Uronic acids are prevalent components of crucial glycoconjugates,pivotal in various biological processes.In nature,NDP-uronic acids,the nucleosides-activated uronic acids,serve as glycosylation donors catalyzed by uro...Uronic acids are prevalent components of crucial glycoconjugates,pivotal in various biological processes.In nature,NDP-uronic acids,the nucleosides-activated uronic acids,serve as glycosylation donors catalyzed by uronosyltransferases(UATs)to construct glycans containing uronic acids.Despite their biological importance,the synthesis of naturally occurring NDP-uronic acids on a large scale remains challenging.Here,we developed an oxidation reaction insertion strategy for the efficient synthesis of NDP-uronic acids,and 11 NDP-uronic acids were successfully prepared in good yield and on a large scale.The prepared NDP-uronic acids can be used to explore new uronosyltransferases and synthesize uronic acids containing carbohydrates for fundamental research.展开更多
Rotavirus(RV)causes acute gastroenteritis in infants and children worldwide.Recent studies showed that glycans such as histo-blood group antigens(HBGAs)function as cell attachment factors affecting RV host susceptibil...Rotavirus(RV)causes acute gastroenteritis in infants and children worldwide.Recent studies showed that glycans such as histo-blood group antigens(HBGAs)function as cell attachment factors affecting RV host susceptibility and prevalence.P[8]is the predominant RV genotype in humans,but the structural basis of how P[8]RVs interact with glycan ligands remains elusive.In this study,we characterized the interactions between P[8]VP8~*s and glycans which showed that VP8~*,the RV glycan binding domain,recognized both mucin core 2 and H type 1 antigens according to the ELISA-based oligosaccharide binding assays.Importantly,we determined the structural basis of P[8]RV-glycans interaction from the crystal structures of a Rotateq P[8]VP8~*in complex with core 2 and H type 1 glycans at 1.82.3?,respectively,revealing a common binding pocket and similar binding mode.Structural and sequence analysis demonstrated that the glycan binding site is conserved among RVs in the P[Ⅱ]genogroup,while genotype-specific amino acid variations determined different glycan binding preference.Our data elucidated the detailed structural basis of the interactions between human P[8]RVs and different host glycan factors,shedding light on RV infection,epidemiology,and development of anti-viral agents.展开更多
Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulator...Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulatory transcriptional loop.The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes.Our in silico analysis of HNF1A,HNF4A.and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrich-ment of these transcription factors specifically in the liver.Our previous studies identified HNF1A as a master regulator of fucosylation,glycan branching,and galactosylation of plasma glycoproteins.Here,we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype.We used the state-of-the-art clus-tered regularly interspaced short palindromic repeats/dead Cas9(CRISPR/dCas9)molecular tool for the downregulation of the HNF1A,HNF4A,and FOXA2 genes in HepG2 cells-a human liver cancer cell line.The results show that the downregulation of all three genes individually and in pairs affects the transcrip-tional activity of many glyco-genes,although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures.The effect is better seen as an overall change in the total HepG2 N-glycome,primarily due to the extension of biantennary glycans.We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure.We also propose a model showing feedback loops with the mutual activation of HNF1A-FOXA2 and HNF4A-FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.展开更多
Immunoglobulin G(IgG)is the most abundant plasma glycoprotein and a prominent humoral immune mediator.Glycan composition affects the affinity of IgG to ligands and consequent immune responses.The modification of IgG N...Immunoglobulin G(IgG)is the most abundant plasma glycoprotein and a prominent humoral immune mediator.Glycan composition affects the affinity of IgG to ligands and consequent immune responses.The modification of IgG N-glycosylation is considered to be one of the various mechanisms by which sex hormones modulate the immune system.Although the menstrual cycle is the central sex hormonerelated physiological process in most women of reproductive age,IgG N-glycosylation dynamics during the menstrual cycle have not yet been investigated.To fill this gap,we profiled the plasma IgG Nglycans of 70 healthy premenopausal women at 12 time points during their menstrual cycles(every 7 days for 3 months)using hydrophilic interaction ultra-performance liquid chromatography(HILIC-UPLC).We observed cyclic periodic changes in the N-glycosylation of IgG in association with the menstrual cycle phase and sex hormone concentration in plasma.On the integrated cohort level,the modeled average menstrual cycle effect on the abundance of IgG N-glycosylation traits was low for each trait,with the highest being 1.1%for agalactosylated N-glycans.However,intrapersonal changes were relatively high in some cases;for example,the largest difference between the minimum and maximum values during the menstrual cycle was up to 21%for sialylated N-glycans.Across all measurements,the menstrual cycle phase could explain up to 0.72%of the variation in the abundance of a single IgG glycosylation trait of monogalactosylation.In contrast,up to 99%of the variation in the abundance of digalactosylation could be attributed to interpersonal differences in IgG N-glycosylation.In conclusion,the average extent of changes in the IgG N-glycopattern that occur during the menstrual cycle is small;thus,the IgG N-glycoprofiling of women in large sample-size studies can be performed regardless of menstrual cycle phase.展开更多
The essential role of immunoglobulin G(IgG)in immune system regulation and combatting infectious diseases cannot be fully recognized without an understanding of the changes in its N-glycans attached to the asparagine ...The essential role of immunoglobulin G(IgG)in immune system regulation and combatting infectious diseases cannot be fully recognized without an understanding of the changes in its N-glycans attached to the asparagine 297 of the fragment crystallizable(Fc)domain that occur under such circumstances.These glycans impact the antibody stability,half-life,secretion,immunogenicity,and effector functions.Therefore,in this study,we analyzed and compared the total IgG glycome—at the level of individual glycan structures and derived glycosylation traits(sialylation,galactosylation,fucosylation,and bisecting Nacetylglucosamine(GlcNAc))—of 64 patients with influenza,77 patients with coronavirus disease 2019(COVID-19),and 56 healthy controls.Our study revealed a significant decrease in IgG galactosylation,sialylation,and bisecting GlcNAc(where the latter shows the most significant decrease)in deceased COVID19 patients,whereas IgG fucosylation was increased.On the other hand,IgG galactosylation remained stable in influenza patients and COVID-19 survivors.IgG glycosylation in influenza patients was more time-dependent:In the first seven days of the disease,sialylation increased and fucosylation and bisecting GlcNAc decreased;in the next 21 days,sialylation decreased and fucosylation increased(while bisecting GlcNAc remained stable).The similarity of IgG glycosylation changes in COVID-19 survivors and influenza patients may be the consequence of an adequate immune response to enveloped viruses,while the observed changes in deceased COVID-19 patients may indicate its deviation.展开更多
Life relies on the coordination of nucleic acids and proteins,yet glycans—one of the four fundamental building blocks of life—are gradually revealing their pivotal role beyond the central dogma of molecular biology,...Life relies on the coordination of nucleic acids and proteins,yet glycans—one of the four fundamental building blocks of life—are gradually revealing their pivotal role beyond the central dogma of molecular biology,linking nucleic acids,proteins,and lipids to regulate life activities.展开更多
Building on coding mutations and splicing variants, post-translational modifications add a final layer to protein diversity that operates at developmental and physiological timescales. Although protein glycosylation i...Building on coding mutations and splicing variants, post-translational modifications add a final layer to protein diversity that operates at developmental and physiological timescales. Although protein glycosylation is one of the most common post-translational modifications, its evolutionary origin remains largely unexplored. Here, we performed a phylostratigraphic tracking of glycosylation machinery(GM) genes and their targets-glycoproteins(GPs)-in a broad phylogenetic context. Our results show that the vast majority of human GM genes trace back to two evolutionary periods: the origin of all cellular organisms and the origin of all eukaryotes. This indicates that protein glycosylation is an ancient process likely common to all life, further elaborated in early eukaryotes. In contrast, human glycoproteins exhibited prominent enrichment signals in more recent evolutionary periods, suggesting an impo rtant role in the transition from metazoans to vertebrates. Focusing specifically on the N-glycosylation(NG) pathway,we noted that the majority of NG genes acting on the cytoplasmic side of the endoplasmic reticulum(ER) trace back to the origin of cellular organisms. This sharply contrasts with the rest of the NG pathway,which is oriented toward the ER lumen, where genes of eukaryotic origin predominate. In the Golgi, we also identified an analogous binary evolutionary origin of GM genes. We discuss these findings in the context of the evolutionary emergence of the eukaryotic endomembrane system and propose that the ER evolved through the invagination of a prokaryotic cell membrane containing an NG pathway.展开更多
基金financially supported by National Natural Science Foundation of China(No.22207113 to J.Zhang)Guangdong Basic and Applied Basic Research Foundation(No.2021A1515110588to J.Zhang)Natural Science Foundation of Shanghai Municipality(No.22ZR1474000 to L.Wen)。
文摘Uronic acids are prevalent components of crucial glycoconjugates,pivotal in various biological processes.In nature,NDP-uronic acids,the nucleosides-activated uronic acids,serve as glycosylation donors catalyzed by uronosyltransferases(UATs)to construct glycans containing uronic acids.Despite their biological importance,the synthesis of naturally occurring NDP-uronic acids on a large scale remains challenging.Here,we developed an oxidation reaction insertion strategy for the efficient synthesis of NDP-uronic acids,and 11 NDP-uronic acids were successfully prepared in good yield and on a large scale.The prepared NDP-uronic acids can be used to explore new uronosyltransferases and synthesize uronic acids containing carbohydrates for fundamental research.
基金This research was supported by grants from the National Science and Technology Major Project(2018ZX10711-001)the National Natural Science Foundation of China(NSFC)(No.81601813).
文摘Rotavirus(RV)causes acute gastroenteritis in infants and children worldwide.Recent studies showed that glycans such as histo-blood group antigens(HBGAs)function as cell attachment factors affecting RV host susceptibility and prevalence.P[8]is the predominant RV genotype in humans,but the structural basis of how P[8]RVs interact with glycan ligands remains elusive.In this study,we characterized the interactions between P[8]VP8~*s and glycans which showed that VP8~*,the RV glycan binding domain,recognized both mucin core 2 and H type 1 antigens according to the ELISA-based oligosaccharide binding assays.Importantly,we determined the structural basis of P[8]RV-glycans interaction from the crystal structures of a Rotateq P[8]VP8~*in complex with core 2 and H type 1 glycans at 1.82.3?,respectively,revealing a common binding pocket and similar binding mode.Structural and sequence analysis demonstrated that the glycan binding site is conserved among RVs in the P[Ⅱ]genogroup,while genotype-specific amino acid variations determined different glycan binding preference.Our data elucidated the detailed structural basis of the interactions between human P[8]RVs and different host glycan factors,shedding light on RV infection,epidemiology,and development of anti-viral agents.
基金the European Structural and Investment Funded Grant"Cardio Metabolic"(#KK.01.2.1.02.0321)the Croatian National Centre of Research Excellence in Personalized Healthcare Grant(#KK.01.1.1.01.0010)+2 种基金the European Regional Development Fund Grant,project"CRISPR/Cas9-CasMouse"(#KK.01.1.1.04.0085)the European Structural and Investment Funded Project of Centre of Competence in Molecular Diagnostics(#KK.01.2.2.03.0006)the Croatian National Centre of Research Excellence in Personalized Healthcare Grant(#KK.01.1.1.01.0010).
文摘Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulatory transcriptional loop.The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes.Our in silico analysis of HNF1A,HNF4A.and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrich-ment of these transcription factors specifically in the liver.Our previous studies identified HNF1A as a master regulator of fucosylation,glycan branching,and galactosylation of plasma glycoproteins.Here,we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype.We used the state-of-the-art clus-tered regularly interspaced short palindromic repeats/dead Cas9(CRISPR/dCas9)molecular tool for the downregulation of the HNF1A,HNF4A,and FOXA2 genes in HepG2 cells-a human liver cancer cell line.The results show that the downregulation of all three genes individually and in pairs affects the transcrip-tional activity of many glyco-genes,although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures.The effect is better seen as an overall change in the total HepG2 N-glycome,primarily due to the extension of biantennary glycans.We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure.We also propose a model showing feedback loops with the mutual activation of HNF1A-FOXA2 and HNF4A-FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.
基金funded by the European Structural and Investment Funds grant for the Croatian National Centre of Research Excellence in Personalized Healthcare(KK.01.1.1.01)Australia-China International Collaborative Grant(NHMRC APP1112767-NSFC 81561128020)+1 种基金National Natural Science Foundation of China(81773527 and 81573215)the European Structural and Investment Funds CEKOM(KK.01.2.2.03.0006).
文摘Immunoglobulin G(IgG)is the most abundant plasma glycoprotein and a prominent humoral immune mediator.Glycan composition affects the affinity of IgG to ligands and consequent immune responses.The modification of IgG N-glycosylation is considered to be one of the various mechanisms by which sex hormones modulate the immune system.Although the menstrual cycle is the central sex hormonerelated physiological process in most women of reproductive age,IgG N-glycosylation dynamics during the menstrual cycle have not yet been investigated.To fill this gap,we profiled the plasma IgG Nglycans of 70 healthy premenopausal women at 12 time points during their menstrual cycles(every 7 days for 3 months)using hydrophilic interaction ultra-performance liquid chromatography(HILIC-UPLC).We observed cyclic periodic changes in the N-glycosylation of IgG in association with the menstrual cycle phase and sex hormone concentration in plasma.On the integrated cohort level,the modeled average menstrual cycle effect on the abundance of IgG N-glycosylation traits was low for each trait,with the highest being 1.1%for agalactosylated N-glycans.However,intrapersonal changes were relatively high in some cases;for example,the largest difference between the minimum and maximum values during the menstrual cycle was up to 21%for sialylated N-glycans.Across all measurements,the menstrual cycle phase could explain up to 0.72%of the variation in the abundance of a single IgG glycosylation trait of monogalactosylation.In contrast,up to 99%of the variation in the abundance of digalactosylation could be attributed to interpersonal differences in IgG N-glycosylation.In conclusion,the average extent of changes in the IgG N-glycopattern that occur during the menstrual cycle is small;thus,the IgG N-glycoprofiling of women in large sample-size studies can be performed regardless of menstrual cycle phase.
基金supported by the European Structural and Investment Funds grant for the Croatian National Centre of Competence in Molecular Diagnostics (KK.01.2.2.03.0006)the Croatian National Centre of Research Excellence in Personalized Healthcare grant (KK.01.1.1.01.0010)supported by the Human Glycome Project。
文摘The essential role of immunoglobulin G(IgG)in immune system regulation and combatting infectious diseases cannot be fully recognized without an understanding of the changes in its N-glycans attached to the asparagine 297 of the fragment crystallizable(Fc)domain that occur under such circumstances.These glycans impact the antibody stability,half-life,secretion,immunogenicity,and effector functions.Therefore,in this study,we analyzed and compared the total IgG glycome—at the level of individual glycan structures and derived glycosylation traits(sialylation,galactosylation,fucosylation,and bisecting Nacetylglucosamine(GlcNAc))—of 64 patients with influenza,77 patients with coronavirus disease 2019(COVID-19),and 56 healthy controls.Our study revealed a significant decrease in IgG galactosylation,sialylation,and bisecting GlcNAc(where the latter shows the most significant decrease)in deceased COVID19 patients,whereas IgG fucosylation was increased.On the other hand,IgG galactosylation remained stable in influenza patients and COVID-19 survivors.IgG glycosylation in influenza patients was more time-dependent:In the first seven days of the disease,sialylation increased and fucosylation and bisecting GlcNAc decreased;in the next 21 days,sialylation decreased and fucosylation increased(while bisecting GlcNAc remained stable).The similarity of IgG glycosylation changes in COVID-19 survivors and influenza patients may be the consequence of an adequate immune response to enveloped viruses,while the observed changes in deceased COVID-19 patients may indicate its deviation.
文摘Life relies on the coordination of nucleic acids and proteins,yet glycans—one of the four fundamental building blocks of life—are gradually revealing their pivotal role beyond the central dogma of molecular biology,linking nucleic acids,proteins,and lipids to regulate life activities.
基金supported by the Croatian Science Foundation (IP-2016-06-5924),the City of Zagreb, and the Adris Foundation to Tomislav Domazet-Lošothe European Regional Development Fund (KK.01.1.1.1.01.0009 DATACROSS) to Mirjana Domazet-Lošo and Tomislav Domazet-Lošo.
文摘Building on coding mutations and splicing variants, post-translational modifications add a final layer to protein diversity that operates at developmental and physiological timescales. Although protein glycosylation is one of the most common post-translational modifications, its evolutionary origin remains largely unexplored. Here, we performed a phylostratigraphic tracking of glycosylation machinery(GM) genes and their targets-glycoproteins(GPs)-in a broad phylogenetic context. Our results show that the vast majority of human GM genes trace back to two evolutionary periods: the origin of all cellular organisms and the origin of all eukaryotes. This indicates that protein glycosylation is an ancient process likely common to all life, further elaborated in early eukaryotes. In contrast, human glycoproteins exhibited prominent enrichment signals in more recent evolutionary periods, suggesting an impo rtant role in the transition from metazoans to vertebrates. Focusing specifically on the N-glycosylation(NG) pathway,we noted that the majority of NG genes acting on the cytoplasmic side of the endoplasmic reticulum(ER) trace back to the origin of cellular organisms. This sharply contrasts with the rest of the NG pathway,which is oriented toward the ER lumen, where genes of eukaryotic origin predominate. In the Golgi, we also identified an analogous binary evolutionary origin of GM genes. We discuss these findings in the context of the evolutionary emergence of the eukaryotic endomembrane system and propose that the ER evolved through the invagination of a prokaryotic cell membrane containing an NG pathway.
