Liraglutide(Lira),a glucagon-like peptide-1(GLP-1)receptor agonist approved for diabetes and obesity,has shown significant potential in treating metabolic dysfunction-associated steatotic liver disease(MASLD).However,...Liraglutide(Lira),a glucagon-like peptide-1(GLP-1)receptor agonist approved for diabetes and obesity,has shown significant potential in treating metabolic dysfunction-associated steatotic liver disease(MASLD).However,its systematic molecular regulation and mechanisms remain underexplored.In this study,a mouse model of MASLD was developed using a high-fat diet(HFD),followed by Lira administration.Proteomics and glycoproteomics were analyzed using label-free liquid chromatography-tandem mass spectrometry(LC-MS/MS),while potential molecular target analysis was conducted via quantitative real-time polymerase chain reaction(qPCR)and Western blotting.Our results revealed that Lira treatment significantly reduced liver weight and serum markers,including alanine aminotransferase(ALT)and others,with glycosylation changes playing a more significant role than overall protein expression.The glycoproteome identified 255 independent glycosylation sites,emphasizing the impact of Lira on amino acid,carbohydrate metabolism,and ferroptosis.Simultaneously,proteomic analysis highlighted its effects on lipid metabolism and fibrosis pathways.21 signature molecules,including 7 proteins and 14 N-glycosylation sites(N-glycosites),were identified as potential targets.A Lira hydrogel formulation(Lira@fibrin(Fib)Gel)was developed to extend drug dosing intervals,offering enhanced therapeutic efficacy in managing chronic metabolic diseases.Our study demonstrated the importance of glycosylation regulation in the therapeutic effects of Lira on MASLD,identifying potential molecular targets and advancing its clinical application for MASLD treatment.展开更多
Aberrations in protein glycosylation and polysaccharides play a pivotal role in pancreatic tumorigenesis, influencing cancer progression, metastasis, immunoresponse and chemoresistance. Abnormal expression in sugar mo...Aberrations in protein glycosylation and polysaccharides play a pivotal role in pancreatic tumorigenesis, influencing cancer progression, metastasis, immunoresponse and chemoresistance. Abnormal expression in sugar moieties can impact the function of various glycoproteins, including mucins, surface receptors, adhesive proteins, proteoglycans, as well as their effectors and binding ligands, resulting in an increase in pancreatic cancer invasiveness and a cancerfavored microenvironment. Recent advance in glycoproteomics, glycomics and other chemical biology techniques have been employed to better understand the complex mechanism of glycosylation events and how they orchestrate molecular activities in genomics, proteomics and metabolomics implicated in pancreatic adenocarcinoma. A variety of strategies have been demonstrated targeting protein glycosylation and polysaccharides for diagnostic and therapeutic development.展开更多
In order to establish the novel high throughput,high efficiency and low cost technological platform for the research of N-glycoproteomics,to resolve the significance of characteristic expression profile of glycoprotei...In order to establish the novel high throughput,high efficiency and low cost technological platform for the research of N-glycoproteomics,to resolve the significance of characteristic expression profile of glycoprotein and to find the proteins with biological functional importance,the glycoproteins with high-mannose core and the two antennary types were purified and enriched by the Con A affinity chromatography.Con A affinity protein expression profiles of normal human liver tissue were gener-ated by using SDS-PAGE,two-dimensional electrophoresis(2-DE)followed by fast fluorescence stain-ing based on multiplexed proteomics(MP)technology.301 visible protein spots on the gel were de-tected and 85 of glycoproteins were further successfully identified via peptide mass fingerprinting(PMF)by a matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS/MS)and annotated to IPI databases.Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes.The glycosylation sites were predicted with NetNGlyc 1.0 and NetOGlyc 3.1 software,meanwhile they were classified according to the geneontology methods.The construction of Con A affinity glycoprotein database of normal human liver tissue would contribute to the subsequent research.展开更多
The first corona-pandemic,coronavirus disease 2019(COVID-19)caused a huge health crisis and incalculable damage worldwide.Knowledge of how to cure the disease is urgently needed.Emerging immune escaping mutants of the...The first corona-pandemic,coronavirus disease 2019(COVID-19)caused a huge health crisis and incalculable damage worldwide.Knowledge of how to cure the disease is urgently needed.Emerging immune escaping mutants of the virus suggested that it may be potentially persistent in human society as a regular health threat as the flu virus.Therefore,it is imperative to identify appropriate biomarkers to indicate pathological and physiological states,and more importantly,clinic outcomes.Proteins are the performers of life functions,and their abundance and modification status can directly reflect the immune status.Protein glycosylation serves a great impact in modulating protein function.The use of both unmodified and glycosylated proteins as biomarkers has also been proved feasible in the studies of SARS,Zika virus,influenza,etc.In recent years,mass spectrometry-based glycoproteomics,as well as proteomics approaches,advanced significantly due to the evolution of mass spectrometry.We focus on the current development of the mass spectrometry-based strategy for COVID-19 biomarkers’investigation.Potential application of glycoproteomics approaches and challenges in biomarkers identification are also discussed.展开更多
Identification evaluation and result dissemination are essential components in mass spectrometry-based proteomics analysis.The visualization of fragment ions in mass spectrum provides strong evidence for peptide ident...Identification evaluation and result dissemination are essential components in mass spectrometry-based proteomics analysis.The visualization of fragment ions in mass spectrum provides strong evidence for peptide identification and modification localization.Here,we present an easy-to-use tool,named GP-Plotter,for ion annotation of tandem mass spectra and corresponding image output.Identification result files of common searching tools in the community and user-customized files are supported as input of GP-Plotter.Multiple display modes and parameter customization can be achieved in GP-Plotter to present annotated spectra of interest.Different image formats,especially vector graphic formats,are available for image generation which is favorable for data publication.Notably,GP-Plotter is also well-suited for the visualization and evaluation of glycopeptide spectrum assignments with comprehensive annotation of glycan fragment ions.With a user-friendly graphical interface,GP-Plotter is expected to be a universal visualization tool for the community.GP-Plotter has been implemented in the latest version of Glyco-Decipher(v1.0.4)and the standalone GP-Plotter software is also freely available at https://github.com/DICP-1809.展开更多
Glycoproteins are complex biological macromolecules composed of proteins and glycans,playing a crucial role in various biological processes and being closely related to human health and diseases.However,the intricate ...Glycoproteins are complex biological macromolecules composed of proteins and glycans,playing a crucial role in various biological processes and being closely related to human health and diseases.However,the intricate structure and function of glycoproteins gives them with a vast capacity for information,which cannot be simply deduced from the genetic code(Di Marco et al.2023).展开更多
Pancreatic cancer is among the most malignant cancers,and thus early intervention is the key to better survival outcomes.However,no methods have been derived that can reliably identify early precursors of development ...Pancreatic cancer is among the most malignant cancers,and thus early intervention is the key to better survival outcomes.However,no methods have been derived that can reliably identify early precursors of development into malignancy.Therefore,it is urgent to discover early molecular changes during pancreatic tumorigenesis.As aberrant glycosylation is closely associated with cancer progression,numerous efforts have been made to mine glycosylation changes as biomarkers for diagnosis;however,detailed glycoproteomic information,especially site-specific N-glycosylation changes in pancreatic cancer with and without drug treatment,needs to be further explored.Herein,we used comprehensive solid-phase chemoenzymatic glycoproteomics to analyze glycans,glycosites,and intact glycopeptides in pancreatic cancer cells and patient sera.The profiling of N-glycans in cancer cells revealed an increase in the secreted glycoproteins from the primary tumor of MIA PaCa-2 cells,whereas human sera,which contain many secreted glycoproteins,had significant changes of glycans at their specific glycosites.These results indicated the potential role for tumor-specific glycosylation as disease biomarkers.We also found that AMG-510,a small molecule inhibitor against Kirsten rat sarcoma viral oncogene homolog(KRAS)G12C mutation,profoundly reduced the glycosylation level in MIA PaCa-2 cells,suggesting that KRAS plays a role in the cellular glycosylation process,and thus glycosylation inhibition contributes to the anti-tumor effect of AMG-510.展开更多
Adoptive cell therapies(ACTs)have achieved remarkable clinical success in treating cancers;however,their broader application is greatly impeded by high cost and restricted antigen specificity.Recently,engineering the ...Adoptive cell therapies(ACTs)have achieved remarkable clinical success in treating cancers;however,their broader application is greatly impeded by high cost and restricted antigen specificity.Recently,engineering the glycocalyx has provided a convenient transgene-free means to design ACTs with highavidity glycan ligands to target CD22,offering a new avenue for B lymphoma immunotherapy.In this work,we perform a comparative analysis of the molecular profiles involved in metabolic or chemoenzymatic glycocalyx engineering and explore their multiplexing capability.The glycoproteomic results revealed content-dependent customization of the natural killer(NK)-92MI glycocalyx.Compared with metabolic engineering,exogenous chemoenzymatic engineering has comparable or even superior ligand-loading efficiency,with some immune synapse components modified to facilitate their spatial recognition against target cells.Next,we tested the orthogonal creation of ligands on NK-92MI cells by further engineering a2,3-sialylated N-acetyllactosamine moieties to produce selectin ligands that are essential for better in vivo eradication of mouse xenograft B lymphoma.Finally,we demonstrate that analogous engineering of CD19-targeted chimeric antigen receptor T(CAR-T)cells to produce CD19/CD22 bitargeted therapy can enhance antigen targeting and tumor cell killing,offering an alternative cost-efficient agent for treating cancer relapse with decreased levels of CD19 antigens.These findings establish a mechanistic foundation for glycocalyx engineering and support the rational design of nextgeneration ACTs against B lymphoma.展开更多
To understand the implications of protein glycosylation for clinical diagnostics and biopharmaceuticals,innovative glycoproteomic technologies are required.Recently,significant advances have been made in such technolo...To understand the implications of protein glycosylation for clinical diagnostics and biopharmaceuticals,innovative glycoproteomic technologies are required.Recently,significant advances have been made in such technologies,particularly in the area of structure-focused N-glyco-proteomic analyses.Mass spectrometric analysis of intact N-glycopeptides using stepped collision fragmentation along with glycan oxonium ion profiling now makes it possible to reliably discriminate between different N-glycan structures.Still,current N-glycoproteomic approaches have weaknesses that must be overcome,namely①the handling of incorrect identifications,②the identification of rare and modified N-glycans,and③insufficient glycoproteomic coverage,especially in complex samples.To address these shortcomings,we have established an innovative N-glycoproteomic workflow that aims to provide comprehensive site-specific and structural N-glycoproteomic data on human blood plasma(HBP)glycoproteins.The workflow features protein depletion plus a fractionation strategy and the use of high-resolution mass spectrometry with stepped collision fragmentation.Furthermore,by including a new decision tree procedure developed for data validation,we can significantly improve the description of N-glycan micro-heterogeneity.Our advanced data analysis workflow allows the reliable differentiation of ambiguous N-glycan structures such as antenna versus core fucosylation,as well as modified and rare N-glycans such as sulfated and glucuronidated ones.With this workflow,we were able to achieve the detection of HBP glycoproteins with reported concentrations within the ng mL^(-1)level.A total of 1929 N-glycopeptides and 942 N-glycosites derived from 805 human middle-to low-abundant glycoproteins were identified.Overall,the presented workflow holds great potential to improve our understanding of protein glycosylation and to foster the discovery of blood plasma biomarkers.展开更多
Protein glycosylation is a critical post-translational modification that influences protein folding,localization,stability,and functional interactions by attaching glycans to specific sites.This process is crucial for...Protein glycosylation is a critical post-translational modification that influences protein folding,localization,stability,and functional interactions by attaching glycans to specific sites.This process is crucial for biological functions of glycoproteins,and aberrant glycosylation can lead to genetic disorders,immune system issues,and multi-organ pathologies.Recent advancements in glycoproteomic technologies have made the study of protein glycosylation a key focus for understanding the pathogenesis of kidney diseases.This review provides a comprehensive overview of protein glycosylation mechanisms,its biological roles,molecular pathways,and significant functions in renal physiology and pathology.It specifically highlights the dynamic changes and regulatory networks associated with aberrant glycosylation in kidney diseases such as immunoglobulin A nephropathy,diabetic kidney disease,autosomal dominant polycystic kidney disease,renal cell carcinoma,and acute kidney injury.It also evaluates the clinical applications of related technologies and biomarkers.Additionally,it discusses the challenges in developing glycosylation-targeted therapeutic strategies.Future research should focus on clarifying cell-specific glycosylation regulatory networks in the kidney,integrating glycobiology with multi-omics approaches,and improving precision diagnostics and treatment for kidney diseases.展开更多
Chicken egg white contains hundreds of proteins that are widely used in the food,biological and pharmaceutical industries.Glycosylation provides additional structural diversity to the already specialized proteins.The ...Chicken egg white contains hundreds of proteins that are widely used in the food,biological and pharmaceutical industries.Glycosylation provides additional structural diversity to the already specialized proteins.The array of glycans on protein surfaces and different glycosylation sites provide sophisticated structures essentials for the multiple functions that glycoproteins assume.Different types of analysis have been performed to determine N-glycosylation in chicken egg white.In different studies,up to 19 N-glycoproteins have been characterized.However,regarding O-glycosylation,there is insufficient knowledge of their structures and abundances.In fact,only ovomucin,a major component of chicken egg white,has been described as bearing O-glycans that consist of 2-6 sugar residues carrying sialic acid and/or sulfate groups.In the present work,the use of a reductive β-elimination reaction followed by a HPAEC-PAD analysis,showed released oligosaccharides suggesting the presence of different O-glycoproteins.Getting deeper into chicken egg white O-glycoproteomics,a nanoHPLC-ESI-Orbitrap-HCD analysis was performed and two different software tools were employed.Under these conditions,at least seven different O-glycosylated proteins were described.In addition,O-glycosylation of isolated ovalbumin was characterized for the first time and in a BEMAD analysis of the isolated glycoprotein,three O-glycosites were detected.Taking into account that glycosylation has been involved in the structure and properties of chicken egg white proteins such as allergenicity,antibacterial action and embryo protection,attention must be paid to explore new properties of this heterogeneous modification in relation to both the food and biopharmaceutical industries.展开更多
The structure of N-glycans on specific proteins can regulate innate and adaptive immunity via sensing environmental signals.Meanwhile,the structural diversity of N-glycans poses analytical challenges that limit the ex...The structure of N-glycans on specific proteins can regulate innate and adaptive immunity via sensing environmental signals.Meanwhile,the structural diversity of N-glycans poses analytical challenges that limit the exploration of specific glycosylation functions.In this work,we used THP-1-derived macrophages as examples to show the vast potential of a N-glycan structural interpretation tool StrucGP in N-glycoproteomic analysis.The intact glycopeptides of macrophages were enriched and analyzed using mass spectrometry(MS)-based glycoproteomic approaches,followed by the large-scale mapping of site-specific glycan structures via StrucGP.Results revealed that bisected GlcNAc,core fucosylated,and sialylated glycans(e.g.,HexNAc4Hex5Fuc1Neu5Ac1,N4H5F1S1)were increased in M1 and M2 macrophages,especially in the latter.The findings indicated that these structures may be closely related to macrophage polarization.In addition,a high level of glycosylated PD-L1 was observed in M1 macrophages,and the LacNAc moiety was detected at Asn-192 and Asn-200 of PD-L1,and Asn-200 contained Lewis epitopes.The precision structural interpretation of site-specific glycans and subsequent intervention of target glycoproteins and related glycosyltransferases are of great value for the development of new diagnostic and therapeutic approaches for different diseases.展开更多
基金supported by the Young Scientists Fund of the National Natural Science Foundation of China(Grant No.:82204513)the Natural Science Foundation of Sichuan Province,China(Grant No.:2023NSFSC1673)+1 种基金the Innovation Guidance Foundation of the Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province,China(Grant No.:SCU2023D005)the Scientific Research Staring Foundation of Sichuan University,China(Grant No.:YJ202165).
文摘Liraglutide(Lira),a glucagon-like peptide-1(GLP-1)receptor agonist approved for diabetes and obesity,has shown significant potential in treating metabolic dysfunction-associated steatotic liver disease(MASLD).However,its systematic molecular regulation and mechanisms remain underexplored.In this study,a mouse model of MASLD was developed using a high-fat diet(HFD),followed by Lira administration.Proteomics and glycoproteomics were analyzed using label-free liquid chromatography-tandem mass spectrometry(LC-MS/MS),while potential molecular target analysis was conducted via quantitative real-time polymerase chain reaction(qPCR)and Western blotting.Our results revealed that Lira treatment significantly reduced liver weight and serum markers,including alanine aminotransferase(ALT)and others,with glycosylation changes playing a more significant role than overall protein expression.The glycoproteome identified 255 independent glycosylation sites,emphasizing the impact of Lira on amino acid,carbohydrate metabolism,and ferroptosis.Simultaneously,proteomic analysis highlighted its effects on lipid metabolism and fibrosis pathways.21 signature molecules,including 7 proteins and 14 N-glycosylation sites(N-glycosites),were identified as potential targets.A Lira hydrogel formulation(Lira@fibrin(Fib)Gel)was developed to extend drug dosing intervals,offering enhanced therapeutic efficacy in managing chronic metabolic diseases.Our study demonstrated the importance of glycosylation regulation in the therapeutic effects of Lira on MASLD,identifying potential molecular targets and advancing its clinical application for MASLD treatment.
文摘Aberrations in protein glycosylation and polysaccharides play a pivotal role in pancreatic tumorigenesis, influencing cancer progression, metastasis, immunoresponse and chemoresistance. Abnormal expression in sugar moieties can impact the function of various glycoproteins, including mucins, surface receptors, adhesive proteins, proteoglycans, as well as their effectors and binding ligands, resulting in an increase in pancreatic cancer invasiveness and a cancerfavored microenvironment. Recent advance in glycoproteomics, glycomics and other chemical biology techniques have been employed to better understand the complex mechanism of glycosylation events and how they orchestrate molecular activities in genomics, proteomics and metabolomics implicated in pancreatic adenocarcinoma. A variety of strategies have been demonstrated targeting protein glycosylation and polysaccharides for diagnostic and therapeutic development.
基金the National Basic Research Program of China(973)(Grant No.2004CB520802)
文摘In order to establish the novel high throughput,high efficiency and low cost technological platform for the research of N-glycoproteomics,to resolve the significance of characteristic expression profile of glycoprotein and to find the proteins with biological functional importance,the glycoproteins with high-mannose core and the two antennary types were purified and enriched by the Con A affinity chromatography.Con A affinity protein expression profiles of normal human liver tissue were gener-ated by using SDS-PAGE,two-dimensional electrophoresis(2-DE)followed by fast fluorescence stain-ing based on multiplexed proteomics(MP)technology.301 visible protein spots on the gel were de-tected and 85 of glycoproteins were further successfully identified via peptide mass fingerprinting(PMF)by a matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS/MS)and annotated to IPI databases.Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes.The glycosylation sites were predicted with NetNGlyc 1.0 and NetOGlyc 3.1 software,meanwhile they were classified according to the geneontology methods.The construction of Con A affinity glycoprotein database of normal human liver tissue would contribute to the subsequent research.
基金funded by SZSTC(SGDX20190816230207535)obtained a donation from Kwok Chung Bo Fun Charitable Fund,which commemorates the establishment of the Kwok Yat Wai Endowed Chair of Environmental and Biological Analysis。
文摘The first corona-pandemic,coronavirus disease 2019(COVID-19)caused a huge health crisis and incalculable damage worldwide.Knowledge of how to cure the disease is urgently needed.Emerging immune escaping mutants of the virus suggested that it may be potentially persistent in human society as a regular health threat as the flu virus.Therefore,it is imperative to identify appropriate biomarkers to indicate pathological and physiological states,and more importantly,clinic outcomes.Proteins are the performers of life functions,and their abundance and modification status can directly reflect the immune status.Protein glycosylation serves a great impact in modulating protein function.The use of both unmodified and glycosylated proteins as biomarkers has also been proved feasible in the studies of SARS,Zika virus,influenza,etc.In recent years,mass spectrometry-based glycoproteomics,as well as proteomics approaches,advanced significantly due to the evolution of mass spectrometry.We focus on the current development of the mass spectrometry-based strategy for COVID-19 biomarkers’investigation.Potential application of glycoproteomics approaches and challenges in biomarkers identification are also discussed.
基金supported in part by funds from the National Key R&D Program of China(Grant Nos.2022YFC3400801 and 2021YFA1302601)the National Natural Science Foundation of China(Grant Nos.22034007,22274014,92153302,22304174)+3 种基金the innovation program of science and research from the Dalian Institute of Chemical Physics(DICP)Chinese Academy of Sciences(CAS)(Grant No.DMU-2&DICP UN202303)the Youth Innovation Promotion Association of CAS(Grant No.Y2022059)the China Postdoctoral Science Foundation(Grant No.2023M743424).
文摘Identification evaluation and result dissemination are essential components in mass spectrometry-based proteomics analysis.The visualization of fragment ions in mass spectrum provides strong evidence for peptide identification and modification localization.Here,we present an easy-to-use tool,named GP-Plotter,for ion annotation of tandem mass spectra and corresponding image output.Identification result files of common searching tools in the community and user-customized files are supported as input of GP-Plotter.Multiple display modes and parameter customization can be achieved in GP-Plotter to present annotated spectra of interest.Different image formats,especially vector graphic formats,are available for image generation which is favorable for data publication.Notably,GP-Plotter is also well-suited for the visualization and evaluation of glycopeptide spectrum assignments with comprehensive annotation of glycan fragment ions.With a user-friendly graphical interface,GP-Plotter is expected to be a universal visualization tool for the community.GP-Plotter has been implemented in the latest version of Glyco-Decipher(v1.0.4)and the standalone GP-Plotter software is also freely available at https://github.com/DICP-1809.
文摘Glycoproteins are complex biological macromolecules composed of proteins and glycans,playing a crucial role in various biological processes and being closely related to human health and diseases.However,the intricate structure and function of glycoproteins gives them with a vast capacity for information,which cannot be simply deduced from the genetic code(Di Marco et al.2023).
基金approved by the Research Ethics Committees of Zhejiang Provincial People’s Hospital(No.QT2022387).
文摘Pancreatic cancer is among the most malignant cancers,and thus early intervention is the key to better survival outcomes.However,no methods have been derived that can reliably identify early precursors of development into malignancy.Therefore,it is urgent to discover early molecular changes during pancreatic tumorigenesis.As aberrant glycosylation is closely associated with cancer progression,numerous efforts have been made to mine glycosylation changes as biomarkers for diagnosis;however,detailed glycoproteomic information,especially site-specific N-glycosylation changes in pancreatic cancer with and without drug treatment,needs to be further explored.Herein,we used comprehensive solid-phase chemoenzymatic glycoproteomics to analyze glycans,glycosites,and intact glycopeptides in pancreatic cancer cells and patient sera.The profiling of N-glycans in cancer cells revealed an increase in the secreted glycoproteins from the primary tumor of MIA PaCa-2 cells,whereas human sera,which contain many secreted glycoproteins,had significant changes of glycans at their specific glycosites.These results indicated the potential role for tumor-specific glycosylation as disease biomarkers.We also found that AMG-510,a small molecule inhibitor against Kirsten rat sarcoma viral oncogene homolog(KRAS)G12C mutation,profoundly reduced the glycosylation level in MIA PaCa-2 cells,suggesting that KRAS plays a role in the cellular glycosylation process,and thus glycosylation inhibition contributes to the anti-tumor effect of AMG-510.
基金financial support from the National Natural Science Foundation of China(32150027 and 22177002)the National Key Research and Development Program of China(2022YFC3400803 and 2023YFC2308003)supported by the Russian Science Foundation(5-74-30002)。
文摘Adoptive cell therapies(ACTs)have achieved remarkable clinical success in treating cancers;however,their broader application is greatly impeded by high cost and restricted antigen specificity.Recently,engineering the glycocalyx has provided a convenient transgene-free means to design ACTs with highavidity glycan ligands to target CD22,offering a new avenue for B lymphoma immunotherapy.In this work,we perform a comparative analysis of the molecular profiles involved in metabolic or chemoenzymatic glycocalyx engineering and explore their multiplexing capability.The glycoproteomic results revealed content-dependent customization of the natural killer(NK)-92MI glycocalyx.Compared with metabolic engineering,exogenous chemoenzymatic engineering has comparable or even superior ligand-loading efficiency,with some immune synapse components modified to facilitate their spatial recognition against target cells.Next,we tested the orthogonal creation of ligands on NK-92MI cells by further engineering a2,3-sialylated N-acetyllactosamine moieties to produce selectin ligands that are essential for better in vivo eradication of mouse xenograft B lymphoma.Finally,we demonstrate that analogous engineering of CD19-targeted chimeric antigen receptor T(CAR-T)cells to produce CD19/CD22 bitargeted therapy can enhance antigen targeting and tumor cell killing,offering an alternative cost-efficient agent for treating cancer relapse with decreased levels of CD19 antigens.These findings establish a mechanistic foundation for glycocalyx engineering and support the rational design of nextgeneration ACTs against B lymphoma.
基金supported by European Commission(EC)Horizon 2020 research and innovation program for Frania J.Zuniga-Banuelos and Erdmann Rapp under the project"IMforFUTURE"(H2020-MSCAITN/721815)by the Deutsche Forschungsgemeinschaft(DFG,German Research Foundation)for Marcus Hoffmann and Erdmann Rapp under the project"The concert of dolichol-based glycosylation:from molecules to disease models"(FOR2509)。
文摘To understand the implications of protein glycosylation for clinical diagnostics and biopharmaceuticals,innovative glycoproteomic technologies are required.Recently,significant advances have been made in such technologies,particularly in the area of structure-focused N-glyco-proteomic analyses.Mass spectrometric analysis of intact N-glycopeptides using stepped collision fragmentation along with glycan oxonium ion profiling now makes it possible to reliably discriminate between different N-glycan structures.Still,current N-glycoproteomic approaches have weaknesses that must be overcome,namely①the handling of incorrect identifications,②the identification of rare and modified N-glycans,and③insufficient glycoproteomic coverage,especially in complex samples.To address these shortcomings,we have established an innovative N-glycoproteomic workflow that aims to provide comprehensive site-specific and structural N-glycoproteomic data on human blood plasma(HBP)glycoproteins.The workflow features protein depletion plus a fractionation strategy and the use of high-resolution mass spectrometry with stepped collision fragmentation.Furthermore,by including a new decision tree procedure developed for data validation,we can significantly improve the description of N-glycan micro-heterogeneity.Our advanced data analysis workflow allows the reliable differentiation of ambiguous N-glycan structures such as antenna versus core fucosylation,as well as modified and rare N-glycans such as sulfated and glucuronidated ones.With this workflow,we were able to achieve the detection of HBP glycoproteins with reported concentrations within the ng mL^(-1)level.A total of 1929 N-glycopeptides and 942 N-glycosites derived from 805 human middle-to low-abundant glycoproteins were identified.Overall,the presented workflow holds great potential to improve our understanding of protein glycosylation and to foster the discovery of blood plasma biomarkers.
基金supported by the National Key Research and Development Program of China(grant Nos.2022YFF0608401,2022YFF0608404,2021YFF0702003-02)the National Natural Science Foundation of China(grant No.92478101).
文摘Protein glycosylation is a critical post-translational modification that influences protein folding,localization,stability,and functional interactions by attaching glycans to specific sites.This process is crucial for biological functions of glycoproteins,and aberrant glycosylation can lead to genetic disorders,immune system issues,and multi-organ pathologies.Recent advancements in glycoproteomic technologies have made the study of protein glycosylation a key focus for understanding the pathogenesis of kidney diseases.This review provides a comprehensive overview of protein glycosylation mechanisms,its biological roles,molecular pathways,and significant functions in renal physiology and pathology.It specifically highlights the dynamic changes and regulatory networks associated with aberrant glycosylation in kidney diseases such as immunoglobulin A nephropathy,diabetic kidney disease,autosomal dominant polycystic kidney disease,renal cell carcinoma,and acute kidney injury.It also evaluates the clinical applications of related technologies and biomarkers.Additionally,it discusses the challenges in developing glycosylation-targeted therapeutic strategies.Future research should focus on clarifying cell-specific glycosylation regulatory networks in the kidney,integrating glycobiology with multi-omics approaches,and improving precision diagnostics and treatment for kidney diseases.
基金supported by CONICET(Grant PIP-11220150100622CO),by ANPCyT(Grant PICT 2018-02222)UBA(Grant 20020170100414BA)supported by ANPCyT Grant PME 2012(CEQUIBIEM).
文摘Chicken egg white contains hundreds of proteins that are widely used in the food,biological and pharmaceutical industries.Glycosylation provides additional structural diversity to the already specialized proteins.The array of glycans on protein surfaces and different glycosylation sites provide sophisticated structures essentials for the multiple functions that glycoproteins assume.Different types of analysis have been performed to determine N-glycosylation in chicken egg white.In different studies,up to 19 N-glycoproteins have been characterized.However,regarding O-glycosylation,there is insufficient knowledge of their structures and abundances.In fact,only ovomucin,a major component of chicken egg white,has been described as bearing O-glycans that consist of 2-6 sugar residues carrying sialic acid and/or sulfate groups.In the present work,the use of a reductive β-elimination reaction followed by a HPAEC-PAD analysis,showed released oligosaccharides suggesting the presence of different O-glycoproteins.Getting deeper into chicken egg white O-glycoproteomics,a nanoHPLC-ESI-Orbitrap-HCD analysis was performed and two different software tools were employed.Under these conditions,at least seven different O-glycosylated proteins were described.In addition,O-glycosylation of isolated ovalbumin was characterized for the first time and in a BEMAD analysis of the isolated glycoprotein,three O-glycosites were detected.Taking into account that glycosylation has been involved in the structure and properties of chicken egg white proteins such as allergenicity,antibacterial action and embryo protection,attention must be paid to explore new properties of this heterogeneous modification in relation to both the food and biopharmaceutical industries.
基金supported by the National Key Research and Development Program of China(No.2019YFA0905200)the National Natural Science Foundation of China(Nos.91853123,81773180,and 21705127).
文摘The structure of N-glycans on specific proteins can regulate innate and adaptive immunity via sensing environmental signals.Meanwhile,the structural diversity of N-glycans poses analytical challenges that limit the exploration of specific glycosylation functions.In this work,we used THP-1-derived macrophages as examples to show the vast potential of a N-glycan structural interpretation tool StrucGP in N-glycoproteomic analysis.The intact glycopeptides of macrophages were enriched and analyzed using mass spectrometry(MS)-based glycoproteomic approaches,followed by the large-scale mapping of site-specific glycan structures via StrucGP.Results revealed that bisected GlcNAc,core fucosylated,and sialylated glycans(e.g.,HexNAc4Hex5Fuc1Neu5Ac1,N4H5F1S1)were increased in M1 and M2 macrophages,especially in the latter.The findings indicated that these structures may be closely related to macrophage polarization.In addition,a high level of glycosylated PD-L1 was observed in M1 macrophages,and the LacNAc moiety was detected at Asn-192 and Asn-200 of PD-L1,and Asn-200 contained Lewis epitopes.The precision structural interpretation of site-specific glycans and subsequent intervention of target glycoproteins and related glycosyltransferases are of great value for the development of new diagnostic and therapeutic approaches for different diseases.
