatments (this principle applies on both chemotherapy and radiotherapy) are those that induce immunogenic cell death and are able to trigger a specific antitumor immune response; and 2) the immunogenicity of cell death depends very closely on the plasma membrane quantity of calreticulin (CRT), an endoplasmic reticulum (ER) stress protein exposed to the cell membrane after immunogenic treatment. Nevertheless, the mechanisms implicated in CRT translocation are unknown. CRT is known to interact in the ER with ERP57, another ER stress protein. I sought to determine whether ERP57 would have any role in tumor immunogenicity. In this article I report that CRT exposure is controlled by ERP57 exposure. CRT and ERP57 are translocated together in the same molecular complex. ERP57 knockdown suppressed CRT exposure as well as phagocytosis by dendritic cells and abolished the immunogenicity in vivo. Knockdown or the absence of CRT abolishes ERP57 exposure. Administration of recombinant ERP57, unlike the administration of recombinant CRT, did not restore the immunogenicity of CRT or ERP57 small interfering RNA-transfected tumor cells. Together, these studies identify ERP57 as a key protein that controls immunogenicity by controlling CRT exposure and illustrate the ability of ERP57 to serve as a new molecular marker of immunogenicity.     

Effects of calreticulin mutations on cell transformation and immunity

Myeloproliferative neoplasms (MPNs) are cancers involving dysregulated production and function of myeloid lineage hematopoietic cells. Among MPNs, Essential thrombocythemia (ET), Polycythemia Vera (PV) and Myelofibrosis (MF), are driven by mutations that activate the JAK–STAT signalling pathway. Somatic mutations of calreticulin (CRT), an endoplasmic reticulum (ER)-localized lectin chaperone, are driver mutations in approximately 25% of ET and 35% of MF patients. The MPN-linked mutant CRT proteins have novel frameshifted carboxy-domain sequences and lack an ER retention motif, resulting in their secretion. Wild type CRT is a regulator of ER calcium homeostasis and plays a key role in the assembly of major histocompatibility complex (MHC) class I molecules, which are the ligands for antigen receptors of CD8⁺ T cells. Mutant CRT-linked oncogenesis results from the dysregulation of calcium signalling in cells and the formation of stable complexes of mutant CRT with myeloproliferative leukemia (MPL) protein, followed by downstream activation of the JAK–STAT signalling pathway. The intricate participation of CRT in ER protein folding, calcium homeostasis and immunity suggests the involvement of multiple mechanisms of mutant CRT-linked oncogenesis. In this review, we highlight recent findings related to the role of MPN-linked CRT mutations in the dysregulation of calcium homeostasis, MPL activation and immunity.     

Heterologous expression of rice calnexin (OsCNX) confers drought tolerance in Nicotiana tabacum

Calnexin (CNX) is an integral membrane protein of endoplasmic reticulum (ER) and is a critical component of ER quality control machinery. It acts as a chaperone and ensures proper folding of newly synthesised glycoproteins. CNX shares a considerable homology with its luminal counterpart calreticulin (CRT). Together, they constitute CNX/CRT cycle which is imperative for proper folding of nascent proteins. CNX deficient organisms develop severe complications because of improper folding of proteins and consequently ER stress. CNX maintains calcium homeostasis by binding to the Ca²⁺ which is a central node in various signaling pathways. Phosphorylation of cytoplasmic tail of CNX controls the sarco endoplasmic reticulum calcium ATPase and thus the movement of Ca²⁺ in and out of its store-house, i.e. ER. Our studies on Oryza sativa CNX (OsCNX) reveal constitutive expression at various developmental stages and various tissues, thereby proving its requirement throughout the plant development. Further, its expression under various stress conditions gives an insight of the crosstalk existing between ER stress and abiotic stress signaling. This was confirmed by heterologous expression of OsCNX (OsCNX-HE) in tobacco and the OsCNX-HE lines were observed to exhibit better germination under mannitol stress and survival under dehydration stress conditions. The dehydration tolerance conferred by OsCNX appears to be ABA-dependent pathway.     

Efficient selection of branch-specific models of sequence evolution

The analysis of extant sequences shows that molecular evolution has been heterogeneous through time and among lineages. However, for a given sequence alignment, it is often difficult to uncover what factors caused this heterogeneity. In fact, identifying and characterizing heterogeneous patterns of molecular evolution along a phylogenetic tree is very challenging, for lack of appropriate methods. Users either have to a priori define groups of branches along which they believe molecular evolution has been similar or have to allow each branch to have its own pattern of molecular evolution. The first approach assumes prior knowledge that is seldom available, and the second requires estimating an unreasonably large number of parameters. Here we propose a convenient and reliable approach where branches get clustered by their pattern of molecular evolution alone, with no need for prior knowledge about the data set under study. Model selection is achieved in a statistical framework and therefore avoids overparameterization. We rely on substitution mapping for efficiency and present two clustering approaches, depending on whether or not we expect neighbouring branches to share more similar patterns of sequence evolution than distant branches. We validate our method on simulations and test it on four previously published data sets. We find that our method correctly groups branches sharing similar equilibrium GC contents in a data set of ribosomal RNAs and recovers expected footprints of selection through dN/dS. Importantly, it also uncovers a new pattern of relaxed selection in a phylogeny of Mantellid frogs, which we are able to correlate to life-history traits. This shows that our programs should be very useful to study patterns of molecular evolution and reveal new correlations between sequence and species evolution. Our programs can run on DNA, RNA, codon, or amino acid sequences with a large set of possible models of substitutions and are available at http://biopp.univ-montp2.fr/forge/testnh.     

YKL-40/c-Met Expression in Rectal Cancer Biopsies Predicts Tumor Regression following Neoadjuvant Chemoradiotherapy: A Multi-Institutional Study

Background

Neoadjuvant chemo-radiotherapy (CRT) followed by surgical resection is the standard treatment for locally advanced rectal cancer, although complete tumor pathological regression is achieved in only up to 30% of cases. A clinicopathological and molecular predictive stratification of patients with advanced rectal cancer is still lacking. Here, c-Met and YKL-40 have been studied as putative predictors of CRT response in rectal cancer, due to their reported involvement in chemoradioresistance in various solid tumors.

Material and Methods

A multicentric study was designed to assess the role of c-Met and YKL-40 expression in predicting chemoradioresistance and to correlate clinical and pathological features with CRT response. Immunohistochemistry and fluorescent in situ hybridization for c-Met were performed on 81 rectal cancer biopsies from patients with locally advanced rectal adenocarcinoma. All patients underwent standard (50.4 gy in 28 fractions + concurrent capecitabine 825 mg/m²) neoadjuvant CRT or the XELOXART protocol. CRT response was documented on surgical resection specimens and recorded as tumor regression grade (TRG) according to the Mandard criteria.

Results

A significant correlation between c-Met and YKL-40 expression was observed (R = 0.43). The expressions of c-Met and YKL-40 were both significantly associated with a lack of complete response (86% and 87% of c-Met and YKL-40 positive cases, p< 0.01 and p = 0.006, respectively). Thirty of the 32 biopsies co-expressing both markers had partial or absent tumor response (TRG 2-5), strengthening their positive predictive value (94%). The exclusive predictive role of YKL-40 and c-Met was confirmed using a multivariate analysis (p = 0.004 and p = 0.007 for YKL-40 and c-Met, respectively). TRG was the sole morphological parameter associated with poor outcome.

Conclusion

c-Met and YKL-40 expression is a reliable predictor of partial/absent response to neoadjuvant CRT in rectal cancer. Targeted therapy protocols could take advantage of prior evaluations of c-MET and YKL-40 expression levels to increase therapeutic efficacy.     

钙网蛋白的免疫生物学活性研究进展

钙网蛋白(calreticulin,CRT)是内质网中的Ca2+结合蛋白,对维持细胞内的Ca2+稳态具有重要作用。CRT还可作为分子伴侣,帮助新合成的内质网蛋白正确折叠。除此之外,CRT还出现在细胞膜表面及细胞外环境中,并发挥重要的免疫调节作用。膜表面的CRT参与凋亡细胞的清除和抗肿瘤免疫应答,在T细胞活化早期也发挥重要作用。可溶性CRT出现在自身免疫病患者的血清中,且其浓度与疾病活动度相关。重组可溶性CRT腹腔注射可显著影响小鼠的Th1/Th2平衡。重组可溶性CRT分子不仅具有超强免疫刺激活性,还表现出很强的佐剂效应,提示可溶性CRT可能在自身免疫性疾病的发生与发展过程中发挥一定作用。可以预见,CRT分子的免疫生物学活性将在不久的将来成为该领域研究的一个热点。     

Hard tick calreticulin (CRT) gene coding regions have only one intron with conserved positions and variable sizes

Calreticulin (CRT) is a unique eukaryotic gene. The CRT gene product, calreticulin, was first identified as a calcium binding protein in 1974, but further investigations have indicated that CRT protein performs many functions in cells, including involvement in evading the host's immune system by parasites. Many studies of CRT have been published since the molecule was first discovered; however, the CRT gene exon-intron structure is only known for a limited number of ectoparasite species. In this study, we compared tick CRT genomic sequences to the corresponding cDNA from 28 species and found that 2 exons and 1 intron are present in the tick CRT gene. The intron position is conserved in 28 hard ticks, but intron size and nucleotide sequences vary. Three tick introns possess duplicated fragments and are twice as long as other introns. All tick CRT introns obey the GT-AG rule in the splice-site junctions and are phase 1 introns. By comparing tick CRT introns to those of fruit fly, mouse, and human, we conclude that tick CRT introns belong to the intron-late type. The number and size of CRT introns have increased through the evolution of eukaryotes.