Lassa virus(LASV)belongs to the Mammarenavirus genus(family Arenaviridae)and causes severe hemorrhagic fever in humans.The glycoprotein complex(GPC)contains eleven N-linked glycans that play essential roles in GPC fun...Lassa virus(LASV)belongs to the Mammarenavirus genus(family Arenaviridae)and causes severe hemorrhagic fever in humans.The glycoprotein complex(GPC)contains eleven N-linked glycans that play essential roles in GPC functionalities such as cleavage,transport,receptor recognition,epitope shielding,and immune response.We used three mutagenesis strategies(asparagine to glutamine,asparagine to alanine,and serine/tyrosine to alanine mutants)to abolish individual glycan chain on GPC and found that all the three strategies led to cleavage inefficiency on the 2nd(N89),5th(N119),or 8th(N365)glycosylation motif.To evaluate N to Q mutagenesis for further research,it was found that deletion of the 2nd(N89Q)or 8th(N365Q)glycan completely inhibited the transduction efficiency of pseudotyped particles.We further investigated the role of individual glycan on GPC-mediated immune response by DNA immunization of mice.Deletion of the individual 1st(N79Q),3rd(N99Q),5th(N119Q),or 6th(N167Q)glycan significantly enhanced the proportion of effector CD4+cells,whereas deletion of the 1st(N79Q),2nd(N89Q),3rd(N99Q),4th(N109Q),5th(N119Q),6th(N167Q),or 9th(N373Q)glycan enhanced the proportion of CD8+effector T cells.Deletion of specific glycan improves the Th1-type immune response,and abolishment of glycan on GPC generally increases the antibody titer to the glycan-deficient GPC.However,the antibodies from either the mutant or WT GPC-immunized mice show little neutralization effect on wild-type LASV.The glycan residues on GPC provide an immune shield for the virus,and thus represent a target for the design and development of a vaccine.展开更多
BACKGROUND Mass spectrometry-based proteomics and glycomics reveal post-translational modifications providing significant biological insights beyond the scope of genomic sequencing.AIM To characterize the N-linked gly...BACKGROUND Mass spectrometry-based proteomics and glycomics reveal post-translational modifications providing significant biological insights beyond the scope of genomic sequencing.AIM To characterize the N-linked glycoproteomic profile in esophageal squamous cell carcinoma(ESCC)via two complementary approaches.METHODS Using tandem multilectin affinity chromatography for enrichment of N-linked glycoproteins,we performed N-linked glycoproteomic profiling in ESCC tissues by two-dimensional gel electrophoresis(2-DE)-based and isobaric tags for relative and absolute quantification(iTRAQ)labeling-based mass spectrometry quantitation in parallel,followed by validation of candidate glycoprotein biomarkers by Western blot.RESULTS 2-DE-based and iTRAQ labeling-based quantitation identified 24 and 402 differentially expressed N-linked glycoproteins,respectively,with 15 in common,demonstrating the outperformance of iTRAQ labeling-based quantitation over 2-DE and complementarity of these two approaches.Proteomaps showed the distinct compositions of functional categories between proteins and glycoproteins with differential expression associated with ESCC.Western blot analysis validated the up-regulation of total procathepsin D and high-mannose procathepsin D,and the downregulation of total haptoglobin,high-mannose clusterin,and GlcNAc/sialic acid-containing fraction of 14-3-3ζ in ESCC tissues.The serum levels of glycosylated fractions of clusterin,prolinearginine-rich end leucine-rich repeat protein,and haptoglobin in patients with ESCC were remarkably higher than those in healthy controls.CONCLUSION Our study provides insights into the aberrant N-linked glycoproteome associated with ESCC,which will be a valuable resource for future investigations.展开更多
The asparagine-X-serine/threonine （NXS/T） motif, where X is any amino acid except proline, is the consensus motif for N-linked glycosylation. Significant numbers of high-resolution crystal structures of glycosylated...The asparagine-X-serine/threonine （NXS/T） motif, where X is any amino acid except proline, is the consensus motif for N-linked glycosylation. Significant numbers of high-resolution crystal structures of glycosylated proteins allow us to carry out structural analysis of the N-linked glycosylation sites （NGS）. Our analysis shows that there is enough structural information from diverse glycoproteins to allow the development of rules which can be used to predict NGS. A Python-based tool was developed to investigate asparagines implicated in N-glycosylation in five species： Homo sapiens, Mus musculus, Drosophila melanogaster, Arabidopsis thaliana and Saccharo- myees cerevisiae. Our analysis shows that 78 % of all asparagines of NXS/T motif involved in N-gly- cosylation are localized in the loop/turn conformation in the human proteome. Similar distribution was revealed for all the other species examined. Comparative analysis of the occurrence of NXS/T motifs not known to be glycosylated and their reverse sequence （S/TXN） shows a similar distribu- tion across the secondary structural elements, indicating that the NXS/T motif in itself is not bio- logically relevant. Based on our analysis, we have defined rules to determine NGS. Using machine learning methods based on these rules we can predict with 93% accuracy if a particular site will be glycosylated. If structural information is not available the tool uses structural prediction results resulting in 74% accuracy. The tool was used to identify glycosylation sites in 108 human proteins with structures and 2247 proteins without structures that have acquired NXS/T site/s due to non-synonymous variation. The tool, Structure Feature Analysis Tool （SFAT）, is freely available to the public at http：//hive.biochemistry.gwu.edu/tools/sfat.展开更多
目的O-GlcNAc糖基化(O-linked beta-N-acetylglucosamine,O-GlcNAc)和Yes相关蛋白1(yes-associated protein 1,YAP1)是否会发生糖基化、在结直肠癌中的表达情况及发挥作用的机制都尚未得到充分阐明。本研究旨在借助癌症基因组图谱(the c...目的O-GlcNAc糖基化(O-linked beta-N-acetylglucosamine,O-GlcNAc)和Yes相关蛋白1(yes-associated protein 1,YAP1)是否会发生糖基化、在结直肠癌中的表达情况及发挥作用的机制都尚未得到充分阐明。本研究旨在借助癌症基因组图谱(the cancer genome atlas,TCGA)数据库、组织标本和细胞系探究O-GlcNAc糖基化和YAP1糖基化在结直肠中所发挥的作用。方法采用免疫组化法检测O-GlcNAc和OGT蛋白(O-linked-N-acetylglucosamine transferase,OGT)在结直肠组织和癌旁组织中的表达情况;运用qPCR检测结直肠组织和癌旁组织中OGT mRNA表达水平;借助在线分析TCGA数据库,了解OGT的表达情况及其对预后产生的影响;通过Western Blot检测OSMI-1对O-GlcNAc糖基化的抑制作用;采用CCK-8和Transwell检测,在抑制O-GlcNAc糖基化后,观察肠癌细胞增殖、侵袭及迁移能力的变化;借助数据库分析YAP1的表达情况及其O-GlcNAc糖基化位点,并经质谱分析确定YAP1是否发生O-GlcNAc糖基化修饰;运用Co-IP和免疫荧光实验验证YAP1和OGT之间的结合关系;采用qPCR和免疫荧光明确抑制O-GlcNAc糖基化后YAP1表达位置的改变以及下游效应基因表达的改变情况。结果研究发现,OGT蛋白和mRNA在结直肠组织中的表达水平明显升高,但对患者的预后并无明显影响;OSMI-1能够抑制肠癌细胞的O-GlcNAc糖基化,并且抑制细胞增殖、侵袭与迁移能力;YAP1可与OGT结合并发生O-GlcNAc糖基化,当抑制O-GlcNAc糖基化后,YAP1入核数量减少,转录活性降低。结论在结直肠癌中,O-GlcNAc糖基化水平呈现升高趋势,OGT和YAP1结合,使YAP1发生O-GlcNAc糖基化修饰,促进其转录活性。展开更多
基金This work was supported by the National Key Research and Development Program of China(2018YFA0507204)the National Natural Sciences Foundation of China(31670165)+1 种基金Wuhan National Biosafety Laboratory,Chinese Academy of Sciences Advanced Customer Cultivation Project(2019ACCP-MS03)the Open Research Fund Program of the State Key Laboratory of Virology of China(2018IOV001).Author Contributions。
文摘Lassa virus(LASV)belongs to the Mammarenavirus genus(family Arenaviridae)and causes severe hemorrhagic fever in humans.The glycoprotein complex(GPC)contains eleven N-linked glycans that play essential roles in GPC functionalities such as cleavage,transport,receptor recognition,epitope shielding,and immune response.We used three mutagenesis strategies(asparagine to glutamine,asparagine to alanine,and serine/tyrosine to alanine mutants)to abolish individual glycan chain on GPC and found that all the three strategies led to cleavage inefficiency on the 2nd(N89),5th(N119),or 8th(N365)glycosylation motif.To evaluate N to Q mutagenesis for further research,it was found that deletion of the 2nd(N89Q)or 8th(N365Q)glycan completely inhibited the transduction efficiency of pseudotyped particles.We further investigated the role of individual glycan on GPC-mediated immune response by DNA immunization of mice.Deletion of the individual 1st(N79Q),3rd(N99Q),5th(N119Q),or 6th(N167Q)glycan significantly enhanced the proportion of effector CD4+cells,whereas deletion of the 1st(N79Q),2nd(N89Q),3rd(N99Q),4th(N109Q),5th(N119Q),6th(N167Q),or 9th(N373Q)glycan enhanced the proportion of CD8+effector T cells.Deletion of specific glycan improves the Th1-type immune response,and abolishment of glycan on GPC generally increases the antibody titer to the glycan-deficient GPC.However,the antibodies from either the mutant or WT GPC-immunized mice show little neutralization effect on wild-type LASV.The glycan residues on GPC provide an immune shield for the virus,and thus represent a target for the design and development of a vaccine.
基金Supported by National Natural Science Foundation of China,No.81072039 and No.81872037.
文摘BACKGROUND Mass spectrometry-based proteomics and glycomics reveal post-translational modifications providing significant biological insights beyond the scope of genomic sequencing.AIM To characterize the N-linked glycoproteomic profile in esophageal squamous cell carcinoma(ESCC)via two complementary approaches.METHODS Using tandem multilectin affinity chromatography for enrichment of N-linked glycoproteins,we performed N-linked glycoproteomic profiling in ESCC tissues by two-dimensional gel electrophoresis(2-DE)-based and isobaric tags for relative and absolute quantification(iTRAQ)labeling-based mass spectrometry quantitation in parallel,followed by validation of candidate glycoprotein biomarkers by Western blot.RESULTS 2-DE-based and iTRAQ labeling-based quantitation identified 24 and 402 differentially expressed N-linked glycoproteins,respectively,with 15 in common,demonstrating the outperformance of iTRAQ labeling-based quantitation over 2-DE and complementarity of these two approaches.Proteomaps showed the distinct compositions of functional categories between proteins and glycoproteins with differential expression associated with ESCC.Western blot analysis validated the up-regulation of total procathepsin D and high-mannose procathepsin D,and the downregulation of total haptoglobin,high-mannose clusterin,and GlcNAc/sialic acid-containing fraction of 14-3-3ζ in ESCC tissues.The serum levels of glycosylated fractions of clusterin,prolinearginine-rich end leucine-rich repeat protein,and haptoglobin in patients with ESCC were remarkably higher than those in healthy controls.CONCLUSION Our study provides insights into the aberrant N-linked glycoproteome associated with ESCC,which will be a valuable resource for future investigations.
基金Support for this work came from the George Washington University funds to RM.RG's participation is supported by RO1 CA135069 and U01 CA168926supported in part by an appointment to the Research Participation Program at the Center for Biologics Evaluation and Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration
文摘The asparagine-X-serine/threonine （NXS/T） motif, where X is any amino acid except proline, is the consensus motif for N-linked glycosylation. Significant numbers of high-resolution crystal structures of glycosylated proteins allow us to carry out structural analysis of the N-linked glycosylation sites （NGS）. Our analysis shows that there is enough structural information from diverse glycoproteins to allow the development of rules which can be used to predict NGS. A Python-based tool was developed to investigate asparagines implicated in N-glycosylation in five species： Homo sapiens, Mus musculus, Drosophila melanogaster, Arabidopsis thaliana and Saccharo- myees cerevisiae. Our analysis shows that 78 % of all asparagines of NXS/T motif involved in N-gly- cosylation are localized in the loop/turn conformation in the human proteome. Similar distribution was revealed for all the other species examined. Comparative analysis of the occurrence of NXS/T motifs not known to be glycosylated and their reverse sequence （S/TXN） shows a similar distribu- tion across the secondary structural elements, indicating that the NXS/T motif in itself is not bio- logically relevant. Based on our analysis, we have defined rules to determine NGS. Using machine learning methods based on these rules we can predict with 93% accuracy if a particular site will be glycosylated. If structural information is not available the tool uses structural prediction results resulting in 74% accuracy. The tool was used to identify glycosylation sites in 108 human proteins with structures and 2247 proteins without structures that have acquired NXS/T site/s due to non-synonymous variation. The tool, Structure Feature Analysis Tool （SFAT）, is freely available to the public at http：//hive.biochemistry.gwu.edu/tools/sfat.
文摘目的O-GlcNAc糖基化(O-linked beta-N-acetylglucosamine,O-GlcNAc)和Yes相关蛋白1(yes-associated protein 1,YAP1)是否会发生糖基化、在结直肠癌中的表达情况及发挥作用的机制都尚未得到充分阐明。本研究旨在借助癌症基因组图谱(the cancer genome atlas,TCGA)数据库、组织标本和细胞系探究O-GlcNAc糖基化和YAP1糖基化在结直肠中所发挥的作用。方法采用免疫组化法检测O-GlcNAc和OGT蛋白(O-linked-N-acetylglucosamine transferase,OGT)在结直肠组织和癌旁组织中的表达情况;运用qPCR检测结直肠组织和癌旁组织中OGT mRNA表达水平;借助在线分析TCGA数据库,了解OGT的表达情况及其对预后产生的影响;通过Western Blot检测OSMI-1对O-GlcNAc糖基化的抑制作用;采用CCK-8和Transwell检测,在抑制O-GlcNAc糖基化后,观察肠癌细胞增殖、侵袭及迁移能力的变化;借助数据库分析YAP1的表达情况及其O-GlcNAc糖基化位点,并经质谱分析确定YAP1是否发生O-GlcNAc糖基化修饰;运用Co-IP和免疫荧光实验验证YAP1和OGT之间的结合关系;采用qPCR和免疫荧光明确抑制O-GlcNAc糖基化后YAP1表达位置的改变以及下游效应基因表达的改变情况。结果研究发现,OGT蛋白和mRNA在结直肠组织中的表达水平明显升高,但对患者的预后并无明显影响;OSMI-1能够抑制肠癌细胞的O-GlcNAc糖基化,并且抑制细胞增殖、侵袭与迁移能力;YAP1可与OGT结合并发生O-GlcNAc糖基化,当抑制O-GlcNAc糖基化后,YAP1入核数量减少,转录活性降低。结论在结直肠癌中,O-GlcNAc糖基化水平呈现升高趋势,OGT和YAP1结合,使YAP1发生O-GlcNAc糖基化修饰,促进其转录活性。
