Tobacco(Nicotiana tabacum)is a widely used platform for producing recombinant proteins for clinical applications.However,achieving mammalian-like glycosylation modifications in plantderived therapeutic proteins remain...Tobacco(Nicotiana tabacum)is a widely used platform for producing recombinant proteins for clinical applications.However,achieving mammalian-like glycosylation modifications in plantderived therapeutic proteins remains challenging,particularly in the case of fucosylation mediated by fucosyltransferases(FUTs).In this study,an RNA interference(RNAi)plasmid targeting the first exon ofα-1,3-fucosyltransferase 4(FUT4)gene was constructed,named as FUT4-RNAi.Using Agrobacterium-mediated transformation with EHA105 harboring the FUT4-RNAi plasmid,we obtained 29 regenerated tobacco lines,17 of which were confirmed as putatively positive by PCR.The mRNA transcript accumulation of the FUT4 gene was significantly reduced in 16 out of the 17 transgenic lines compared to the negative control,cv.Yunyan 87.Among these,11 lines(4^(#),6,7,11^(#),12^(#),15^(#),19^(#),22^(#),26^(#),28^(#) and 29^(#))showed FUT4 transcript levels below 25%of those in cv.Yunyan 87.Four lines(7^(#),12^(#),15^(#),and 29^(#))with the lowest mRNA levels were selected for further analysis by western blotting(WB)and enzyme-linked immunosorbent assay(ELISA).The results confirmed a significant decrease in FUT4 protein levels in these lines compared with that in cv.Yunyan 87,with line 29*showing less than 13%of the FUT4 protein content compared to cv.Yunyan 87.This study successfully developed a tobacco chassis with severely downregulated FUT4 expression,laying an important foundation for the production of human therapeutic proteins using a plant expression system.展开更多
AIM:To investigate the contribution of fucosyltransferase 2 (FUT2) variants to the genetic susceptibility and clinical heterogeneity of ulcerative colitis (UC) between Han and Uyghur patients in Xinjiang, China. METHO...AIM:To investigate the contribution of fucosyltransferase 2 (FUT2) variants to the genetic susceptibility and clinical heterogeneity of ulcerative colitis (UC) between Han and Uyghur patients in Xinjiang, China. METHODS:A total of 102 UC patients (53 Han patients including 22 men and 31 women, and 49 Uyghur patients including 25 men and 24 women; aged 48 ± 16 years) and 310 age-and sex-matched healthy controls were enrolled from January 2010 to May 2011 in Xinjiang People's Hospital of China. UC was diagnosed based on the clinical, endoscopic and histological findings following Lennard-Jones criteria. Blood samples were collected and genomic DNA was extracted by the routine laboratory methods. Polymerase chain reaction-sequence-based typing method was used to identify FUT2 variants rs281377, rs1047781, rs601338 and rs602662. Genotypic and allelic frequencies were documented and compared between the UC patients and the healthy controls. Genotypic frequencies were also compared between Han and Uyghur patients. Potential association of genetic variation and UC between Han and Uyghur patients was examined. RESULTS: rs281377 was found significantly associated with UC in the Han population as compared with the controls (P = 0.011) while rs281377 was not associated with UC in the Uyghur population (P = 0.06). TT homozygous rs281377 frequencies were higher in the UC groups than in the controls (88.7% vs 68.7% and 55.1% vs 50.3%). rs1047781 was specifically associated with UC in the Uyghur population (P = 0.001), but not associated with UC in the Han population (P = 0.13). TT homozygous rs1047781 frequencies were lower in the UC groups than in the controls (9.5% vs 11.8% and 4.0% vs 6.7%). rs601338 was statistically related to UC in both populations (Han, P = 0.025; Uyghur, P = 8.33 × 10 -5 ). AA homozygous rs601338 frequencies were lower in the UC groups than in the controls (0% vs 1.8% and 12.2% vs 13.4%). No association was found between rs602662 and UC in both Han and the Uyghur populations. Allelic analysis showed that rs281377 allele was significantly associated with UC in the Han population as compared with the controls [P = 0.001, odd ratio (OR) = 0.26], however, it was not associated with UC in the Uyghur population (P = 0.603, OR = 1.14), and rs1047781 allele was associated with UC in the Uyghur population (P = 0.001, OR = 0.029) while it was not associated with UC in the Han population (P = 0.074, OR = 0.62). Moreover, rs601338 was associated with UC in both Han (P = 0.005, OR = 0.1) and Uyghur pop- ulations (P = 0.002, OR = 0.43). Meta analysis showed that rs1047781 and rs601338 conferred risk of UC as compared with the controls [P = 0.005, OR = 0.47; P = 0.0003, OR = 0.35; 95% confidence interval (CI) = 0.31-0.72 and 0.21-0.58], but rs281377 and rs602662 showed no statistically significant differences betweenpatients with UC and controls (P = 0.10, OR = 0.71; P = 0.68, OR = 0.09; 95% CI = 0.47-1.07 and 0.56-1.47). CONCLUSION:Functionally relevant FUT2 gene variants are associated with UC, suggesting that they play a potential role in the pathogenesis of UC and may contribute to the clinical heterogeneity of UC between Han and Uyghur patients.展开更多
Glycosylation is a common post-translational modification in eukaryotic cells.It is involved in the production of many biologically active glycoproteins and the regulation of protein structure and function.Core fucosy...Glycosylation is a common post-translational modification in eukaryotic cells.It is involved in the production of many biologically active glycoproteins and the regulation of protein structure and function.Core fucosylation plays a vital role in the immune response.Most immune system molecules are core fucosylated glycoproteins such as complements,cluster differentiation antigens,immunoglobulins,cytokines,major histocompatibility complex molecules,adhesion molecules,and immune molecule synthesis-related transcription factors.These core fucosylated glycoproteins play important roles in antigen recognition and clearance,cell adhesion,lymphocyte activation,apoptosis,signal transduction,and endocytosis.Core fucosylation is dominated by fucosyltransferase 8(Fut8),which catalyzes the addition ofα-1,6-fucose to the innermost GlcNAc residue of N-glycans.Fut8 is involved in humoral,cellular,and mucosal immunity.Tumor immunology is associated with aberrant core fucosylation.Here,we summarize the roles and potential modulatory mechanisms of Fut8 in various immune processes of the gastrointestinal system.展开更多
Objective: To study the correlation of FUT3 gene polymorphism with immune response and inflammatory response in patients with ulcerative colitis (UC). Methods: Patients with UC and patients with irritable bowel syndro...Objective: To study the correlation of FUT3 gene polymorphism with immune response and inflammatory response in patients with ulcerative colitis (UC). Methods: Patients with UC and patients with irritable bowel syndrome who accepted endoscopic biopsy in the First Affiliated Hospital of Jiamusi University between May 2014 and April 2017 were selected as UC group and control group respectively, and the FUT3 gene polymorphism, immune transcription factor and inflammation molecule expression as well as immune cell contents in intestinal mucosa were determined. Results: FUT3 gene rs28362459 and rs3894326 locus genotype constituent ratio in intestinal mucosal tissue of UC group were not significantly different from those of control group whereas rs3745635 locus GA+AA genotype constituent ratio was significantly higher than that of control group;CD4+CD29+ and CD4+IL17A+ cell contents as well as HSF-2, NF-kB, Bax, TNF-α and IL-1β mRNA expression in mucosal tissue of UC group were significantly higher than those of control group whereas CD4+CD25+Foxp3+ cell content as well as SOCS2 and SOCS3 mRNA expression was significantly lower than those of control group;CD4+CD29+ and CD4+IL17A+ cell contents as well as HSF-2, NF-kB, Bax, TNF-α and IL-1β mRNA expression in UC group of mucosal tissue with FUT2 rs3745635 locus GA+AA genotype were significantly higher than those of UC group of mucosal tissue with GG genotype whereas CD4+CD25+Foxp3+ cell content as well as SOCS2 and SOCS3 mRNA expression was significantly lower than those in UC group of mucosal tissue with GG genotype. Conclusion: The FUT3 gene rs3745635 locus polymorphism in intestinal mucosa of UC is closely related to immune response and inflammatory response disorders.展开更多
Fucosylation is a post-translational modification that attaches fucose to glycoproteins or glycolipids,thereby influencing their biological functions.Consequently,fucosylation proves indispensable for many biological ...Fucosylation is a post-translational modification that attaches fucose to glycoproteins or glycolipids,thereby influencing their biological functions.Consequently,fucosylation proves indispensable for many biological processes,such as ligand–receptor interaction,cell adhesion,and signal transduction,holding critical clinical significance in the genesis and development of diseases.Recent studies further unveiled the clinical significance and molecular mechanism underlying the pathogenetic role of aberrant fucosylation.Herein,we summarize the effects of fucosylation in digestive system inflammatory diseases and cancers,primarily concentrating on the intestine,stomach,liver,and pancreas,from the aspects of the genetic risks of fucosyltransferase mutation,the roles of aberrant fucosylated glycans as diagnostic biomarkers,and the molecular mechanisms of fucosylation-related gene-induced disorders.Finally,we discuss therapeutic strategies targeting fucosylation by fucose or fucosylation inhibitors.We aim to elaborate on the current understanding and provide novel insights into the role of fucosylation in digestive diseases,hoping to facilitate future studies and resolve clinical issues.展开更多
Background：Core fucosylation （CF）,catalyzed by α-1,6 fucosyltransferase （Fut8） in mammals,plays an important role in pathological processes through posttranslational modification of key signaling receptor protei...Background：Core fucosylation （CF）,catalyzed by α-1,6 fucosyltransferase （Fut8） in mammals,plays an important role in pathological processes through posttranslational modification of key signaling receptor proteins,including transforming growth factor （TGF）-β receptors and platelet-derived growth factor （PDGF） receptors.However,its effect on peritoneal fibrosis is unknown.Here,we investigated its influence on epithelial-mesenchymal transition （EMT） of rat peritoneal mesothelial cells （PMCs） in vitro induced by a high-glucose （HG） culture solution.Methods：Rat PMCs were first cultured in a HG （2.5%） culture solution to observe the CF expression level （fluorescein isothiocyanate-lens culinaris agglutinin）,we next established a knockdown model of rat PMCs in vitro with Fut8 small interfering RNA （siRNA） to observe whether inhibiting CF decreases the messenger RNA （mRNA） expression and protein expression of Fut8 and reverses EMT status.Rat PMCs were randomly divided into control group,mock group （transfected with scrambled siRNA）,Fut8 siRNA group,HG group,HG ＋ mock group,and HG ＋ Fut8 siRNA group.Finally,we examined the activation of TGF-β/Smad2/3 signaling and PDGF/extracellular signal-regulated kinase （ERK） signaling to observe the influence of CF on them.Results：CF,Fut8 mRNA,and protein expression were all significantly upregulated in HG-induced EMT model than those in the control rat PMCs （P 〈 0.05）.Fut8 siRNA successfully blocked CF of TGF-β receptors and PDGF receptors and attenuated the EMT status （E-cadherin and α-SMA and phenotypic changes） in HG-induced rat PMCs.In TGF-β/Smad2/3 signaling,Fut8 siRNA did not suppress the protein expression of TGF-3 receptors and Smad2/3;however,it significantly suppressed the phosphowlation of Smad2/3 （relative expression folds of HG ＋ Fut8 group vs.HG group：7.6 ± 0.4 vs.15.1 ± 0.6,respectively,P 〈 0.05）.In PDGF/ERK signaling,Fut8 siRNA did not suppress the protein expression of PDGF receptors and ERK,but it significantly suppressed the phosphorylation of ERK （relative expression folds of HG ＋ Fut8 group vs.HG group：8.7 ± 0.9 vs.15.6 ± 1.2,respectively,P 〈 0.05）.Blocking CF inactivated the activities of TGF-β and PDGF signaling pathways,and subsequently blocked EMT.Conclusions：These results demonstrate that CF contributes to rat PMC EMT.and that blocking it attenuates EMT.CF regulation is a potential therapeutic target of peritoneal fibrosis.展开更多
基金supported by Key Research and Development Program of China National Tobacco Corporation (2021-150-110202102026)。
文摘Tobacco(Nicotiana tabacum)is a widely used platform for producing recombinant proteins for clinical applications.However,achieving mammalian-like glycosylation modifications in plantderived therapeutic proteins remains challenging,particularly in the case of fucosylation mediated by fucosyltransferases(FUTs).In this study,an RNA interference(RNAi)plasmid targeting the first exon ofα-1,3-fucosyltransferase 4(FUT4)gene was constructed,named as FUT4-RNAi.Using Agrobacterium-mediated transformation with EHA105 harboring the FUT4-RNAi plasmid,we obtained 29 regenerated tobacco lines,17 of which were confirmed as putatively positive by PCR.The mRNA transcript accumulation of the FUT4 gene was significantly reduced in 16 out of the 17 transgenic lines compared to the negative control,cv.Yunyan 87.Among these,11 lines(4^(#),6,7,11^(#),12^(#),15^(#),19^(#),22^(#),26^(#),28^(#) and 29^(#))showed FUT4 transcript levels below 25%of those in cv.Yunyan 87.Four lines(7^(#),12^(#),15^(#),and 29^(#))with the lowest mRNA levels were selected for further analysis by western blotting(WB)and enzyme-linked immunosorbent assay(ELISA).The results confirmed a significant decrease in FUT4 protein levels in these lines compared with that in cv.Yunyan 87,with line 29*showing less than 13%of the FUT4 protein content compared to cv.Yunyan 87.This study successfully developed a tobacco chassis with severely downregulated FUT4 expression,laying an important foundation for the production of human therapeutic proteins using a plant expression system.
基金Supported by National Natural Science Foundation of China,No. 30871148 and No. 81160052Natural Science Foundation of Xinjiang Uyghur Autonomous Region of China, No.2009211A26
文摘AIM:To investigate the contribution of fucosyltransferase 2 (FUT2) variants to the genetic susceptibility and clinical heterogeneity of ulcerative colitis (UC) between Han and Uyghur patients in Xinjiang, China. METHODS:A total of 102 UC patients (53 Han patients including 22 men and 31 women, and 49 Uyghur patients including 25 men and 24 women; aged 48 ± 16 years) and 310 age-and sex-matched healthy controls were enrolled from January 2010 to May 2011 in Xinjiang People's Hospital of China. UC was diagnosed based on the clinical, endoscopic and histological findings following Lennard-Jones criteria. Blood samples were collected and genomic DNA was extracted by the routine laboratory methods. Polymerase chain reaction-sequence-based typing method was used to identify FUT2 variants rs281377, rs1047781, rs601338 and rs602662. Genotypic and allelic frequencies were documented and compared between the UC patients and the healthy controls. Genotypic frequencies were also compared between Han and Uyghur patients. Potential association of genetic variation and UC between Han and Uyghur patients was examined. RESULTS: rs281377 was found significantly associated with UC in the Han population as compared with the controls (P = 0.011) while rs281377 was not associated with UC in the Uyghur population (P = 0.06). TT homozygous rs281377 frequencies were higher in the UC groups than in the controls (88.7% vs 68.7% and 55.1% vs 50.3%). rs1047781 was specifically associated with UC in the Uyghur population (P = 0.001), but not associated with UC in the Han population (P = 0.13). TT homozygous rs1047781 frequencies were lower in the UC groups than in the controls (9.5% vs 11.8% and 4.0% vs 6.7%). rs601338 was statistically related to UC in both populations (Han, P = 0.025; Uyghur, P = 8.33 × 10 -5 ). AA homozygous rs601338 frequencies were lower in the UC groups than in the controls (0% vs 1.8% and 12.2% vs 13.4%). No association was found between rs602662 and UC in both Han and the Uyghur populations. Allelic analysis showed that rs281377 allele was significantly associated with UC in the Han population as compared with the controls [P = 0.001, odd ratio (OR) = 0.26], however, it was not associated with UC in the Uyghur population (P = 0.603, OR = 1.14), and rs1047781 allele was associated with UC in the Uyghur population (P = 0.001, OR = 0.029) while it was not associated with UC in the Han population (P = 0.074, OR = 0.62). Moreover, rs601338 was associated with UC in both Han (P = 0.005, OR = 0.1) and Uyghur pop- ulations (P = 0.002, OR = 0.43). Meta analysis showed that rs1047781 and rs601338 conferred risk of UC as compared with the controls [P = 0.005, OR = 0.47; P = 0.0003, OR = 0.35; 95% confidence interval (CI) = 0.31-0.72 and 0.21-0.58], but rs281377 and rs602662 showed no statistically significant differences betweenpatients with UC and controls (P = 0.10, OR = 0.71; P = 0.68, OR = 0.09; 95% CI = 0.47-1.07 and 0.56-1.47). CONCLUSION:Functionally relevant FUT2 gene variants are associated with UC, suggesting that they play a potential role in the pathogenesis of UC and may contribute to the clinical heterogeneity of UC between Han and Uyghur patients.
基金Supported by the National Natural Science Foundation of China,No.32171279Natural Science Foundation of Liaoning Province,No.2022-BS-254,and No.2022-MS-317the Project of Dalian Medical Science Research,No.2012026.
文摘Glycosylation is a common post-translational modification in eukaryotic cells.It is involved in the production of many biologically active glycoproteins and the regulation of protein structure and function.Core fucosylation plays a vital role in the immune response.Most immune system molecules are core fucosylated glycoproteins such as complements,cluster differentiation antigens,immunoglobulins,cytokines,major histocompatibility complex molecules,adhesion molecules,and immune molecule synthesis-related transcription factors.These core fucosylated glycoproteins play important roles in antigen recognition and clearance,cell adhesion,lymphocyte activation,apoptosis,signal transduction,and endocytosis.Core fucosylation is dominated by fucosyltransferase 8(Fut8),which catalyzes the addition ofα-1,6-fucose to the innermost GlcNAc residue of N-glycans.Fut8 is involved in humoral,cellular,and mucosal immunity.Tumor immunology is associated with aberrant core fucosylation.Here,we summarize the roles and potential modulatory mechanisms of Fut8 in various immune processes of the gastrointestinal system.
基金Project of Heilongjiang Provincial Natural Science Foundation(No.:H201367).Scientific Research Project of Health Department of Heilongjiang Province(:2013245).
文摘Objective: To study the correlation of FUT3 gene polymorphism with immune response and inflammatory response in patients with ulcerative colitis (UC). Methods: Patients with UC and patients with irritable bowel syndrome who accepted endoscopic biopsy in the First Affiliated Hospital of Jiamusi University between May 2014 and April 2017 were selected as UC group and control group respectively, and the FUT3 gene polymorphism, immune transcription factor and inflammation molecule expression as well as immune cell contents in intestinal mucosa were determined. Results: FUT3 gene rs28362459 and rs3894326 locus genotype constituent ratio in intestinal mucosal tissue of UC group were not significantly different from those of control group whereas rs3745635 locus GA+AA genotype constituent ratio was significantly higher than that of control group;CD4+CD29+ and CD4+IL17A+ cell contents as well as HSF-2, NF-kB, Bax, TNF-α and IL-1β mRNA expression in mucosal tissue of UC group were significantly higher than those of control group whereas CD4+CD25+Foxp3+ cell content as well as SOCS2 and SOCS3 mRNA expression was significantly lower than those of control group;CD4+CD29+ and CD4+IL17A+ cell contents as well as HSF-2, NF-kB, Bax, TNF-α and IL-1β mRNA expression in UC group of mucosal tissue with FUT2 rs3745635 locus GA+AA genotype were significantly higher than those of UC group of mucosal tissue with GG genotype whereas CD4+CD25+Foxp3+ cell content as well as SOCS2 and SOCS3 mRNA expression was significantly lower than those in UC group of mucosal tissue with GG genotype. Conclusion: The FUT3 gene rs3745635 locus polymorphism in intestinal mucosa of UC is closely related to immune response and inflammatory response disorders.
基金supported by the National Natural Science Foundation of China(No.92268108,82170570,81974062).
文摘Fucosylation is a post-translational modification that attaches fucose to glycoproteins or glycolipids,thereby influencing their biological functions.Consequently,fucosylation proves indispensable for many biological processes,such as ligand–receptor interaction,cell adhesion,and signal transduction,holding critical clinical significance in the genesis and development of diseases.Recent studies further unveiled the clinical significance and molecular mechanism underlying the pathogenetic role of aberrant fucosylation.Herein,we summarize the effects of fucosylation in digestive system inflammatory diseases and cancers,primarily concentrating on the intestine,stomach,liver,and pancreas,from the aspects of the genetic risks of fucosyltransferase mutation,the roles of aberrant fucosylated glycans as diagnostic biomarkers,and the molecular mechanisms of fucosylation-related gene-induced disorders.Finally,we discuss therapeutic strategies targeting fucosylation by fucose or fucosylation inhibitors.We aim to elaborate on the current understanding and provide novel insights into the role of fucosylation in digestive diseases,hoping to facilitate future studies and resolve clinical issues.
基金This work was supported by a grant from the National Natural Science Foundation of China （No. 81530021）.
文摘Background：Core fucosylation （CF）,catalyzed by α-1,6 fucosyltransferase （Fut8） in mammals,plays an important role in pathological processes through posttranslational modification of key signaling receptor proteins,including transforming growth factor （TGF）-β receptors and platelet-derived growth factor （PDGF） receptors.However,its effect on peritoneal fibrosis is unknown.Here,we investigated its influence on epithelial-mesenchymal transition （EMT） of rat peritoneal mesothelial cells （PMCs） in vitro induced by a high-glucose （HG） culture solution.Methods：Rat PMCs were first cultured in a HG （2.5%） culture solution to observe the CF expression level （fluorescein isothiocyanate-lens culinaris agglutinin）,we next established a knockdown model of rat PMCs in vitro with Fut8 small interfering RNA （siRNA） to observe whether inhibiting CF decreases the messenger RNA （mRNA） expression and protein expression of Fut8 and reverses EMT status.Rat PMCs were randomly divided into control group,mock group （transfected with scrambled siRNA）,Fut8 siRNA group,HG group,HG ＋ mock group,and HG ＋ Fut8 siRNA group.Finally,we examined the activation of TGF-β/Smad2/3 signaling and PDGF/extracellular signal-regulated kinase （ERK） signaling to observe the influence of CF on them.Results：CF,Fut8 mRNA,and protein expression were all significantly upregulated in HG-induced EMT model than those in the control rat PMCs （P 〈 0.05）.Fut8 siRNA successfully blocked CF of TGF-β receptors and PDGF receptors and attenuated the EMT status （E-cadherin and α-SMA and phenotypic changes） in HG-induced rat PMCs.In TGF-β/Smad2/3 signaling,Fut8 siRNA did not suppress the protein expression of TGF-3 receptors and Smad2/3;however,it significantly suppressed the phosphowlation of Smad2/3 （relative expression folds of HG ＋ Fut8 group vs.HG group：7.6 ± 0.4 vs.15.1 ± 0.6,respectively,P 〈 0.05）.In PDGF/ERK signaling,Fut8 siRNA did not suppress the protein expression of PDGF receptors and ERK,but it significantly suppressed the phosphorylation of ERK （relative expression folds of HG ＋ Fut8 group vs.HG group：8.7 ± 0.9 vs.15.6 ± 1.2,respectively,P 〈 0.05）.Blocking CF inactivated the activities of TGF-β and PDGF signaling pathways,and subsequently blocked EMT.Conclusions：These results demonstrate that CF contributes to rat PMC EMT.and that blocking it attenuates EMT.CF regulation is a potential therapeutic target of peritoneal fibrosis.
