Neem tree serves as a cornucopia for triterpenoids called limonoids that are of profound interest to humans due to their diverse biological activities. However, the biosynthetic pathway that plant employs for the production of limonoids remains unexplored for this wonder tree.13C isotopologues of glucose in neem cell suspension. Growth and development specific limonoid spectrum of neem seedling and time dependent limonoid biosynthetic characteristics of cell lines were established. Further to understand the role of mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways in limonoid biosynthesis, Ultra Performance Liquid Chromatography (UPLC)- tandem mass spectrometry based structure-fragment relationship developed for limonoids and their isotopologues have been utilized. Analyses of labeled limonoid extract lead to the identification of signature isoprenoid units involved in azadirachtin and other limonoid biosynthesis, which are found to be formed through mevalonate pathway. This was further confirmed by treatment of cell suspension with mevinolin, a specific inhibitor for MVA pathway, which resulted in drastic decrease in limonoid levels whereas their biosynthesis was unaffected with fosmidomycin mediated plastidial methylerythritol 4-phosphate (MEP) pathway inhibition. This was also conspicuous, as the expression level of genes encoding for the rate-limiting enzyme of MVA pathway, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) was comparatively higher to that of deoxyxylulose-phosphate synthase (DXS) of MEP pathway in different tissues and also in the in vitro grown cells. Thus, this study will give a comprehensive understanding of limonoid biosynthetic pathway with differential contribution of MVA and MEP pathways.Herein, we report the tracing of limonoid biosynthetic pathway through feeding experiments using 13C limonoid isotopologues analysis. The undifferentiated cell lines of neem suspension produced a spectrum of C-seco limonoids, similar to parental tissue, kernel. Azadirachtin, a C-seco limonoid is produced in young tender leaves of plant whereas in the hard mature leaves of tree, ring intact limonoid nimocinol accumulates in high level. Furthermore, mevalonate pathway exclusively contributes for isoprene units of limonoids as evidenced through stable isotope labeling and no complementation of MEP pathway was observed with mevalonate pathway dysfunction, using chemical inhibitors.Limonoid biosynthesis of neem tree and cell lines have been unraveled through comparative quantification of limonoids with that of neem tree and through The online version of this article (10.1186/s12870-018-1447-6) contains supplementary material, which is available to authorized users. Azadirachta indica A. Juss (Indian Lilac), a member of Mahogany family (Meliaceae) is a medicinal tree of Indian subcontinent. Different parts of the tree have been used in traditional Ayurvedic and Unani medicine for the treatment of myriads of human ailments , [2-13C] glucose (Glc) isotopomers and its isotopologue,  Glc in Azadirachta indica suspension cell cultures, which served as an experimental system for studying the biosynthesis of limonoids. We have utilized high resolution-tandem Mass spectrometry to study the limonoids produced in cell culture followed by the study of 13C labeling pattern of the limonoids obtained through feeding experiment. The dissection of the chemical complexity of limonoids by rendition through the isotopologues and isotopomers obtained with tandem MS was made possible because of high mass resolution and accuracy of MS. The isotope labeling experiments were carried out subsequent to the inhibition of isoprenoid biosynthetic pathways to study the complementation of MVA and MEP pathways for the biosynthesis of limonoids in neem cell culture. Spatial expression levels of genes involved in the rate-limiting step of the MVA and MEP pathways were studied in different tissues of neem tree. To further comprehend their regulatory roles during the inhibition of MVA pathway, the expression level of the same genes were examined through quantitative polymerase chain reaction (qPCR) experiments.To gain insight into the isoprenoid biosynthetic pathway(s) involved in the contribution of isoprene units into limonoid skeleton, we carried out labeling experiment with  Glc, [2-13C] Glc or  Glc. The control experiment was carried out using MS media containing unlabeled glucose under similar conditions. At the end of 19 days of incubation, cell biomass were extracted for limonoids and subjected to UPLC-HRMS analysis. Azadirachtin A, nimbin, 6-deacetylnimbin, nimbinene, 6-deacetylnimbinene, salannin, 3-deacetylsalannin, salannolacetate and nimbanal were identified in the cell suspension generated 13C isotopologues, which showed normal distribution pattern in their relative intensity. Azadirachtin A, salannin, salannolacetate, 6-deacetylnimbinene were identified in abundance with high intensity of isotopologues. The isotopologues varied in their number and intensity for each of the studied limonoid, upon labeling with different positional 13C-Glc tracers. High resolution MS/MS serves as a powerful technique, and helped us for the determination of intramolecular distribution of 13C in the isotopologues of the metabolite. Natural abundance of isotopologues was observed in MS for limonoids obtained from unlabeled control cell suspension . However, in 13C enrichment experiments, molecular ions obtained in MS are represented by different isotopologues.The isoprene units synthesized through MVA and MEP pathway will have different labeling patterns when cell suspension culture is fed with ose Fig. . This prm/z 703.2588 ([M-H2O + H]+). Neem cell suspension enriched by feeding [1-13C] Glc and  Glc yielded with 14 and 19 isotopologues, respectively (m/z 703.2588 to 717.3056 and 722.3222) . Whereas, 13C enrichment with [2-13C] Glc labeling culminates with 10 isotopologues  and 16 mass units (m/z 597.3050 to 613.3582) were generated from  Glc as isotopic tracer. With the use of [2-13C] Glc as tracer, 12 isotopologues were observed . In case of [2-13C] Glc labeling, isotopologues with 13 mass units were observed in addition to the parent ion (m/z 599.3209 to 612.3672)  however had the fragment ion with a loss of water molecule, i.e. [M-H2O + H]+ and sodium adduct [M + Na]+. [M-H2O + H]+ adduct was considered as parent ion and was subjected to MS/MS fragmentation at various NCEs such as 10, 15, 20 and 25% -quadrupole/orbitrap-MS/MS study. Mass spectral traces of azadirachtin A and its derivatives did not show molecular ion peak , 607.2174 [M-3H2O-AcOH+H]+ (1.2%), 585.1967 [M-2H2O-Tiglic acid+H]+, 567.1861 [M-3H2O-Tiglic acid+H]+ (62.2%), 525.1755 [M-2H2O-Tiglic acid-AcOH+H]+ (30.4%), and 507.1650 [M-3H2O-Tiglic acid-AcOH+H]+ (85.6%). Hydrolysis of one of the ester group and/or removal of one or two CH2O molecules from m/z 507.1650 gave rise to the fragments m/z 493.1493 [M-3H2O-Tiglic acid-AcOH-CH3 + H]+ (15%), 463.1387 [M-3H2O-Tiglic acid-AcOH-CH2O-CH3 + H] (28.9%) and 447.1438 [M-3H2O-Tiglic acid-AcOH-2CH2O + H]+ (50%), respectively. A low relative ion intensity (13%) fragment of m/z 555.1861 [M-2H2O-Tiglic acid-CH2O + H]+ was formed after removal of a H2O, tiglic acid and a CH2O group from the parent ion.Probable fragmentation pathway for the formation of daughter ions of azadirachtin A, when the parent ion, + was subjected to fragmentation at NCE of 20% and each daughter fragment obtained were probed for its molecular formula and corresponding structure. The daughter ions of m/z 643.2382 [M-2H2O + H]+, 625.2281 [M-3H2O + H]+ and 607.2174 [M-4H2O + H]+ were obtained after successive removal of H2O molecules following protonation. Fragments such as m/z 525.1758, 507.1655, 465.1543, 447.1436 and 329.1013 obtained were in common with azadirachtin A fragmentation pattern  yielded the fragment m/z 545.2017 [M-H2O-Tiglic acid+H]+. The fragments, m/z 509.1806 [M-3H2O-Tiglic acid-AcOH+H]+, 527.1912 [M-2H2O-Tiglic acid-AcOH+H]+ and 271.0965 obtained were on par with m/z 507.1650, m/z 525.1755 and m/z 269.0809 of azadirachtin A, respectively . Higher mass fragments such as m/z 545.2017 [M-H2O-Tiglic acid+H]+, 509.1806 [M-3H2O-Tiglic acid+H]+, 467.1700 [M-2H2O-Tiglic acid-AcOH+H]+ and 449.1595 [M-3H2O-Tiglic acid-AcOH+H]+ were obtained at ≤15% NCE.Azadirachtin H is a decarboxymethyl derivative of azadirachtin A at 11-C position and MS/MS at NCE 20% fragmented the parent ion to greater extent such that only lower m/z 659.2704 [M-H2O + H]+ and 641.2598 [M-2H2O + H]+. The following fragments m/z 285.1121 and 345.1333 were formed from the decalin portion of the molecule (Table S1). The subsequent removal of the functional groups such as hydroxyl, tigloyl, acetyl groups generated fragments such as m/z 541.2068 [M-2H2O-Tiglic acid+H]+, 523.1963 [M-3H2O-Tiglic acid+H]+, 481.1857 [M-2H2O-Tiglic acid-AcOH+H]+, 463.1751 [M-3H2O-Tiglic acid-AcOH+H]+, m/z 451.1751 [M-2H2O-Tiglic acid-AcOH-CH2O + H]+ from both the parent ions.11-epi-azadirachtin D lacks one of the two methylester groups of azadirachtin A and produced two types of protonated ions with m/z 161.0597 was identified in fragmentation pattern of most of the azadirachtin derivatives. The fragments, m/z 161.0597 and m/z 83.0491 corresponding to hydrofuran acetal moiety and tiglate group. Therefore either one acts as a signature fragment for azadirachtin class of limonoids from neem tree.The fragment 13C labels incorporated into it, but also serves as a sensitive method for identification of azadirachtin in environmental as well as biological samples. We have studied already, the structure-fragment relationship for C-seco limonoids such as salannin, salannolacetate, nimbin, nimbinene, 6-deacetylnimbinene and 6-deacetylnimbin, isolated and purified from neem oil ,  and [2-13C] Glc feeding experiments, in order to identify the position of each 13C label in the skeleton of limonoids. MS/MS traces obtained with NCE of 20% for each of the limonoid isotopologue were compared with that of unlabeled fragments to establish the number of 13C labels present in that fragment   corresponding to salannin and salannolacetete   Fig. . Isovaleine Fig. . Isovaleues Fig. .Fig. 8Trm/z 269.0808 was corresponding to the decalin moiety. Three isoprene units and a single carbon from fourth isoprene contribute to this moiety. 13C enrichment through [1-13C] Glc labeling showed additional 9 mass units to this fragment suggesting that each of the three 13C labels may have originated from a single isoprene unit. The same experiment with [2-13C] Glc labeling gave 7 additional mass units further corroborating its route of origin  were generated after fragmentation of labeled azadirachtin A from [1-13C] / Glc labeling whereas two mass units increase were observed through [2-13C] Glc labeling experiment. The earlier report supports the formation of tiglic acid through isoleucine biosynthetic pathway so its origami was traced through multiple primary metabolic pathways  in order to trace the limonoid biosynthetic pathway and with 1,2-13C glucose to track limonoid formation during the blockage of either of the isoprene biosynthetic pathway. In Croton sublyratus and Withania somnifera, sterols and triterpenes are biosynthesized through both the pathways  Glc, D-[2-13C] Glc and D- Glc  were performed separately in 25 ml Erlenmeyer flasks containing 5 ml MS liquid with 3% of labeled Glucose in duplicates.For feeding experiments, cells from 2 days old suspension was harvested from media and sub-cultured into media with labeled carbon source. Feeding experiments with D- Glc  was supplemented to the 5 ml culture after inhibitor treatment. Two independent experiments were performed each in duplicates. Limonoid extraction was carried out from the cells after ten days of fosmidomycin addition. To further analyze the total carotenoid content, the cells were frozen in liquid nitrogen, made into fine powder and extracted with acetone in the dark. The extract was centrifuged to remove debris and concentrated to the volume of 3 mL. It was read spectrometrically at 470 nm and the total carotenoid contents were calculated as described earlier [Mevinolin  and fosmidomycin  were added to the cell suspension culture after 2 days of initiation of culture after replacing the cells with fresh media containing no sugars. 3% D- [1,2- earlier .T method. PCR efficiencies were calculated based on the slope of dilution curve of control cDNA of cell suspension. The two housekeeping genes gave same level of expression and the results were normalized with ELF4A.For quantitative PCR analysis, RNA isolation was carried out using Spectrum™ plant total RNA isolation kit (Sigma) from neem callus, suspension, leaf, flower, pericarp, kernel tissues and also from the control and 0.05 mM mevinolin treated suspension cells. RNA isolation was followed by DNase treatment by using DNase I Amplification Grade kit (Sigma). cDNA was synthesized using SuperScript® III First-Strand Synthesis System (Invitrogen) from 3 μg of total RNA. Through transcriptome analysis, we could identify two ORFs encoding for HMG-CoA reductase (HMGR) and 4 for deoxyxylulose-phosphate synthase (DXS), the enzyme involved in the catalysis of rate limiting step in MVA and MEP pathway respectively ) for norp < 0.05, 0.01 and 0.001, respectively).Asterisks on the tops of bar denotes the values that were determined by Student’s t-test using Microsoft Office Excel 2007. They were found to be significantly different from their respective controls  with that of the control. Figure S4. Comparison of MS/MS of limonoids obtained from feeding experiment (with different 13C labeled glucose) with that of the control. Figure S5. Relative isotopologue distribution and Heatmap for distribution of isotopologues for specific fragments of 6-deacetylnimbinene. Figure S6. Relative isotopologue distribution and Heatmap for distribution of isotopologues for specific fragments of salannin. Figure S7. Relative expression level of genes of MVA and MEP pathway in different neem tissues. Table S1. MS and MS/MS data for azadirachtin A and its derivatives. Table S2. Primers used in real time PCR analysis. (PDF 2627 kb)This file contains MS data of limonoids, MS/MS data for confirmation of limonoids from cell culture,


Clostridium perfringens enterotoxin (CPE) is a common cause of food poisoning and hyperkalemia-associated death. Previously, we reported that fusion of pneumococcal surface protein A (PspA) to C-terminal fragment of CPE (C-CPE) efficiently bound mucosal epithelium so that PspA-specific immune responses could be provoked. In this study, we found that fusion of C-CPE with PspA augmented the antigenicity of C-CPE itself. These findings allowed us to hypothesize that fusion of C-CPE and another food poisoning vaccine act as a bivalent food poisoning vaccine. Therefore, we constructed an adjuvant-free bivalent vaccine against CPE and cholera toxin (CT), which is a major food poisoning in developing country, by genetically fusing CT B subunit to C-CPE. Because of the low antigenicity of C-CPE, immunization of mice with C-CPE alone did not induce C-CPE-specific immune responses. However, immunization with our vaccine induced both C-CPE- and CT-specific neutralizing antibody. The underlying mechanism of the augmented antigenicity of C-CPE included the activation of T cells by CTB. Moreover, neutralizing antibodies lasted for at least 48 weeks and the quality of the antibody was dependent on the binding activity of CTB–C-CPE to its receptors. These findings suggest that our fusion protein is a potential platform for the development of an adjuvant-free bivalent vaccine against CPE and CT. Salmonella spp. infections alone in the US  and occasionally death (C. perfringens-associated food poisoning causes an estimated economic burden of $382 million per year in the US (C. perfringens has not changed in recent years (C. perfringens produces four main toxins and one enterotoxin (CPE)  . Strainsoduction . C. perfted food . C. perf-forming , which m2+ ion and fluids, leading to the development of diarrhea  were purchased from CLEA, Inc. . Mice were housed with a 12-h light/12-h dark cycle and were allowed free access to food and water. All experiments and protocols were approved by the Animal Care and Use Committee of the National Institutes of Biomedical Innovation, Health and Nutrition , and conducted in accordance with the guideline of the Animal Care and Use Committee of National Institutes of Biomedical Innovation, Health and Nutrition.2 atmosphere at 37°C. Parent and claudin-4-expressing human sarcoma cell lines (HT1080 cells)  were cultured in Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum in a 5% CO2 atmosphere at 37°C (2 atmosphere at 37°C.Parent and claudin-4-expressing mouse fibroblast cell lines (L cells) were kindly provided by Dr. S. Tsukita  . Both ce at 37°C . AfricanpET16b plasmids encoding C-CPE were prepared as previously described . pET16b-ggtaccacacctcaaaatattact-3′, KpnI site is underlined; reverse primer: 5′-agaattcttaatttgccatactaattgc-3′, EcoRI site is underlined). pCold II DNA  and CTB PCR products were digested with KpnI and EcoRI. The CTB fragment was inserted into pCold II DNA to yield pCold II-CTB.CTB cDNA was amplified by means of PCR  . CTB–C-CPE mutant cDNA was amplified by means of PCR to yield pCold II-CTB Y12D–C-CPE , pCold II-CTB–C-CPE Y306A/L315A , and pCold II-CTB Y12D–C-CPE Y306A/L315A .Escherichia coli strain BL21 (DE3) (Toyobo) and pCold II plasmids were transformed into pG-Tf2/E. coli strain BL21 (DE3) (Takara). To induce the production of recombinant protein, isopropyl-D-thiogalactopyranoside  was added to the culture medium. The culture pellet was sonicated in buffer A . The supernatant was loaded onto a HiTrap HP column . Recombinant protein was eluted with buffer A containing 100–500 mM imidazole. The solvent was exchanged with phosphate-buffered saline (PBS) by using a PD-10 column . The concentration of recombinant protein was measured by using a BCA Protein Assay Kit . The purity of eluted protein was confirmed by using a NuPAGE electrophoresis system (Life Technologies) followed by staining with Coomassie Brilliant Blue. CTB, C-CPE, or CTB–C-CPE was biotinylated by using a biotinylation kit . For the T-cell proliferation assay, endotoxin was removed from the recombinant protein by using an endotoxin removal kit . Endotoxin contaminants in the recombinant protein were measured using ToxinSensor™ Chromatogenic LAL Endotoxin Assay Kit . Recombinant protein contained around 300 EU/ml, and recombinant protein treated with endotoxin removal kit contained <5 EU/ml.To obtain recombinant protein, pET16b plasmids were transformed into Mouse claudin-4-expressing L cells and human claudin-4-expressing HT1080 cells were incubated with recombinant protein for 1 h at 4°C. The cells were washed with 2% newborn calf serum in PBS and incubated with mouse anti-histidine (His) tag antibody  for 1 h at 4°C. After washing with 2% newborn calf serum in PBS, the cells were incubated with fluorescein isothiocyanate-labeled goat anti-mouse IgG1 antibody  for 30 min at 4°C. After washing with 2% newborn calf serum in PBS, the cells were stained with 7-aminoactinomycin D (BioLegend) for 10 min at 4°C. The cells were washed with 2% newborn calf serum in PBS, and then analyzed by means of flow cytometry .Peyer's patches isolated from the mice were embedded in Tissue-Tek optimal cutting temperature compound  and cut into 6-μm sections by using a cryostat. Sections were fixed in 100% acetone for 1 min at 4°C. To prevent non-specific binding, sections were treated with 2% fetal calf serum in PBS for 30 min at room temperature. Sections were washed with PBS and incubated with biotinylated-CTB, -C-CPE, or -CTB–C-CPE at 4°C overnight. After washing with PBS, sections were stained with Alexa Fluor 546-conjugated streptavidin for 30 min at room temperature. After washing with PBS, sections were stained with 4′,6-diamidino-2-phenylindole (DAPI). The sections were then washed with PBS, mounted in Fluoromount , and observed under a fluorescence microscope .Surgically constructed intestinal loop was fixed in 4% paraformaldehyde, embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek Japan), and cut into 6-μm sections by using a cryostat. Sections were stained with hematoxylin for 10 min and eosin for 3 min. The sections were then fixed in ethanol and xylene, mounted in Permount (Thermo Fisher Scientific), and observed under an optical microscope .Mice were intraperitoneally immunized with C-CPE, a mixture of C-CPE and Imject Alum adjuvant (Thermo Fisher Scientific), or PspA–C-CPE  once a week for two weeks. One week after the final immunization, serum was collected.Mice were subcutaneously immunized on the back with vehicle, CTB, C-CPE, a mixture of C-CPE and CT , or CTB–C-CPE . One week after immunization, fasted mice (>12 h~) were orally administered sodium bicarbonate . After 15–20 min, the mice were orally immunized with vehicle, CTB, C-CPE, a mixture of C-CPE and CT, or CTB–C-CPE  once a week for 3 weeks. One week ~ forty-eight weeks after the final immunization, serum, feces, and intestinal wash were collected. Feces was suspended in PBS (100 mg/mL) and vortexed for 10 min at 4°C. Small intestine was washed with PBS. The samples were centrifuged at 3,000 g for 10 min and the supernatants were collected.Ninety-six-well immunoplates were coated with CT (5 μg/mL), C-CPE (10 μg/mL), or CTB–C-CPE (10 μg/mL) and incubated at 4°C overnight. After incubation, the plates were blocked with 1% bovine serum albumin in PBS for 2 h at room temperature. After washing with 0.05% Tween 20 in PBS, two-fold serial diluted serum, fecal extract, or intestinal wash was added to the wells and the plates were incubated for 2 h at room temperature. After washing with 0.05% Tween 20 in PBS, goat anti-mouse IgG, IgG1, IgG2b, or IgG3 conjugated horseradish peroxidase  was added to the wells and the plates were incubated for 1 h at room temperature. After washing with 0.05% Tween 20 in PBS, CT-, C-CPE-, or CTB–C-CPE-specific antibodies were detected by using 3,3′,5,5′-tetramethylbenzidine peroxide substrate and measuring absorbance at a wavelength of 450 nm.Mice immunized with vehicle, CTB, C-CPE, a mixture of C-CPE and CT, or CTB–C-CPE were fasted for 24 h. Under anesthesia by isoflurane, the small intestine was withdrawn and a 5- to 9-cm loop was made 6 to 18 cm from the pylorus. CPE  was injected into the loop, and the abdomen was closed. After 90 min, the loop was collected and its weight and length measured.4 cells) were inoculated into a 96-well plate and cultured in a 5% CO2 atmosphere at 37°C. The next day, CPE (0.1 μg) and sera from immunized mice (40 μL) were incubated for 1 h at 37°C. The mixture of CPE and serum was added to the wells and the plates were incubated for 30 min in a 5% CO2 atmosphere at 37°C. After gently washing with PBS, Cell Count Reagent SF  was added to the wells for the detection of living cells. After 1 h, absorbance at a wavelength of 450 nm was measured.Vero cells . After 30 min, symptom of mice was monitored. After 4 h, serum was collected and serum potassium was measured by using a DRI-CHEM NX500 dry chemistry analyzer .Ninety-six-well immunoplates were coated with GM1-ganglioside (5 μg/mL)  and incubated at 4°C overnight. After incubation, the plates were blocked with 1% bovine serum albumin in PBS for 2 h at room temperature. After washing with 0.05% Tween 20 in PBS, a mixture of CT (2.5 ng) and serum (4 μl), which had been preincubated for 1 h at 37°C, was added to the wells and the plates were incubated for 2 h at room temperature. After washing with 0.05% Tween 20 in PBS, rabbit anti-CTB antibody  was added to the wells and the plates were incubated for 2 h at room temperature. After washing with 0.05% Tween 20 in PBS, donkey anti-rabbit IgG conjugated horseradish peroxidase  was added to the wells and the plates were incubated for 1 h at room temperature. After washing with 0.05% Tween 20 in PBS, the binding of CT to GM1–ganglioside was detected by using 3,3′,5,5′-tetramethylbenzidine peroxide substrate and measuring absorbance at a wavelength of 450 nm.Mice immunized with vehicle, CTB, C-CPE, or CTB–C-CPE were fasted for 12 h and then orally administered sodium bicarbonate. After 15–20 min, the mice were orally challenged with CT (25 μg). After 13–14 h, intestinal fluid volume was measured.+ T cells were isolated from the spleen of mice immunized with vehicle, C-CPE, or CTB–C-CPE by using anti-CD4 microbeads (Miltenyi Biotec). Antigen-presenting cells were isolated from the spleen of naïve mice and then irradiated (30 Gy) to inhibit growth, but still maintained an ability to process and present antigens to CD4+ T cells. The purified CD4+ T cells and antigen-presenting cells were cocultured with vehicle, CTB, C-CPE, or CTB–C-CPE (5 μM) in a 5% CO2 atmosphere at 37°C. Five days after culture, proliferation was measured by using a CyQUANT Direct Cell Proliferation Assay Kit (Thermo Fisher Scientific).CD4Biotinylated CTB–C-CPE was pre-incubated with serum from immunized mice for 1 h at 37°C and applied to mouse claudin-4-expressing L cells for 1 h incubation at 4°C. The cells were washed with 2% newborn calf serum in PBS and incubated with Alexa Fluor 488-conjugated streptavidin for 1 h at 4°C. After washing with 2% newborn calf serum in PBS, the cells were stained with 7-aminoactinomycin D (BioLegend) for 10 min at 4°C. The cells were washed with 2% newborn calf serum in PBS, and then analyzed by flow cytometry (MACSQuant).U-test .Data are presented as mean ± SD or SEM. Statistical analyses were performed by using the non-parametric Mann–Whitney We previously reported that C-CPE was a useful vehicle for the delivery of a genetically fused antigen  to the mucosal epithelium to induce antigen-specific immune responses without the need for an adjuvant , 20. In Vibrio cholera –C-CPE and CTB–C-CPE (Y306A/L315A) were comparable with parent CTB–C-CPE, but significantly decreased in mice immunized with double mutant CTB (Y12D)–C-CPE (Y306A/L315A) Figure . We thenWe then checked the activity of neutralizing antibodies induced by the mutants against CPE and CT. Serum from mice immunized with CTB–C-CPE (Y306A/L315A) or CTB (Y12D)–C-CPE (Y306A/L315A) failed to protect against CPE-induced cell death in Vero cells Figure . MoreoveHere, we demonstrated that fusion of an antigen to C-CPE increased the antigenicity of C-CPE. A previous report has indicated that CPE contains at least 10 immunogenic domains, with the N-terminal containing more immunogenic domains than the C-terminal . Among tIn general, antibodies in the serum and feces are mainly produced by plasma cells at spleen and lamina propria, respectively –49. MoreSome enterotoxins including CPE migrate from intestine into systemic compartment, and cause systemic pathological responses. On this point, IgG plays an important role in the protection against systemic pathological responses. IgG binds to toxin in the blood stream, resulted in prevention of the binding between toxin and cellular receptor. IgG also interacts with IgG-Fc receptors (FcγRs), which are expressed on macrophages, and induces phagocytosis and degradation of toxin . Indeed,IgA plays a pivotal role in intestinal immune responses by binding to and neutralizing toxins. Therefore, the induction of neutralizing IgA is an important part of host protection against CT- and CPE-mediated diarrhea because they are toxin-mediated conditions. Indeed, mice deficient in polymeric immunoglobulin receptor (a key factor in the secretion of IgA into the lumen) immunized with CTB vaccine produce CTB-specific serum IgG, but not fecal IgA, resulting in the development of diarrhea . MoreoveThe results of the present study show that the binding of CTB–C-CPE to either claudin-4 or GM1–ganglioside was important for the induction of antibodies to C-CPE or CT. There are several pathways through which these antigen-specific antibody responses could be induced.In terms of the C-CPE-mediated immune response induction pathway, the main receptor for CTB–C-CPE is claudin-4. We previously showed that claudin-4 was expressed on epithelial cells, including on M cells, of nasopharyngeal-associated lymphoid tissue and Peyer's patches , 25, 54.Furthermore, claudin-4 has clathrin-sorting signal sequences in its C-terminal intracellular domain , 59. We + dendritic cells located on the interior of Peyer's patches take up antigen by elongating their membrane protrusions into the lumen for antigen sampling –C-CPE and CTB–C-CPE (Y306A/L315A) induced immune responses via CTB- or C-CPE-mediated immune induction pathways, depending on whether they were able to bind claudin-4 or GM1–ganglioside. As expected, the double-mutant CTB (Y12D)–C-CPE (Y306A/L315A) showed less immune induction than the other mutants. However, this double mutant did not completely attenuate immune induction. These findings suggest that CTB–C-CPE has dual roles as a vaccine. Firstly, CTB–C-CPE acts as an antigen and second one includes delivery function. We suppose that the former role may be enhanced in the presence of specific antibodies . On the other hand, the latter role is likely to be attenuated by the specific antibodies. These effects may influence the efficacy of repeated immunization with CTB–C-CPE and therefore we have to design immunization protocol carefully .In the present study, we found that the binding activities of C-CPE–claudin-4 and CTB–GM1–ganglioside were crucial for the induction of antigen-specific antibodies with neutralizing activity. Indeed, a previous report indicated that mice immunized with a tetanus toxin heavy-chain mutant that lacked binding activity to its receptor exhibited low levels of anti-tetanus toxin antibody, including neutralizing antibody, because the mutant lacked an essential protective epitope, and binding activity . Thus, tThe order in which the proteins are fused is also important for maximizing the induction of immune responses by fusion proteins. A previous study indicated that a fusion protein comprising CTB and the rotavirus globular protein VP8-1 (CTB–VP8-1) showed greater binding affinity for GM1–ganglioside compared with the reverse fusion protein, VP8-1–CTB. Moreover, CTB–VP8-1 elicited a higher titer of neutralizing antibodies against rotavirus and showed a higher antigenicity than did VP8-1–CTB. Because the N-terminus of CTB contains the GM1–ganglioside-binding residues, and N-terminus of VP8-1 extends from globular domain and N-terminus is flexible domain of VP8-1. Thereby, it is plausible that the binding activity, antigenicity and conformation of the antigens were disrupted more in VP8-1–CTB compared with in CTB–VP8-1 . SimilarFor clinical application of CTB–C-CPE, the expression of claudin-4 and GM1–ganglioside in the human intestine is important. Claudin-4 is expressed on human intestinal epithelial cells including M cells , 70. We The safety of CTB–C-CPE is also important for its clinical application. Previously, we reported that mice intravenously immunized with C-CPE showed no hepatic or renal side effects , even thLong-term immune induction is also important factor for the vaccine development and we noted that CTB–C-CPE could induce long-term protective immunity against both CPE and CT. A previous study indicated that antigen-specific antibody could be detected longer than 1 year together with long-lived plasma cells even if half-life of IgG is generally considered to be 21 days , 77. TheIn summary, genetic fusion of CTB to C-CPE increased the antigenicity of C-CPE. The fusion protein induced both C-CPE- and CT-specific immune responses in the intestinal and systemic immune compartments in mice, suggesting that CTB–C-CPE could be potentially useful for the development of a bivalent vaccine against CPE- and CT-mediated food poisoning.All experiments and protocols were approved by the Animal Care and Use Committee of the National Institutes of Biomedical Innovation, Health and Nutrition , and conducted in accordance with the guideline of the Animal Care and Use Committee of National Institutes of Biomedical Innovation, Health and Nutrition. This guideline has been established based on Act on Welfare and Management of Animals, and Standards Relating to the Care and Keeping and Reducing Pain of Laboratory Animals, established by the Ministry of the Environment in Japan, and Basic policies for the conduct of animal experimentation in the Ministry of Health, Labor and Welfare, established by Ministry of Health, Labor and Welfare in Japan, and Guidelines for Proper Conduct of Animal Experiments, established by Science Council of Japan.HS planned and performed the experiments, analyzed the data, and wrote the manuscript. KH and AN performed the experiments and analyzed the data. MK provided helpful discussion. JK planned the experiments and wrote the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


High-grade endometrioid and serous endometrial cancers (ECs) are an aggressive subtype of ECs without effective therapies. The reciprocal communication between tumor cells and their surrounding microenvironment drives tumor progression. Long noncoding RNAs (lncRNAs) are key mediators of tumorigenesis and metastasis. However, little is known about the role of lncRNAs in aggressive EC progression and tumor microenvironment remodeling.We performed an array-based lncRNA analysis of a parental HEC-50 EC cell population and derivatives with highly invasive, sphere-forming, and paclitaxel (TX)-resistant characteristics. We characterized the roles of the lncRNA NEAT1 in mediating aggressive EC progression in vitro and in vivo and explored the molecular events downstream of NEAT1.MEF2D, ROCK1, WNT7A, VEGF-A, PDE4B, and KPNA4.We identified 10 lncRNAs with upregulated expression  and 10 lncRNAs with downregulated expression  in the highly invasive, sphere-forming and TX-resistant derivatives. NEAT1 expression was markedly upregulated in early-stage EC tissue samples, and high NEAT1 expression predicted a poor prognosis. Inhibiting NEAT1 expression with small hairpin RNAs (shRNAs) diminished cellular proliferation, invasion, sphere formation, and xenograft tumor growth and improved TX response in aggressive EC cells. We showed that NEAT1 functions as an oncogenic sponge for the tumor suppressor microRNA-361 (miR-361), which suppresses proliferation, invasion, sphere formation and TX resistance by directly targeting the oncogene STAT3. Furthermore, miR-361 also suppressed the expression of multiple prometastatic genes and tumor microenvironment-related genes, including NEAT1 initiates a miR-361-mediated network to drive aggressive EC progression. These data support a rationale for inhibiting NEAT1 signaling as a potential therapeutic strategy for overcoming aggressive EC progression and chemoresistance. Endometrial cancer (EC) is the most frequently diagnosed gynecological cancer in developed countries and the second leading cause of gynecological cancer-related death among females . Unlike Tumor progression is a complex process that involves tumor cell proliferation, detachment, invasiveness, blood and lymphatic vessel entry, and eventual extravasation at distant sites . Tumor pLong noncoding RNAs (lncRNAs) are regulatory RNA transcripts longer than 200 nucleotides with no or limited protein-coding capacity . LncRNAsTo elucidate the exact roles and underlying mechanisms of NEAT1 in aggressive ECs, we used the representative aggressive EC cell line HEC-50 to generate derivatives with high invasiveness, sphere-forming ability and paclitaxel (TX) resistance. Knocking down NEAT1 expression attenuated cellular proliferation, invasion, and sphere formation and sensitized aggressive EC cells to TX treatment. Mechanistically, NEAT1 serves as a molecular sponge for miR-361, a pivotal tumor suppressor that suppresses proliferation, invasion, sphere formation and TX resistance by directly targeting the oncogene STAT3. We further showed that miR-361 downregulates the expression of multiple prometastatic, proangiogenic and inflammation-related genes. Our findings identified an essential NEAT1-miR-361 axis that contributes to aggressive EC progression and chemoresistance, producing potential translational implications for retarding aggressive EC progression and improving the efficiency of chemotherapy for aggressive EC.The human EC cell line HEC-50  was derived from poorly differentiated endometrioid EC and cultured in DMEM/F12  supplemented with 10% fetal bovine serum (FBS). The human serous EC cell line SPAC-1-L was obtained from Dr. Fumihiko Suzuki  and maintained in RPMI-1640 medium  supplemented with 10% FBS. The immortalized human endometrial epithelial cell line EM was kindly provided by Dr. Satoru Kyo  and grown in DMEM/F12 supplemented with 15% FBS. The STAT3 inhibitor BP-1-102 was purchased from Merck Millipore (Germany).Highly invasive subpopulations of HEC-50 cells (referred to as HI cells) have been described previously . We usedwww.sirnawizard.com; InvivoGen, San Diego, CA, USA) and cloned into the BbsI sites of the psiRNA-h7SKneo G1 expression vector (InvivoGen). The NEAT1 shRNA-1 sequences were as follows: Oligo 1, 5′-ACCTCGATGCCACAACGCAGATTGATTCAAGAGATCAATCTGCGTTGTGGCATCTT-3′ and Oligo 2, 5′-CAAAAAGATGCCACAACGCAGATTGATCTCTTGAATCAATCTGCGTTGTGGCATCG-3′. The NEAT1 shRNA-2 sequences were as follows: Oligo 1, 5′- ACCTCGAAGATCCCTAAGCTGTAGAATCAAGAGTTCTACAGCTTAGGGATCTTCTT-3′ and Oligo 2, 5′-CAAAAAGAAGATCCCTAAGCTGTAGAACTCTTGATTCTACAGCTTAGGGATCTTCG-3′. A premade plasmid DNA vector (InvivoGen) expressing shRNA for enhanced green fluorescent protein (EGFP) was used as a negative control. HEC-50 cells were transfected with the above DNA plasmids and selected with G418  for 4 weeks.Two short hairpin RNAs (shRNAs) targeting NEAT1_2  were designed using the web-based software siRNA Wizard (http://pga.mgh.harvard.edu/primerbank/). The PCR primers for lncRNAs were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The expression of mature miRNAs was measured using NCode miRNA qRT-PCR analysis . The forward primers for miRNA detection had the same sequences as the mature miRNAs. The results were normalized to the levels of GAPDH for mRNA and lncRNA measurement or the levels of U6 for miRNA detection.Total RNA was isolated from cells or tumor xenografts using TRIzol reagent  and subjected to reverse transcription using a Reverse Transcription Kit . The mRNA levels were determined using Takara SYBR Premix Ex Taq II  and an ABI7300 PCR instrument. For mRNA detection, all primers were obtained from the PrimerBank database  and purified with an RNeasy Mini Kit . RNA samples were subjected to the human lncRNA microarray V3 , which includes 30,600 lncRNA probes. The Agilent Array platform was used for the microarray analysis. Sample preparation, microarray hybridization, slide washing and scanning were performed according to standard Arraystar protocols. Agilent Feature Extraction software  was used to analyze the acquired array images. Subsequent data processing was performed with the GeneSpring GX v11.5 software package (Agilent Technologies). Differentially expressed lncRNAs were identified with the criteria of log2 (fold change) > 2 and 3) were plated in 96-well plates for 24 h and then treated with dimethyl sulfoxide (DMSO) or varying doses of TX . After 24 h, cell viability was assessed using a Cell Counting Kit-8 assay . Absorbance was determined at 450 nm using a microplate reader, and relative survival was calculated by comparing TX-treated cells with DMSO-treated cells.Cells (5 × 106) were injected subcutaneously into the flanks of nude mice under sterile conditions. Xenograft tumor formation was monitored using the following formula: tumor volume (mm3) = width (mm2) × length (mm) × 0.5. After 24 days, all mice were sacrificed, and the tumors were harvested for RNA isolation.For tumorigenesis assessment, female BALB/c-nu mice (aged 4 weeks) were purchased from Guangdong Medical Laboratory Animal Center . All mice were maintained under specific pathogen-free conditions, and experimental procedures were performed according to the institutional animal welfare guidelines approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University Cancer Center. As described previously , 11, EC 6) were injected subcutaneously into the flanks of nude mice. After the formation of palpable tumors, the mice were divided randomly into two groups, saline and TX groups, and treated via intraperitoneal injection twice a week for 24 days. Then, the tumors were weighed and analyzed by immunohistochemistry. Cell proliferation was measured in paraffin-embedded tumor tissue with immunohistochemical (IHC) staining using an anti-Ki-67 antibody .To determine the in vivo antitumor activity of TX, EC cells . Microarray analysis was performed using the Superprint G3 Human GE 8 × 60 k Microarray (Agilent Technologies) as described previously . VolcanoMEF2D 3′-UTR containing the miR-361 binding site were synthesized and cloned into the pGL3-basic vector . The 3′-UTR reporter vectors for human ROCK1, WNT7A, KNP4A, PDE4B and VEGF-A were purchased from OriGene . A luciferase reporter assay was performed as described previously [A wild-type (WT) or mutant (MUT) human NEAT1 fragment and the eviously . In brieAn RNA immunoprecipitation (RIP) assay was performed with the Magna RIP RNA-Binding Protein Immunoprecipitation Kit  following the manufacturer’s protocol. Briefly, EC cells were transfected with miR-361 mimic or control mimic. After 48 h, the EC cells were collected and lysed using RIP lysis buffer. An anti-Argonaute2 (Ago2) antibody  or negative control normal mouse IgG  was conjugated to magnetic beads and incubated with whole cell extract. The immunoprecipitated RNAs were isolated, and a qRT-PCR assay was used to detect the expression of NEAT1.Cells were lysed with M-Per Mammalian Protein Extraction Reagent . Equal amounts of the extracts were loaded, subjected to 10% SDS-PAGE, transferred onto nitrocellulose membranes, and probed by antibodies against STAT3 , E-cadherin , CD133 (Abcam), MEF2D (Abcam), ROCK1 (Abcam), WNT7A (Abcam), KPNA4 (Abcam), VEGF-A (Santa Cruz), Snail (Santa Cruz), Survivin (Santa Cruz) and GAPDH (Santa Cruz) at 4 °C overnight. After incubation with the corresponding secondary antibodies, signals were detected with an ECL detection kit .Expression vectors encoding STAT3, MED2D, ROCK1, WNT7A and KPNA4 and the corresponding empty control vector  were transfected using Lipofectamine 2000  according to the manufacturer’s protocol.t-tests were used to compare the means of two groups, and ANOVA was used to compare the differences among multiple groups. A P value < 0.05 was considered statistically significant.The results are shown as the mean ± standard deviation of triplicate experiments. Statistical evaluations were carried out with SPSS 13.0 software . Two-tailed Student’s To identify the lncRNAs that govern invasion, sphere formation and TX resistance in aggressive EC, we established HEC-50 human EC cell derivatives that display a high invasive ability as measured by Matrigel penetration , exhibitLncRNA microarray profiling was performed to identify lncRNAs with differential expression between the parental cells and their derivatives. Ten lncRNAs, including known oncogenic lncRNAs involved in EC , NEAT1 levels were found to be significantly higher in lung, pancreatic and kidney cancer and EC samples than in normal tissue samples  and KM Plotter , which integrated gene expression and clinical data from EC patients and divided the patients into a NEAT1-high group and a NEAT1-low group. Overall survival was relatively lower in the NEAT1-high group than in the NEAT1-low group  database. NEAT1 was highly expressed in EC tissue samples but rarely expressed in normal endometrium samples , and we found that the longer variant of NEAT1 (NEAT1_2), rather than NEAT1_1, was expressed in EC tissue  and NEAT1_2  [56 bp andWe validated the expression of NEAT1 in the normal endometrial epithelial cell line EM and in parental HEC-50 cells and their derivatives using qRT-PCR analysis. NEAT1 expression was higher in EC cells than in EM cells, and SPAC-1-L and HI cells exhibited higher levels of NEAT1 than the other EC cells Fig. b. To invTo identify miRNAs that might be regulated by NEAT1, we used a miRNA microarray to compare miRNA levels between the SPAC-1-L cells with NEAT1 expression knockdown and control cells. The expression of a number of miRNAs, including known tumor suppressor miRNAs (let-7a and miR-34a) and miR-361, was markedly upregulated in NEAT1-knockdown SPAC-1-L cells , we predicted miRNAs with the potential to interact with NEAT1 and identified a putative binding site for miR-361 in the NEAT1 sequence  and identified a set of common genes targeted by miR-361; these genes included STAT3  were significantly overexpressed in TCGA EC samples compared with those in normal samples . STAT3, VEGF-A, MEF2D, ROCK1, KPNA4 and PDE4B were expressed mainly in the nucleus, cytoplasm and membrane of EC cells but rarely or not expressed in adjacent normal tissues . The patients with high levels of MEF2D, ROCK1, WNT7A or KPNA4 mRNA displayed shorter overall survival periods (Fig. To examine the clinical importance of the miR-361-mediated signaling pathway in EC samples, we searched for the mRNA expression of miR-361 target genes in the MethHC database. We found that miR-361-regulated genes (les Fig. a. In addues Fig. b. Finallods Fig. c. Taken Aggressive EC tends to invade adjacent normal tissue and be more resistant to chemotherapy . The eluTo study the molecular and genetic mechanisms underlying the invasiveness, CSC-like features and TX resistance of aggressive EC, we established subpopulations that all originated from the same EC cell line, HEC-50, but showed differences in invasiveness, sphere formation and drug resistance to TX between the subpopulations and the parental cells. These sublines with different malignant potentials might provide suitable cell models for screening the key molecules involved in the regulation of tumorigenesis and metastasis in aggressive EC.By comparing the lncRNA expression profiles of these subpopulations with the profile of the parental cell line via a lncRNA microarray assay, we identified a set of lncRNAs, including H19, PVT1, UCA1, MEG3, GAS5, and LINC00261, that promote or suppress EC growth and progression , 14, 25 NEAT1 levels were found to be significantly higher in endometrioid EC tissue than in normal endometrial tissue, and higher levels of NEAT1 were positively associated with advanced tumor stage and lymph node metastasis . Our datMetastasis is a complicated process that consists of a series of multiple sequential and interrelated steps that include tumor cell detachment from the primary tumor, extracellular matrix invasion, blood or lymphatic vessel penetration, survival within and exit from the circulation and finally proliferation in a distant organ . Tumor cAmong the miR-361 target genes identified in this study, MEF2D is involved in tumor promotion in pancreatic cancer , hepatocOur data provide important advances in understanding the functional importance and mechanisms of NEAT1 in driving aggressive EC progression. NEAT1 sponges miR-361 to initiate a prometastatic network involving STAT3 signaling, prometastatic genes and tumor microenvironment-related genes. The components of this regulatory network, including NEAT1, miR-361, STAT3, MEF2D, ROCK1, WNT7A and KPNA4, could be considered potential targets for the development of new treatments for aggressive EC.


Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders. Yet the structural underpinnings of activity and specificity in this crucial ligase are incompletely understood. Here, we unravel the determinants of ubiquitin recognition by the catalytic domain of E6AP and assign them to particular steps in the catalytic cycle. We identify a functionally critical interface that is specifically required during the initial formation of a thioester-linked intermediate between the C terminus of ubiquitin and the ligase-active site. This interface resembles the one utilized by NEDD4-type enzymes, indicating that it is widely conserved across HECT ligases, independent of their linkage specificities. Moreover, we uncover surface regions in ubiquitin and E6AP, both in the N- and C-terminal portions of the catalytic domain, that are important for the subsequent reaction step of isopeptide bond formation between two ubiquitin molecules. We decipher key elements of linkage specificity, including the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site Lys-48. Intriguingly, mutation of Glu-51, a single residue within this region, permits formation of alternative chain types, thus pointing to a key role of ubiquitin in conferring linkage specificity to E6AP. We speculate that substrate-assisted catalysis, as described previously for certain RING-associated ubiquitin–conjugating enzymes, constitutes a common principle during linkage-specific ubiquitin chain assembly by diverse classes of ubiquitination enzymes, including HECT ligases. Ubiquitination controls the lifetimes, conformational dynamics, as well as the localization and interaction patterns of eukaryotic proteins, thereby directing cellular functions at all levels. The molecular basis for this regulatory potential lies in the finely-tuned interplay of ubiquitinating and de-ubiquitinating activities, which modify substrate proteins with ubiquitin in reversible and highly-specific manners. The structural mechanisms encoding specificity in ubiquitination reactions, however, are incompletely understood.Ubiquitination is driven by a cascade of ubiquitin-activating enzymes E1),,1)—have Really Interesting New Gene)- and U-box ligases catalyze the direct transfer of ubiquitin from a thioester-linked complex with an E2 to a substrate. In contrast, HECT (Homologous to E6AP Cterminus) and RBR (RING-between-RING) ligases act through a catalytic cysteine and form a thioester-linked intermediate with ubiquitin before modifying the substrate .HECT ligases share a C-terminal catalytic “HECT” domain (∼45 kDa), which is composed of two lobes : the N-t30–34–30–In this study, we combine NMR spectroscopy with mutational analyses and absolute quantification (AQUA) MS to decipher the mechanism of ubiquitin recognition by E6AP. This ligase regulates key cellular processes, including translation, DNA replication, and intracellular trafficking , and is 43–Here, we demonstrate that the HECT domain of E6AP relies on canonical, “NEDD4-type” contacts with the donor ubiquitin during thioester formation. We also identify surface patches on ubiquitin and E6AP critical for the subsequent step of isopeptide bond formation, and we uncover determinants of the Lys-48 linkage specificity of E6AP. Intriguingly, these determinants reside in both the ligase and ubiquitin itself, which underscores the widespread role of substrate-assisted catalysis in ubiquitination reactions. Finally, we reveal that the N-lobe of E6AP interacts with ubiquitin and that the exosite region is required for isopeptide bond formation and influences ubiquitin binding, in a similar yet not identical manner as characterized for NEDD4 ligases.trans . The affected resonances map to mostly continuous surface areas on the two binding partners. On ubiquitin, this area includes several hydrophobic residues  as well as residues in the flexible C-terminal tail (Arg-72 and Arg-74) (C). Perturbed resonances of E6AP map primarily to residues in the active-site region surrounding the catalytic cysteine (Cys-820), such as His-818, Thr-819, Phe-821, Asn-822, Val-823, and Leu-824, with additional perturbations seen for Gly-755, Lys-801, Ile-803, and Ala-805 (C).During the catalytic cycle of HECT ligases, the C-lobe recognizes both the donor and acceptor ubiquitin in trans , 31. Howtrans , 31, 37.eraction , A and B Arg-74) C. Pertur Ala-805 C.DK, of 400 ± 20 μm for the C-lobe–ubiquitin interaction (D). This rather weak affinity explains why the interaction had not been directly detected in previous assays  measurements with fluorophore-labeled ubiquitin, we determined an apparent dissociation constant, eraction D. This rs assays . Moreovecritical .A and B). Based on time-course experiments, monitoring both the formation of the thioester-linked HECT domain–ubiquitin conjugate (“E6AP∼Ub”) and the concomitant loss of the E2–ubiquitin conjugate (“UBE2L3∼Ub”), we found that the mutations I803A, H818A, and T819A in E6AP do not impair thioester transfer . In contrast, the F821A substitution adjacent to the catalytic cysteine causes a strong defect, as seen from a delay in the formation of the E6AP∼Ub product and the prolonged presence of the UBE2L3∼Ub precursor. The V823A variant is also compromised in thioester transfer, albeit to a smaller degree than F821A .To interrogate the functional significance of the identified E6AP–ubiquitin interaction, we introduced individual alanine mutations at those positions that displayed the largest binding-induced chemical shift perturbations. Those include Ile-803, His-818, Thr-819, Phe-821, and Val-823 of E6AP  and Thr-14, Glu-34, Ile-36, Leu-71, and Arg-74 of ubiquitin. The purified HECT domain variants were tested for their ability to receive the donor ubiquitin from the cognate E2 (UBE2L3) in thioester transfer assays , A and Btransfer A, left. an F821A A, right.B). Notably, these defects are specific to the transfer of ubiquitin from the E2 to the E3, because the preceding reaction step, as monitored by the E1-mediated formation of UBE2L3∼Ub, is unaffected by the mutations , in line with previous analyses . Consistently, thioester formation is also delayed upon mutation of Gln-40 in ubiquitin (Q40A), which makes key contacts at the NEDD4-type donor ubiquitin interface . However, Gln-40 is not required for thioester transfer of ubiquitin from the E1 to the E2 .Interestingly, the residues of ubiquitin required for thioester transfer to E6AP coincide with a surface of the donor ubiquitin that engages the C-lobe of NEDD4-type ligases , 40, 41.Fig. 3A). To this end, we monitored the second step in the catalytic cycle of HECT ligases, isopeptide bond formation, by virtue of auto-ubiquitination (“E6APUb”) and di-ubiquitin formation (“Ub2”) (B). These studies show that the mutation of residues in close proximity to the catalytic cysteine (H818A and T819A) causes defects in isopeptide bond formation activity compared with the WT. In contrast, Ile-803, which borders a β-strand flanking the active-site region, has similar activity to the WT.We also studied the functional significance of those residues of E6AP that displayed significant ubiquitin-induced chemical shift perturbations in our NMR experiments but are not required for thioester transfer of ubiquitin  B. These trans.These observations are consistent with the notion that the ubiquitin-induced chemical shift perturbations of E6AP backbone amide resonances report on both direct contacts with ubiquitin, which occur predominantly at the active-site region, and conformational changes propagated to the nearby β-sheet. Similarly, solution studies of donor ubiquitin conjugated to the C-lobe of SMURF2 or of HUWE1 revealed conformational changes in the C-lobes remote from the crystallographic donor interface . HoweverTaken together, we conclude that the C-lobe of E6AP relies on similar contacts with ubiquitin as NEDD4-type ligases for thioester formation with ubiquitin , 40, 41.A, left) and compared the pattern of chemical shift perturbations of C-lobe resonances induced by ubiquitin in cis with those seen in trans (see A). In quantitative terms, this analysis yields overall rather similar chemical shift perturbations . This is consistent with the FP-derived affinity for the interaction (see D). At the given protein concentrations  and a DK value of 400 μm, a complex saturation of ∼85% is expected. However, the generally small amplitudes and limited number of chemical shift perturbations of E6AP resonances indicate that the donor ubiquitin retains considerable flexibility upon conjugation to the C-lobe.We next set out to analyze the interaction of the E6AP C-lobe with the donor ubiquitin in the context of a covalently linked intermediate, as formed during the reaction cycle. We thus prepared a stable mimic of the conjugate by replacing the native thioester with a disulfide bond A, left arans see A. In quarbations A, right.tion see D. At them) of unlabeled ubiquitin to the C-lobe–donor conjugate slightly enhances the chemical shift perturbations of C-lobe resonances . This likely reflects an increased saturation of the C-lobe–donor complex in the presence of added ubiquitin concentrations that approach the local concentrations of the covalently linked donor at the C-lobe. A second ubiquitin-binding site on the C-lobe is not detected, indicating that interactions with the acceptor ubiquitin are extremely weak under these conditions and require additional components to be stabilized. As noted above, however, it is impossible to decide unequivocally whether the observed chemical shift perturbations result from a single or several dynamic binding modes.Addition of high concentrations  and at the rim of the flanking β-sheet (residues 755 and 802–807) . Several resonances undergo pronounced chemical shift perturbations in cis: those correspond to Thr-786, Thr-787, Lys-801, Met-802, and Phe-821, all of which are located in structural elements near the catalytic cysteine where the C-terminal tail of ubiquitin is attached; and Leu-814 and Thr-816, which fall into the canonical donor interface, in line with our model of thioester formation  (A and B), indicating that the C-tail contributes to interactions with the donor, once a covalent linkage between the C-lobe and ubiquitin has been established.As seen in 802–807) A, right.ormation . Notably845–852) , A and BAFig. S3)  , or the four C-terminal residues are deleted (“Δ4”). In line with previous mutational analyses, both variants promote thioester formation with the donor ubiquitin to a similar degree as the WT (ig. S3A) , 51. Morig. S3B) . In regas the WT . Howevers the WT .A–C). These studies reveal that the C-tail variants promote Ub2 formation with each of the single Lys-to-Arg variants of ubiquitin, including K48R. In contrast, the WT HECT domain is highly selective for the formation of Lys-48 linkages, in line with previous studies (–A–C).These observations prompted us to examine whether the C-tail influences the recognition of the acceptor ubiquitin and thus the linkage specificity of E6AP. We compared the activities of the HECT domain variants WT, F849A, and Δ4) toward WT ubiquitin, individual Lys-to-Arg variants , and a lysine-free variant , respectively , A–C. ThA, and Δ4studies –, A–C.2 species assembled by the WT, F849A, and Δ4 HECT domain variants by AQUA MS (absolute quantification MS) . These analyses confirm that the WT HECT domain is Lys-48–specific. Alteration of the C-tail, however, results in a loss of specificity, as evidenced by the formation of Lys-11 and Lys-63 linkages. Notably, these data illustrate also that studies on Lys-to-Arg variants of ubiquitin do not exactly mirror the results of Ub-AQUA MS analyses (performed in the context of WT ubiquitin), consistent with previous reports, cautioning direct correlation of these two types of measurements , we hypothesized that they may be required for alternative interactions of the donor or the acceptor ubiquitin. In addition, we decided to interrogate the functional significance of a series of hydrophilic residues of ubiquitin in proximity to the Lys-48–acceptor site, including Arg-42, Gln-49, and Glu-51 .To further decipher the requirements of acceptor ubiquitin recognition during E6AP-mediated ubiquitin linkage formation, we focused on an array of hydrophobic residues of ubiquitin , often referred to as the “hydrophobic patch” that is critical for E6AP activity . BecauseFig. 9B and Fig. S5). Surprisingly, the E51A variant is a better substrate than WT ubiquitin . None of the tested mutations interfere with thioester transfer of ubiquitin to the E2 or E3 .We generated ubiquitin variants with individual alanine substitutions at the selected sites and monitored isopeptide bond formation, as catalyzed by the E6AP HECT domain. These studies confirm that residues in the hydrophobic patch of ubiquitin, as well as Arg-42 and Gln-49, are important for E6AP activity , which cannot be activated by the E1 and thus solely acts as an acceptor . When supplied with a mixture of UbΔGG and full-length ubiquitin, the E6AP HECT domain assembles two distinct Ub2 species, which contain either two full-length ubiquitin molecules or a full-length ubiquitin molecule linked distally to a UbΔGG variant. If one ubiquitin variant carries a His6-tag, these alternative reaction products can be readily separated by SDS-PAGE and provide a selective readout of mutational effects on the donor and acceptor functions during ubiquitin linkage formation. A proof-of-concept experiment is shown in Fig. 9C (right).To understand the precise roles of the mutated residues in ubiquitin during E6AP-mediated isopeptide bond formation and to discriminate donor from acceptor functions, we adopted a strategy previously used for the analysis of E2–ubiquitin interactions . This stacceptor C, left. D and E). Because these residues are dispensable for thioester formation, this implies that they mediate alternative interactions of the donor during isopeptide bond formation. Mutation of Arg-42 interferes with both the donor and the acceptor ubiquitin functions (D and E). In contrast, mutation of Glu-51 in the acceptor ubiquitin enhances the formation of Ub2 (D and E).Using this setup, we demonstrate that residues in the hydrophobic patch , as well as Gln-49, are exclusively required by the donor ubiquitin , D and Eunctions , D and En of Ub2 , D and E2 species assembled from WT and E51A ubiquitin by Western blotting using two anti-ubiquitin antibodies, which recognize all linkage types (P4D1) and Lys-48 linkages (D9D5), respectively (A). Quantification of the relative amounts of Ub2 detected by these antibodies suggests that the E51A mutation in ubiquitin triggers a loss of Lys-48 specificity in E6AP and enables the formation of alternative linkage types. AQUA analysis identifies Lys-11 as the alternative linkage . We could not accurately quantify the proportion of Lys-48 linkages formed with the E51A variant, due to the impact of the mutation on the ionization. With WT ubiquitin, however, Lys-48 linkages are exclusively formed .We therefore speculated that the E51A mutation may alter the recognition of the acceptor ubiquitin by E6AP and allow for the formation of linkages other than Lys-48. To test this idea, we analyzed the Ubectively A. QuantiTaken together, these studies assign distinct roles to individual surface regions of ubiquitin during E6AP-mediated isopeptide bond formation. (i) The hydrophobic patch, along with Arg-42 and Gln-49, is required by the donor ubiquitin for interactions that are distinct from those formed with the C-lobe during thioester formation; these interactions may involve the N-lobe, the acceptor ubiquitin, or alternatively, an alternative interface with the C-lobe, following structural rearrangements. (ii) A hydrophilic patch adjacent to Lys-48 is utilized by the acceptor ubiquitin, with Glu-51 making critical contributions to the Lys-48 linkage specificity of E6AP.m, is similar to the one we measured for the full HECT domain (83 ± 6 μm) (A) and is significantly tighter than that of the isolated C-lobe .To test for interactions between ubiquitin and the N-lobe of E6AP, we performed FP experiments, analogous to our studies of the C-lobe. These experiments reveal that the isolated N-lobe indeed binds to ubiquitin. The dissociation constant, 70 ± 6 μ ± 6 μm) A and is m for the HECT domains of NEDD4 and RSP5, respectively ; for example, a particular helical linker (Asn-621–Glu-629 of NEDD4) that contains a functionally critical hydrophobic residue  is shortened in E6AP (Asn-625–Glu-584). We introduced nonconservative amino acid substitutions in E6AP at positions homologous to key residues in the exosite of NEDD4-type enzymes . Compared with the WT, ubiquitin binding to the F665D and I564D/F583D variants is only slightly weakened with DK values of 120 ± 4 and 110 ± 4 μm, respectively. In contrast, the equivalent mutations in NEDD4-type enzymes were reported to result in a drastic reduction in ubiquitin binding ectively ) and detectively , B and CD), while leaving thioester formation intact (E). These observations mirror the effect of the exosite in certain NEDD4-type enzymes . Consistently, residues in the NEDD4-type donor interface are at least partially conserved in E6AP, and functionally critical residues are largely identical, despite the HECT domains being relatively distant in phylogenetic terms . Taken together, these observations support the notion that thioester transfer of ubiquitin from the E2 to the E3 occurs through a structurally conserved interface between ubiquitin and the C-lobe of HECT ligases (A) ..Fig. S9AB). Alternatively, the hydrophobic patch may mediate alternative interactions of the donor with the C-lobe, following structural rearrangements in the course of isopeptide bond formation . Because our NMR data point to considerable flexibility within the donor–C-lobe conjugate and the HECT domain harbors additional inter-lobe flexibility  were subcloned into pET-28a.The genes encoding the HECT domain  and the C-lobe (residues 741–852) of E6AP were cloned into a pET-28a vector  that had been modified to encode an N-terminal 3C protease–cleavable Hisescribed . The genΔGG variants were purified as His6-tagged proteins from Escherichia coli BL21 (DE3). Cells were grown at 37 °C in TB medium, supplemented with the appropriate antibiotics. At an A600 value of 0.6, protein expression was induced by the addition of 0.5 mm isopropyl 1-thio-β-d-galactopyranoside. After 12 h at 18 °C, cells were harvested by centrifugation and lysed in 50 mm Tris, pH 8.0, 100 mm NaCl, 5 mm benzamidine, 4% glycerol, 0.1% Triton X-100, 8 mm β-mercaptoethanol (β-ME), and protease inhibitor mixture . After centrifugation the supernatant was applied to a HisTrap HP column , washed with 50 mm Tris, pH 8.0, 400 mm NaCl, and 8 mm β-ME and eluted in the presence of 500 mm imidazole. 3C protease–mediated cleavage of the His6-tag was performed at 4 °C in 50 mm Tris, pH 7.5, 200 mm NaCl, and 2 mm DTT overnight. To remove the tag and the protease, a second nickel–affinity chromatography (HisTrap HP column) was performed, followed by a gel filtration  in 50 mm Tris, pH 7.5, 75 mm NaCl, and 2 mm DTT.E6AP and Ub15N-Enriched proteins were expressed in M9-medium supplemented with 15N-enriched ammonium chloride (Sigma) and purified as described above.Full-length ubiquitin variants, UBA1 and UBE2L3, were prepared as described previously , 69. 15Nm sodium phosphate, pH 7.5, and ubiquitin-activated by incubation with a 9-fold molar excess of DTNB at room temperature for 30 min. Excess DTNB was then removed , and the DTNB-modified ubiquitin was incubated with a sub-stoichiometric amount of the E6AP C-lobe at room temperature for 30 min. After buffer exchange into 25 mm Tris, pH 6.5, the disulfide-linked conjugate was purified by cation-exchange chromatography , using a gradient of 0–500 mm NaCl and size-exclusion chromatography  in 75 mm sodium phosphate, pH 7.5, and 1 mm EDTA.To obtain a stable mimic of the E6AP C-lobe–ubiquitin complex, we replaced the native thioester linkage by a disulfide bond. To this end, we activated a G76C variant of ubiquitin by 5,5′-dithiobisnitrobenzoic acid (DTNB), followed by a disulfide exchange reaction with the single cysteine-containing C-lobe, as described in previous studies on E2s . In shor1H/15N/13C cryo-probe. Backbone resonance assignments for the E6AP C-lobe and ubiquitin were taken from the Biological Magnetic Resonance Bank , were calculated according to Data were recorded at 25 °C, using a Bruker DRX 700 MHz spectrometer, equipped with a ers 5013  and 174315N HSQC  and BEST15N HSQC  spectra m UBA1, 2 μm UBE2L3, and 200 μm ubiquitin WT or variants were preincubated in 50 mm Tris, pH 7.0, 75 mm NaCl, 10 mm MgCl2, and 2 mm ATP at 30 °C for 30 min; then 2 μm E6AP HECT domain WT or variants were added for the indicated reaction times.To monitor the formation of unattached ubiquitin chains (readout by virtue of di-ubiquitin) and E6AP auto-ubiquitination, 0.2 μ6-UbΔGG variants were mixed at 100 μm concentration (each) with 0.2 μm UBA1, 2 μm UBE2L3, and 2 μm E6AP HECT domain in 50 mm Tris, pH 7.0, 75 mm NaCl, 10 mm MgCl2, and 2 mm ATP at 30 °C for 60 min.To test the effects of mutations in the donor and acceptor ubiquitin, full-length ubiquitin and Hism UBA1, 10 μm UBE2L3, and 200 μm ubiquitin WT or variants thereof were incubated in 50 mm Tris, pH 7.0, 75 mm NaCl, 0.1 mm DTT, 10 mm MgCl2, and 2 mm ATP at 30 °C for 30 min; the reactions were quenched by 4-fold dilution in the same buffer, but including 25 mm EDTA and stored on ice for 30 min. The UBE2L3–ubiquitin conjugate was then incubated with 5 μm E6AP for the indicated reaction times.To monitor ubiquitin thioester transfer from UBE2L3 to E6AP, we performed single-turnover, pulse-chase assays. Here, 1 μm UBA1, 10 μm UBE2L3, and 200 μm ubiquitin WT or variants thereof in 50 mm Tris, pH 7.0, 75 mm NaCl, 0.1 mm DTT, 10 mm MgCl2, and 2 mm ATP at 30 °C for the indicated reaction times.Thioester transfer of ubiquitin from the E1 to the E2 was monitored by incubating 1 μAll reactions were quenched by addition of reducing and nonreducing SDS loading dye, respectively, as specified and analyzed by SDS-PAGE, followed by either Coomassie staining, silver staining or Western blotting with the following antibodies: anti-UBE3A ; anti-UBE2L3 ; anti-ubiquitin P4D1 SC-8017 ; or anti-Lys-48 linkage .Where appropriate, reaction input and products were quantified with ImageJ (RRID: SCR_003070) . The meam HEPES, pH 7.2, 200 mm NaCl, 5% glycerol, and 1 mm TCEP) at 4 °C overnight. The separation of the unreacted fluorophore was achieved by three rounds of dialysis, followed by a size-exclusion chromatography  in reaction buffer.FP measurements were performed analogously to previous studies . In shorm HEPES, pH 7.2, 200 mm NaCl, 1 mm TCEP, and 0.01% Triton X-100 in 96-well flat-bottom microplates , using a Clariostar microplate reader  at 540 nm excitation and 590 nm emission wavelengths. The peptide concentration was 50 nm, whereas that of the E6AP C-lobe ranged from 0 to 500 μm. FP reads from three independent experiments were averaged and fitted to a single-site binding model, as described previously , and in-gel trypsin digestion was performed as described previously . ExtractTM TribidTM mass spectrometer (ThermoFisher Scientific). The resolving power of the mass analyzer was set to 60,000; spectra were recorded over a range of 300–1500 m/z. For data-dependent MS/MS, the top four most intense ions with charge states of 2–5 were selected using an isolation window of 2 m/z. Fragmentation was achieved by collision-induced dissociation at 35% nominal energy with product ion detection in the linear ion trap. Ion chromatograms were extracted for each ubiquitin peptide of interest with a window of 5 ppm. Chromatograms were smoothed using the Boxcar algorithm with a 7-point window. Integration was performed using default parameters with manual adjustment, as appropriate. Results were normalized to the total amount of ubiquitin for each linkage type detected and represented from three replicates.Peptides were separated using an Easy nLC 1000 UHPLC (ThermoFisher Scientific) equipped with a homemade ProntoSIL C18 (75 μm × 15 cm) column. A linear gradient of 0–50% of solvent B over solvent A for 30 min, 50–95% of solvent B for 3 min, and 95% hold of solvent B for 7 min . For HR/AM AQUA analysis the UHPLC system was coupled with an Orbitrap FusionaK values for the thiol group of the catalytic cysteine (Cys-820) in E6AP C-lobe variants (WT and Δ4) were derived from the reaction kinetics of 100 μm DTNB and 14.4 μm protein at 10 °C, as monitored by UV absorbance (λ = 412 nm) using an SFM-3000S stopped-flow spectrometer . Proteins were in 100 mm NaCl, 1 mm EDTA, and one of the following buffers covering a pH range from 5 to 10.5: 20 mm sodium acetate; 20 mm sodium phosphate; 20 mm Tris; or 20 mm CAPSO. Each reaction was measured at least five times under identical conditions, and the data were averaged and fitted with single exponential functions, using the BioKine (Bio-Logic) software. Three independent experimental replicates were performed, and the resulting rate constants were averaged, plotted against the pH value, and fitted with OriginPro 2017  to a two-state model, as given by kapp is the experimentally derived apparent rate constant; and kSH and kS− are the rate constants of the reaction of fully protonated and fully deprotonated cysteines, respectively (pectively .All structural figures were generated with the PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.L. K. R., B. S., K. K. D., M.-A. L., E. R. S., and S. L. formal analysis; L. K. R., K. K. D., E. R. S., and S. L. funding acquisition; L. K. R., B. S., K. K. D., M.-A. L., E. R. S., and S. L. validation; L. K. R., B. S., K. K. D., M.-A. L., and S. L. investigation; L. K. R., B. S., K. K. D., M.-A. L., E. R. S., and S. L. visualization; L. K. R., K. K. D., and S. L. writing-original draft; L. K. R., B. S., E. R. S., and S. L. writing-review and editing; E. R. S. and S. L. supervision; S. L. conceptualization; S. L. methodology; S. L. project administration.


A cross-linked structure (Q3) appeared when magnesium uptake in C-S-H. The carbonation product of C-S-H was vaterite if the Ca/Si ratio was lower than 0.87. The carbonation products of C-S-H were vaterite and calcite if the Ca/Si ratio was higher than 1.02. C-M-S-H had more polymerized units, stronger bond strength and better carbonation resistance than C-S-H. The effects of Ca/Si ratio, aluminum and magnesium on the carbonation behavior of calcium silicate hydrate (C-S-H) were investigated by using X-ray powder diffraction (XRD), nuclear magnetic resonance (NMR) and thermogravimetric analyzer (TGA). The results showed that the Ca/Si ratio, Al/Si ratio and Mg/Si ratio had a significant influence on the structure, carbonation products and carbonation resistance of C-(M)-(A)-S-H. The mean chain length of silicate chains in C-S-H increased as the Ca/Si ratio decreased. Aluminum uptake in C-S-H increased the content of bridging silicate tetrahedron (Q The care to AAS ,4. Theree to AAS ,6. Many researchers studied the carbonation behavior of C-S-H to improve the carbonation resistance of cementitious materials. According to literatures, the carbonation behavior of C-S-H mainly depends on its structure, which is affected by the Ca/Si ratio, uptake of aluminum and magnesium in C-S-H ,8,9,10.2) or bridging tetrahedra (Q3). If Al (IV) occurs in Q3 sites (bridging position), cross-linked structure will appear. Al (V) represents the aluminum enters the position of interlayer. Al (VI-1) represents the aluminum that enters the position of interlayer or in the C-S-H structure by replacing calcium [C-S-H is a kind of amorphous phase with layer structure . An incr calcium . Al . The carbonation products of C-S-H were analyzed by using X-ray powder diffraction. The content of carbonation products of C-S-H were investigated by means of thermogravimetric analyzer (TGA), XRD and the Rietveld method. In this way, insights into the effects of Ca/Si ratio, aluminum and magnesium on the carbonation behavior of C-S-H were provided.In this study, C-(M)-(A)-S-H with different Ca/Si ratio, Al/Si ratio and Mg/Si ratio were synthesized by means of the chemical precipitation method. The effects of the Ca/Si ratio, aluminum and magnesium on the structure of C-S-H were investigated by means of X-ray powder diffraction (XRD) and 3)2·4H2O, 150 mL) with molarity of 0.067 M, 0.10 M, 0.12 M, 0.14 M and 0.16 M were added drop by drop into sodium silicate solution with molarity of 0.1 M , respectively. Sodium hydroxide particles (NaOH) were added into sodium silicate solution to keep the pH value of the mixed solution over 13. Ultrapure water was boiled for 10 min to remove the dissolved carbon dioxide (CO2). The mixed solution was stirred constantly during the whole process (1 day). The entire synthesizing process was under a N2 atmosphere at a room temperature. A white gel was precipitated immediately as the calcium nitrate solution drops to the sodium silicate solution. Separation of gel from the liquid was conducted by using a centrifugal machine . After that, the gel was washed twice with ultrapure and decarbonized water to remove the sodium and nitrate ions. Finally, the obtained gel was dried at 60 °C for 7 days in a vacuum drying chamber .Preparation of C-S-H with different Ca/Si ratio by using chemical precipitation method. The chemical reagents used in the following work were the analytical grade. The C-S-H with initial Ca/Si ratio from 0.67 to 1.6 were prepared by using the chemical precipitation method ,15. Calc2SiO3·5H2O, 100 mL) was used as the source of silicon, a 0.12 M calcium nitrate solution (Ca(NO3)2·4H2O, 100 mL) was used as the source of calcium, and 0.00125 M, 0.0025 M, 0.005 M, 0.01 M, and 0.02 M aluminum nitrate solution (Al(NO3)3·9H2O, 100 mL) were used as the source of silicon aluminum.Preparation of C-A-S-H with different Al/Si ratio by using chemical precipitation method. The procedures to synthesize C-A-S-H with different Al/Si ratio were the same as those to synthesize C-S-H. A 0.10 M sodium silicate solution  was used as the source of silicon, a 0.12 M calcium nitrate solution (Ca(NO3)2·4H2O, 100 mL) was used as the source of calcium, and 0.00125 M, 0.0025 M, 0.005 M, 0.01 M, and 0.02 M magnesium nitrate solution (Mg(NO3)2·6H2O, 100 mL) were used as the source of silicon magnesium.Preparation of C-M-S-H with different Mg/Si ratio by using chemical precipitation method. The procedures to synthesize C-M-S-H with a different Mg/Si ratio were the same as those to synthesize C-S-H. A 0.10 M sodium silicate solution  were recorded on an AVANCE III 400 NMR spectrometer . The mean silicate chain length (CL) of C-S-H was calculated as follows [b2 is the bridging tetrahedron with calcium; Qu2 is the bridging tetrahedron with an H+; Qp2 is the pairing tetrahedron. The data of  follows : (1)CL¯ The mineralogical identification of C-(M)-(A)-S-H before and after carbonation was analyzed by XRD . XRD patterns were recorded on a Panlytical X’Pert PRO X-ray diffraction with Cu-Kα radiation. The X-ray tube was operated at 40 kV and 40 mA. Values for qualitative analysis were recorded at 2θ intervals from 5° to 70° with a step size of 0.033° for 10 s.2 atmosphere from room temperature to 1000 °C. Samples were characterized by mean of TGA with and without carbonation. The amounts of CaCO3 is determined directly via the weight loss of samples at the temperature of 500–900 °C instead of tangent method [1 is the weight loss of a sample in decomposition peak of CaCO3 before carbonation; T1 is the total weight of corresponding sample before carbonation in TGA test; WL2 is the weight loss of a sample in decomposition peak of CaCO3 after carbonation; T2 is the total weight of corresponding sample after carbonation in TGA test.The quantitative analysis of carbonation products of C-S-H with different Ca/Si ratio was carried out by using a thermogravimetric analyzer  with a heating rate of 10 °C per minute in Nt method ,39. The The quantification of carbonation products of C-A-S-H and C-M-S-H was studied by means of XRD and Rietveld method. Values for quantitative analysis were recorded at 2θ intervals from 10° to 70° with a step size of 0.017° for 20 s The internal standard substance was brucite with content of 15 wt. %.The real Ca/Si ratio, Al/Si ratio and Mg/Si ratio of synthesized C-(M)-(A)-S-H were presented in The effect of Ca/Si ratio on the structure of C-S-H were analyzed by comparing samples CSH_0.64, CSH_0.87, CSH_1.02, CSH_1.14 and CSH_1.25 see a. As the29Si NMR results of synthesized samples. The Ca/Si ratio, Al/Si ratio and Mg/Si ratio of C-(M)-(A)-S-H had a direct influence on the structure of C-(M)-(A)-S-H. n, and silicate chain length. The main characteristic peaks appeared at around 79 ppm (Q1), 83 ppm , 85 ppm , 88 ppm , 95 cm−1 (Q3), which were typical bands of C-S-H. To further characterize the structure of C-S-H, parts of samples were then analyzed by using NMR. 1 site represented the lower polymerization degree and short silicate chain, and vice versa. As shown in 3 site existed in pure C-S-H. The content of silicate tetrahedron in bridging the position increased when aluminum was added, indicating that part of aluminum enters the structure of C-S-H by replacing the bridge silicium. 29Si NMR results also showed that cross-linked silicate groups (Q3) existed in sample CMSH_0.16. Reference [3 site in C-(M)-S-H was the signal for the formation of M-S-H.The high content of the Si-O band in the Qeference  believed3. The intensity of the diffraction peaks of the carbonation products became stronger as the carbonation time prolonged, indicating the carbonation degree of C-S-H became higher. But the type of carbonation product remain unchanged. At the same carbonation time, C-S-H with a low Ca/Si ratio may have a better carbonation resistance by comparing the intensity of diffraction peaks of CaCO3. Besides, in sample CSH_1.02, the formation of calcite was earlier than the formation of vaterite. Therefore, vaterite might not be the transition phase of calcite.3 varies with the Ca/Si ratio of C-S-H. The Ca/Si ratio of C-S-H had a crucial influence on the type of carbonation products. When the Ca/Si ratio of C-S-H was lower than 0.87, i.e., CSH_0.87, the carbonation product was vaterite. When the Ca/Si ratio was higher than 1.02, i.e., CSH_1.02, both vaterite and calcite appeared as carbonation products. It is suggest that C-S-H with low Ca/Si ratio have a calcium octahedral sheets structure, similar symmetries, and a positive charge of the gel surface to vaterite . As for 3 was determined by means of TGA. 3 content is given in The content of CaCO2 increased as the carbonation time prolonged. The CCP of C-S-H initially decreased and then increased with the increasing Ca/Si ratio. Sample CSH_0.87 had the smallest value of CCP (6.43), while sample CSH_1.25 had the biggest value of CCP (14.85). The order of carbonation resistance of C-S-H with different Ca/Si ratio was as follows: CSH_0.87 (6.43) > CSH_1.02 (8.70) > CSH_0.64 (13.21) > CSH_1.14 (14.02) > CSH_1.25 (14.85). C-S-H with Ca/Si ratio around 0.9 had a better carbonation resistance which agreed well with the XRD results . tion see , it was 3. It was clear that no significant changes in the total content of CaCO3 as the Al/Si ratio increased, indicating the carbonation resistance of C-S-H was not affected by the aluminum uptake in C-S-H obviously. The effect of Al/Si ratio on the carbonation behavior of C-(A)-S-H was studied by means of XRD and the Rietveld method. The XRD patterns of C-(A)-S-H after carbonation for 28 days are presented in 2 in bridging site increased when Al enters the structure of C-S-H, which means C-A-S-H had more polymerized units and stronger bond strength. However, the entrance of aluminum into C-S-H also enlarged the interlayer distance, which increased the porosity of the gel. Therefore, the carbonation resistance of C-S-H has not improved obviously when aluminum entered the structure of C-S-H. According to 3 as a function of the Mg/Si ratio of C-(M)-S-H. The total content of CaCO3 reduced from 49.94 wt. % to 21.22 wt. % with increasing Mg/Si ratio. The results implied that the magnesium uptake in C-S-H could improve its carbonation resistance, especially when the Mg/Si ratio was bigger than 0.08.The effect Mg/Si ratio on the carbonation behavior of C-(M)-S-H after carbonation for 28 days was also studied by using XRD, as shown in 3) when the magnesium uptake in C-S-H occurred, indicating a higher degree of polymerization of silicon chain. Besides, reference [3 is the signal of the formation of M-S-H with a clay-like structure, therefore the mixture of C-S-H and M-S-H may be more compact. Both of these two reasons can make the C-(M)-S-H have a better carbonation resistance than C-S-H.According to eference  believed2) increased obviously with aluminum uptake in C-S-H, indicating long silicate chains and large interlayer distance. An obvious peak of band Si-O in Q3 site appeared with magnesium uptake in C-S-H before carbonation, which was a signal of the formation of M-S-H. Generally speaking, the length of silicate chains in C-(M)-(A)-S-H increased as the Ca/Si ratio decreased and Al/Si ratio and Mg/Si ratio increased.The effects of Ca/Si ratio, Al/Si ratio and Mg/Si ratio on the structure and carbonation behavior of C-(M)-(A)-S-H were investigated by means of XRD, NMR and TGA. The results showed that the polymerization degree of silicon chain decreased as the Ca/Si ratio of C-S-H increased, which means C-S-H with a low Ca/Si ratio had a higher bond strength and shorter bond length. The content of the Si-O band in bridging site (Q3 content of CMSH_0.16 after carbonation for 28 days: 21.22 wt. %) has a better carbonation resistance than C-(A)-S-H . Due to the differences in the structure of C-S-H, the carbonation products and carbonation resistance were various. For the samples with different Ca/Si ratio, the main carbonation product was vaterite, if the Ca/Si ratio was lower than 0.87. When the Ca/Si ratio was bigger than 1.02, the main products were vaterite and calcite. The C-S-H with a low Ca/Si ratio had a better carbonation resistance. For the samples with a different Al/Si ratio, the carbonation resistance of C-(A)-S-H did not change visibly as the Al/Si ratio increased. Moreover, the content of calcite decreased as the Al/Si ratio increased. For the samples with different Mg/Si ratio, the carbonation resistance of C-(M)-S-H improved obviously, especially when the Mg/Si ratio was bigger than 0.08. Moreover, the content of vaterite decreased as the Al/Si ratio increased. In general, C-(M)-S-H (total CaCO


Retinoic acid (RA) plays a key role in pluripotent cell differentiation. In F9 embryonic carcinoma cells, RA can induce differentiation towards somatic lineages via the Ras-extracellular signal-regulated kinase (Ras/Erk) pathway, but the mechanism through which it induces the Erk1/2 phosphorylation is unclear. Here, we show that miR-485 is a positive regulator that targets α/β-hydrolase domain-containing protein 2 (Abhd2), which can result in Erk1/2 phosphorylation and triggers differentiation. RA up-regulates miR-485 and concurrently down-regulates Abhd2. We verified that Abhd2 is targeted by miR-485 and they both can influence the phosphorylation of Erk1/2. In summary, RA can mediate cell differentiation by phosphorylating Erk1/2 via miR-485 and Abhd2. The ability to differentiate into all cell types is an important characteristic of embryonic stem cells (ESCs) that can be used for the treatment of diseases ,2. BeforPrevious reports have suggested that endogenous small-molecule signaling pathways can maintain the pluripotency of mouse embryonic stem cells (mESCs) ,12 whileMicroRNAs (miRNAs) are small noncoding RNAs that consists of 21–25 nucleotides and are assembled into RNA-induced silencing complexes, which target mRNAs for silencing via the base pairing of the miRNA sequence and mRNA 3′ untranslated region (3′-UTR ). They are evolutionarily conserved and play important roles in differentiation ,25. For MiR-485 is known to be involved in cancer progression. It can suppress breast cancer cells  and targIn this study, we discovered that miR-485 plays a role in RA-induced F9 EC cell differentiation. Our data demonstrate that Abhd2 is a downstream target of miR-485 while both are associated with the regulation of Erk1/2 phosphorylation in F9 EC cells. Additionally, PD can induce the transcription of miR-485 to elevate Abhd2 levels and RA can induce Abhd2 by improving the transcription of miR-485. These findings imply that RA promotes F9 EC cell differentiation at least partly through miR-485 via Abhd2-regulated Erk1/2 phosphorylation.To identify the miRNAs that are involved in the MAPK/Erk pathway, which is involved in differentiation, the small RNA sequences were analyzed from J1 mESCs treated for 24 h with 1 μM PD or from control cells treated with an equal volume of DMSO. This showed that miR-485 is significantly down-regulated by PD  and we iTo identify the target genes of miR-485, the miRNA target-prediction programs, namely miRbase, miRanda, PicTar and TargetScan, were used. Using these different algorithms, nine genes were selected as possible miR-485 targets and their 3′-UTRs containing the complementary base pairs to the seed sequence of miR-485 were cloned for further study B. We verTo determine whether the targets of miR-485 were down-regulated by miR-485 at the transcriptional and post-transcriptional levels, mimics and antisense oligonucleotides of miR-485 were transfected into F9 ECs. These samples were subsequently analyzed by immunoblotting and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). As shown in To confirm that miR-485 and Abhd2 is involved in the differentiation process of F9 ECs, the small molecules of RA and PD were used to induce the cell processes. F9 ECs were treated with DMSO, RA or PD. After 36 h, the total protein or RNA was collected for immunoblotting and RT-qPCR, respectively. As expected, Abhd2 was up-regulated by PD and down-regulated by RA D. Those To confirm that miR-485 is involved in the MAPK/Erk pathway, we first tested whether RA and PD could influence the signaling pathway using the firefly luciferase vector pSRE-TA-luc, which is the signaling pathway vector of MAPK/Erk pathway. F9 ECs were treated with DMSO, RA or PD for 36 h before being transfected with pSRE-TA-luc. This showed that RA stimulated the MAPK/Erk pathway whereas PD antagonized it A. SimilaA previous study showed that RA can modulate the phosphorylation of Erk1/2, which subsequently accelerates the cell differentiation. Furthermore, they found that the phosphorylation of Erk1/2 can be up-regulated by inhibiting Abhd2 in cancer cells ,36. TherUndifferentiated pluripotent cells exhibit higher levels of alkaline phosphatase (AP) activity and thus, AP staining was used to test cellular pluripotency. The AP activity of F9 ECs was lower after RA compared to a DMSO control. Furthermore, this activity improved after PD, indicating that the pluripotency of F9 ECs was blocked by RA while it was promoted by PD in the presence of RA A. SimilaNanog and Klf4 is core pluripotent markers of ESC and ECs, which is involved in the MAPK/Erk pathway ,37. PrevRA treatment can lead to pluripotent cell differentiation and this process involves a variety of genes ,38,39. FRA induces numerous alterations in microRNAs. For example, miR-10a targets histone deacetylase 4 to influence the NF-kB pathway during smooth muscle cell (SMC) differentiation from embryonic stem cells . FurthermiR-485 is involved in the suppression of tumors by inhibiting cell growth  and RA cNanog occupies a central position in the pluripotency network and is a highly expressed pluripotency marker in ES and EC cells. During cellular differentiation, its level decreases rapidly ,49 and RWe have shown that Abhd2 acts as a target of miR-485 in the MAPK pathway and that it regulates the phosphorylation of Erk1/2 in F9 ECs. Furthermore, Abhd2 is a factor that promotes the maintenance of pluripotency. Collectively, our results provide a new insight into the function of miRNAs in the MAPK pathway during RA-induced cell differentiation.Our results indicated that miR-485 participates in several mechanisms of differentiation. However, there needs to be further studies on how miR-485 and Abhd2 regulate the phosphorylation of Erk1/2 and in the future, the mechanisms through which Abhd2 influences differentiation also need to be clarified in order for RA-induced cell differentiation to be fully understood.v/v) FBS. All cells were maintained at 37 °C and 5% CO2 in a humidified incubator.HEK293T cells and F9 teratoma cells were obtained from the American Type Culture Collection  and cultured in Dulbecco’s modified Eagle’s medium (DMEM) in the sterile cell wells which were purchased from Nunclon . The cells were supplemented with 10%  according to the manufacturer’s instructions. All cell culture reagents were purchased from Gibco  unless indicated.PD  was dissolved in dimethyl sulfoxide (DMSO) and added to cell medium at a final concentration of 2 μM. RA  was dissolved in DMSO at a final concentration of 2 μM. An equal volume of DMSO added in another well was used as a control.http://pga.mgh.harvard.edu/primerbank/) and shown in the Total RNA was extracted from treated cells using Trizol Reagent (Life Technologies) according to the manufacturer’s protocol. Total RNA (1 μg) was reverse-transcribed using a PrimeScript RT reagents kit  with the protocol: Reactions were carried out at 37 °C for 15 min and 85 °C for 5 s. The amount of RNA was determined by real-time PCR with an ABI StepOne Plus PCR system  using SYBR Premix Ex Taq II (TaKaRa). The following conditions were used for qPCR: 30 s at 95 °C, and 40 cycles of 5 s at 95 °C and 30 s at 60 °C, and the melting curve was analyzed to ensure that a single PCR product was obtained. Data were collected after each annealing step. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an endogenous control to normalize for the differences in the amount of total RNA in each sample. All primer sequences used for qPCR examination were obtained from the PrimerBank  was reverse-transcribed using a miScript II RT kit  to obtain mature miRNA. Real-time PCR was performed in triplicate for each sample by using miScript SYBR Green PCR kit (Qiagen). The endogenous reference RNA RNU6B was used to normalize the amount of template added for each sample. The following primers were used: q-miR485-5p: GGAGAGGCTGGCCGTGA and Universal miRNA Forward: TGAATCGAGCACCAGTTACGCATGCCGAGGTCGACTTCCTAGA.GCCACCATGGACTACAAAGACGATGACGACAAGATGAATGCCATGCTAGAGACCC-3′ (underlined letters indicate an EcoRI restriction site) and reverse primer: 5′-ATAAGAATGCGGCCGCTCATTCCAACTCGGCCTCCATCTGCTCC-3′ (underlined letters indicate a NotI restriction site). The PCR sample was ligated into the pcDNA 3.1. a fragment carrying pre-miR-485 was amplified from genomic DNA of J1 ESCs and cloned into the pCDH-CMV-MCS-EF1-coGFP vector . The PCR primers were as follows: forward primer: 5′-GCTCTAGACTACCACAGGAGCTTCCAGAATA-3′ (underlined letters indicate a XbaI restriction site) and reverse primer: 5′-ATAAGAATGCGGCCGCTAGCTTGGACACTGGGATAACTG-3′ (underlined letters indicate a NotI restriction site).CDS sequence of Abhd2 was amplified from the genomic DNA of ES cells by PCR. The PCR primers were as follows: forward primer: 5′-CGGAATTCThe 3′-UTR of genes that contain putative miRNA binding sites were amplified from J1 ESC cDNA through PCR and then cloned into psiCHECK-2 Vector (Promega) for Luciferase assays. All primer sequences used were shown in the Short interfering RNAs (siRNAs) that target mouse Abhd2 and negative control siRNA-NC were purchased from Shanghai GenePharma . siRNA sequences were shown in the Luciferase assays were performed by using a Dual-Luciferase Reporter Assay System . The luciferase reporter and pCDH-miR-485 were cotransfected into HEK293T cells using Lipofectamine 2000. After 36 h, transfected cells were lysed in passive lysis buffer for 15 min with shaking by Rockers. Firefly luciferase activity in cell lysates was measured on a VICTOR X5 Multilabel Plate Reader  and normalized to Renilla luciferase.Pathway reporter vector pSRE-TA-luc was purchased from Beyotime. The negative control vectors pTA-luc and the JAK/STAT pathway reporter vector pISRE-TA-luc were obtained from Clonetech . The Wnt signaling pathway reporter vector pSuperTOPFlash luciferase reporter was constructed by inserting seven copies of the TCF/LEF binding site (AGATCAAAGG) into the pTA-luc vector, The pRL-SV40 (Promega) was used to normalized to Renilla luciferase. they were cotransfected with vectors like pcDNA3.1-Abhd2 or mimic of miR-485-5p into F9 ECs by using Lipofectamine 2000 for 36 h, Luciferase activity was detected as above.Cells were lysed with RIPA buffer  The BCA Protein Assay Reagent (Pierce) was used to quantify the protein concentration of lysates. Equal amounts of protein were resolved by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then the proteins were transferred to the polyvinylidene fluoride membranes . After blocking for 4 h at room temperature in 10% nonfat dry milk in TBST containing 0.05% Tween-20, the membranes were incubated with primary antibodies overnight at 4 °C and then after washes using TBST, it was incubated with HRP-conjugated secondary antibodies for 2 h at room temperature.Primary antibodies included rabbit anti-Klf4  Rabbit monoclonal anti-GAPDH  rabbit anti-Nanog , Anti-ERK1/2 rabbit polyclonal antibody , Anti-ERK1/2(Thr202/Tyr204)) rabbit polyclonal antibody , Rabbit Polyclonal anti-ABHD2  and anti-rabbit/mouse horseradish peroxidase-conjugated secondary antibody were obtained from the Beyotime Institute of Biotechnology .t-test, and significance was set at p-values < 0.05. The difference between three or more groups was compared by an analysis of variance (ANOVA), and significance of differences was determined by post hoc testing. Multiple comparisons between the groups were performed using the S-N-K method.The data in graphs are expressed as the mean ± SD. The difference between two groups was compared by a two-tailed paired Student’s We explored the mechanisms by which RA induces cell differentiation via miRNA. Our results suggest that RA-induced miR-485 is involved in the F9 EC differentiation and that miR-485 plays its role by targeting Abhd2. This process modulates Erk1/2 phosphorylation and ultimately Nanog activity. Ultimately, RA regulates miR-485 to induce embryonal carcinoma cell differentiation through the MAPK/Erk pathway B.