Full-length core sequence dependent complex-type glycosylation of hepatitis C virus E2 glycoprotein

AIM: To study HCV polyprotein processing is important for the understanding of the natural history of HCV and the design of vaccines against HCV. The purpose of this study is to investigate the affection of context sequences on hepatitis C virus (HCV) E2 processing. METHODS: HCV genes of different lengths were expressed and compared in vaccinia virus/T7 system with homologous patient serum S94 and mouse anti-serum M( E2116) raised against E.coli -derived E2 peptide, respectively.Deglycosylation analysis and GNA ( Galanthus nivalus ) lectin binding assay were performed to study the post-translational processing of the expressed products. RESULTS: E2 glycoproteins with different molecular weights (-75 kDa and -60 kDa) were detected using S94 and M( E2116), respectively. Deglycosylation analysis showed that this difference was mainly due to different glycosylation. Endo H resistance and its failure to bind to GNA lectin demonstrated that the higher molecular weight form (75 kDa) of E2 was complex-type glycosylated, which was readily recognized by homologous patient serum S94. Expression of complex-type glycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected in constructs encoding full-length core sequences. CONCLUSION: The upstream conserved full-length core coding sequence was required for the production of E2 glycoproteins carrying complex-type N-glycans which reacted strongly with homologous patient serum and therefore possibly represented more mature forms of E2. As complex-type N-glycans indicated modification by Golgi enzymes, the results suggest that the presence of full-length core might be critical for E1/E2 complex to leave ER. Our data may contribute to a better understanding of the processing of HCV structural proteins as well as HCV morphogenesis.

Serum Mac-2 binding protein glycosylation isomer level predicts hepatocellular carcinoma development in E-negative chronic hepatitis B patients

BACKGROUND Liver cirrhosis is a major risk factor for hepatocellular carcinoma(HCC)development in chronic hepatitis B(CHB). Serum Mac-2 binding protein glycosylation isomer(M2 BPGi) is a novel serological marker for fibrosis. The role of M2 BPGi in prediction of HCC is unknown.AIM To examine the role of serum M2 BPGi in predicting HCC development in hepatitis B e antigen(HBeAg)-negative patients.METHODS Treatment-naive CHB patients with documented spontaneous HBeAg seroconversion were recruited. Serum M2 BPGi was measured at baseline(within3 years from HBeAg seroconversion), at 5 years and 10 years after HBeAg seroconversion and expressed as cut-off index(COI). Multivariate cox regression was performed to identify predictors for HCC development. ROC analysis was used to determine the cut-off value of M2 BPGi.RESULTS Among 207 patients(57% male, median age at HBeAg seroconversion 40 years old) with median follow-up of 13.1(11.8-15.5) years, the cumulative incidence of HCC at 15 years was 7%. Median M2 BPGi levels were significantly higher in patients with HCC compared to those without HCC(baseline: 1.39 COI vs 0.38 COI, P < 0.001; 5-year: 1.45 COI vs 0.47 COI, P < 0.001; 10-year: 1.20 COI vs 0.55 COI, P = 0.001). Multivariate analysis revealed age at HBeAg seroconversion[odds ratio(OR) = 1.196, 95% confidence interval(CI): 1.034-1.382, P = 0.016] and baseline M2 BPGi(OR = 4.666, 95%CI: 1.296-16.802, P = 0.018) were significant factors predictive of HCC. Using a cut-off value of 0.68 COI, baseline M2 BPGi yielded AUROC of 0.883 with 91.7% sensitivity and 80.8% specificity.CONCLUSION High serum M2 BPGi within 3 years after HBeAg seroconversion was a strong predictor for subsequent HCC development in treatment-naive HBeAg-negative CHB patients.

Usefulness of Mac-2 binding protein glycosylation isomer in noninvasive probing liver disease in the Vietnamese population

BACKGROUND Early diagnosis is critical for successful intervention before liver disease progresses to cirrhosis and hepatocellular carcinoma.AIM To examine a novel biomarker for probing early liver disease quickly using an automated immunology system.METHODS This was a cross-sectional study.140 patients at various stages of liver disease were randomly selected.The cohort consisted of patients who were treatment naïve and currently undergoing therapy.We included patients with diverse liver disease etiologies.Mac-2 binding protein glycosylation isomer(M2BPGi)levels in addition to different clinical parameters,co-morbidities and transient elastography results were collected and compared.RESULTS M2BPGi levels were significantly correlated with transient elastography for liver fibrosis staging across all disease etiologies.Statistically significant differences were observed in patients with F0-1;F2 and>F3 liver fibrosis.Further examination showed that M2BPGi levels were two-fold higher in F4 than F3 hepatitis C(HCV)patients.M2BPGi was observed to be etiology-specific and HCV patients had higher mean M2BPGi levels.We also observed significant correlations with aspartate aminotransferase to platelet ratio index and fibrosis-4 index as well as HBV DNA levels.Mean M2BPGi levels for HBV patients with a viral load lower than 2000 IU/mL was 1.75-fold lower than those with a viral load greater than 2000 IU/mL.CONCLUSION M2BPGi was observed to be a good indicator of early liver disease in patients with different etiologies.Our results provide reference cut-offs for different causes of liver disease and demonstrated the utility of this marker for early disease monitoring.This is useful for remote regions in developing countries.

Pre-sarcopenia and Mac-2 binding protein glycosylation isomer as predictors of recurrence and prognosis of early-stage hepatocellular carcinoma

BACKGROUND The Mac-2 binding protein glycosylation isomer(M2BPGi),a fibrosis marker in various liver diseases,is reportedly a prognostic marker in patients with hepatocellular carcinoma(HCC)who underwent hepatectomy.AIM To evaluate whether the M2BPGi value,M2BP,and pre-sarcopenia before radiofrequency ablation(RFA)could be useful recurrence and prognostic markers in patients with early-stage HCC.METHODS In total,160 patients with early-stage primary HCC treated with RFA were separately analyzed as hepatitis C virus(HCV)-positive and HCV-negative.Factors contributing to recurrence and liver-related death,including M2BP,M2BPGi,and skeletal muscle mass index,were statistically analyzed.Eighty-three patients were HCV-positive and 77 were HCV-negative.RESULTS In HCV-positive patients,only des-γ-carboxy-prothrombin≥23 mAU/mL was a significant poor prognostic factor affecting survival after RFA.In HCV-negative patients,M2BPGi≥1.86 cutoff index was significantly associated with tumor recurrence,while M2BP was not.M2BPGi≥1.86 cutoff index(hazard ratio,4.89;95%confidence interval:1.97-12.18;P<0.001)and pre-sarcopenia(hazard ratio,3.34,95%confidence interval:1.19-9.37;P=0.022)were independent significant poor prognostic factors in HCV-negative patients.CONCLUSION In HCV-negative patients with primary HCC treated with RFA,lower M2BPGi contributed to a lower tumor recurrence rate and longer survival period.Pre-sarcopenia contributed to the poor prognosis independently in HCV-negative patients.These factors might be useful recurrence and prognostic markers for early-stage primary HCC.

Preterminal protein,the achilles heel of adenoviridae:Implications for adenoviral infections

BACKGROUND Adenoviruses pose a serious health risk particularly in the absence of any clinically approved treatment.As adenoviral infections are quite frequent and recent outbreaks manifest more virulent variant strains,the need to develop an effective treatment remains a priority.The adenoviral protein,preterminal protein(pTP),is one of the key common products of the viral lifecycle as it is necessary to initiate viral replication and hence the infection process.This makes pTP a potential chemotherapeutic target in the search for and development of an effective treatment for adenoviral induced infections.Here we report,for the first time,that glycosylation of pTP in situ prevents binding to ssDNA in vitro.AIM To explore whether specific structural tailoring of the adenoviral protein pTP,imparts the potential to scupper the viral replication process.METHODS All chemicals used were of reagent grade.Overexpression of pTP was achieved using the‘BAC to BAC’expression system.The presence and relative concentration of the protein was determined throughout the incubation period by the Bradford assay.The pTP was identified by MALDI-TOFF and sodium dodecyl sulphate polyacrylamide gel electrophoresis.For the removal of the aminosugar,a deglycosylase enzyme kit from PROZYME was used.Purification of cloned pTP(6xHis)was done with a ssDNA cellulose column followed by a Ni-NTA column.His-tags were excised with the Tobacco etch virus protease.Protein fractionation was performed with a fraction collector coupled to a UV detector(280 nm)from Pharmacia.RESULTS The pTP overexpressed in insect cells(Spodoptera frugiperda)(>96 hours),is unable to bind to ssDNA in vitro.Treatment of this unbound protein with a deglycosidase enzyme that is specific for the removal of truncated unsubstituted O-linked Galβ(1-3)GalNAc-α1 disaccharides bound to Thr or Ser in a glycoprotein,restores binding to ssDNA.Data is presented as a linegraph for both the glycosylated and the deglycosylated proteins.Each point represents the mean of triplicate experiments(from different batches).Means and standard deviation were calculated and plotted on a line graph(with error bars).CONCLUSION The finding that glycosylation of cloned pTP in situ prevents binding to ssDNA in vitro could aid in the development of an effective treatment of adenoviral infections and/or as an adjunct to complement other antiadenoviral chemotherapeutic strategies.

红曲发酵物抑制蛋白质非酶糖基化作用研究

蛋白质非酶糖基化反应导致晚期糖基化终产物(advanced glycation end products,AGEs)的形成。研究表明,AGEs的积累与糖尿病及其并发症的发生和发展密切相关。通过构建牛血清白蛋白-果糖体外模拟体系,考察红曲发酵物(Monascus fermented products,MFP)对蛋白糖基化产物及其结构的影响。结果表明:MFP对蛋白糖基化早期、中期和晚期产物的生成均具有抑制作用,尤其对晚期产物即AGEs的抑制率最高(87.80%);当MFP质量浓度由0.05 mg/mL增加至0.55 mg/mL时,糖基化蛋白中羰基质量摩尔浓度由32.50μmol/g降低至28.10μmol/g,游离巯基质量摩尔浓度由2.60μmol/g增加至3.50μmol/g,蛋白氧化产物二酪氨酸和犬尿氨酸的最大生成抑制率分别为66.84%和59.37%,表明MFP可通过保护巯基、抑制蛋白氧化等途径发挥AGEs抑制作用;与天然蛋白相比,糖基化蛋白中α-螺旋结构含量减少,添加MFP后,蛋白中α-螺旋结构的含量均增加,说明MFP可借助稳定蛋白天然构象来抑制AGEs的生成;糖基化蛋白中淀粉样β-交联结构含量较高,添加MFP后其含量显著减少,且随着MFP添加量的增加,淀粉样β-交联结构生成抑制率逐渐增大,表明MFP可抑制蛋白质交联从而阻断AGEs的形成。研究旨在为红曲源AGEs抑制剂的开发和利用提供数据支持和理论基础。

Recombinant Expression and Purification of Mouse Nectin-like 4 Glycoprotein in 293ET Cell Line

Objective To screen the transient and stable cell lines with high production of Nectin-like 4(Necl-4)protein.Methods First,c DNA sequences encoding the extracellular domain of Necls were cloned into the modified vector p APtag at the N terminus of alkaline phosphatase(AP)for fusion expression.Next,293ET cells stably expressed Necls-AP fusion protein and secreted it into the culture medium which were detected by the AP activity assay and Western blot analysis.Then,by adding N-glycosylation processing inhibitor kifunensine into the medium,complex glycan was inhibited to generate.The residual glycan of purified protein was removed by endoglycosidase H.Finally,AP protein was removed by using human rhinovirus protease and size exclusion chromatography.The concentration of purified Necl-4 protein was monitored by measuring the absorbance at280 nm and analyzed by SDS-PAGE.Results The transient and stable cell lines with high production of Necl-4 protein were screened by the color reaction with the AP-tag in the recombinant vector.The soluble and active form of purified Necl-4 protein was obtained after deglycosylation of native N-glycan protein with an expression level of 4 mg/L culture and purity of 95%.Conclusions By using modified AP mammalian protein expression system,we can easily screen the high productive stable cell lines by using AP activity assay.By adding mannosidase inhibitor kifunensine into the medium and cutting purified protein by using endoglycosidase H,we can obtain deglycosylated Necl-4 protein in milligram quantities.Our method might throw a light on the expression and purification of glycoprotein for structural and functional studies.

Recombinant human zona pellucida proteins ZP1, ZP2 and ZP3 co-expressed in a human cell line

Aim: To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests. Methods: The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed. Results: RhZP2 and rhZP3 were secreted into the culture medium, whereas rhZPl was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZPl was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed. Conclusion: RhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZPl from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.

Aberrant post-translational protein modifications in the pathogenesis of alcohol-induced liver injury

It is likely that the majority of proteins will undergo post-translational modification, be it enzymatic or non-enzymatic. These modified protein(s) regulate activity, localization and interaction with other cellular molecules thereby maintaining cellular hemostasis. Alcohol exposure significantly alters several of these post-translational modifications leading to impairments of many essential physiological processes. Here, we present new insights into novel modifications following ethanol exposure and their role in the initiation and progression of liver injury. This critical review condenses the proceedings of a symposium at the European Society for the Biomedical Research on Alcoholism Meeting held September 12-15, 2015, in Valencia, Spain.

INTERACTION BETWEEN THE SURFACE GLYCOSYLATED POLYPROPYLENE MEMBRANE AND LECTIN

A glycopolymer bearing glucose residues was tethered onto the surface of polypropylene microporous membrane by UV-induced graft polymerization ofα-allyl glucoside.Concanavalin A (Con A),a glucose recognizing lectin,could be specifically adsorbed to the membrane surface.On the other hand,the membrane surface showed no recognition ability to another lectin peanut agglutinin.Moreover,the recognition complex between the glycosylated membrane surface and Con A could be inhibited by glucose and mannose solution.T...

The Comparison of Genetic Variation in the Envelope Protein Between Various Immunodeficiency Viruses and Equine Infectious Anemia Virus

The envelope protein （Env） of lentiviruses such as HIV, SIV, FIV and EIAV is larger than that of other retroviruses. The Chinese EIAV attenuated vaccine is based on Env and has helped to successfully control this virus, demonstrating that envelope is crucial for vaccine. We compared Env variation of the four kinds of lentiviruses. Phylogenetic analysis showed that the evolutionary relationship of Env between HIV and SIV was the closest and they appeared to descend from a common ancestor, and the relationship of HIV and EIAV was the furthest. EIAV had the shortest Env length and the least number of potential N-linked glyeosylation sites （PNGS） as well as glyeosylation density compared to various immunodefieiency viruses. However, HIV had the longest Env length and the most PNGS. Moreover, the alignment of HIV and SIV showed that PNGS were primarily distributed within extraeellular membrane protein gp120 rather than transmembrane gp41. It implies that the size difference among these viruses is associated with a lentivirus specific function and also the diversity of env. There arc low levels of modification of glycosylation sites of Env and selection of optimal protective epitopes might be useful for development of an effective vaccine against HIV/AIDS.

限制性酶解结合糖基化改性对大豆分离蛋白乳化性质的影响

目的:开发基于大豆分离蛋白(soybean protein isolate,SPI)的新型乳化剂。方法:采用限制性酶解结合糖基化处理对SPI进行结构修饰,研究协同改性对SPI乳化特性的影响。结果:SPI水解物(soybean protein isolate hydrolysate,SPIH)中的相对分子质量较大组分(F30)的乳化性最佳,且糖基化反应4 h的F30-葡聚糖轭合物乳化稳定性相对最好。相较于SPI,SPIH与F30,F30-葡聚糖轭合物稳定的乳液表现出最低的初始平均粒径,并且具有最佳的贮藏稳定性。当pH接近SPI等电点或体系处于高盐浓度时,所有乳液均出现不稳定聚集现象。与SPI相比,SPIH和F30稳定乳液的聚集程度更高,而F30-葡聚糖轭合物由于共价结合的葡聚糖提供了额外的空间位阻和亲水性,使得轭合物稳定的乳液能够耐受离子强度和温度的变化,在不利环境条件下表现出更高的抵抗力。结论:限制性酶解结合糖基化改性是开发SPI基乳化配料的潜在可靠途径。

血清D-D、GSP、HbA1c水平对妊娠糖尿病的临床意义

目的 探究检测血清D-二聚体(D-D)、糖化血清蛋白(GSP)、糖化血红蛋白(HbA1c)表达水平在妊娠糖尿病中的临床意义。方法 选取2020年1月至2022年12月于郑州大学第二附属医院就诊117例妊娠糖尿病患者为研究组,同期选择117例体检健康孕妇作为对照组进行研究,比较两组血清D-D、GSP、HbA1c水平及稳态模型胰岛素抵抗指数(HOMA-IR),采用Pearson分析入院时血清D-D、GSP、HbA1c水平与HOMA-IR相关性,受试者工作特征(ROC)曲线分析血清D-D、GSP联合HbA1c水平检测对妊娠糖尿病病情程度的评估价值。结果 与对照组相比,研究组血清D-D、GSP、HbA1c水平及HOMA-IR均升高(P<0.05)。不同病情程度孕妇的血清D-D、GSP、HbA1c水平及HOMA-IR指数比较:重度>中度>轻度(P<0.05)。血清D-D、GSP、HbA1c水平与HOMA-IR均呈正相关(r=0.671、0.715、0.696,P<0.05)。产检时血清D-D、GSP、HbA1c水平联合诊断中度、重度妊娠糖尿病的曲线下面积(AUC)分别为0.859、0.873,最佳敏感度分别为95.64%、96.30%,特异度分别为76.12%、78.26%。结论 血清D-D、GSP、HbA1c水平联合检测可为临床评估妊娠糖尿病病情程度提供可靠参考依据。

库尔勒垦区健康孕妇妊娠24~28周GSP、HbA1c参考区间的建立

目的 建立库尔勒垦区健康孕妇妊娠24~28周糖化血清蛋白(glycosylated serum protein,GSP)、糖化血红蛋白(glycosylated hemoglobin,HbA1c)的参考区间,为临床筛查妊娠糖尿病(gestational diabetes mellitus,GDM)提供依据。方法 选取2019年1月至2021年2月于新疆生产建设兵团第二师库尔勒医院产检并分娩的795名孕妇为研究对象,根据24~28周时口服葡萄糖耐量试验结果将孕妇分为健康研究组(n=627)和GDM组(n=168),健康研究组孕妇按分娩年龄又分为适龄组(年龄<35岁,n=491)和高龄组(年龄≥35岁,n=136);选取同期131名健康未孕女性为对照组。检测各组研究对象的GSP、HbA1c水平,采用百分位数法(P2.5~P97.5)建立参考区间。采用受试者操作特征曲线(receiver operating characteristic curve,ROC曲线)评价GSP、HbA1c筛查GDM的效能。结果 适龄组、高龄组和对照组研究对象的GSP、HbA1c比较差异均有统计学意义(P<0.05)。适龄组和高龄组孕妇的GSP、HbA1c比较差异均无统计学意义(P>0.05)。健康孕妇妊娠24~28周时的GSP参考区间为188.15~257.75μmol/L,HbA1c参考区间为4.50%~5.70%。ROC曲线结果显示,GSP、HbA1c单独筛查GDM的曲线下面积分别为0.803、0.708。结论 库尔勒垦区健康孕妇妊娠24~28周的GSP、HbA1c参考区间分别为188.15~257.75μmol/L、4.5%~5.7%,且GSP、HbA1c对筛查GDM人群有较大应用价值。

糖基化结合单宁酸改性对熔球态蛋清蛋白乳化特性的影响

[目的]开发基于熔球态蛋清蛋白(molten-globule state egg white protein,MGEWP)稳定乳液的修饰方法。[方法]采用pH诱导法使蛋清蛋白部分变性生成MGEWP,并研究D-木糖与单宁酸改性对MGEWP乳化特性的协同增强作用。[结果]随着单宁酸浓度的增加,D-木糖糖基化修饰的MGEWP(X-MGEWP)浊度与褐变程度显著增加(P<0.05),且单宁酸导致X-MGEWP发生解折叠行为,暴露更多游离巯基与活性基团,并显著提高X-MGEWP的分子柔度、表观黏度、弹性模量和黏性模量(P<0.05)。在极端pH处理下,MGEWP的有效反应基团钝化,与D-木糖的美拉德反应被抑制,导致X-MGEWP的乳化特性与未经糖基化处理的MGEWP无显著差异(P>0.05),而添加单宁酸可显著增强X-MGEWP的乳化活性(P<0.05),且与单宁酸表现出剂量依赖性。[结论]D-木糖/单宁酸的协同改性是提高MGEWP乳化性的潜在方法之一。

湿法糖基化改性大豆分离蛋白在低致敏千页豆腐中的应用

为寻求高效的糖基化改性条件和良好的应用途径,本文以葡萄糖、木糖两种单糖为糖基供体,探究反应时间、反应温度、蛋白与糖比例、蛋白浓度对大豆分离蛋白湿法糖基化反应的影响,分析大豆分离蛋白-葡萄糖复合物(SPI-葡萄糖)、大豆分离蛋白-木糖复合物(SPI-木糖)致敏性的变化,并将SPI-葡萄糖、SPI-木糖应用于低致敏千页豆腐生产。结果表明,木糖相较于葡萄糖更易与大豆分离蛋白发生糖基化反应,但也更容易有类黑素产生。在蛋白浓度为25 mg/mL时,糖基化反应程度最高,并且糖基化反应程度与反应温度、反应时间、糖添加量呈正相关。SDS-PAGE和傅里叶变换红外光谱分析结果表明糖基化改性使大豆分离蛋白和糖分子发生了共价结合,SPI的自由氨基减少。糖基化反应程度越高,SPI致敏性越低,SPI-木糖接枝物致敏性降低程度最高可达50%,以其为原料经谷氨酰胺转氨酶(TG酶)交联所制作的千页豆腐有良好的弹性和咀嚼性。

芦丁调控miR-155和HMGB1/RAGE通路对糖尿病视网膜病变大鼠炎性反应的影响

目的:探讨芦丁对糖尿病视网膜病变大鼠炎性反应的影响及其机制。方法:采用腹腔注射链脲佐菌素的方法制备糖尿病大鼠模型,将造模成功的大鼠随机分为模型组(0.9%氯化钠溶液灌胃)和芦丁低、中、高剂量组[分别以50、100、150 mg/(kg·d)芦丁灌胃],并取正常大鼠作为对照组(0.9%氯化钠溶液灌胃),每组20只,持续给药12周后,比较各组大鼠视网膜组织中伊凡斯兰渗透量、神经节细胞凋亡和微小RNA(miR)-155、高迁移率族蛋白1(HMGB1)、晚期糖基化终产物受体(RAGE)mRNA表达水平,以及炎症因子水平。结果:对照组、模型组和芦丁低、中、高剂量组中伊凡斯兰渗透量分别为(1.61±0.31)μg/g、(12.84±1.24)μg/g、(9.85±0.62)μg/g、(6.80±0.53)μg/g、(2.87±0.51)μg/g,神经节细胞凋亡指数分别为(1.09±0.45)%、(30.96±2.50)%、(24.65±2.48)%、(18.61±1.41)%、(8.36±0.54)%,miR-155表达水平分别为(0.38±0.03)、(1.92±0.15)、(1.55±0.12)、(1.09±0.10)、(0.55±0.05),HMGB1 mRNA表达水平分别为(0.26±0.03)、(0.77±0.05)、(0.68±0.04)、(0.57±0.04)、(0.34±0.03),RAGE mRNA表达水平分别为(0.18±0.02)、(0.67±0.04)、(0.58±0.03)、(0.45±0.03)、(0.23±0.02)。模型组和对照组比较,伊凡斯兰渗透量、凋亡指数和miR-155、HMGB1、RAGE mRNA水平均升高(均P<0.05);芦丁低、中、高剂量组中上述指标呈剂量依赖性降低(均P<0.05)。模型组、对照组与芦丁低、中、高剂量组,芦丁各组的炎症因子水平低于模型组(均P<0.05)。结论:芦丁可保护糖尿病大鼠视网膜损伤,延缓视网膜炎症效应,其作用机制可能与抑制miR-155和HMGB1/RAGE通路有关。

鸡蛋清蛋白质糖肽的结构鉴定

鸡蛋清中主要蛋白质均为糖蛋白,经胃肠消化后将在消化道内产生大量糖肽,这些糖肽的结构需要被鉴定和明确。本研究采用胰蛋白酶将鸡蛋清消化,然后从水解物中通过亲水相互作用层析富集N-糖肽、以Retain AX层析结合AminoLink树脂吸附富集O-糖肽,运用液相色谱-质谱联用技术对糖肽结构进行解析,并借助生物信息学分析探究蛋清糖蛋白对肠道的潜在作用。经质谱解析,共鉴定到来自于49种N-糖蛋白的1720个N-糖肽结构,以及来自29种O-糖蛋白的565个O-糖肽结构。其中,卵黏蛋白糖肽结构最为多样,共有490个N-糖肽结构和216个O-糖肽结构。京都基因与基因组百科全书通路富集分析发现,含唾液酸修饰的N-糖蛋白主要涉及白细胞介素-17信号通路和铁死亡,而含唾液酸修饰的O-糖蛋白主要涉及胆固醇代谢、维生素消化吸收、低氧诱导因子-1信号通路和肾素-血管紧张素系统。本研究系统解析了鸡蛋清蛋白质的糖肽结构,为后续探究蛋清糖肽对肠道的潜在作用提供了重要的结构信息。

促糖基化对香菇蛋白质消化特性及消化产物抗氧化活性的影响

为探讨促糖基化对香菇(Lentinula edodes)蛋白质消化特性及消化产物抗氧化活性的影响,采用碱溶酸沉法提取子实体蛋白质,并制备糖基化和谷氨酰胺转氨酶(transglutaminase, TGase)促糖基化蛋白质,通过模拟胃肠消化分别测定蛋白质组(C)、糖基化蛋白质组(G)和促糖基化蛋白质组(TG)的蛋白质消化率、水解度、蛋白质消化产物中的游离氨基酸和多肽含量,以及蛋白质消化产物的DPPH自由基清除率、超氧阴离子自由基清除率、羟基自由基清除率和总抗氧化能力。结果表明:与C组相比,G组在模拟消化90、120、180、240、270min时和TG组在模拟消化90、120、210、240、270min时消化率显著降低,且G与TG组间无显著性差异;在模拟消化60 min时G组水解度显著增加,在90~360 min时显著降低,TG组在模拟消化30、60、150~240、300 min时显著增加,且在模拟消化60~360 min时显著高于G组;G组在模拟消化60、210~360 min时和TG组在模拟消化0、60~360 min时游离氨基酸含量显著增加,且在模拟消化0、120、300~360 min时TG组显著高于G组;在模拟消化所有时间点,G组和TG组多肽含量均显著降低,且在模拟消化0、300~360 min时TG组显著高于G组;在模拟消化所有时间点,G组DPPH自由基清除率均显著增加,且TG组在模拟消化0、150、210~360 min时显著增加,但在模拟消化120、150、180、330、360 min时TG组显著低于G组;G组在模拟消化240~360 min时和TG组在模拟消化330、360 min时超氧阴离子清除率显著增加,且在模拟消化270min时,TG组显著低于G组;在模拟消化所有时间点,G组和TG组羟基自由基清除率和总抗氧化能力均显著增加;但在模拟消化0~180、240 min时TG组羟基自由基清除率显著高于G组,在模拟消化0~180、240~360 min时TG组总抗氧化能力显著低于G组。

基于分子库策略的蛋白质O⁃糖基化研究

糖以共价键的形式连接到蛋白质氨基酸的侧链上可以形成糖基化。糖基化在许多情况下能够显著增加蛋白质结构、性质和功能的多样性。特定的糖基化模式还能够赋予蛋白质和酶特异的性能,同时异常的糖基化模式也可能导致疾病。因此,全面深入地了解蛋白质糖基化的作用,无论是对于基础研究还是应用研究,都有重要的意义。然而,由于难以获得适于研究的样品,这方面的进展一直异常缓慢。近年来,研究人员逐渐开始探索使用一个基于糖基化异形体分子库(glycoform library)的策略来改变这一现状。这一策略以使用合成的方法制备的具有高纯度且在结构上存在系统差异的一系列分子作为研究对象,通过对它们结构、性质和功能的比较,相对快速而准确地获得蛋白质糖基化的作用和作用机制,进而更好地提高糖基化在改善蛋白质和酶的性能方面的应用水平。本综述旨在通过对分子库策略在O-糖基化研究方向上的进展,以大致按照时间顺序进行的总结、概述和简单讨论,实现对现有不多的实例的研究方法、每个实例取得的研究成果的系统展示,从而能够帮助研究人员更清晰地了解该策略目前的发展状况和不足,帮助他们在今后更好地利用该策略进行蛋白质糖基化的研究和应用,更好地提高该策略的使用深度和广度。