N-glycans in many proteins are of great concern because of their strong association with food allergies. Triticum aestivum(bread wheat), a major food crop, is known as one of the “Big Eight” allergenic groups. Howev...N-glycans in many proteins are of great concern because of their strong association with food allergies. Triticum aestivum(bread wheat), a major food crop, is known as one of the “Big Eight” allergenic groups. However, little research has been done about N-glycans in wheat glycoproteins. In this study, a soluble wheat glycoprotein was purified from wheat and further identified as globulin-1 S allele(GSA). The wheat GSA displayed significant IgE-binding activity. Moreover, one N-glycosylation site and 6 kinds of N-glycans were identified by mass spectrometry, including 3 high mannose types and 3 complex types. Furthermore, the IgE-binding activity of wheat GSA is proved to be reduced by the removal of N-glycan, thermal treatment(temperatures > 80 ℃), and strong acidic treatment(pH 3.0). These findings would provide a better understanding of the effects of N-glycosylation, thermal treatment, and acidic treatment on the molecular characteristics of GSA, and further provide new insights into the development of hypoallergenic wheat products.展开更多
Despite advances in understanding the development and progression of cancer in recent years,there remains a lack of comprehensive characterization of the cancer glycoproteome.Glycoproteins play an important role in me...Despite advances in understanding the development and progression of cancer in recent years,there remains a lack of comprehensive characterization of the cancer glycoproteome.Glycoproteins play an important role in medicine and are involved in various human disease conditions including cancer.Glycan-moieties participate in fundamental cancer processes like cell signaling,invasion,angiogenesis,and metastasis.Aberrant N-glycosylation significantly impacts cancer processes and targeted therapies in clinic.Therefore,understanding N-glycosylation in a tumor is essential for comprehending disease progression and discovering anti-cancer targets and biomarkers for therapy monitoring and diagnosis.This review presents the fundamental process of protein N-glycosylation and summarizes glycosylation changes in tumor cells,including increased terminal sialylation,N-glycan branching,and corefucosylation.Also,the role of N-glycosylation in tumor signaling pathways,migration,and metabolism are discussed.Glycoproteins and glycopeptides as potential biomarkers for early detection of cancer based on site specificity have been introduced.Collectively,understanding and exploring the cancer glycoproteome,along with its role in medicine,implication in cancer and other human diseases,highlights the significance of N-glycosylation in tumor processes,necessitating further research for potential anticancer targets and biomarkers.展开更多
BACKGROUND Wnt/FZD-mediated signaling pathways are activated in more than 90%of hepatocellular carcinoma(HCC)cell lines.As a well-known secretory glycoprotein,Wnt3 can interact with FZD receptors on the cell surface,t...BACKGROUND Wnt/FZD-mediated signaling pathways are activated in more than 90%of hepatocellular carcinoma(HCC)cell lines.As a well-known secretory glycoprotein,Wnt3 can interact with FZD receptors on the cell surface,thereby activating the Wnt/β-catenin signaling pathway.However,the N-glycosylation modification site of Wnt3 and the effect of this modification on the biological function of the protein are still unclear.AIM To investigate the effect of Wnt3 N-glycosylation on the biological function of HCC cells.METHODS Site-directed mutagenesis was used to verify the Wnt3 N-glycosylation sites,actinomycin D treatment was used to detect the stability of Wnt3 after site-directed mutation,the binding of the N-glycosylation site-directed mutant Wnt3 to FZD7 was observed by laser confocal microscopy,and the effects of the N-glycosylation site-directed mutation of Wnt3 on the Wnt/β-catenin signaling pathway and the progression of HCC cells were detected by western blot and cell function experiments.RESULTS Wnt3 has two N-glycosylation-modified sites(Asn90 and Asn301);when a single site at amino acid 301 is mutated,the stability of Wnt3 is weakened;the binding ability of Wnt3 to FZD7 decreases when both sites are mutated simultaneously;and the level of proteins related to the Wnt/β-catenin signaling pathway is downregulated.Cell proliferation,migration and invasion are also weakened in the case of single 301 site and double-site mutations.CONCLUSION These results indicate that by inhibiting the N-glycosylation of Wnt3,the proliferation,migration,invasion and colony formation abilities of liver cancer cells can be weakened,which might provide new therapeutic strategies for clinical liver cancer in the future.展开更多
Background:We are using genetics to identify genes specifically involved in hearing regeneration.In a large-scale genetic screening,we identified mgat5a,a gene in the N-glycosylation biosynthesis pathway whose activit...Background:We are using genetics to identify genes specifically involved in hearing regeneration.In a large-scale genetic screening,we identified mgat5a,a gene in the N-glycosylation biosynthesis pathway whose activity negatively impacts hair cell regeneration.Methods:We used a combination of mutant analysis in zebrafish and a hair cell regeneration assay to phenotype the loss of Mgat5a activity in zebrafish.We used pharmacological inhibition of N-glycosylation by swansonine.We also used over-expression analysis by mRNA injections to demonstrate how changes in N-glycosylation can alter cell signaling.Results:We found that mgat5a was expressed in multiple tissues during zebrafish embryo development,particularly enriched in neural tissues including the brain,retina,and lateral line neuromasts.An mgat5a insertional mutation and a CRISPR/Cas9-generated truncation mutation both caused an enhancement of hair cell regeneration which could be phenocopied by pharmacological inhibition with swansonine.In addition to hair cell regeneration,inhibition of the N-glycosylation pathway also enhanced the regeneration of lateral line axon and caudal fins.Further analysis showed that N-glycosylation altered the responsiveness of TGF-beta signaling.Conclusions:The findings from this study provide experimental evidence for the involvement of N-glycosylation in tissue regeneration and cell signaling.展开更多
Most human-secreted and membrane-bound proteins have covalently attached oligosaccharide chains or glycans.Glycosylation influences the physical and chemical properties of proteins,as well as their biological function...Most human-secreted and membrane-bound proteins have covalently attached oligosaccharide chains or glycans.Glycosylation influences the physical and chemical properties of proteins,as well as their biological functions.Unsurprisingly,alterations in protein glycosylation have been implicated in a growing number of human diseases,and glycans are increasingly being considered as potential therapeutic targets,an essential part of therapeutics,and biomarkers.Although glycosylation pathways are biochemically well-studied,little is known about the networks of genes that guide the cell-and tissue-specific regulation of these biochemical reactions in humans in vivo.The lack of a detailed understanding of the mechanisms regulating glycome variation and linking the glycome to human health and disease is slowing progress in clinical applications of human glycobiology.Two of the tools that can provide much sought-after knowledge of human in vivo glycobiology are human genetics and genomics,which offer a powerful data-driven agnostic approach for dissecting the biology of complex traits.This review summarizes the current state of human populational glycogenomics.In Section 1,we provide a brief overview of the N-glycan’s structural organization,and in Section 2,we give a description of the major blood plasma glycoproteins.Next,in Section 3,we summarize,systemize,and generalize the results from current N-glycosylation genome-wide association studies(GWASs)that provide novel knowledge of the genetic regulation of the populational variation of glycosylation.Until now,such studies have been limited to an analysis of the human blood plasma N-glycome and the N-glycosylation of immunoglobulin G and transferrin.While these three glycomes make up a rather limited set compared with the enormous multitude of glycomes of different tissues and glycoproteins,the study of these three does allow for powerful analysis and generalization.Finally,in Section 4,we turn to genes in the established loci,paying particular attention to genes with strong support in Section 5.At the end of the review,in Sections 6 and 7,we describe special cases of interest in light of new discoveries,focusing on possible mechanisms of action and biological targets of genetic variation that have been implicated in human protein N-glycosylation.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 coronavirus disease 2019(COVID-19)pandemic has led to worldwide efforts to understand the biological traits of the newly identified human coronavirus(HCoV-19)virus.In this mass spectrometry(MS)-based study,we reve...The coronavirus disease 2019(COVID-19)pandemic has led to worldwide efforts to understand the biological traits of the newly identified human coronavirus(HCoV-19)virus.In this mass spectrometry(MS)-based study,we reveal that out of 21 possible glycosites in the HCoV-19 spike protein(S protein),20 are completely occupied by N-glycans,predominantly of the oligomannose type.All seven glycosylation sites in human angiotensin I converting enzyme 2(hACE2)were found to be completely occupied,mainly by complex N-glycans.However,glycosylation did not directly contribute to the binding affinity between HCoV-19 S protein and hACE2.Additional post-translational modification(PTM)was identified,including multiple methylated sites in both proteins and multiple sites with hydroxylproline in hACE2.Refined structural models of HCoV-19 S protein and hACE2 were built by adding N-glycan and PTMs to recently published cryogenic electron microscopy structures.The PTM and glycan maps of HCoV-19 S protein and hACE2 provide additional structural details for studying the mechanisms underlying host attachment and the immune response of HCoV-19,as well as knowledge for developing desperately needed remedies and vaccines.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
T-cell development ensures the formation of diverse repertoires of T-cell receptors(TCRs)that recognize a variety of antigens.Glycosylation is a major posttranslational modification present in virtually all cells,incl...T-cell development ensures the formation of diverse repertoires of T-cell receptors(TCRs)that recognize a variety of antigens.Glycosylation is a major posttranslational modification present in virtually all cells,including T-lymphocytes,that regulates activity/functions.Although these structures are known to be involved in TCR-selection in DP thymocytes,it is unclear how glycans regulate other thymic development processes and how they influence susceptibility to disease.Here,we discovered stage-specific glycome compositions during T-cell development in human and murine thymocytes,as well as dynamic alterations.After restricting the N-glycosylation profile of thymocytes to high-mannose structures,using specific glycoengineered mice(Rag1CreMgat1fl/fl),we showed remarkable defects in key developmental checkpoints,includingß-selection,regulatory T-cell generation andγδT-cell development,associated with increased susceptibility to colon and kidney inflammation and infection.We further demonstrated that a single N-glycan antenna(modeled in Rag1CreMgat2fl/fl mice)is the sine-qua-non condition to ensure normal development.In conclusion,we revealed that mannosylated thymocytes lead to a dysregulation in T-cell development that is associated with inflammation susceptibility.展开更多
Glycosylation is a common posttranslational modification on membrane-associated and secreted proteins that is of pivotal importance for regulating cell functions.Aberrant glycosylation can lead to uncontrolled cell pr...Glycosylation is a common posttranslational modification on membrane-associated and secreted proteins that is of pivotal importance for regulating cell functions.Aberrant glycosylation can lead to uncontrolled cell proliferation,cell-matrix interactions,migration and differentiation,and has been shown to be involved in cancer and other diseases.The epithelial-to-mesenchymal transition is a key step in the metastatic process by which cancer cells gain the ability to invade tissues and extravasate into the bloodstream.This cellular transformation process,which is associated by morphological change,loss of epithelial traits and gain of mesenchymal markers,is triggered by the secreted cytokine transforming growth factor-β(TGF-β).TGF-βbioactivity is carefully regulated,and its effects on cells are mediated by its receptors on the cell surface.In this review,we first provide a brief overview of major types of glycans,namely,N-glycans,O-glycans,glycosphingolipids and glycosaminoglycans that are involved in cancer progression.Thereafter,we summarize studies on how the glycosylation of TGF-βsignaling components regulates TGF-βsecretion,bioavailability and TGF-βreceptor function.Then,we review glycosylation changes associated with TGF-β-induced epithelial-to-mesenchymal transition in cancer.Identifying and understanding the mechanisms by which glycosylation affects TGF-βsignaling and downstream biological responses will facilitate the identification of glycans as biomarkers and enable novel therapeutic approaches.展开更多
The interaction among type Ⅱ collagen(CⅡ),human DR4 major histocompatibility complex type Ⅱ molecule(MHC Ⅱ)and T-cell receptor(TCR)is associated with the development of rheumatoid arthritis(RA).The activation of T...The interaction among type Ⅱ collagen(CⅡ),human DR4 major histocompatibility complex type Ⅱ molecule(MHC Ⅱ)and T-cell receptor(TCR)is associated with the development of rheumatoid arthritis(RA).The activation of T cells can be reduced through exposure to modified CⅡ(263-272)glycopeptide fragment via competitive inhibition with self-antigen.In this work,30 peptides based on the sequence of CⅡ(263-272)were prepared and evaluated for their binding to DR4 protein by surface plasmon resonance(SPR)assay.The effect on the secretion of pro-inflammatory factors by the spleen cells in collagen induced rheumatoid arthritis(CIA)mouse was also investigated.Two N-glycosylated CⅡ peptides were identified to have strong binding to the human recombinant DR4 protein and weak proinflammatory effect.These glycopeptides could be developed as therapeutic saccharide vaccines for the treatment of rheumatoid arthritis(RA).展开更多
Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization.However,only a few secretory proteins were functionally characterized in Magnaporthe ory...Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization.However,only a few secretory proteins were functionally characterized in Magnaporthe oryzae,the fungal pathogen causing rice blast disease worldwide.Asparagine-linked glycosylation 3(Alg3)is an a-1,3-mannosyltransferase functioning in the Nglycan synthesis of N-glycosylated secretory proteins.Fungal pathogenicity and cell wall integrity are impaired inΔalg3 mutants,but the secreted proteins affected inΔalg3 mutants are largely unknown.In this study,we compared the secretomes of the wild-type strain and theΔalg3 mutant and identified 51 proteins that require Alg3 for proper secretion.These proteins were predicted to be involved in metabolic processes,interspecies interactions,cell wall organization,and response to chemicals.Nine proteins were selected for further validation.We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae.Moreover,the Nglycosylation of these proteins was significantly changed in theΔalg3 mutant,leading to the decreased protein secretion and abnormal protein localization.Furthermore,we tested the biological functions of two genes,INV1(encoding invertase 1,a secreted invertase)and AMCase(encoding acid mammalian chinitase,a secreted chitinase).The fungal virulence was significantly reduced,and the cell wall integrity was altered in theΔinv1 andΔamcase mutant strains.Moreover,the N-glycosylation was essential for the function and secretion of AMCase.Taken together,our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.展开更多
Currently,drug resistance of anti-cancer therapy has become the main cause of low survival rate and poor prognosis.Full understanding of drug resistance mechanisms is an urgent request for further development of anti-...Currently,drug resistance of anti-cancer therapy has become the main cause of low survival rate and poor prognosis.Full understanding of drug resistance mechanisms is an urgent request for further development of anti-cancer therapy and improve-ment of prognosis.Here we present our N-glycoproteomics study of putative N-glycoprotein biomarkers of drug resistance in doxorubicin resistance breast cancer cell line michigan cancer foundation-7(MCF-7/ADR)relative to parental michigan cancer foundation-7(MCF-7)cells.Intact N-glycopeptides(IDs)from MCF-7/ADR and MCF-7 cells were enriched with zwitterionic hydrophilic interaction liquid chromatography(ZIC-HILIC),labeled with stable isotopic diethylation(SIDE),and analyzed with C18-RPLC-MS/MS(HCD with stepped normalized collision energies);these IDs were identified with database search engine GPSeeker,and the differentially expressed intact N-glycopeptides(DEGPs)were quantified with GPSeekerQuan.With target-decoy searches and control of spectrum-level FDR≤1%,322 intact N-glycopeptides were identified;these intact N-glycopeptides come from the combination of 249 unique peptide backbones(corresponding to 234 intact N-glycoproteins)and 90 monosaccharide compositions(corresponding to 248 putative N-glycosites).The sequence structures of 165 IDs were confirmed with structure-diagnostic fragment ions.With the criteria of observation at least twice among the three technical replicates,≥1.5-fold change and p value<0.05,20 DEGPs were quantified,where five of them were up-regulated and 15 of them were down-regulated;the corresponding intact N-glycoproteins as putative markers of drug resistance were discussed.展开更多
Glucoamylase from Monascus rubiginosus has been found to exist in multiple forms on PAGE pattern （E<sub>1</sub>-E<sub>5</sub>）. Both major molecular forms E<sub>3</sub> and E<s...Glucoamylase from Monascus rubiginosus has been found to exist in multiple forms on PAGE pattern （E<sub>1</sub>-E<sub>5</sub>）. Both major molecular forms E<sub>3</sub> and E<sub>4</sub> are glycoproteins.Sugar content as mannose （%） is 7 for E<sub>3</sub> and 9 for E<sub>4</sub>, respectively,展开更多
Plants are known to be efficient hosts for the production of mammalian therapeutic proteins.However,plants produce complex N-glycans bearingβ1,2-xylose and coreα1,3-fucose residues,which are absent in mammals.The im...Plants are known to be efficient hosts for the production of mammalian therapeutic proteins.However,plants produce complex N-glycans bearingβ1,2-xylose and coreα1,3-fucose residues,which are absent in mammals.The immunogenicity and allergenicity of plant-specific Nglycans is a key concern in mammalian therapy.In this study,we amplified the sequences of 2 plant-specific glycosyltransferases from Nicotiana tabacum L.cv Bright Yellow 2(BY2),which is a well-established cell line widely used for the expression of therapeutic proteins.The expression of the endogenous xylosyltranferase(XylT)and fucosyltransferase(FucT)was downregulated by using RNA interference(RNAi)strategy.The xylosylated and core fucosylated N-glycans were significantly,but not completely,reduced in the glycoengineered lines.However,these RNAi-treated cell lines were stable and viable and did not exhibit any obvious phenotype.Therefore,this study may provide an effective and promising strategy to produce recombinant glycoproteins in BY2 cells with humanized N-glycoforms to avoid potential immunogenicity.展开更多
The Coronavirus disease 2019(COVID-19)has posed a serious threat to global health and the world economy.Antiviral therapies targeting coronavirus are urgently required.The Cepharanthine(CEP)is a traditional Chinese he...The Coronavirus disease 2019(COVID-19)has posed a serious threat to global health and the world economy.Antiviral therapies targeting coronavirus are urgently required.The Cepharanthine(CEP)is a traditional Chinese herbal extract.Our previous research revealed that CEP has a very potent anti-coronavirus effect,but its mechanism of action was not fully understood.To investigate the effect of novel coronavirus on protein glycosylation in infected cells and to further investigate the mechanism of action of CEP against coronavirus,a cellular model using coronavirus GX_P2V infection of Vero E6 cells was established.The effect of coronavirus GX_P2V on host cell protein glycosylation was investigated by N-glycoproteomic analysis,and the antagonistic effect of CEP on the abnormal protein glycosylation caused by coronavirus was analyzed.The results showed that GX_P2V could cause abnormal changes in protein glycosylation levels in host cells,while CEP could partially antagonize the abnormal protein glycosylation caused by GX_P2V.In addition,we also found that CEP could regulate the glycosylation level of coronavirus S protein.In conclusion,this article provides important ideas about the infection mechanism of novel coronaviruses,providing evidence for CEP as a promising therapeutic option for coronavirus infection.展开更多
基金financially supported by the National Natural Science Foundation of China (31871735)。
文摘N-glycans in many proteins are of great concern because of their strong association with food allergies. Triticum aestivum(bread wheat), a major food crop, is known as one of the “Big Eight” allergenic groups. However, little research has been done about N-glycans in wheat glycoproteins. In this study, a soluble wheat glycoprotein was purified from wheat and further identified as globulin-1 S allele(GSA). The wheat GSA displayed significant IgE-binding activity. Moreover, one N-glycosylation site and 6 kinds of N-glycans were identified by mass spectrometry, including 3 high mannose types and 3 complex types. Furthermore, the IgE-binding activity of wheat GSA is proved to be reduced by the removal of N-glycan, thermal treatment(temperatures > 80 ℃), and strong acidic treatment(pH 3.0). These findings would provide a better understanding of the effects of N-glycosylation, thermal treatment, and acidic treatment on the molecular characteristics of GSA, and further provide new insights into the development of hypoallergenic wheat products.
基金support of this work from the National Cancer Institute under Grants 1R01 CA160254 and U01 CA225753(David M.Lubman,USA)。
文摘Despite advances in understanding the development and progression of cancer in recent years,there remains a lack of comprehensive characterization of the cancer glycoproteome.Glycoproteins play an important role in medicine and are involved in various human disease conditions including cancer.Glycan-moieties participate in fundamental cancer processes like cell signaling,invasion,angiogenesis,and metastasis.Aberrant N-glycosylation significantly impacts cancer processes and targeted therapies in clinic.Therefore,understanding N-glycosylation in a tumor is essential for comprehending disease progression and discovering anti-cancer targets and biomarkers for therapy monitoring and diagnosis.This review presents the fundamental process of protein N-glycosylation and summarizes glycosylation changes in tumor cells,including increased terminal sialylation,N-glycan branching,and corefucosylation.Also,the role of N-glycosylation in tumor signaling pathways,migration,and metabolism are discussed.Glycoproteins and glycopeptides as potential biomarkers for early detection of cancer based on site specificity have been introduced.Collectively,understanding and exploring the cancer glycoproteome,along with its role in medicine,implication in cancer and other human diseases,highlights the significance of N-glycosylation in tumor processes,necessitating further research for potential anticancer targets and biomarkers.
基金Supported by National Natural Science Foundation of China,No.81560390the Guizhou Medical University Cultivation Project of the National Natural Science Foundation of China,No.22NSFCP02Basic Research Project of Science and Technology Department of Guizhou Province,No.ZK[2024]General 136.
文摘BACKGROUND Wnt/FZD-mediated signaling pathways are activated in more than 90%of hepatocellular carcinoma(HCC)cell lines.As a well-known secretory glycoprotein,Wnt3 can interact with FZD receptors on the cell surface,thereby activating the Wnt/β-catenin signaling pathway.However,the N-glycosylation modification site of Wnt3 and the effect of this modification on the biological function of the protein are still unclear.AIM To investigate the effect of Wnt3 N-glycosylation on the biological function of HCC cells.METHODS Site-directed mutagenesis was used to verify the Wnt3 N-glycosylation sites,actinomycin D treatment was used to detect the stability of Wnt3 after site-directed mutation,the binding of the N-glycosylation site-directed mutant Wnt3 to FZD7 was observed by laser confocal microscopy,and the effects of the N-glycosylation site-directed mutation of Wnt3 on the Wnt/β-catenin signaling pathway and the progression of HCC cells were detected by western blot and cell function experiments.RESULTS Wnt3 has two N-glycosylation-modified sites(Asn90 and Asn301);when a single site at amino acid 301 is mutated,the stability of Wnt3 is weakened;the binding ability of Wnt3 to FZD7 decreases when both sites are mutated simultaneously;and the level of proteins related to the Wnt/β-catenin signaling pathway is downregulated.Cell proliferation,migration and invasion are also weakened in the case of single 301 site and double-site mutations.CONCLUSION These results indicate that by inhibiting the N-glycosylation of Wnt3,the proliferation,migration,invasion and colony formation abilities of liver cancer cells can be weakened,which might provide new therapeutic strategies for clinical liver cancer in the future.
基金This research was supported by the Intramural Research Program of the National Human Genome Research Institute(ZIAHG200386-05).
文摘Background:We are using genetics to identify genes specifically involved in hearing regeneration.In a large-scale genetic screening,we identified mgat5a,a gene in the N-glycosylation biosynthesis pathway whose activity negatively impacts hair cell regeneration.Methods:We used a combination of mutant analysis in zebrafish and a hair cell regeneration assay to phenotype the loss of Mgat5a activity in zebrafish.We used pharmacological inhibition of N-glycosylation by swansonine.We also used over-expression analysis by mRNA injections to demonstrate how changes in N-glycosylation can alter cell signaling.Results:We found that mgat5a was expressed in multiple tissues during zebrafish embryo development,particularly enriched in neural tissues including the brain,retina,and lateral line neuromasts.An mgat5a insertional mutation and a CRISPR/Cas9-generated truncation mutation both caused an enhancement of hair cell regeneration which could be phenocopied by pharmacological inhibition with swansonine.In addition to hair cell regeneration,inhibition of the N-glycosylation pathway also enhanced the regeneration of lateral line axon and caudal fins.Further analysis showed that N-glycosylation altered the responsiveness of TGF-beta signaling.Conclusions:The findings from this study provide experimental evidence for the involvement of N-glycosylation in tissue regeneration and cell signaling.
基金an output of a research project implemented as part of the Research Program at the Moscow State University (MSU) Institute for Artificial Intelligence.
文摘Most human-secreted and membrane-bound proteins have covalently attached oligosaccharide chains or glycans.Glycosylation influences the physical and chemical properties of proteins,as well as their biological functions.Unsurprisingly,alterations in protein glycosylation have been implicated in a growing number of human diseases,and glycans are increasingly being considered as potential therapeutic targets,an essential part of therapeutics,and biomarkers.Although glycosylation pathways are biochemically well-studied,little is known about the networks of genes that guide the cell-and tissue-specific regulation of these biochemical reactions in humans in vivo.The lack of a detailed understanding of the mechanisms regulating glycome variation and linking the glycome to human health and disease is slowing progress in clinical applications of human glycobiology.Two of the tools that can provide much sought-after knowledge of human in vivo glycobiology are human genetics and genomics,which offer a powerful data-driven agnostic approach for dissecting the biology of complex traits.This review summarizes the current state of human populational glycogenomics.In Section 1,we provide a brief overview of the N-glycan’s structural organization,and in Section 2,we give a description of the major blood plasma glycoproteins.Next,in Section 3,we summarize,systemize,and generalize the results from current N-glycosylation genome-wide association studies(GWASs)that provide novel knowledge of the genetic regulation of the populational variation of glycosylation.Until now,such studies have been limited to an analysis of the human blood plasma N-glycome and the N-glycosylation of immunoglobulin G and transferrin.While these three glycomes make up a rather limited set compared with the enormous multitude of glycomes of different tissues and glycoproteins,the study of these three does allow for powerful analysis and generalization.Finally,in Section 4,we turn to genes in the established loci,paying particular attention to genes with strong support in Section 5.At the end of the review,in Sections 6 and 7,we describe special cases of interest in light of new discoveries,focusing on possible mechanisms of action and biological targets of genetic variation that have been implicated in human protein N-glycosylation.
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funded by National Natural Science Foundation of China(8177120753)China-Australia International Collaborative Grant(NHMRC APP1112767,NSFC 81561128020)to Wei Wang+1 种基金Zhiyuan Wu was supported by the China Scholarship Council(201908110447)Yulu Zheng and Zheng Guo were supported by the Edith Cowan University Higher Degree by Research Scholarship(ECU-HDR ST10469322 and ST10468211).
基金supported by grants from the National Natural Science Foundation of China(81872682)the Young Taishan Scholars Program of Shandong Province of China(tsqn20161046)+2 种基金the Academic Promotion Programme of Shandong First Medical University(2019RC010)the Shandong Province Higher Educational Young and Innovation Technology Supporting Program(2019KJL004)the Doctoral Scientific Research Foundation of Shandong First Medical University.
基金This work was supported by the National Key Research and Development Program(2017YFC1200204,2017YFA0504803,and 2018YFA0507700)Emergency Project of Zhejiang Provincial Department of Science and Technology(2020C03123-1)+1 种基金Fundamental Research Funds for the Central Universities(2018XZZX001-13)Independent Project Fund of the State Key Laboratory for Diagnosis and Treatment of Infectious Disease.
文摘The coronavirus disease 2019(COVID-19)pandemic has led to worldwide efforts to understand the biological traits of the newly identified human coronavirus(HCoV-19)virus.In this mass spectrometry(MS)-based study,we reveal that out of 21 possible glycosites in the HCoV-19 spike protein(S protein),20 are completely occupied by N-glycans,predominantly of the oligomannose type.All seven glycosylation sites in human angiotensin I converting enzyme 2(hACE2)were found to be completely occupied,mainly by complex N-glycans.However,glycosylation did not directly contribute to the binding affinity between HCoV-19 S protein and hACE2.Additional post-translational modification(PTM)was identified,including multiple methylated sites in both proteins and multiple sites with hydroxylproline in hACE2.Refined structural models of HCoV-19 S protein and hACE2 were built by adding N-glycan and PTMs to recently published cryogenic electron microscopy structures.The PTM and glycan maps of HCoV-19 S protein and hACE2 provide additional structural details for studying the mechanisms underlying host attachment and the immune response of HCoV-19,as well as knowledge for developing desperately needed remedies and vaccines.
基金the European Structural and Invest-ment Funded Grant"CardioMetabolic"(#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“2022 Lupus Research Alliance(LRA)Lupus Innovation Award”.Institutional funding from the Portuguese Foundation for Science and Technology(FCT):projects NORTE-01-0145-FEDER-000029,POCI-01/0145-FEDER-016601,POCI-01-0145-FEDER-028772,and PTDC/MEC-REU/28772/2017(SSP)This study was co-funded by the European Union(ERC Synergy,GlycanSwitch,101071386)+1 种基金Views and opinions expressed are,however,those of the author(s)only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency.The study was also co-funded by the European Union,GlycanTrigger project,Grant Agreement No:101093997Views and opinions expressed are,however,those of the author(s)only and do not necessarily reflect those of the European Union or European Health and Digital Executive Agency.Neither the European Union nor the granting authority can be held responsible for them.A grant was received from the Portuguese group of study in autoimmune diseases(NEDAI)to SSP.MMV(PD/BD/135452/2017,COVID/BD/152488/2022)received funding from the FCT.
文摘T-cell development ensures the formation of diverse repertoires of T-cell receptors(TCRs)that recognize a variety of antigens.Glycosylation is a major posttranslational modification present in virtually all cells,including T-lymphocytes,that regulates activity/functions.Although these structures are known to be involved in TCR-selection in DP thymocytes,it is unclear how glycans regulate other thymic development processes and how they influence susceptibility to disease.Here,we discovered stage-specific glycome compositions during T-cell development in human and murine thymocytes,as well as dynamic alterations.After restricting the N-glycosylation profile of thymocytes to high-mannose structures,using specific glycoengineered mice(Rag1CreMgat1fl/fl),we showed remarkable defects in key developmental checkpoints,includingß-selection,regulatory T-cell generation andγδT-cell development,associated with increased susceptibility to colon and kidney inflammation and infection.We further demonstrated that a single N-glycan antenna(modeled in Rag1CreMgat2fl/fl mice)is the sine-qua-non condition to ensure normal development.In conclusion,we revealed that mannosylated thymocytes lead to a dysregulation in T-cell development that is associated with inflammation susceptibility.
文摘Glycosylation is a common posttranslational modification on membrane-associated and secreted proteins that is of pivotal importance for regulating cell functions.Aberrant glycosylation can lead to uncontrolled cell proliferation,cell-matrix interactions,migration and differentiation,and has been shown to be involved in cancer and other diseases.The epithelial-to-mesenchymal transition is a key step in the metastatic process by which cancer cells gain the ability to invade tissues and extravasate into the bloodstream.This cellular transformation process,which is associated by morphological change,loss of epithelial traits and gain of mesenchymal markers,is triggered by the secreted cytokine transforming growth factor-β(TGF-β).TGF-βbioactivity is carefully regulated,and its effects on cells are mediated by its receptors on the cell surface.In this review,we first provide a brief overview of major types of glycans,namely,N-glycans,O-glycans,glycosphingolipids and glycosaminoglycans that are involved in cancer progression.Thereafter,we summarize studies on how the glycosylation of TGF-βsignaling components regulates TGF-βsecretion,bioavailability and TGF-βreceptor function.Then,we review glycosylation changes associated with TGF-β-induced epithelial-to-mesenchymal transition in cancer.Identifying and understanding the mechanisms by which glycosylation affects TGF-βsignaling and downstream biological responses will facilitate the identification of glycans as biomarkers and enable novel therapeutic approaches.
基金supported by the National Natural Science Foundation of China(Nos.81930097,21977005,82151223)supported by the National Key R&D Program of China(No.2022YFF1203005).
文摘The interaction among type Ⅱ collagen(CⅡ),human DR4 major histocompatibility complex type Ⅱ molecule(MHC Ⅱ)and T-cell receptor(TCR)is associated with the development of rheumatoid arthritis(RA).The activation of T cells can be reduced through exposure to modified CⅡ(263-272)glycopeptide fragment via competitive inhibition with self-antigen.In this work,30 peptides based on the sequence of CⅡ(263-272)were prepared and evaluated for their binding to DR4 protein by surface plasmon resonance(SPR)assay.The effect on the secretion of pro-inflammatory factors by the spleen cells in collagen induced rheumatoid arthritis(CIA)mouse was also investigated.Two N-glycosylated CⅡ peptides were identified to have strong binding to the human recombinant DR4 protein and weak proinflammatory effect.These glycopeptides could be developed as therapeutic saccharide vaccines for the treatment of rheumatoid arthritis(RA).
基金supported by the National Key R&D Plan of China(Grant No.2016YFD0300703)the China Agricultural Research System(Grant No.CARS-01-33)+2 种基金the Program for Changjiang Scholars and Innovative Research Team in University(Grant No.IRT1042)the 111 Project(Grant No.B13006)to YLPthe National Natural Science Foundation of China(Grant No.32001848)to XL。
文摘Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization.However,only a few secretory proteins were functionally characterized in Magnaporthe oryzae,the fungal pathogen causing rice blast disease worldwide.Asparagine-linked glycosylation 3(Alg3)is an a-1,3-mannosyltransferase functioning in the Nglycan synthesis of N-glycosylated secretory proteins.Fungal pathogenicity and cell wall integrity are impaired inΔalg3 mutants,but the secreted proteins affected inΔalg3 mutants are largely unknown.In this study,we compared the secretomes of the wild-type strain and theΔalg3 mutant and identified 51 proteins that require Alg3 for proper secretion.These proteins were predicted to be involved in metabolic processes,interspecies interactions,cell wall organization,and response to chemicals.Nine proteins were selected for further validation.We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae.Moreover,the Nglycosylation of these proteins was significantly changed in theΔalg3 mutant,leading to the decreased protein secretion and abnormal protein localization.Furthermore,we tested the biological functions of two genes,INV1(encoding invertase 1,a secreted invertase)and AMCase(encoding acid mammalian chinitase,a secreted chitinase).The fungal virulence was significantly reduced,and the cell wall integrity was altered in theΔinv1 andΔamcase mutant strains.Moreover,the N-glycosylation was essential for the function and secretion of AMCase.Taken together,our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.
基金supported by National Natural Science Foundation of China(21775110,22074105)Shanghai Science and Technology Commission(14DZ2261100).
文摘Currently,drug resistance of anti-cancer therapy has become the main cause of low survival rate and poor prognosis.Full understanding of drug resistance mechanisms is an urgent request for further development of anti-cancer therapy and improve-ment of prognosis.Here we present our N-glycoproteomics study of putative N-glycoprotein biomarkers of drug resistance in doxorubicin resistance breast cancer cell line michigan cancer foundation-7(MCF-7/ADR)relative to parental michigan cancer foundation-7(MCF-7)cells.Intact N-glycopeptides(IDs)from MCF-7/ADR and MCF-7 cells were enriched with zwitterionic hydrophilic interaction liquid chromatography(ZIC-HILIC),labeled with stable isotopic diethylation(SIDE),and analyzed with C18-RPLC-MS/MS(HCD with stepped normalized collision energies);these IDs were identified with database search engine GPSeeker,and the differentially expressed intact N-glycopeptides(DEGPs)were quantified with GPSeekerQuan.With target-decoy searches and control of spectrum-level FDR≤1%,322 intact N-glycopeptides were identified;these intact N-glycopeptides come from the combination of 249 unique peptide backbones(corresponding to 234 intact N-glycoproteins)and 90 monosaccharide compositions(corresponding to 248 putative N-glycosites).The sequence structures of 165 IDs were confirmed with structure-diagnostic fragment ions.With the criteria of observation at least twice among the three technical replicates,≥1.5-fold change and p value<0.05,20 DEGPs were quantified,where five of them were up-regulated and 15 of them were down-regulated;the corresponding intact N-glycoproteins as putative markers of drug resistance were discussed.
文摘Glucoamylase from Monascus rubiginosus has been found to exist in multiple forms on PAGE pattern （E<sub>1</sub>-E<sub>5</sub>）. Both major molecular forms E<sub>3</sub> and E<sub>4</sub> are glycoproteins.Sugar content as mannose （%） is 7 for E<sub>3</sub> and 9 for E<sub>4</sub>, respectively,
基金This work was supported by grants from National Natural Science Foundation of China(Grant No.31030047)the European Commission Sixth Framework Program(Grant No.SP22-CT-2004-511060).
文摘Plants are known to be efficient hosts for the production of mammalian therapeutic proteins.However,plants produce complex N-glycans bearingβ1,2-xylose and coreα1,3-fucose residues,which are absent in mammals.The immunogenicity and allergenicity of plant-specific Nglycans is a key concern in mammalian therapy.In this study,we amplified the sequences of 2 plant-specific glycosyltransferases from Nicotiana tabacum L.cv Bright Yellow 2(BY2),which is a well-established cell line widely used for the expression of therapeutic proteins.The expression of the endogenous xylosyltranferase(XylT)and fucosyltransferase(FucT)was downregulated by using RNA interference(RNAi)strategy.The xylosylated and core fucosylated N-glycans were significantly,but not completely,reduced in the glycoengineered lines.However,these RNAi-treated cell lines were stable and viable and did not exhibit any obvious phenotype.Therefore,this study may provide an effective and promising strategy to produce recombinant glycoproteins in BY2 cells with humanized N-glycoforms to avoid potential immunogenicity.
基金the National Key Research and Development Program of China(2018YFA0903000)the National Natural Science Foundation of China(81672001)+2 种基金China MoST Emergency Project on COVID-19(2020YFC0840800)Guangdong Basic and Applied Basic Research Foundation(2021A1515110619)the Fundamental Research Funds for the Central Universities(KG16052901).
文摘The Coronavirus disease 2019(COVID-19)has posed a serious threat to global health and the world economy.Antiviral therapies targeting coronavirus are urgently required.The Cepharanthine(CEP)is a traditional Chinese herbal extract.Our previous research revealed that CEP has a very potent anti-coronavirus effect,but its mechanism of action was not fully understood.To investigate the effect of novel coronavirus on protein glycosylation in infected cells and to further investigate the mechanism of action of CEP against coronavirus,a cellular model using coronavirus GX_P2V infection of Vero E6 cells was established.The effect of coronavirus GX_P2V on host cell protein glycosylation was investigated by N-glycoproteomic analysis,and the antagonistic effect of CEP on the abnormal protein glycosylation caused by coronavirus was analyzed.The results showed that GX_P2V could cause abnormal changes in protein glycosylation levels in host cells,while CEP could partially antagonize the abnormal protein glycosylation caused by GX_P2V.In addition,we also found that CEP could regulate the glycosylation level of coronavirus S protein.In conclusion,this article provides important ideas about the infection mechanism of novel coronaviruses,providing evidence for CEP as a promising therapeutic option for coronavirus infection.
