O-linked N-acetylglucosamine modification induced by lipopolysaccharide is involved in inflammatory signaling pathway in endothelial cells / 中华危重病急救医学

Objective:To explore whether the lipopolysaccharide (LPS)-induced modification of O-linked N-acetylglucosamine (O-GlcNAc) is involved in the inflammatory signaling pathway of endothelial cells.Methods:Human umbilical vein endothelial cells (HUVEC) were cultured in vitro, and cells in logarithmic growth phase were used for experiments. Cells were divided into blank control group, LPS group (2 000 mg/L LPS), O-GlcNAc transferase (OGT) overexpression (OGT-OE)+LPS group (plasmid transfection OGT+2 000 mg/L LPS), protein kinase C (PKC) inhibitor+LPS group (10 μmol/L Go 6983+2 000 mg/L LPS), RhoA inhibitor+LPS group (40 μmol/L Rhoin hydrochloride+2 000 mg/L LPS), phosphatidylinositol-3-kinase (PI3K) inhibitor+LPS group (1 μmol/L SL-2052+2 000 mg/L LPS), serine/threonine kinase (Akt) inhibitor+LPS group (10 μmol/L PP2+2 000 mg/L LPS) and small interfering RNA (siRNA) treated Akt (si-AKT)+LPS group (si-Akt+2 000 mg/L LPS). After 24 hours of LPS treatment, real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the transcription levels of inflammatory cytokines [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. The protein expression or phosphorylation of OGT, O-GlcNAc, Akt, extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK), nuclear factor-κB p65 (NF-κB p65), and signal transducer and activator of transcription 3 (STAT3) were determined by Western blotting. Results:Compared with the blank control group, the expression of OGT and the modification of O-GlcNAc in the LPS group were decreased, while the expressions of phosphorylated ERK, p38MAPK, and STAT3 were increased, and the transcript levels of inflammatory cytokines were also significantly increased [IL-6 mRNA (2 -ΔΔCt): 4.71±0.60 vs. 1.03±0.29, TNF-α mRNA (2 -ΔΔCt): 1.89±0.11 vs. 1.04±0.35, ICAM-1 mRNA (2 -ΔΔCt): 2.06±0.18 vs. 1.02±0.21, VCAM-1 mRNA (2 -ΔΔCt): 2.94±0.57 vs. 1.01±0.17, all P < 0.05], indicating that LPS could decrease O-GlcNAc modification, activate inflammatory signaling pathways and increase inflammatory cytokines expression. Compared with the LPS group, the expressions of phosphorylated ERK, p38MAPK, NF-κB p65, and STAT3 in the endothelial cells of the OGT-OE+LPS group were decreased, and the expression of inflammatory factors were significantly decreased [IL-6 mRNA (2 -ΔΔCt): 0.12±0.01 vs. 0.90±0.17, TNF-α mRNA (2 -ΔΔCt): 0.31±0.01 vs. 0.91±0.14, ICAM-1 mRNA (2 -ΔΔCt): 0.64±0.02 vs. 1.13±0.16, VCAM-1 mRNA (2 -ΔΔCt): 0.11±0.01 vs. 0.93±0.11, all P < 0.05], indicating that the increase of OGT level could inhibit the partial activation of the endothelial inflammatory signal pathway under the LPS stimulation. Compared with the blank control group, the phosphorylation level of Akt in the LPS group was increased. Compared with the LPS group, both OGT expression and O-GlcNAc modification were down-regulated after pretreatment of PKC inhibitor, RhoA inhibitor, PI3K inhibitor, or Akt inhibitor. Compared with the LPS group, the transcript levels of IL-6, TNF-α and ICAM-1 in the PP2+LPS group were significantly decreased [IL-6 mRNA (2 -ΔΔCt): 1.46±0.16 vs. 3.55±0.87, TNF-α mRNA (2 -ΔΔCt): 0.98±0.14 vs. 1.76±0.10, ICAM-1 mRNA (2 -ΔΔCt): 1.39±0.24 vs. 2.04±0.13, all P < 0.05], but there was no significant change in VCAM-1. Compared with the LPS group, the expression of OGT and O-GlcNAc modification in the si-Akt+LPS group were decreased, while the transcript levels of inflammatory cytokines were also significantly decreased [IL-6 mRNA (2 -ΔΔCt): 0.75±0.03 vs. 0.99±0.09, TNF-α mRNA (2 -ΔΔCt): 0.69±0.01 vs. 1.10±0.08, ICAM-1 mRNA (2 -ΔΔCt): 0.76±0.01 vs. 0.99±0.02, VCAM-1 mRNA (2 -ΔΔCt): 0.93±0.08 vs. 1.20±0.21, all P < 0.05], indicating that Akt participated in the action process of LPS on OGT and affected the inflammatory factor expression. Conclusions:The decreased level of O-GlcNAc modification in endothelial cells stimulated with LPS promotes partial activation of inflammatory signaling pathways, mainly involving ERK, p38MAPK, and STAT3, and affects the expression of inflammatory factors. AKT may be involved in the effect of LPS on the inhibition of O-GlcNAc modification.

The Effect of EOGT-Mediated O-Glcnac Modification on Notch Signaling Pathway and Its Role in Diseases / 中国生物化学与分子生物学报

O-linked-N-acetylglucosamine (O-GlcNAc) modification is a unique post-translational modification that plays a regulatory role in many cellular processes, such as transcription, intracellular signaling, endocytosis, and protein stability. Epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER) resident protein which can glycosylate the residues of Ser or Thr of secreted or membrane (transmembrane) glycoproteins containing EGF domain. Notch signaling pathway is involved in cell-to-cell communication which regulates cell biological processes through interactions between adjacent cells. To date, EOGT-mediated O-GlcNAc modification has been found to be involved in many human diseases, and shown significant relation with Notch signaling pathway. However, the specific molecular mechanisms have not been fully elucidated. In this review, we briefly introduce recent studies regarding to the roles of EOGT-mediated O-GlcNAc modification and its correlation with Notch signaling pathway in human diseases.

Upregulation of OGT by Caveolin-1 Promotes Mouse Hepatocellular Carcinoma Cells Migration and Invasion / 中国生物化学与分子生物学报

Caveolin-1 (Cav-1), a major structural protein of caveolae, is implicated in the vesicular uptake processes of transcytosis and cell signaling. However, its role in modulating protein glycosylation and tumor metastasis remains to be further elucidated. In the present study, it was shown that Cav-1 promotes the expression of O-GlcNAcylation and O-GlcNAc transferase (OGT), and triggers the invasion and metastasis of hepatocellular carcinoma (HCC) cells. The results of RT-qPCR, Western blot and dual lucif-erase reporter assay showed that Cav-1 negatively regulated the expression of transcription factor RUNX2 in HCC. Subsequently, this results in attenuate RUNX2-induced transcription of miR24. miR24 suppresses mouse HCC cells invasion and metastasis via directly targeting Ogt mRNA 3′UTR. This research provides evidence of Cav-1-mediated OGT expression and O-GlcNAc (O-linked N-acetylglucosamine) elevation. These data give insight into a novel mechanism of HCC occurrence and development.

Expression and characterization of β-N-acetylglucosaminidases from Bacillus coagulans DSM1 for N-acetyl-β-D glucosamine production / 生物工程学报

β-N-acetylglucosaminidases (NAGases) can convert natural substrates such as chitin or chitosan to N-acetyl-β-D glucosamine (GlcNAc) monomer that is wildly used in medicine and agriculture. In this study, the BcNagZ gene from Bacillus coagulans DMS1 was cloned and expressed in Escherichia coli. The recombinant protein was secreted into the fermentation supernatant and the expression amount reached 0.76 mg/mL. The molecular mass of purified enzyme was 61.3 kDa, and the specific activity was 5.918 U/mg. The optimal temperature and pH of the BcNagZ were 75 °C and 5.5, respectively, and remained more than 85% residual activity after 30 min at 65 °C. The Mie constant Km was 0.23 mmol/L and the Vmax was 0.043 1 mmol/(L·min). The recombinant BcNagZ could hydrolyze colloidal chitin to obtain trace amounts of GlcNAc, and hydrolyze disaccharides to monosaccharide. Combining with the reported exochitinase AMcase, BcNagZ could produce GlcNAc from hydrolysis of colloidal chitin with a yield over 86.93%.

Inhibitory effect of resveratrol on fat synthesis in liver cancer HepG2 cells and its mechanism / 吉林大学学报(医学版)

Objective:To investigate the effect of resveratrol (Res) on the fat synthesis in the liver cancer HepG2cells, and to elucidate its possible mechanism.Methods:The HepG2cells were cultured in vitro and divided into Res group (treated with 40μmol·L-1 DMSO-diluted Res for 24h) and control group (treated with the same concentration of DMSO for 24h) .The cell supernatant was collected, and the levels of triglyceride (TG) and total cholesterol (TC) in the cells in various groups were measured by ELISA.The mRNA and protein expression levels of lipase synthase acetyl-CoA carboxylase (ACC1) , fatty acid synthetase (FASN) and stearoyl-CoA desaturase (SCD1) in the cells in various groups were detected by qRT-PCR and Western blotting method.The levels of O-linked N-acetylglucosamine (O-GlcNAc) glycosylation in the cells in various groups were detected by Western blotting method.Results:Compared with control group, the levels of TG and TC in the cells in Res group were decreased, but the difference was not statistically significant (t1=1.886, P＞0.05;t2=2.457, P＞0.05) .Compared with control group, the levels of expressions of ACC1, FASN and SCD1mRNA and proteins in the cells in Res group were significantly decreased (P＜0.05or P＜0.01) ;the O-GlcNAc glycosylation level in the cells in Res group was significantly decreased (t=2.87, P＜0.05) .Conclusion:Res has the effect of inhibiting the fat synthesis in the liver cancer HepG2 cells.Its mechanism may be related to the reduction of cellular O-GlcNAc glycosylation level and the reduction of the expression of FASN.

Inhibitory effect of resveratrol on fat synthesis in liver cancer HepG2 cells and its mechanism / 吉林大学学报(医学版)

Objective: To investigate the effect of resveratrol (Res) on the fat synthesis in the liver cancer HepG cells, and to elucidate its possible mechanism. Methods: The HepG cells were cultured in vitro and divided into Res group (treated with 40 umol • L-1 DMSO- diluted Res for 24 h) and control group (treated with the same concentration of DMSO for 24 h). The cell supernatant was collected, and the levels of triglyceride (TG) and total cholesterol (TO in the cells in various groups were measured by ELISA. The mRNA and protein expression levels of lipase synthase acetyl-CoA carboxylase (ACCl), fatty acid synthetase (FASN) and stearoyl-CoA desaturase (SCD1) in the cells in various groups were detected by qRT-PCR and Western blotting method. The levels of O-linked N-acetylglucosamine (O-GIcNAc) glycosylation in the cells in various groups were detected by Western blotting method. Results: Compared with control group, the levels of TG and TC in the cells in Res group were decreased, but the difference was not statistically significant (t1=1.886, P>0.05; t2=2.457,P>0.05). Compared with control group, the levels of expressions of ACCl, FASN and SCD1 mRNA and proteins in the cells in Res group were significantly decreased ( P<0.05 or P<0. 01); the O-GlcNAc glycosylation level in the cells in Res group was significantly decreased (t=2. 87, P<0.05). Conclusion: Res lias the effect of inhibiting the fat synthesis in the liver cancer HepG cells. Its mechanism may be related to the reduction of cellular O-GlcNAc glycosylation level and the reduction of the expression of FASN.

Glucose-Insulin-Potassium Solution Protects Ventricular Myocytes of Neonatal Rat in an In Vitro Coverslip Ischemia/Reperfusion Model

BACKGROUND AND OBJECTIVES: The benefit of high glucose-insulin-potassium (GIK) solution in clinical applications is controversial. We established a neonatal rat ventricular myocyte (NRVM) in vitro coverslip ischemia/reperfusion (I/R) model and investigated the effects of GIK solution on suppressing reactive oxygen species (ROS) and upregulating O-GlcNacylation, which protects cells from ischemic injury. MATERIALS AND METHODS: NRVMs were isolated from postnatal day 3-4 Sprague-Dawley rat pups and grown in Dulbecco's modified Eagle's medium containing high glucose (4.5 g/L), fetal bovine serum, and penicillin/streptomycin. The effects of the GIK solution on ROS production, apoptosis, and expression of O-GlcNAc and O-GlcNAc transferase (OGT) were investigated in the coverslip I/R model. RESULTS: Covering the 24-well culture plates for 3 hr with 12 mm diameter coverslips resulted in the appropriate ischemic shock. Glucose and insulin synergistically reduced ROS production, protected NRVM dose-dependently from apoptosis, and altered O-GlcNAc and OGT expression. CONCLUSION: The high GIK solution protected NRVM from I/R injury in vitro by reducing ROS and altering O-GlcNacylation.

Immunohistochemical Study of O-GlcNAcylation in Human Skin Tumors / 체질인류학회지

O-linked beta-N-acetylglucosamine modification is an important post-translational modification, emerging as a novel regulatory mechanism in various cellular events. Recently, several studies have shown that O-GlcNAcylation plays an essential role in human breast, lung, and colon cancers. With regard to skin cancers, the role of O-GlcNAcylation has yet to be elucidated. To investigate whether O-GlcNAcylation is linked to human skin tumor development, immunohistochemical analysis was performed to investigate the presence of O-GlcNAcylation in various skin tumors. We evaluated the levels of O-GlcNAcylation, O-GlcNAc transferase, and O-GlcNAcase in 29 benign tumors, 12 premalignant tumors, and 26 malignant tumors in skin. Compared to the benign tumors, premalignant and malignant tumors had increased patterns of O-GlcNAcylation. In addition, the O-GlcNAc transferase and O-GlcNAcase levels were higher in premalignant and malignant tumors than in benign tumors. Interestingly, O-GlcNAcase levels were significantly increased in premalignant tumors compared to benign and malignant tumors. These results suggest that O-GlcNAcylation of proteins may play an important role in the development of human skin tumors.

Location and expression of galactose β-1,4-glycosidic bonds in keloid tissue / 中华皮肤科杂志

Objective To observe the expression and location of galactose β-1,4-glycosidic bonds (Gal β-1,4-GlcNAc) in keloid tissue,and to investigate the role of glycoprotein galactosylation in the formation of keloid.Methods This study included 10 keloid tissue specimens,7 hyperplastic scar tissue specimens,and 6 normal skin specimens.Lectin blot analysis was performed to measure the glycosylation level of glycoproteins,saturated picric acid-Sirius red staining followed by polarization microscopy to observe the type,expression and distribution of collagens in these specimens.The type Ⅰ/type Ⅲ collagen ratio was calculated.Immunofluorescence-based histochemistry was carried out by using Ricinus communis agglutinin I to analyze the expression and location of Gal β-1,4-GlcNAc in these skin samples,and double immunofluorescent staining to observe the colocalization of Gal β-1,4-GlcNAc and type Ⅰ procollagen α1.Results Compared with the normal skin tissue,the keloid tissue showed a significant increase in the expression of Gal β-1,4-GlcNAc in glycoproteins with a relative molecular mass of 30 000 to 40 000.Polarization microscopy revealed that there was a considerable expression of type Ⅰ collagen fibers,which amounted to (71.53 ± 4.03)％ in all the collagen fibers.The type Ⅰ/type Ⅲ collagen ratio was significantly higher in keloid tissue specimens than in normal tissue specimens (2.56 ± 0.53 vs.0.91 ± 0.11,P ＜0.05).Fluorescence microscopy showed that Gal β-1,4-GlcNAc was uniformly distributed in the membrane and cytoplasm of fibroblasts in keloid tissue,and the expression intensity of Gal β-1,4-GlcNAc in keloid tissue was notably stronger than that in normal skin tissue.There was a colocalization between Gal β-1,4-GlcNAc and type Ⅰ procollagen α1 in keloid tissue.Conclusions The expression of Gal β-1,4-GlcNAc,which is mainly observed in fibroblasts,is upregulated in keloid tissue,suggesting that Gal β-1,4-GlcNAc may be involved in the modulation of factors responsible for excessive fibre formation during the repair process of keloid.

Role of O-GlcNAc protein modification in attenuation of brain damage by glutamine in septic rats / 中华麻醉学杂志

Objective To evaluate the role of O-GlcNAc protein modification in attenuation of brain damage by glutamine in septic rats.Methods Sixty male SD rata weighing 180-240 g were randomly divided into 4 groups:sham operation group(group S,n =12),sepsis group(group CLP,n =16),glutamine group(group G,n =16),an inhibitor of O-linked-N-acetyl glucosamine transferase Alloxan + glutamine group(group G + A,n =16).Rats were submitted to sepsis by cecal ligation and perforation(CLP).Glutamine(Gln)0.75 g/kg was injected iv after CLP in group G.Gln 0.75 g/kg was injected iv and Alloxan 90 mg/kg was injected ip after CLP in group G + A.Equal volume of normal saline was given in group S and group CLP.A1 24 h afler CLP,the neural reflex score was evaluated,then rat was sacrificed.The brain was removed for measurement of brain water content,observation of histopathology and determination of O-GlcNAc-modified protein expression.Results Compared with group S,neural reflex score and brain water content were significantly increased in groups CLP,G and G + A(P ＜ 0.05).Compared with group CLP,neural reflex score and brain water content were significantly decreased in groups G and G + A(P ＜ 0.05),and the expression of O-GlcNAc-modified protein was upregulated in group G(P ＜ 0.05),Compared with groups G,neural reflex score and brain water content were significantly increased,and the expression of O-GlcNAc-modified protein downregulated in group G + A(P ＜ 0.05).There was no significant difference in O-GlcNAc-modified protein expression among groups S,CLP and G + A.Conclusion Glutamine attenuates brain damage through O-GlcNAc protein modification in septic rats.

O-GlcNAc modification on IRS-1 and Akt2 by PUGNAc inhibits their phosphorylation and induces insulin resistance in rat primary adipocytes

It has been known that O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of proteins plays an important role in transcription, translation, nuclear transport and signal transduction. The increased flux of glucose through the hexosamine biosynthetic pathway (HBP) and increased O-GlcNAc modification of protein have been suggested as one of the causes in the development of insulin resistance. However, it is not clear at the molecular level, how O-GlcNAc protein modification results in substantial impairment of insulin signaling. To clarify the association of O-GlcNAc protein modification and insulin resistance in rat primary adipocytes, we treated the adipocytes with O-(2-acetamido-2deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), a potent inhibitor of O-GlcNAcase that catalyzes removal of O-GlcNAc from proteins. Prolonged treatment of PUGNAc (100 micrometer for 12 h) increased O-GlcNAc modification on proteins in adipocytes. PUGNAc also drastically decreased insulin-stimulated 2-deoxyglucose (2DG) uptake and GLUT4 translocation in adipocytes, indicating that PUGNAc developed impaired glucose utilization and insulin resistance in adipocytes. Interestingly, the O-GlcNAc modification of IRS-1 and Akt2 was increased by PUGNAc, accompanied by a partial reduction of insulin-stimulated phosphorylations of IRS-1 and Akt2. The PUGNAc treatment has no effect on the expression level of GLUT4, whereas O-GlcNAc modification of GLUT4 was increased. These results suggest that the increase of O-GlcNAc modification on insulin signal pathway intermediates, such as IRS-1 and Akt2, reduces the insulin-stimulated phosphorylation of IRS-1 and Akt2, subsequently leading to insulin resistance in rat primary adipocytes.

Expressions of peripheral lymph node addressin and GlcNAc-6-sulfotransferase in endometrium and their impacts on implantation / 北京大学学报(医学版)

Objective:To investigate the expressions of peripheral lymph node addressin(PNAd)andGlcNAc-6-sulfotransferase(GlcNAc6ST)in endometrium and their impacts on implantation.Methods:PNAd expression in endometrium was examined by immunohistochemistry and Western Blot from 75women(12 from healthy women,in proliferative phase;63 from sterile women,of whom,27 were inearly-secretory and 36 in mid-secretory phase).GlcNAc6ST mRNA was examined by real-time PCR in41 sterile women.The 63 sterile women had underwent ⅣF-ET and were consequently divided into clini-cal pregnant(29 cases)and nonpregnant(34 cases)groups.Results:(1)PNAd localized to the mem-brane and cytoplasm of luminal and glandular epithelia.Staining was patchy and much less intense duringthe proliferative phase than during the secretory phase.In Western Blot of PNAd,four bands appeared,which were Sgp200,CD34,MAdCAM-1,GlyCAM-1 respectively,and each was positively correlatedwith the others significantly.The former three molecular levels were significantly higher during the secre-tory phase as compared with the proliferative phase.Message RNA of GlcNAe6ST was positive in all ca-ses and showed no correlation with any component of PNAd.(2)The expressions of CD34 and GlyCAM-1,but not Sgp200 and MAdCAM-1,were significantly higher in pregnant women than in nonpregnantones.However,the GlcNAc6ST mRNA level did not differ between groups.(3)No significant differ-ence was found in female age,methods of fertilization,thickness of endometrium on day hCG,cumulativeembryo score(CES)and mean score of transferred embryo(MSTE)between the groups.Conclusion:PNAd expression in the human endometrium fluctuates with the menstrual cycle.Elevated CD34 and Gly-CAM-1 during the secretory phase might be stimulative factors for embryo implantation.Defect in PNAdexpression may account for a portion of unexplained infertility.

EXPRESSION OF GAL?-1,4-GlcNAc GROUP IN ASTROCYTOMA / 解剖学报

Objective To study the expression of Gal?-1,4-GlcNAc group in human normal brains and astrocytomas. Methods The protein extracts from human normal brains and astrocytomas were subjected to sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE),then were stained with Coomassie Brilliant blue(CBB).In addition,the expression of Gal?-1,4-GlcNAc group was studied by ricinus communis agglutinin-I(RCA-I) lectin blot and lectin histochemistry with RCA-I respectively. Results By comparison,CBB staining showed that the protein components from normal brains were similar to those from astrocytomas.However,by RCA-I lectin blot and histochemistry assay,it was found that the expression of Gal?-1,4-GlcNAc was more extensive in astrocytomas than normal brains.It was surprising that a major 61?kD protein was galactosylated in astrocytomas but not in normal brains.Ratio of RCA-I stained cells was 8% in human normal brains,while 15%,21%,28% and 41% in astrocytomas Ⅰ,Ⅱ,Ⅲ and Ⅳ respectively.Conclusion The expressions of Gal?-1,4-GlcNAc groups in astrocytomas were more than those in normal brains,which suggested that Gal?-1,4-GlcNAc group might be related to the malignant degree of astrocytomas.