GlcNAc6ST2/CHST4 Is Essential for the Synthesis of R-10G-Reactive Keratan Sulfate/Sulfated N-Acetyllactosamine Oligosaccharides in Mouse Pleural Mesothelium.

We recently showed that 6-sulfo sialyl N-acetyllactosamine (LacNAc) in O-linked glycans recognized by the CL40 antibody is abundant in the pleural mesothelium under physiological conditions and that these glycans undergo complementary synthesis by GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5) in mice. GlcNAc6ST3 is essential for the synthesis of R-10G-positive keratan sulfate (KS) in the brain. The predicted minimum epitope of the R-10G antibody is a dimeric asialo 6-sulfo LacNAc. Whether R-10G-reactive KS/sulfated LacNAc oligosaccharides are also present in the pleural mesothelium was unknown. The question of which GlcNAc6STs are responsible for R-10G-reactive glycans was an additional issue to be clarified. Here, we show that R-10G-reactive glycans are as abundant in the pulmonary pleura as CL40-reactive glycans and that GlcNAc6ST3 is only partially involved in the synthesis of these pleural R-10G glycans, unlike in the adult brain. Unexpectedly, GlcNAc6ST2 is essential for the synthesis of R-10G-positive KS/sulfated LacNAc oligosaccharides in the lung pleura. The type of GlcNAc6ST and the magnitude of its contribution to KS glycan synthesis varied among tissues in vivo. We show that GlcNAc6ST2 is required and sufficient for R-10G-reactive KS synthesis in the lung pleura. Interestingly, R-10G immunoreactivity in KSGal6ST (encoded by Chst1) and C6ST1 (encoded by Chst3) double-deficient mouse lungs was markedly increased. MUC16, a mucin molecule, was shown to be a candidate carrier protein for pleural R-10G-reactive glycans. These results suggest that R-10G-reactive KS/sulfated LacNAc oligosaccharides may play a role in mesothelial cell proliferation and differentiation. Further elucidation of the functions of sulfated glycans synthesized by GlcNAc6ST2 and GlcNAc6ST3, such as R-10G and CL40 glycans, in pathological conditions may lead to a better understanding of the underlying mechanisms of the physiopathology of the lung mesothelium.

Sulfated glycosaminoglycans mediate prion-like behavior of p53 aggregates.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) are heteropolysaccharides implicated in the pathology of protein aggregation diseases including localized and systemic forms of amyloidosis. Among subdomains of sulfated GAGs, highly sulfated domains of HS, called HS S-domains, have been highlighted as being critical for HS function in amyloidoses. Recent studies suggest that the tumor suppressor p53 aggregates to form amyloid fibrils and propagates in a prion-like manner; however, molecules and mechanisms that are involved in the prion-like behavior of p53 aggregates have not been addressed. Here, we identified sulfated GAGs as molecules that mediate prion-like behavior of p53 aggregates. Sulfated GAGs at the cell surface were required for cellular uptake of recombinant and cancer cell-derived p53 aggregates and extracellular release of p53 from cancer cells. We further showed that HS S-domains accumulated within p53 deposits in human ovarian cancer tissues, and enzymatic remodeling of HS S-domains by Sulf-2 extracellular sulfatase down-regulated cellular uptake of p53 aggregates. Finally, sulfated GAG-dependent cellular uptake of p53 aggregates was critical for subsequent extracellular release of the aggregates and gain of oncogenic function in recipient cells. Our work provides a mechanism of prion-like behavior of p53 aggregates and will shed light on sulfated GAGs as a common mediator of prions.

Thrombospondin type 1 repeat-derived C-mannosylated peptide attenuates synaptogenesis of cortical neurons induced by primary astrocytes via TGF-ß.

C-Mannosylation is a rare type of protein glycosylation and is reportedly critical for the proper folding and secretion of parental proteins. Still, the effects of C-mannosylation on the biological functions of these modified proteins remain to be elucidated. The Trp-x-x-Trp (WxxW) sequences, whose first tryptophan (Trp) can be C-mannosylated, constitute the consensus motifs for this glycosylation modification and are commonly found in thrombospondin type 1 repeats that regulate molecular functions of thrombospondin 1 in binding and activation of transforming growth factor ß (TGF-ß). TGF-ß plays critical roles in the control of the central nervous system including synaptogenesis. Here, we investigated whether C-mannosylation of the synthetic Trp-Ser-Pro-Trp (WSPW) peptide may confer certain functions to this peptide in TGF-ß-mediated synaptogenesis. By using primary cultured rat astrocytes and cortical neurons, we found that the C-mannosylated WSPW (C-Man-WSPW) peptide, but not non-mannosylated WSPW peptide, suppressed astrocyte-conditioned medium (ACM)-stimulated synaptogenesis. C-Man-WSPW peptide inhibited both ACM- and recombinant mature TGF-ß1-induced activations of Smad 2, an important mediator in TGF-ß signaling. Interactions of recombinant mature TGF-ß with the C-Man-WSPW peptide were similar to those with non-C-mannosylated WSPW peptide. Taken together, our results reveal a novel function of C-mannosylation of the WxxW motif in signaling and synaptogenesis mediated by TGF-ß. Molecular details of how C-mannosylation affects the biological functions of WxxW motifs deserve future study for clarification.

Complementary Role of GlcNAc6ST2 and GlcNAc6ST3 in Synthesis of CL40-Reactive Sialylated and Sulfated Glycans in the Mouse Pleural Mesothelium.

Sialyl 6-sulfo Lewis X (6-sulfo sLeX) and its derivative sialyl 6-sulfo N-acetyllactosamine (LacNAc) are sialylated and sulfated glycans of sialomucins found in the high endothelial venules (HEVs) of secondary lymphoid organs. A component of 6-sulfo sLeX present in the core 1-extended O-linked glycans detected by the MECA-79 antibody was previously shown to exist in the lymphoid aggregate vasculature and bronchial mucosa of allergic and asthmatic lungs. The components of 6-sulfo sLeX in pulmonary tissues under physiological conditions remain to be analyzed. The CL40 antibody recognizes 6-sulfo sLeX and sialyl 6-sulfo LacNAc in O-linked and N-linked glycans, with absolute requirements for both GlcNAc-6-sulfation and sialylation. Immunostaining of normal mouse lungs with CL40 was performed and analyzed. The contribution of GlcNAc-6-O-sulfotransferases (GlcNAc6STs) to the synthesis of the CL40 epitope in the lungs was also elucidated. Here, we show that the expression of the CL40 epitope was specifically detected in the mesothelin-positive mesothelium of the pulmonary pleura. Moreover, GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5), but not GlcNAc6ST1 (encoded by Chst2) or GlcNAc6ST4 (encoded by Chst7), are required for the synthesis of CL40-positive glycans in the lung mesothelium. Furthermore, neither GlcNAc6ST2 nor GlcNAc6ST3 is sufficient for in vivo expression of the CL40 epitope in the lung mesothelium, as demonstrated by GlcNAc6ST1/3/4 triple-knock-out and GlcNAc6ST1/2/4 triple-knock-out mice. These results indicate that CL40-positive sialylated and sulfated glycans are abundant in the pleural mesothelium and are synthesized complementarily by GlcNAc6ST2 and GlcNAc6ST3, under physiological conditions in mice.

Protein C-Mannosylation and C-Mannosyl Tryptophan in Chemical Biology and Medicine.

C-Mannosylation is a post-translational modification of proteins in the endoplasmic reticulum. Monomeric α-mannose is attached to specific Trp residues at the first Trp in the Trp-x-x-Trp/Cys (W-x-x-W/C) motif of substrate proteins, by the action of C-mannosyltransferases, DPY19-related gene products. The acceptor substrate proteins are included in the thrombospondin type I repeat (TSR) superfamily, cytokine receptor type I family, and others. Previous studies demonstrated that C-mannosylation plays critical roles in the folding, sorting, and/or secretion of substrate proteins. A C-mannosylation-defective gene mutation was identified in humans as the disease-associated variant affecting a C-mannosylation motif of W-x-x-W of ADAMTSL1, which suggests the involvement of defects in protein C-mannosylation in human diseases such as developmental glaucoma, myopia, and/or retinal defects. On the other hand, monomeric C-mannosyl Trp (C-Man-Trp), a deduced degradation product of C-mannosylated proteins, occurs in cells and extracellular fluids. Several studies showed that the level of C-Man-Trp is upregulated in blood of patients with renal dysfunction, suggesting that the metabolism of C-Man-Trp may be involved in human kidney diseases. Together, protein C-mannosylation is considered to play important roles in the biosynthesis and functions of substrate proteins, and the altered regulation of protein C-manosylation may be involved in the pathophysiology of human diseases. In this review, we consider the biochemical and biomedical knowledge of protein C-mannosylation and C-Man-Trp, and introduce recent studies concerning their significance in biology and medicine.

Hsc70 is required for E-cadherin expression in epithelial-like NRK-52E cells.

Heat-shock cognate protein 70 (Hsc70), a molecular chaperone, is involved in multiple cellular functions. We previously demonstrated that Hsc70 is required for TGF-ß-induced Smad signaling in mesenchymal-like NRK-49F cells. In the present study, to compare the Hsc70 functions in TGF-ß-related signaling between epithelial and mesenchymal cells, we examined the effect of Hsc70 downregulation on TGF-ß-induced signaling in epithelial-like NRK-52E cells. TGF-ß-induced Smad signaling was suppressed in cells treated with small interfering RNA (siRNA) for Hsc70. Interestingly, despite interference with TGF-ß signaling, TGF-ß-induced suppression of E-cadherin expression was not affected by Hsc70 knockdown. Instead, Hsc70 knockdown itself caused the suppression of E-cadherin expression at the transcription level in cells treated with Hsc70 siRNA. We also examined the effects of Hsc70 knockdown on the level of E-cadherin-gene repressors, such as Snail1, Slug, Zeb1, Zeb2, and Twist1, and found that transcription of the repressors was upregulated after 24- or 36-h treatment with Hsc70 siRNA. Collectively, these results indicate that, in addition to a supportive role in TGF-ß-induced signaling, Hsc70 supports E-cadherin expression through downregulation of the E-cadherin-gene repressors in NRK-52E cells, suggesting that Hsc70 plays a functional role to maintain the epithelial cell phenotype.

Hsc70 facilitates TGF-ß-induced activation of Smad2/3 in fibroblastic NRK-49F cells.

Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-ß-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-ß signaling, was also suppressed in the early phase of TGF-ß stimulation. Upon stimulation with TGF-ß, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-ß-induced Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-ß-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-ß-induced Smad signaling in NRK-49F cells.

Beta3Gn-T7 Is a Keratan Sulfate ß1,3 N-Acetylglucosaminyltransferase in the Adult Brain.

Keratan sulfate (KS) glycan is covalently attached to a core protein of proteoglycans. KS is abundant in neuropils and presents densely in close proximity to the perineuronal region of the perineuronal net-positive neurons in the adult brain under physiological conditions. We previously showed that the synthesis of KS positive for the R-10G antibody in the adult brain is mediated by GlcNAc-6-sulfotransferase 3 (GlcNAc6ST3; encoded by Chst5). Deficiency in both GlcNAc6ST3 and GlcNAc6ST1, encoded by Chst2, completely abolished KS. Protein-tyrosine phosphatase receptor type z1 (Ptprz1)/phosphacan was identified as a KS scaffold. KS requires the extension of GlcNAc by ß1,3 N-acetylglucosaminyltransferase (Beta3Gn-T). Members of the Beta3Gn-T family involved in the synthesis of adult brain KS have not been identified. In this study, we show by a method of gene targeting that Beta3Gn-T7, encoded by B3gnt7, is a major Beta3Gn-T for the synthesis of KS in neuropils and the perineuronal region in the adult brain. Intriguingly, the B3gnt7 gene is selectively expressed in oligodendrocyte precursor cells (OPCs) and oligodendrocytes similar to that of GlcNAc6ST3. These results indicate that Beta3Gn-T7 in oligodendrocyte lineage cells may play a role in the formation of neuropils and perineuronal nets in the adult brain through the synthesis of R-10G-positive KS-modified proteoglycan.

C-Mannosylated tryptophan-containing WSPW peptide binds to actinin-4 and alters E-cadherin subcellular localization in lung epithelial-like A549 cells.

The Trp-x-x-Trp (W-x-x-W) peptide motif, a consensus site for C-mannosylation, is the functional motif in cytokine type I receptors or thrombospondin type I repeat (TSR) superfamily proteins. W-x-x-W motifs are important for physiological and pathological functions of their parental proteins, but effects of C-mannosylation on protein functions remain to be elucidated. By using chemically synthesized WSPW peptides and C-mannosylated WSPW peptides (C-Man-WSPW), we herein investigated whether C-mannosylation of WSPW peptides confer additional biological functions to WSPW peptides. C-Man-WSPW peptide, but not non-mannosylated WSPW, reduced E-cadherin levels in A549 cells. Via peptide mass fingerprinting analysis, we identified actinin-4 as a C-Man-WSPW-binding protein in A549 cells. Actinin-4 partly co-localized with E-cadherin or ß-catenin, despite no direct interaction between actinin-4 and E-cadherin. C-Man-WSPW reduced co-localization of E-cadherin and actinin-4; non-mannosylated WSPW had no effect on localization. In actinin-4-knockdown cells, E-cadherin was upregulated and demonstrated a punctate staining pattern in the cytoplasm, which suggests that actinin-4 regulated cell-surface E-cadherin localization. Thus, C-mannosylation of WSPW peptides is required for interaction with actinin-4 that subsequently alters expression and subcellular localization of E-cadherin and morphology of epithelial-like cells. Our results therefore suggest a regulatory role of C-mannosylation of the W-x-x-W motif in interactions between the motif and its binding partner and will thereby enhance understanding of protein C-mannosylation.

Calnexin Is Involved in Forskolin-Induced Syncytialization in Cytotrophoblast Model BeWo Cells.

Calnexin (CNX), a membrane-bound molecular chaperone, is involved in protein folding and quality control of nascent glycoproteins in the endoplasmic reticulum. We previously suggested critical roles of calreticulin, a functional paralogue of CNX, in placentation, including invasion of extravillous trophoblasts and syncytialization of cytotrophoblasts. However, the roles of CNX in placentation are unclear. In human choriocarcinoma BeWo cells, which serve as an experimental model of syncytialization, CNX knockdown suppressed forskolin-induced cell fusion and ß-human chorionic gonadotropin (ß-hCG) induction. Cell-surface luteinizing hormone/chorionic gonadotropin receptor, a ß-hCG receptor, was significantly down-regulated in CNX-knockdown cells, which suggested the presence of a dysfunctional autocrine loop of ß-hCG up-regulation. In this study, we also found abundant CNX expression in normal human placentas. Collectively, our results revealed the critical role of CNX in the syncytialization-related signaling in a villous trophoblast model and suggest a link between CNX expression and placenta development.

Cytoplasmic p53 aggregates accumulated in p53-mutated cancer correlate with poor prognosis.

Recent studies suggested that aggregates of mutant p53 proteins may propagate and impair normal p53 functioning in recipient cells. Our previous study showed that cancer cell-derived p53 aggregates that cells internalized interfered with p53-dependent apoptosis in recipient cells. However, involvement of p53 aggregate propagation in cancer pathology has not been fully elucidated. Here, we screened patients with high-grade serous ovarian carcinoma, which is characterized by an extremely high frequency of TP53 gene mutations, to show that patients with cytoplasmic p53 deposits have a poor prognosis compared with patients with complete p53 absence or strong nuclear p53 positivity. Cytoplasmic p53 in the patients with poor prognosis consisted of protein aggregates, which suggests that p53 aggregates are oncogenic drivers. Indeed, an inhibitor of p53 aggregation restored cellular apoptosis, a proper p53 function, in p53 aggregate-bearing patient-derived tumor organoids. In cell-based assays, endogenous and exogenous mutant p53 aggregates hindered chemotherapeutic activity of cisplatin, which depends on normal p53 functions. This inhibition was reduced by blocking p53 aggregation or internalization of p53 aggregates. Our study, thus indicates the involvement of p53 aggregate transmission in poor prognosis and in chemotherapy resistance in cancers.

Alteration of integrin-dependent adhesion and signaling in EMT-like MDCK cells established through overexpression of calreticulin.

Calreticulin (CRT) is a multi-functional Ca(2+) -binding molecular chaperone in the endoplasmic reticulum. We previously reported that kidney epithelial cell-derived Madin-Darby Canine Kidney cells were transformed into mesenchymal-like cells by gene transfection of CRT. In this study, we investigated the altered characteristics of cell adhesion in these epithelial-mesenchymal transition (EMT)-like cells. Several extracellular matrix substrata were tested, and cell adhesion to fibronectin was found to be specifically increased in the CRT-overexpressing cells compared to controls. The expression of integrins was significantly up-regulated in subunits α5 and αV, resulting in an increase in the formation of complexes such as α5ß1 and αVß3. These integrins also contributed to the enhanced binding of fibronectin. In the CRT-overexpressing cells, the phosphorylation of Akt, a downstream target of integrin-linked kinase (ILK), was up-regulated on attachment to fibronectin or collagen IV. Integrin-associated signaling through ILK was also promoted on attachment to fibronectin, suggesting some of the correlation between ILK and Akt in the CRT-overexpressing cells. Furthermore, on treatment with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester, a membrane-permeable Ca(2+) chelator, the enhanced Akt signaling was suppressed with a concomitant decrease in the formation of complexes between integrins and ILK in the CRT-overexpressing cells. In conclusion, these findings demonstrate that CRT regulates cell-substratum adhesion by modulating integrin-associated signaling through altered Ca(2+) homeostasis in the CRT-overexpressing EMT-like cells, suggesting a novel regulatory role for CRT in EMT.

Cell-to-cell transmission of p53 aggregates: a novel player in oncology?

The mutants of the tumor suppressor protein p53 form protein aggregates. It has been proposed that these aggregates propagate like prions, albeit the detailed mechanism of the propagation is unclear. Our recent study revealed that sulfated glycosaminoglycans, especially highly sulfated domains of heparan sulfate (heparan sulfate S-domains), participate in cancer pathology by mediating transcellular propagation of p53 aggregates.

Lipid Droplet Accumulation Independently Predicts Poor Clinical Prognosis in High-Grade Serous Ovarian Carcinoma.

High-grade serous ovarian carcinoma (HGSOC) is an epithelial cancer that accounts for most ovarian cancer deaths. Metabolic abnormalities such as extensive aerobic glycolysis and aberrant lipid metabolism are well-known characteristics of cancer cells. Indeed, accumulation of lipid droplets (LDs) in certain types of malignant tumors has been known for more than 50 years. Here, we investigated the correlation between LD accumulation and clinical prognosis. In 96 HGSOC patients, we found that high expression of the LD marker adipophilin was associated with poor progression-free and overall survival (p = 0.0022 and p = 0.014, respectively). OVCAR-3 ovarian carcinoma cells accumulated LDs in a glucose-dependent manner, which suggested the involvement of aerobic glycolysis and subsequently enhanced lipogenesis, with a result being LD accumulation. The acyl-CoA: cholesterol acyltransferase 1 inhibitor K604 and the hydroxymethylglutaryl-CoA reductase inhibitor pitavastatin blocked LD accumulation in OVCAR-3 cells and reduced phosphorylation of the survival-related kinases Akt and ERK1/2, both of which have been implicated in malignancy. Our cell-based assays thus suggested that enhanced aerobic glycolysis resulted in LD accumulation and activation of survival-related kinases. Overall, our results support the idea that cancers with lipogenic phenotypes are associated with poor clinical prognosis, and we suggest that adipophilin may serve as an independent indicator of a poor prognosis in HGSOC.

Extracellularly Released Calreticulin Induced by Endoplasmic Reticulum Stress Impairs Syncytialization of Cytotrophoblast Model BeWo Cells.

The pregnancy-specific syndrome preeclampsia is a major cause of maternal mortality throughout the world. The initial insult resulting in the development of preeclampsia is inadequate trophoblast invasion, which may lead to reduced maternal perfusion of the placenta and placental dysfunction, such as insufficient trophoblast syncytialization. Endoplasmic reticulum (ER) stress has been implicated in the pathology of preeclampsia and serves as the major risk factor. Our previous studies suggested critical roles of calreticulin (CRT), which is an ER-resident stress response protein, in extravillous trophoblast invasion and cytotrophoblast syncytialization. Here, we studied the mechanism by which ER stress exposes the placenta to the risk of preeclampsia. We found that CRT was upregulated in the serum samples, but not in the placental specimens, from preeclamptic women. By using BeWo cells, an established model of cytotrophoblasts that syncytialize in the presence of forskolin, we demonstrated that thapsigargin-induced ER stress caused extracellular release of CRT from BeWo cells and that the extracellular CRT suppressed forskolin-induced release of ß-human chorionic gonadotropin and altered subcellular localization of E-cadherin, which is a key adhesion molecule associated with syncytialization. Our results together provide evidence that induction of ER stress leads to extracellular CRT release, which may contribute to placental dysfunction by suppressing cytotrophoblast syncytialization.

C-Mannosylated peptides derived from the thrombospondin type 1 repeat interact with Hsc70 to modulate its signaling in RAW264.7 cells.

The thrombospondin type 1 repeat (TSR) is a functional module of proteins called TSR superfamily proteins (e.g., thrombospondin, F-spondin, mindin, etc.) and includes a conserved Trp-x-x-Trp (W-x-x-W) motif, in which the first Trp residue is preferably modified by C-mannosylation. We previously reported that synthesized C-mannosylated TSR-derived peptides (e.g., C-Man-WSPW) specifically enhanced lipopolysaccharide-induced signaling in macrophage-like RAW264.7 cells. In this study, we searched for the proteins that bind to C-mannosylated TSR-derived peptides in RAW264.7 cells and identified heat shock cognate protein 70 (Hsc70). The binding affinity of Hsc70 for C-mannosylated peptides in solution was higher than that for the peptides without C-mannose. The binding was influenced by a nucleotide-induced conformational change of Hsc70, and C-mannosylated peptides preferred the substrate-binding domain of Hsc70. Furthermore, in RAW264.7 cells, addition of Hsc70 stimulated cellular signaling to produce tumor necrosis factor-alpha, via transforming growth factor-beta-activated kinase 1, and the Hsc70-induced signaling was enhanced more in the presence of the peptides with C-mannose than that without C-mannose, suggesting functional interaction between Hsc70 and the C-mannosylated peptides in the cells. Together, these results demonstrate a novel function of the C-mannosylation of TSR-derived peptides in terms of interaction with Hsc70 to regulate cellular signaling.

Calnexin/Calreticulin and Assays Related to N-Glycoprotein Folding In Vitro.

Calnexin (CNX) and calreticulin (CRT) are ER-resident lectin-like molecular chaperones involved in the quality control of secretory or membrane glycoproteins. They can exert molecular chaperone functions via specific binding to the early processing intermediates of Glc1Man9GlcNAc2 oligosaccharides of N-glycoproteins. CNX and CRT have similar N-terminal luminal domains and share the same jelly roll tertiary structure as legume lectins. In addition to the lectin-like interactions, CNX and CRT also suppress the aggregation of non-glycosylated substrates through interaction with hydrophobic peptide parts, suggesting a general chaperone function in glycan-dependent and glycan-independent manners. This chapter describes the isolation and purification of CRT produced in a bacterial expression system. We also introduce in vitro assays to estimate the molecular chaperone functions of CRT via the interaction with monoglucosylated N-glycans using Jack bean α-mannosidase as a target substrate. These assays are valuable in assessing quality control events related to the CNX/CRT chaperone cycle and lectin functions.

Calreticulin protects insulin against reductive stress in vitro and in MIN6 cells.

Oxidative folding of proinsulin in the endoplasmic reticulum (ER) is critical for the proper sorting and secretion of insulin from pancreatic ß-cells. Here, by using non-cell-based insulin aggregation assays and mouse insulinoma-derived MIN6 cells, we searched for a candidate molecular chaperone for (pro)insulin when its oxidative folding is compromised. We found that interaction between insulin and calreticulin (CRT), a lectin that acts as an ER-resident chaperone, was enhanced by reductive stress in MIN6 cells. Co-incubation of insulin with recombinant CRT prevented reductant-induced aggregation of insulin. Furthermore, lysosomal degradation of proinsulin, which was facilitated by dithiothreitol-induced reductive stress, depended on CRT in MIN6 cells. Together, our results suggest that CRT may be a protective molecule against (pro)insulin aggregation when oxidative folding is defective, e.g. under reductive stress conditions, in vitro and in cultured cells. Because CRT acts as a molecular chaperone for not only glycosylated proteins but also non-glycosylated polypeptides, we also propose that (pro)insulin is a novel candidate client of the chaperone function of CRT.

Calreticulin Regulates Syncytialization Through Control of the Synthesis and Transportation of E-Cadherin in BeWo Cells.

During placental development, mononuclear cytotrophoblasts differentiate and fuse to syncytiotrophoblasts (STBs) to form syncytia, which secrete human chorionic gonadotropin (hCG). Decreased maternal perfusion of the placenta, which leads to placental dysfunction, induces changes in trophoblast syncytialization. Our previous study showed that calreticulin (CRT), a Ca2+-binding molecular chaperone found in the endoplasmic reticulum, is expressed in the human placenta and is involved in regulating extravillous trophoblast invasion, although its role in villous trophoblasts remains unclear. In this study, we investigated the functional role of CRT in trophoblast differentiation using the human trophoblast-like cell line BeWo, in which CRT gene expression was knocked down. We found that CRT was highly expressed in human third-trimester placentas and mainly localized in STBs. The fetal growth restriction group exhibited significantly lower CRT expression levels than did the control group. In BeWo cells, CRT knockdown markedly suppressed forskolin-induced cell fusion and ß-hCG secretion. As for the mechanism responsible for these effects, the cell surface expression of E-cadherin, a key adhesion molecule related to syncytialization, was decreased, and E-cadherin accumulated adjacent to the Golgi apparatus in the CRT-knockdown cells, which led to dysfunctional cell-to-cell adhesion. Additionally, metabolic labeling and a pulse-chase study revealed that the protein expression of E-cadherin was suppressed at the translational level in the CRT-knockdown cells. Collectively, these results demonstrate that CRT regulates syncytialization by ensuring appropriate control of both the synthesis and transportation of E-cadherin, suggesting that CRT expression is important for placental development during pregnancy.

Calreticulin Is Involved in Invasion of Human Extravillous Trophoblasts Through Functional Regulation of Integrin ß1.

Calreticulin (CRT), a molecular chaperone in the endoplasmic reticulum (ER), plays a variety of roles in cell growth, differentiation, apoptosis, immunity, and cancer biology. It has been reported that CRT is expressed in the human placenta, although its function in placental development is poorly understood. Appropriate invasion of extravillous trophoblasts (EVTs) into the maternal decidua is necessary for successful pregnancy. The objective of the present study was to investigate the expression and functional role of CRT in EVTs using the human EVT cell line HTR8/SVneo, in which CRT gene expression was knocked down. We found that CRT was highly expressed in the human placenta in the early stage of pregnancy and localized to the EVTs. CRT knockdown markedly suppressed the invasion ability of HTR8/SVneo cells. Furthermore, the adhesion to fibronectin was suppressed in the CRT-knockdown cells via the dysfunction of integrin α5ß1. In the CRT-knockdown cells, terminal sialylation and fucosylation were decreased, and the core galactose-containing structure was increased in the N-glycans of integrin ß1. In addition, the expression levels of several critical glycosyltransferases were changed in the CRT-knockdown cells, consistent with the changes in the N-glycans. These results showed that CRT regulates the function of integrin ß1 by affecting the synthesis of N-glycans in HTR8/SVneo cells. Collectively, the results of the present study demonstrate that the ER chaperone CRT plays a regulatory role in the invasion of EVTs, suggesting the importance of CRT expression in placental development during early pregnancy.