BACKGROUND Hepatitis B virus (HBV) infection is the primary cause of hepatitis with chronic HBV infection,which may develop into liver fibrosis,cirrhosis and hepatocellular carcinoma.Detection of early-stage fibrosis ...BACKGROUND Hepatitis B virus (HBV) infection is the primary cause of hepatitis with chronic HBV infection,which may develop into liver fibrosis,cirrhosis and hepatocellular carcinoma.Detection of early-stage fibrosis related to HBV infection is of great clinical significance to block the progression of liver lesion.Direct liver biopsy is regarded as the gold standard to detect and assess fibrosis;however,this method is invasive and prone to clinical sampling error.In order to address these issues,we attempted to find more convenient and effective serum markers for detecting HBV-induced early-stage liver fibrosis.AIM To investigate serum N-glycan profiling related to HBV-induced liver fibrosis and verify multiparameter diagnostic models related to serum N-glycan changes.METHODS N-glycan profiles from the sera of 432 HBV-infected patients with liver fibrosis were analyzed.Significant changed N-glycan levels (peaks)(P <0.05) in differentfibrosis stages were selected in the modeling group,and multiparameter diagnostic models were established based on changed N-glycan levels by logistic regression analysis.The receiver operating characteristic (ROC) curve analysis was performed to evaluate diagnostic efficacy of N-glycans models.These models were then compared with the aspartate aminotransferase to platelet ratio index (APRI),fibrosis index based on the four factors (FIB-4),glutamyltranspeptidase platelet albumin index (S index),GlycoCirrho-test,and GlycoFibro-test.Furthermore,we combined multiparameter diagnostic models with alanine aminotransferase (ALT) and platelet (PLT) tests and compared their diagnostic power.In addition,the diagnostic accuracy of N-glycan models was also verified in the validation group of patients.RESULTS Multiparameter diagnostic models constructed based on N-glycan peak 1,3,4and 8 could distinguish between different stages of liver fibrosis.The area under ROC curves (AUROCs) of Model A and Model B were 0.890 and 0.752,respectively differentiating fibrosis F0-F1 from F2-F4,and F0-F2 from F3-F4,and surpassing other serum panels.However,AUROC (0.747) in Model C used for the diagnosis of F4 from F0-F3 was lower than AUROC (0.795) in FIB-4.In combination with ALT and PLT,the multiparameter models showed better diagnostic power (AUROC=0.912,0.829,0.885,respectively) when compared with other models.In the validation group,the AUROCs of the three combined models (0.929,0.858,and 0.867,respectively) were still satisfactory.We also applied the combined models to distinguish adjacent fibrosis stages of 432patients (F0-F1/F2/F3/F4),and the AUROCs were 0.917,0.720 and 0.785.CONCLUSION Multiparameter models based on serum N-glycans are effective supplementary markers to distinguish between adjacent fibrosis stages of patients caused by HBV,especially in combination with ALT and PLT.展开更多
With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driv...With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships.In monoclonal antibodies,many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known.The importance of their function focuses analytical research efforts on the development of robust,accurate and fast methods to support drug development and quality control.Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however,it is not the only method for quantitative analysis of glycoform heterogeneity.In this study,ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies.While observing good comparability between the quantitative results generated,it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow according to application and the desired depth of data generated.展开更多
Sialylation is one of important glycosylation in human beings and plays an important role in cancer development. α2,3-Linked and α2,6-linked sialic acids are normally observed on the end of N-glycans and have differ...Sialylation is one of important glycosylation in human beings and plays an important role in cancer development. α2,3-Linked and α2,6-linked sialic acids are normally observed on the end of N-glycans and have different functions. Derivatization on sialic acid was designed to detect the different linkages by MALDI-TOF MS. In this study, a two-step derivatization by dimethylamine and ammonium hydroxide was improved to modify the sialic acid and made it easier to detect the different linkages of sialic acids on MALDI-TOF MS. Using this derivatization method, specific sialic acids linkages on N-glycans of protein samples such as fetuin and lactoferrin were detected. For complex cell samples, increased a2,3-linked and a2,6-linked sialic acids on bi-antennary and tri-antennary N-glycans were observed in A549 cells induced by hypoxia environment. Taken together, our two-step derivatization of sialic acids offers a simple and accurate way to detect specific linkages on N-glycans with MALDI-TOF mass spectrometer.展开更多
Nonalcoholic fatty liver disease（NAFLD） is a major public health issue worldwide. Immunoglobulin G（IgG） N-glycans are associated with risk factors for NAFLD, such as obesity and diabetes. A cross-sectional study i...Nonalcoholic fatty liver disease（NAFLD） is a major public health issue worldwide. Immunoglobulin G（IgG） N-glycans are associated with risk factors for NAFLD, such as obesity and diabetes. A cross-sectional study involving 500 Han Chinese adults recruited from a community in Beijing was carried out to explore the association between IgG N-glycans and NAFLD. IgG N-glycosylation was significantly associated with NAFLD, with the disease showing a negative correlation with galactosylation（GP14, GP14n, and G2n）, positive correlation with fucosylation（FBG2n/G2n）, and positive correlation with bisecting N-acetylglucosamine（Glc NAc） [FBG2n/FG2n and FBG2n/（FG2n＋FBG2n）], after controlling age, gender, and prevalence of obesity, type 2 diabetes mellitus, hypertension, and hyperlipidemia. In other words, the present study showed a possible association between NAFLD and the loss of galactose and elevations of fucose and bisecting GlcNAc. Aberrant IgG glycosylation might therefore be a potential biomarker for the primary or secondary prevention of NAFLD.展开更多
Objective This study prospectively investigates the association between immunoglobulin G(IgG)N-glycan traits and ischemic stroke(IS) risk.Methods A nested case-control study was conducted in the China suboptimal healt...Objective This study prospectively investigates the association between immunoglobulin G(IgG)N-glycan traits and ischemic stroke(IS) risk.Methods A nested case-control study was conducted in the China suboptimal health cohort study,which recruited 4,313 individuals in 2013–2014. Cases were identified as patients diagnosed with IS, and controls were 1:1 matched by age and sex with cases. Ig G N-glycans in baseline plasma samples were analyzed.Results A total of 99 IS cases and 99 controls were included, and 24 directly measured glycan peaks(GPs) were separated from Ig G N-glycans. In directly measured GPs, GP4, GP9, GP21, GP22, GP23, and GP24 were associated with the risk of IS in men after adjusting for age, waist and hip circumference,obesity, diabetes, hypertension, and dyslipidemia. Derived glycan traits representing decreased galactosylation and sialylation were associated with IS in men(FBG2S2/(FBG2 + FBG2S1 + FBG2S2): odds ratio(OR) = 0.92, 95% confidence interval(CI): 0.87–0.97;G1n: OR = 0.74, 95% CI: 0.63–0.87;G0n: OR =1.12, 95% CI: 1.03–1.22). However, these associations were not found among women.Conclusion This study validated that altered Ig G N-glycan traits were associated with incident IS in men, suggesting that sex discrepancies might exist in these associations.展开更多
β-Conglycinin is one of the major allergens existed in soybean.N-Glycans attached to theβ-conglycinin influenced the immunoreactivity and antigen presenting efficiency ofβ-conglycinin.In this study,we described a n...β-Conglycinin is one of the major allergens existed in soybean.N-Glycans attached to theβ-conglycinin influenced the immunoreactivity and antigen presenting efficiency ofβ-conglycinin.In this study,we described a new method used to release and collect the N-glycans fromβ-conglycinin,and the N-glycans existed in linear epitopes ofβ-conglycinin were identified.Glycopeptides hydrolyzed fromβ-conglycinin were purified by cotton hydrophilic chromatography.Trifluoromethylsulfonic acid was then used to release glycans from glycopeptides,and new glycopeptides containing one single N-acety1-D-glucosamine(G1 cNAc)moiety were then utilized for mass spectrometry.Five glycosylation sites(Asn-199,Asn-455,Asn-215,Asn-489 and Asn-326)and 22 kinds of glycopeptides were identified.It is noteworthy that the peptide VVN^(#)ATSNL(where^(#)represents for the glycosylation site)was analyzed to be both glycopeptide and linear epitope.Our results provided a new method for the N-glycoform analysis of food allergens,and laid a foundation for understanding the relationship between glyco sylation and food allergy.展开更多
BACKGROUND Pancreatic ductal adenocarcinoma(PDAC)has a poor prognosis,with a 5-year survival rate of less than 10%,owing to its late-stage diagnosis.Early detection of pancreatic cancer(PC)can significantly increase s...BACKGROUND Pancreatic ductal adenocarcinoma(PDAC)has a poor prognosis,with a 5-year survival rate of less than 10%,owing to its late-stage diagnosis.Early detection of pancreatic cancer(PC)can significantly increase survival rates.AIM To identify the serum biomarker signatures associated with early-stage PDAC by serum N-glycan analysis.METHODS An extensive patient cohort was used to determine a biomarker signature,in-cluding patients with PDAC that was well-defined at an early stage(stages I and II).The biomarker signature was derived from a case-control study using a case-cohort design consisting of 29 patients with stage I,22 with stage II,4 with stage III,16 with stage IV PDAC,and 88 controls.We used multiparametric analysis to identify early-stage PDAC N-glycan signatures and developed an N-glycan sig-nature-based diagnosis model called the“Glyco-model”.RESULTS The biomarker signature was created to discriminate samples derived from patients with PC from those of controls,with a receiver operating characteristic area under the curve of 0.86.In addition,the biomarker signature combined with cancer antigen 19-9 could discriminate patients with PDAC from controls,with a receiver operating characteristic area under the curve of 0.919.Glyco-model demonstrated favorable diagnostic performance in all stages of PC.The diagnostic sensitivity for stage I PDAC was 89.66%.Core Tip:This study employed a patient cohort to investigate the N-glycan signature of early-stage pancreatic cancer(PC).Serum N-glycans analysis was conducted to identify the serum biomarker signature associated with early-stage pancreatic ductal adenocarcinoma(PDAC),resulting in the identification of nine early-stage PDAC N-glycan signatures.Subsequently,utilizing these biosignatures,a diagnostic model named the“Glyco-model”was developed,demonstrating promising diagnostic performance across all stages of PC.The study revealed that the diagnostic sensitivity for stage I PDAC was determined to be 89.66%.Consequently,this diagnostic model exhibits potential as a prospective strategy for the early detection of PDAC.展开更多
The glycosylation of proteins is responsible for their structural and functional roles in many cellular activities.This work describes a strategy that combines an efficient release, labeling and liquid chromatography...The glycosylation of proteins is responsible for their structural and functional roles in many cellular activities.This work describes a strategy that combines an efficient release, labeling and liquid chromatography–mass spectral analysis with the use of a comprehensive database to analyze N-glycans. The analytical method described relies on a recently commercialized kit in which quick deglycosylation is followed by rapid labeling and cleanup of labeled glycans. This greatly improves the separation, mass spectrometry（MS） analysis and fluorescence detection of N-glycans. A hypothetical database, constructed using Glyc Resoft, provides all compositional possibilities of N-glycans based on the common sugar residues found in N-glycans. In the initial version this database contains ＆gt; 8,700 N-glycans, and is compatible with MS instrument software and expandable. N-glycans from four different well-studied glycoproteins were analyzed by this strategy. The results provided much more accurate and comprehensive data than that had been previously reported. This strategy was then used to analyze the N-glycans present on the membrane glycoproteins of gastric carcinoma cells with different degrees of differentiation. Accurate and comprehensive N-glycan data from those cells was obtained efficiently and their differences were compared corresponding to their differentiation states. Thus, the novel strategy developed greatly improves accuracy, efficiency and comprehensiveness of N-glycan analysis.展开更多
Asparagine （Asn/N）-Iinked glycans are important for protein folding, trafficking, and endoplasmic reticulum-associated degradation in eukaryotes. The maturation of glycoproteins involves the trimming of mannosyl res...Asparagine （Asn/N）-Iinked glycans are important for protein folding, trafficking, and endoplasmic reticulum-associated degradation in eukaryotes. The maturation of glycoproteins involves the trimming of mannosyl residues by mannosidases and addition of other sugar molecules to three-branched N-glycans in the Golgi. However, the biological importance of Golgi-mediated mannose trimming is not fully understood. Here, we show that abolishment of two functionally redundant mannosidases, MNS1 and MNS2, responsible for α-1,2-mannose trimming on the A and C branches of plant N-glycans lead to severe root growth inhibition under salt stress conditions in Arabidopsis. In contrast, mutants with defects in the biosynthesis of the oligosaccharide precursor displayed enhanced salt tolerance in the absence of mannose trimming. However, mutation in EBS3, which is required for the formation of the branched N-glycan precursor, suppressed the salt-sensitive phenotype of mnsl mns2 double mutant. Interestingly, we observed that cellulose biosynthesis was compromised in mnsl mns2 roots under high salinity. Consistently, abundance of a membrane anchored endo-13-1,4-endoglucanase （RSW2/KOR） that plays a key role in cellulose biosynthesis and its mutant variant rsw2-1 were modulated by α-1,2-mannose trimming under salt stress. Overexpression of RSW2 could partially rescue the salt-sensitive phenotype of mnsl mns2. Taken together, these results suggest that MNS1/2-mediated mannose trimming of N-glycans is crucial in modulating glycoprotein abundance to withstand salt stress in plants.展开更多
Plants possess some desirable characteristics to synthesize recombinant glycoproteins for pharma-ceutical application.However,the mammalian glycoproteins produced in plants are somewhat different from their natural co...Plants possess some desirable characteristics to synthesize recombinant glycoproteins for pharma-ceutical application.However,the mammalian glycoproteins produced in plants are somewhat different from their natural counterparts in terms of N-glycoforms.The immunogenicity of plant-specific glyco-epitopes is the major concern in human therapy.Here,the distribution of N-glycans in different growth phases of tobacco BY2 cells and their immunogenicity in mice were determined.It was ob-served that the percentage of β1,2-xylose and α1,3-fucose in proteins of growing cells increased and the corresponding protein extracts caused accelerated immune response in mice.Based on this observation,the recombinant erythropoietin in BY2 cells was expressed and characterized,and Western blot analysis showed that the recombinant erythropoietin contained a relatively small amount of plant-specific glyco-epitopes in the early phase of culture growth.This study may provide a simple but effective strategy for the production of therapeutic glycoproteins with human-like N-glycan structures in plant hosts to avoid a great allergenic risk.展开更多
Aim:This study explored the prognostic value of N-glycan biosynthesis(NGB)in lower-grade glioma(LGG)and aimed to develop a machine learning model for enhanced prognostic accuracy.Method:LGG patient transcriptome data ...Aim:This study explored the prognostic value of N-glycan biosynthesis(NGB)in lower-grade glioma(LGG)and aimed to develop a machine learning model for enhanced prognostic accuracy.Method:LGG patient transcriptome data were analyzed to identify NGB-related genes.Consensus clustering identified subgroups based on NGB expression.A prognostic NGB signature(pNGB)was developed using machine learning.The pNGB score's association with cell proliferation,inflammation,treatment response,tumor recurrence,and the immune microenvironment was also explored.Results:A 22-gene pNGB signature was identified,with MGAT1 and TUSC3 having the highest and lowest hazard ratios,respectively.Two distinct clusters(C1 and C2)with differential pNGB expression and survival outcomes were revealed.NGB pathway analysis indicated an overall poor prognosis,except for MGAT4C and TUSC3.The Enet-based survival model showed superior discriminatory power and reliability.The NGB risk score correlated with increased cell proliferation,inflammation,and altered immune landscape.Additionally,the score is linked to treatment response and tumor recurrence.Conclusion:This study highlights the critical role of NGB in LGG progression and proposes a pNGB-based model for prognosis.The NGB risk score shows promise as a prognostic biomarker and potential therapeutic target in LGG.展开更多
Background:Microvascular invasion(MVI)was a critical high-risk factor for postoperative recurrence and adverse prognosis in patients with hepatocellular carcinoma(HCC),and there were no reliable non-invasive pre-opera...Background:Microvascular invasion(MVI)was a critical high-risk factor for postoperative recurrence and adverse prognosis in patients with hepatocellular carcinoma(HCC),and there were no reliable non-invasive pre-operative diagnostic markers.The exosomal N-glycan profile was closely related to the invasion and immune escape of HCC.Therefore,this article investigated the expression of N-glycan profiles in serum exosomes of patients with HCC and its clinical significance for MVI.Methods:Serum samples from 210 patients with HCC were collected and randomly divided into modeling and validation cohorts.The abundances of N-glycans in serum exosomes with different MVI grades were determined.A diagnostic model for MVI in HCC based on N-glycosylation was constructed and the diagnostic value was analyzed.Results:In the modeling cohort,comparing groups M0 with M2,the area under the receiver operating characteristic(AUC)of the diagnostic model namely SUM(AUCSUM)was 0.861,the sensitivity of SUM was 92.68%,and the specificity of SUM was 79.41%,all of which were higher than the seven individual indexes(containing Peak 1,Peak 6,Peak 9,Peak 10,Peak 12,AFP,and PIVKA-II).In the comparison between the M1 and M2 groups,the AUCSUM was 0.749,the sensitivity of SUM was 79.07%,and the specificity of SUM was 76.60%,all of which were higher than the seven individual indexes.When comparing the M0 and M1 groups,the AUCSUM was 0.712,the sensitivity of SUM was 88.57%,and the specificity of SUM was 65.00%.The AUCSUM and sensitivity of SUM were higher than the seven individual indexes and the specificity of SUM was slightly lower than that of AFP(68.18%)but higher than other individual indexes.The results of the validation cohort were similar to those of the modeling cohort.Conclusion:The SUM model of serum exosomes can serve as an auxiliary diagnostic index for MVI staging in patients with HCC.展开更多
本研究目的是探讨血清N-聚糖模型在285例丙氨酸转移酶(alanine aminotransferase,ALT)水平正常(<40 U·L^(-1))的慢性乙型肝炎(慢性乙肝)患者中诊断显著肝纤维化和肝硬化的临床意义。入组患者均进行肝组织活检,并使用Ishak评分...本研究目的是探讨血清N-聚糖模型在285例丙氨酸转移酶(alanine aminotransferase,ALT)水平正常(<40 U·L^(-1))的慢性乙型肝炎(慢性乙肝)患者中诊断显著肝纤维化和肝硬化的临床意义。入组患者均进行肝组织活检,并使用Ishak评分系统评估患者肝组织纤维化程度。应用基于DNA测序仪的荧光糖电泳技术检测患者血清N-聚糖图谱,每例患者的血清样本中共鉴定出9个N-聚糖峰。利用机器学习算法,即随机森林(random forest,RF)构建更理想的血清N-聚糖模型,以诊断显著肝纤维化(≥F3)和肝硬化(≥F5),并比较血清N-聚糖模型和其他纤维化标志物的诊断效能。肝组织活检结果显示,有显著肝纤维化和肝硬化患者分别占63.86%(182/285)和16.49%(47/285),有显著炎症患者为4.91%(14/285)。血清N-聚糖RF-A模型具有很好的诊断显著肝纤维化(≥F3)的效能,其受试者工作特征曲线下面积(area under receiver operating characteristic curve,AUROC)为0.94,与肝活检的符合率为90.45%。在诊断肝硬化(≥F5)时,血清N-聚糖RF-B模型的AUROC为0.97,与肝组织活检的符合率为88.94%。血清N-聚糖模型(RF-A和RF-B)的诊断效能优于肝硬度值测量(liver stiffness measurement,LSM)、基于4因子的纤维化指数(fibrosis index based on the four factors,FIB-4)和天冬氨酸转氨酶与血小板比率指数(aspartate aminotransferase-to-platelet ratio index,APRI)。在ALT水平正常的慢性乙肝患者中,血清N-聚糖模型可作为诊断显著肝纤维化或肝硬化的潜在生物标志物。展开更多
基金Supported by Major Science and Technology Special Project of China Thirteenth Five-Year Plan,No.2018ZX10732401-003-015；Guangxi Key Laboratory for the Prevention and Control of Viral Hepatitis,No.GXCDCKL201901
文摘BACKGROUND Hepatitis B virus (HBV) infection is the primary cause of hepatitis with chronic HBV infection,which may develop into liver fibrosis,cirrhosis and hepatocellular carcinoma.Detection of early-stage fibrosis related to HBV infection is of great clinical significance to block the progression of liver lesion.Direct liver biopsy is regarded as the gold standard to detect and assess fibrosis;however,this method is invasive and prone to clinical sampling error.In order to address these issues,we attempted to find more convenient and effective serum markers for detecting HBV-induced early-stage liver fibrosis.AIM To investigate serum N-glycan profiling related to HBV-induced liver fibrosis and verify multiparameter diagnostic models related to serum N-glycan changes.METHODS N-glycan profiles from the sera of 432 HBV-infected patients with liver fibrosis were analyzed.Significant changed N-glycan levels (peaks)(P <0.05) in differentfibrosis stages were selected in the modeling group,and multiparameter diagnostic models were established based on changed N-glycan levels by logistic regression analysis.The receiver operating characteristic (ROC) curve analysis was performed to evaluate diagnostic efficacy of N-glycans models.These models were then compared with the aspartate aminotransferase to platelet ratio index (APRI),fibrosis index based on the four factors (FIB-4),glutamyltranspeptidase platelet albumin index (S index),GlycoCirrho-test,and GlycoFibro-test.Furthermore,we combined multiparameter diagnostic models with alanine aminotransferase (ALT) and platelet (PLT) tests and compared their diagnostic power.In addition,the diagnostic accuracy of N-glycan models was also verified in the validation group of patients.RESULTS Multiparameter diagnostic models constructed based on N-glycan peak 1,3,4and 8 could distinguish between different stages of liver fibrosis.The area under ROC curves (AUROCs) of Model A and Model B were 0.890 and 0.752,respectively differentiating fibrosis F0-F1 from F2-F4,and F0-F2 from F3-F4,and surpassing other serum panels.However,AUROC (0.747) in Model C used for the diagnosis of F4 from F0-F3 was lower than AUROC (0.795) in FIB-4.In combination with ALT and PLT,the multiparameter models showed better diagnostic power (AUROC=0.912,0.829,0.885,respectively) when compared with other models.In the validation group,the AUROCs of the three combined models (0.929,0.858,and 0.867,respectively) were still satisfactory.We also applied the combined models to distinguish adjacent fibrosis stages of 432patients (F0-F1/F2/F3/F4),and the AUROCs were 0.917,0.720 and 0.785.CONCLUSION Multiparameter models based on serum N-glycans are effective supplementary markers to distinguish between adjacent fibrosis stages of patients caused by HBV,especially in combination with ALT and PLT.
文摘With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships.In monoclonal antibodies,many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known.The importance of their function focuses analytical research efforts on the development of robust,accurate and fast methods to support drug development and quality control.Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however,it is not the only method for quantitative analysis of glycoform heterogeneity.In this study,ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies.While observing good comparability between the quantitative results generated,it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow according to application and the desired depth of data generated.
基金supported by the National Natural Science Foundation of China (Nos. 81672537 and 81470294)the National Science and Technology Major Project of China (No. 2018ZX10302205)Hundred-Talent Program of Shaanxi Province
文摘Sialylation is one of important glycosylation in human beings and plays an important role in cancer development. α2,3-Linked and α2,6-linked sialic acids are normally observed on the end of N-glycans and have different functions. Derivatization on sialic acid was designed to detect the different linkages by MALDI-TOF MS. In this study, a two-step derivatization by dimethylamine and ammonium hydroxide was improved to modify the sialic acid and made it easier to detect the different linkages of sialic acids on MALDI-TOF MS. Using this derivatization method, specific sialic acids linkages on N-glycans of protein samples such as fetuin and lactoferrin were detected. For complex cell samples, increased a2,3-linked and a2,6-linked sialic acids on bi-antennary and tri-antennary N-glycans were observed in A549 cells induced by hypoxia environment. Taken together, our two-step derivatization of sialic acids offers a simple and accurate way to detect specific linkages on N-glycans with MALDI-TOF mass spectrometer.
基金supported by grants from the National Natural Science Foundation of China[81673247]the National Natural Sciences Foundation of China(NSFC)-Australian National Health and Medical Research Council(NHMRC) Joint Research Project[NSFC 81561128020-NHMRC APP1112767]
文摘Nonalcoholic fatty liver disease（NAFLD） is a major public health issue worldwide. Immunoglobulin G（IgG） N-glycans are associated with risk factors for NAFLD, such as obesity and diabetes. A cross-sectional study involving 500 Han Chinese adults recruited from a community in Beijing was carried out to explore the association between IgG N-glycans and NAFLD. IgG N-glycosylation was significantly associated with NAFLD, with the disease showing a negative correlation with galactosylation（GP14, GP14n, and G2n）, positive correlation with fucosylation（FBG2n/G2n）, and positive correlation with bisecting N-acetylglucosamine（Glc NAc） [FBG2n/FG2n and FBG2n/（FG2n＋FBG2n）], after controlling age, gender, and prevalence of obesity, type 2 diabetes mellitus, hypertension, and hyperlipidemia. In other words, the present study showed a possible association between NAFLD and the loss of galactose and elevations of fucose and bisecting GlcNAc. Aberrant IgG glycosylation might therefore be a potential biomarker for the primary or secondary prevention of NAFLD.
基金supported by grants from the National Natural Science Foundation of China [No.81673247, 8187268281903401]。
文摘Objective This study prospectively investigates the association between immunoglobulin G(IgG)N-glycan traits and ischemic stroke(IS) risk.Methods A nested case-control study was conducted in the China suboptimal health cohort study,which recruited 4,313 individuals in 2013–2014. Cases were identified as patients diagnosed with IS, and controls were 1:1 matched by age and sex with cases. Ig G N-glycans in baseline plasma samples were analyzed.Results A total of 99 IS cases and 99 controls were included, and 24 directly measured glycan peaks(GPs) were separated from Ig G N-glycans. In directly measured GPs, GP4, GP9, GP21, GP22, GP23, and GP24 were associated with the risk of IS in men after adjusting for age, waist and hip circumference,obesity, diabetes, hypertension, and dyslipidemia. Derived glycan traits representing decreased galactosylation and sialylation were associated with IS in men(FBG2S2/(FBG2 + FBG2S1 + FBG2S2): odds ratio(OR) = 0.92, 95% confidence interval(CI): 0.87–0.97;G1n: OR = 0.74, 95% CI: 0.63–0.87;G0n: OR =1.12, 95% CI: 1.03–1.22). However, these associations were not found among women.Conclusion This study validated that altered Ig G N-glycan traits were associated with incident IS in men, suggesting that sex discrepancies might exist in these associations.
基金funded by National Natural Science Foundation of China(31870798,31972024)Shaanxi Province Innovation Capability Support Plan-Science and Technology Innovation Team(2020TD-044)+2 种基金Key Laboratory of Glycobiology and Glycoengineering of Xi’an(2019219514SYS010CG032)Natural Science Project of Shaanxi Provincial Department of Education(21JK0929)Science and Technology Resources Sharing Platform Project of Science and Technology Department in Shaanxi Province(2022PT-46)。
文摘β-Conglycinin is one of the major allergens existed in soybean.N-Glycans attached to theβ-conglycinin influenced the immunoreactivity and antigen presenting efficiency ofβ-conglycinin.In this study,we described a new method used to release and collect the N-glycans fromβ-conglycinin,and the N-glycans existed in linear epitopes ofβ-conglycinin were identified.Glycopeptides hydrolyzed fromβ-conglycinin were purified by cotton hydrophilic chromatography.Trifluoromethylsulfonic acid was then used to release glycans from glycopeptides,and new glycopeptides containing one single N-acety1-D-glucosamine(G1 cNAc)moiety were then utilized for mass spectrometry.Five glycosylation sites(Asn-199,Asn-455,Asn-215,Asn-489 and Asn-326)and 22 kinds of glycopeptides were identified.It is noteworthy that the peptide VVN^(#)ATSNL(where^(#)represents for the glycosylation site)was analyzed to be both glycopeptide and linear epitope.Our results provided a new method for the N-glycoform analysis of food allergens,and laid a foundation for understanding the relationship between glyco sylation and food allergy.
基金fundings for Clinical Trials from the Affiliated Drum Tower Hospital,Medical School of Nanjing University,No.2021-LCYJ-MS-11.
文摘BACKGROUND Pancreatic ductal adenocarcinoma(PDAC)has a poor prognosis,with a 5-year survival rate of less than 10%,owing to its late-stage diagnosis.Early detection of pancreatic cancer(PC)can significantly increase survival rates.AIM To identify the serum biomarker signatures associated with early-stage PDAC by serum N-glycan analysis.METHODS An extensive patient cohort was used to determine a biomarker signature,in-cluding patients with PDAC that was well-defined at an early stage(stages I and II).The biomarker signature was derived from a case-control study using a case-cohort design consisting of 29 patients with stage I,22 with stage II,4 with stage III,16 with stage IV PDAC,and 88 controls.We used multiparametric analysis to identify early-stage PDAC N-glycan signatures and developed an N-glycan sig-nature-based diagnosis model called the“Glyco-model”.RESULTS The biomarker signature was created to discriminate samples derived from patients with PC from those of controls,with a receiver operating characteristic area under the curve of 0.86.In addition,the biomarker signature combined with cancer antigen 19-9 could discriminate patients with PDAC from controls,with a receiver operating characteristic area under the curve of 0.919.Glyco-model demonstrated favorable diagnostic performance in all stages of PC.The diagnostic sensitivity for stage I PDAC was 89.66%.Core Tip:This study employed a patient cohort to investigate the N-glycan signature of early-stage pancreatic cancer(PC).Serum N-glycans analysis was conducted to identify the serum biomarker signature associated with early-stage pancreatic ductal adenocarcinoma(PDAC),resulting in the identification of nine early-stage PDAC N-glycan signatures.Subsequently,utilizing these biosignatures,a diagnostic model named the“Glyco-model”was developed,demonstrating promising diagnostic performance across all stages of PC.The study revealed that the diagnostic sensitivity for stage I PDAC was determined to be 89.66%.Consequently,this diagnostic model exhibits potential as a prospective strategy for the early detection of PDAC.
基金the National Natural Science Foundation of China (81473179 and 81673388)Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, YX13200111)the funding for Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-PsychoDiseases (BM2013003)
文摘The glycosylation of proteins is responsible for their structural and functional roles in many cellular activities.This work describes a strategy that combines an efficient release, labeling and liquid chromatography–mass spectral analysis with the use of a comprehensive database to analyze N-glycans. The analytical method described relies on a recently commercialized kit in which quick deglycosylation is followed by rapid labeling and cleanup of labeled glycans. This greatly improves the separation, mass spectrometry（MS） analysis and fluorescence detection of N-glycans. A hypothetical database, constructed using Glyc Resoft, provides all compositional possibilities of N-glycans based on the common sugar residues found in N-glycans. In the initial version this database contains ＆gt; 8,700 N-glycans, and is compatible with MS instrument software and expandable. N-glycans from four different well-studied glycoproteins were analyzed by this strategy. The results provided much more accurate and comprehensive data than that had been previously reported. This strategy was then used to analyze the N-glycans present on the membrane glycoproteins of gastric carcinoma cells with different degrees of differentiation. Accurate and comprehensive N-glycan data from those cells was obtained efficiently and their differences were compared corresponding to their differentiation states. Thus, the novel strategy developed greatly improves accuracy, efficiency and comprehensiveness of N-glycan analysis.
基金This work was partly supported by grants from the National Natural Science Foundation--Outstanding Youth Foundation of China （grant no. 31322008） and the National Basic Research Program of China （grant no. 2014CB542300） to Z.H.
文摘Asparagine （Asn/N）-Iinked glycans are important for protein folding, trafficking, and endoplasmic reticulum-associated degradation in eukaryotes. The maturation of glycoproteins involves the trimming of mannosyl residues by mannosidases and addition of other sugar molecules to three-branched N-glycans in the Golgi. However, the biological importance of Golgi-mediated mannose trimming is not fully understood. Here, we show that abolishment of two functionally redundant mannosidases, MNS1 and MNS2, responsible for α-1,2-mannose trimming on the A and C branches of plant N-glycans lead to severe root growth inhibition under salt stress conditions in Arabidopsis. In contrast, mutants with defects in the biosynthesis of the oligosaccharide precursor displayed enhanced salt tolerance in the absence of mannose trimming. However, mutation in EBS3, which is required for the formation of the branched N-glycan precursor, suppressed the salt-sensitive phenotype of mnsl mns2 double mutant. Interestingly, we observed that cellulose biosynthesis was compromised in mnsl mns2 roots under high salinity. Consistently, abundance of a membrane anchored endo-13-1,4-endoglucanase （RSW2/KOR） that plays a key role in cellulose biosynthesis and its mutant variant rsw2-1 were modulated by α-1,2-mannose trimming under salt stress. Overexpression of RSW2 could partially rescue the salt-sensitive phenotype of mnsl mns2. Taken together, these results suggest that MNS1/2-mediated mannose trimming of N-glycans is crucial in modulating glycoprotein abundance to withstand salt stress in plants.
基金Supported by the European Commission Sixth Framework Program (Grant No. SP22-CT-2004-511060)National Natural Science Foundation of China (Grant No. 30325030/30530400)
文摘Plants possess some desirable characteristics to synthesize recombinant glycoproteins for pharma-ceutical application.However,the mammalian glycoproteins produced in plants are somewhat different from their natural counterparts in terms of N-glycoforms.The immunogenicity of plant-specific glyco-epitopes is the major concern in human therapy.Here,the distribution of N-glycans in different growth phases of tobacco BY2 cells and their immunogenicity in mice were determined.It was ob-served that the percentage of β1,2-xylose and α1,3-fucose in proteins of growing cells increased and the corresponding protein extracts caused accelerated immune response in mice.Based on this observation,the recombinant erythropoietin in BY2 cells was expressed and characterized,and Western blot analysis showed that the recombinant erythropoietin contained a relatively small amount of plant-specific glyco-epitopes in the early phase of culture growth.This study may provide a simple but effective strategy for the production of therapeutic glycoproteins with human-like N-glycan structures in plant hosts to avoid a great allergenic risk.
基金supported by the General Project of Nanjing Medical Science and Technology Development(Grant No.YKK22142)the Youth Talent Project of Nanjing Brain Hospital(Grant No.23-25-2R7).
文摘Aim:This study explored the prognostic value of N-glycan biosynthesis(NGB)in lower-grade glioma(LGG)and aimed to develop a machine learning model for enhanced prognostic accuracy.Method:LGG patient transcriptome data were analyzed to identify NGB-related genes.Consensus clustering identified subgroups based on NGB expression.A prognostic NGB signature(pNGB)was developed using machine learning.The pNGB score's association with cell proliferation,inflammation,treatment response,tumor recurrence,and the immune microenvironment was also explored.Results:A 22-gene pNGB signature was identified,with MGAT1 and TUSC3 having the highest and lowest hazard ratios,respectively.Two distinct clusters(C1 and C2)with differential pNGB expression and survival outcomes were revealed.NGB pathway analysis indicated an overall poor prognosis,except for MGAT4C and TUSC3.The Enet-based survival model showed superior discriminatory power and reliability.The NGB risk score correlated with increased cell proliferation,inflammation,and altered immune landscape.Additionally,the score is linked to treatment response and tumor recurrence.Conclusion:This study highlights the critical role of NGB in LGG progression and proposes a pNGB-based model for prognosis.The NGB risk score shows promise as a prognostic biomarker and potential therapeutic target in LGG.
基金Fuzhou“14th Five-Year”Clinical Key Specialty,Grant/Award Number:20220203Natural Science Foundation of Fujian Province,Grant/Award Number:2020J011169。
文摘Background:Microvascular invasion(MVI)was a critical high-risk factor for postoperative recurrence and adverse prognosis in patients with hepatocellular carcinoma(HCC),and there were no reliable non-invasive pre-operative diagnostic markers.The exosomal N-glycan profile was closely related to the invasion and immune escape of HCC.Therefore,this article investigated the expression of N-glycan profiles in serum exosomes of patients with HCC and its clinical significance for MVI.Methods:Serum samples from 210 patients with HCC were collected and randomly divided into modeling and validation cohorts.The abundances of N-glycans in serum exosomes with different MVI grades were determined.A diagnostic model for MVI in HCC based on N-glycosylation was constructed and the diagnostic value was analyzed.Results:In the modeling cohort,comparing groups M0 with M2,the area under the receiver operating characteristic(AUC)of the diagnostic model namely SUM(AUCSUM)was 0.861,the sensitivity of SUM was 92.68%,and the specificity of SUM was 79.41%,all of which were higher than the seven individual indexes(containing Peak 1,Peak 6,Peak 9,Peak 10,Peak 12,AFP,and PIVKA-II).In the comparison between the M1 and M2 groups,the AUCSUM was 0.749,the sensitivity of SUM was 79.07%,and the specificity of SUM was 76.60%,all of which were higher than the seven individual indexes.When comparing the M0 and M1 groups,the AUCSUM was 0.712,the sensitivity of SUM was 88.57%,and the specificity of SUM was 65.00%.The AUCSUM and sensitivity of SUM were higher than the seven individual indexes and the specificity of SUM was slightly lower than that of AFP(68.18%)but higher than other individual indexes.The results of the validation cohort were similar to those of the modeling cohort.Conclusion:The SUM model of serum exosomes can serve as an auxiliary diagnostic index for MVI staging in patients with HCC.
基金supported by the Major Science and Technology Special Project of China Thirteenth Five-year Plan(2018ZX10732401-003-015)the National Science and Technology Major Project(2013ZX10002005 and 2017ZX10203202)。
文摘本研究目的是探讨血清N-聚糖模型在285例丙氨酸转移酶(alanine aminotransferase,ALT)水平正常(<40 U·L^(-1))的慢性乙型肝炎(慢性乙肝)患者中诊断显著肝纤维化和肝硬化的临床意义。入组患者均进行肝组织活检,并使用Ishak评分系统评估患者肝组织纤维化程度。应用基于DNA测序仪的荧光糖电泳技术检测患者血清N-聚糖图谱,每例患者的血清样本中共鉴定出9个N-聚糖峰。利用机器学习算法,即随机森林(random forest,RF)构建更理想的血清N-聚糖模型,以诊断显著肝纤维化(≥F3)和肝硬化(≥F5),并比较血清N-聚糖模型和其他纤维化标志物的诊断效能。肝组织活检结果显示,有显著肝纤维化和肝硬化患者分别占63.86%(182/285)和16.49%(47/285),有显著炎症患者为4.91%(14/285)。血清N-聚糖RF-A模型具有很好的诊断显著肝纤维化(≥F3)的效能,其受试者工作特征曲线下面积(area under receiver operating characteristic curve,AUROC)为0.94,与肝活检的符合率为90.45%。在诊断肝硬化(≥F5)时,血清N-聚糖RF-B模型的AUROC为0.97,与肝组织活检的符合率为88.94%。血清N-聚糖模型(RF-A和RF-B)的诊断效能优于肝硬度值测量(liver stiffness measurement,LSM)、基于4因子的纤维化指数(fibrosis index based on the four factors,FIB-4)和天冬氨酸转氨酶与血小板比率指数(aspartate aminotransferase-to-platelet ratio index,APRI)。在ALT水平正常的慢性乙肝患者中,血清N-聚糖模型可作为诊断显著肝纤维化或肝硬化的潜在生物标志物。
