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1.
Zhongguo Zhong Yao Za Zhi ; 46(1): 86-93, 2021 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-33645056

RESUMO

Caffeic acid and its oligomers are the main water-soluble active constituents of the traditional Chinese medicine(TCM) Arnebiae Radix. These compounds possess multiple biological activities such as antimicrobial, antioxidant, cardiovascular protective, liver protective, anti-liver fibrosis, antiviral and anticancer activities. The phenylpropanoid pathway in plants is responsible for the biosynthesis of caffeic acid and its oligomers. Glycosylation can change phenylpropanoid solubility, stability and toxic potential, as well as influencing compartmentalization and biological activity. In view of the important role played by de-glycosylation in the regulation of phenylpropanoid homeostasis, the biosynthesis of caffeic acid and its oligomers are supposed to be under the control of relative UDP-glycosyltransferases(UGTs). Through the data mining of Arnebia euchroma transcriptome, we cloned 15 full-length putative UGT genes. After recombinant expression using the prokaryotic system, the crude enzyme solution of the putative UGTs was examined for the glycosylation activities towards caffeic acid and rosmarinic acid in vitro. AeUGT_01, AeUGT_02, AeUGT_03, AeUGT_04 and AeUGT_10 were able to glycosylate caffeic acid and/or rosmarinic acid resulting in different mono-and/or di-glycosylated products in the UPLC-MS analyses. The characterized UGTs were distantly related to each other and divided into different clades of the phylogenetic tree. Based on the observation that each characterized UGT exhibited substrate or catalytic similarity with the members in their own clade, we supposed the glycosylation abilities towards caffeic acid and/or rosmarinic acid were evolved independently in different clades. The identification of caffeic acid and rosmarinic acid UGTs from A. euchroma could lead to deeper understanding of the caffeic acid oligomers biosynthesis and its regulation. Furthermore, these UGTs might be used for regiospecific glycosylation of caffeic acid and rosmarinic acid to produce bioactive compounds for potential therapeutic applications.


Assuntos
Boraginaceae , Glicosiltransferases , Boraginaceae/genética , Ácidos Cafeicos , Cromatografia Líquida , Cinamatos , Clonagem Molecular , Depsídeos , Glicosiltransferases/genética , Filogenia , Espectrometria de Massas em Tandem
2.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(3): 278-281, 2021 Mar 10.
Artigo em Chinês | MEDLINE | ID: mdl-33751542

RESUMO

OBJECTIVE: To explore the molecular basis for an individual with Bw subtype. METHODS: Routine serological reactions were used to determine the surface antigens of erythrocytes and antibodies in serum. PCR-sequence-based typing (PCR-SBT) was used to analyze the coding regions of the ABO gene and erythroid-specific regulatory element in its intron 1. Amplicons for exons 5 to 7 containing the variant site were subjected to TA cloning for the isolation of the haploid and verification of the sequence. The 3D structure of mutant protein was predicted with Pymol software. Changes of amino acid residues and structural stability were also analyzed. RESULTS: Serological assay showed that the individual had weakened B antigen and anti-B antibody in his serum. His genotype was determined as ABO*B.01/ABO*O.01.01. Sequencing of the entire coding region of the ABO gene identified an additional heterozygous c.734C/T variant. No variant was found in the erythroid-specific regulatory element of intron 1. Haploid cloning and isolation has obtained an ABO*O.01.01 allele and a ABO*B.01 allele containing a c.734T variant, which has led to substitution of Thr by Ile at position 245 in the functional center of glycosyltransferase. Based on the 3D structure of the protein, the residues binding with the mutation were unchanged, but the bonding distance between the hydrogens was changed with the amino acid substitution. Meanwhile, the connections with water molecules were increased. CONCLUSION: The c.734C>T variant of the GTB gene can lead to an amino acid substitution in the functional center of the enzyme, which in turn may affect the stability of glycosyltransferase B protein and reduceits enzymatic activity.


Assuntos
Sistema ABO de Grupos Sanguíneos , Glicosiltransferases , Sistema ABO de Grupos Sanguíneos/genética , Alelos , Éxons/genética , Genótipo , Glicosiltransferases/genética , Humanos , Masculino , Fenótipo
3.
Molecules ; 26(4)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562410

RESUMO

Notch signaling receptors, ligands, and their downstream target genes are dysregulated in pancreatic ductal adenocarcinoma (PDAC), suggesting a role of Notch signaling in pancreatic tumor development and progression. However, dysregulation of Notch signaling by post-translational modification of Notch receptors remains poorly understood. Here, we analyzed the Notch-modifying glycosyltransferase involved in the regulation of the ligand-dependent Notch signaling pathway. Bioinformatic analysis revealed that the expression of epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine (EOGT) and Lunatic fringe (LFNG) positively correlates with a subset of Notch signaling genes in PDAC. The lack of EOGT or LFNG expression inhibited the proliferation and migration of Panc-1 cells, as observed by the inhibition of Notch activation. EOGT expression is significantly increased in the basal subtype, and low expression of both EOGT and LFNG predicts better overall survival in PDAC patients. These results imply potential roles for EOGT- and LFNG-dependent Notch signaling in PDAC.


Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Glicosiltransferases/genética , N-Acetilglucosaminiltransferases/genética , Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Biologia Computacional , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Receptores Notch/genética , Transdução de Sinais/genética
4.
Sheng Wu Gong Cheng Xue Bao ; 37(1): 112-129, 2021 Jan 25.
Artigo em Chinês | MEDLINE | ID: mdl-33501794

RESUMO

Water solubility, stability, and bioavailability, can be substantially improved after glycosylation. Glycosylation of bioactive compounds catalyzed by glycoside hydrolases (GHs) and glycosyltransferases (GTs) has become a research hotspot. Thanks to their rich sources and use of cheap glycosyl donors, GHs are advantageous in terms of scaled catalysis compared to GTs. Among GHs, sucrose phosphorylase has attracted extensive attentions in chemical engineering due to its prominent glycosylation activity as well as its acceptor promiscuity. This paper reviews the structure, catalytic characteristics, and directional redesign of sucrose phosphorylase. Meanwhile, glycosylation of diverse chemicals with sucrose phosphorylase and its coupling applications with other biocatalysts are summarized. Future research directions were also discussed based on the current research progress combined with our working experience.


Assuntos
Glucosiltransferases , Glicosiltransferases , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação , Glicosiltransferases/genética
5.
GM Crops Food ; 12(1): 242-250, 2021 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-33393843

RESUMO

Since the successful creation of DJ-526, a resveratrol-enriched rice cultivar, research has focused on resveratrol production because of its great potential in pharmaceutical applications. However, the utilization of resveratrol in DJ-526 is limited by glycosylation, which converts resveratrol to its glucoside (piceid), in a process driven by glycosyltransferase. The verification of resveratrol-dependent glycosyltransferase activity is an essential strategy for improving resveratrol production in DJ-526 rice. In this study, 27 candidate glycosyltransferases were evaluated in germinated seeds. Among the candidates, only R12 exhibited upregulation related to increased resveratrol and piceid content during seed germination, whereas various effects on the activity of glycosyltransferase were observed by the presence of a bio-elicitor. Yeast extract tended to enhance glycosyltransferase activity by seven candidates, and a specific peak for an unknown compound production was identified. Conversely, chitosan acted as a glycosyltransferase inhibitor. Our results suggested that R12 and R19 are the most relevant candidate resveratrol-dependent glycosyltransferases in DJ-526 seeds during germination and elicitation. Future research should assess the possibility of silencing these candidate genes in an effort to improve resveratrol levels in DJ-526 rice.


Assuntos
Oryza , Germinação , Glicosiltransferases/genética , Oryza/genética , Resveratrol , Sementes
6.
Plant Physiol Biochem ; 159: 226-233, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33387851

RESUMO

Plants can reduce or eliminate the damage caused by herbicides and gain herbicide resistance, which is an important theoretical basis for the development of herbicide-resistant crops at this stage. Thus, discovering novel herbicide-resistant genes to produce diverse herbicide-resistant crop species is of great value. The glycosyltransferases that commonly exist in plant kingdom modify the receptor molecules to change their physical characteristics and biological activities, and thus possess an important potential to be used in the herbicide-resistance breeding. Here, we identified a novel herbicide-induced UDP-glycosyltransferase 91C1 (UGT91C1) from Arabidopsis thaliana and demonstrated its glucosylating activity toward sulcotrione, a kind of triketone herbicides widely used in the world. Overexpression of UGT91C1 gene enhanced the Arabidopsis tolerance to sulcotrione. While, ugt91c1 mutant displayed serious damage and reduced chlorophyll contents in the presence of sulcotrione, suggesting an important role of UGT91C1 in herbicide detoxification through glycosylation. Moreover, it was also noted that UGT91C1 can affect tyrosine metabolism by reducing the sulcotrione toxicity. Together, our identification of glycosyltransferase UGT91C1, as a potential gene conferring herbicide detoxification through glucosylation, may open up a new possibility for herbicide resistant breeding of crop plants and environmental phytoremediation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Glicosiltransferases/metabolismo , Resistência a Herbicidas , Inativação Metabólica , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Glicosiltransferases/genética , Resistência a Herbicidas/genética , Herbicidas/metabolismo , Herbicidas/toxicidade , Inativação Metabólica/genética
7.
J Agric Food Chem ; 69(5): 1714-1726, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33512142

RESUMO

Ginseng contains a variety of flavonol glycosides that possess diverse biological activities; however, scant information of flavonoid glycosylation was reported in ginseng. We found that panasenoside and kaempferol 3-O-glucoside were commonly accumulated along with cultivation years in leaves. In order to explore the procedure of flavonol glycosylation in ginseng, 50 UDP-glycosyltransferases (UGTs) were screened out using differentiated data-independent acquisition (DIA) proteomics and phylogenetic analysis. UGT92A10 and UGT94Q4 were found contributing to the formation of kaempferol 3-O-glucoside. UGT73A18, UGT74T4, and UGT75W1 could catalyze galactosylation of kaempferol 3-O-glucoside. Ser278, Trp335, Gln338, and Val339 were found forming hydrogen bonds with UDP-galactose in UGT75W1 by docking. MeJA induced transcripts of UGT73A18 and UGT74T4 by over fourfold, consistent with the decrease of kaempferol 3-O-glucoside, which indicated that these genes may be related to resisting adversity stress in ginseng. These results highlight the significance of integrative metabolite profiles, proteomics, and phylogenetic analysis for exploring flavonol glycosylation in ginseng.


Assuntos
Flavonoides/biossíntese , Glucosídeos/biossíntese , Glicosiltransferases/metabolismo , Panax/genética , Proteínas de Plantas/metabolismo , Flavonoides/química , Regulação da Expressão Gênica de Plantas , Glucosídeos/química , Glicosilação , Glicosiltransferases/genética , Panax/classificação , Panax/enzimologia , Panax/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteômica
8.
Plant Physiol Biochem ; 159: 28-36, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33321375

RESUMO

Salt stress is a serious abiotic stressor impeding plant growth and crop production around the world. Plant glycosyltransferases are thought to serve important roles in dealing with stress conditions, however, the functional role of how UGTs cope with salt stress is not well understood. Carex rigescens (Franch.) V. Krecz, is a widely distributed species of turfgrass with strong salinity tolerance found in northern China. To investigate how the glycosyltransferase gene, CrUGT87A1, functions in C. rigescens, we performed analyses of cloning, transcriptional expression, subcellular localization, and overexpression. The full-length sequence of CrUGT87A1 is 1455 bp with a 1338 bp length ORF, which encodes 445 amino acids, while CrUGT87A1 was found to be a nuclear and plasmalemma-localized protein. We found that the transcriptional expression of CrUGT87A1 was up-regulated under ABA, heat, salt, and drought treatments in leaf tissues. CrUGT87A1 overexpression in Arabidopsis plants had a significantly higher germination rate, better growth and physiology, and a higher expression levels of transcripts related to salt stress-related genes under high-salinity conditions, suggesting that CrUGT87A1 is involved in salt tolerance. The transcriptional expression of genes related to flavonoid-synthesis related and the flavonoid content reflected higher accumulations of flavonoids in transgenic plants. Our study demonstrated that CrUGT87A1 could play an important role in resisting salt stress due to increased flavonoid accumulation, which can promote antioxidation when dealing with high-salinity conditions. This study advances our collective understanding of the functional role of UGTs and can be used to improve the salt tolerance and breeding of crops and plants.


Assuntos
Arabidopsis , Carex (Planta) , Flavonoides , Glicosiltransferases , Proteínas de Plantas , Proteínas Recombinantes , Tolerância ao Sal , Arabidopsis/enzimologia , Arabidopsis/genética , Carex (Planta)/genética , Carex (Planta)/metabolismo , China , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas , Glicosiltransferases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tolerância ao Sal/genética
9.
Chemosphere ; 263: 128269, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33297213

RESUMO

Increasing evidence indicates that insect resistance to synthesized insecticides is regulated by the nuclear receptors. However, the underlying mechanisms of this regulation are not clear. Here, we demonstrate that inhibition of hepatocyte nuclear factor 4 (HNF4) confers imidacloprid resistance in the brown planthopper (BPH) Nilaparvata lugens by regulating cytochrome P450 and UDP-glycosyltransferase (UGT) genes. An imidacloprid-resistant strain (Res) exhibited a 251.69-fold resistance to imidacloprid in comparison to the susceptible counterpart (Sus) was obtained by successive selection with imidacloprid. The expression level of HNF4 in the Res strain was lower than that in Sus, and knockdown of HNF4 by RNA interference significantly enhanced the resistance of BPH to imidacloprid. Comparative transcriptomic analysis identified 1400 differentially expressed genes (DEGs) in the HNF4-silenced BPHs compared to controls. Functional enrichment analysis showed that cytochrome P450- and UGT-mediated metabolic detoxification pathways were enriched by the up-regulated DEGs after HNF4 knockdown. Among of them, UGT-1-7, UGT-2B10 and CYP6ER1 were found to be over-expressed in the Res strain, and knockdown of either gene significantly decreased the resistance of BPH to imidacloprid. This study increases our understanding of molecular mechanisms involved in the regulation of insecticide resistance and also provides potential targets for pest management.


Assuntos
Hemípteros , Inseticidas , Animais , Sistema Enzimático do Citocromo P-450/genética , Glicosiltransferases/genética , Hemípteros/genética , Fator 4 Nuclear de Hepatócito , Imidazóis , Resistência a Inseticidas/genética , Inseticidas/toxicidade , Neonicotinoides/toxicidade , Nitrocompostos/toxicidade , Difosfato de Uridina
10.
J Sci Food Agric ; 101(2): 564-572, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32672847

RESUMO

BACKGROUND: Fruit softening is a major determinant of commercial value and shelf life. A transcriptomic analysis of 'Golden Delicious' and 'Golden Del. Reinders' (a bud mutation of 'Golden Delicious' that readily softens) apple fruit was conducted during storage. RESULTS: A comparative analysis of the obtained expression profiles of fruit between two cultivars identified 1345 upregulated and 3475 downregulated differentially expressed genes (DEGs). The DEGs identified were associated with cellular processes and carbohydrate metabolism and were especially enriched in cell-wall-related genes. Among the cell-wall-related genes, the xyloglucan endotransglucosylase/hydrolases (XTH) gene MdXTHB was significantly upregulated and exhibited high expression levels in 'Golden Del. Reinders' fruit, which had a lower level of firmness relative to 'Golden Delicious'. Overexpression of MdXTHB in both 'Golden Delicious' and 'Fuji', which typically maintain high levels of firmness in storage, exhibited faster rates of softening and an earlier peak of ethylene production than empty-vector-infiltrated fruit did. CONCLUSION: The results of this study indicate that MdXTHB potentially promotes apple fruit softening by degrading the fruit cell wall. This result is also useful to designing further experiments on the molecular regulation of fruit softening in apple. © 2020 Society of Chemical Industry.


Assuntos
Frutas/crescimento & desenvolvimento , Glicosiltransferases/metabolismo , Malus/genética , Proteínas de Plantas/metabolismo , Etilenos/metabolismo , Frutas/química , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Glicosiltransferases/genética , Malus/química , Malus/crescimento & desenvolvimento , Malus/metabolismo , Proteínas de Plantas/genética
11.
Biochem Biophys Res Commun ; 534: 73-78, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33310191

RESUMO

Glycosylation catalyzed by uridine diphosphate-dependent glycosyltransferases (UGT) contributes to the chemical and functional diversity of a number of natural products. Bacillus subtilis Bs-YjiC is a robust and versatile UGT that holds potentials in the biosynthesis of unnatural bioactive ginsenosides. To understand the molecular mechanism underlying the substrate promiscuity of Bs-YjiC, we solved crystal structures of Bs-YjiC and its binary complex with uridine diphosphate (UDP) at resolution of 2.18 Å and 2.44 Å, respectively. Bs-YjiC adopts the classical GT-B fold containing the N-terminal and C-terminal domains that accommodate the sugar acceptor and UDP-glucose, respectively. Molecular docking indicates that the spacious sugar-acceptor binding pocket of Bs-YjiC might be responsible for its broad substrate spectrum and unique glycosylation patterns toward protopanaxadiol-(PPD) and PPD-type ginsenosides. Our study reveals the structural basis for the aglycone promiscuity of Bs-YjiC and will facilitate the protein engineering of Bs-YjiC to synthesize novel bioactive glycosylated compounds.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Ginsenosídeos/química , Ginsenosídeos/metabolismo , Glicosilação , Glicosiltransferases/genética , Modelos Moleculares , Simulação de Acoplamento Molecular , Domínios Proteicos , Sapogeninas/metabolismo , Especificidade por Substrato , Difosfato de Uridina/química , Difosfato de Uridina/metabolismo , Uridina Difosfato Glucose/metabolismo
12.
Zhongguo Zhong Yao Za Zhi ; 45(19): 4574-4581, 2020 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-33164420

RESUMO

Glycosyltransferases(UGTs) are key enzymes in the biosynthesis of ginsenosides, which can catalyze the transfer of glycans from donor molecules to acceptor molecules, and form a variety of biologically active glycoside compounds. With the development of high-throughput sequencing technology, a large number of medicinal plant genome and transcriptome sequences have been catalyzed, while more and more UGT genes have been discovered and verified. The methods of discovering UGT genes include differential expression analysis, homologous sequence screening, gene cluster screening, chemical proteome screening. This paper summarized the research progress of UGT genes screening and functional verification of three valuable Panax medicinal materials P. notoginseng, P. ginseng, and P. quinquefolius, so as to provide a reference for the study of UGT genes in natural products.


Assuntos
Ginsenosídeos , Panax notoginseng , Panax , Genoma de Planta , Glicosiltransferases/genética , Família Multigênica , Panax/genética
13.
Nat Commun ; 11(1): 5162, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33056984

RESUMO

Bioactive natural C-glycosides are rare and chemical C-glycosylation faces challenges while enzymatic C-glycosylation catalyzed by C-glycosyltransferases provides an alternative way. However, only a small number of C-glycosyltransferases have been found, and most of the discovered C-glycosyltransferases prefer to glycosylate phenols with an acyl side chain. Here, a promiscuous C-glycosyltransferase, AbCGT, which is capable of C-glycosylating scaffolds lacking acyl groups, is identified from Aloe barbadensis. Based on the substrate promiscuity of AbCGT, 16 C-glycosides with inhibitory activity against sodium-dependent glucose transporters 2 are chemo-enzymatically synthesized. The C-glycoside 46a shows hypoglycemic activity in diabetic mice and is biosynthesized with a cumulative yield on the 3.95 g L‒1 scale. In addition, the key residues involved in the catalytic selectivity of AbCGT are explored. These findings suggest that AbCGT is a powerful tool in the synthesis of lead compounds for drug discovery and an example for engineering the catalytic selectivity of C-glycosyltransferases.


Assuntos
Aloe/enzimologia , Glicosídeos/biossíntese , Glicosiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/metabolismo , Aloxano/toxicidade , Aloe/genética , Animais , Biocatálise , Glicemia/análise , Glicemia/efeitos dos fármacos , Clonagem Molecular , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/tratamento farmacológico , Descoberta de Drogas/métodos , Feminino , Glicosídeos/farmacologia , Glicosídeos/uso terapêutico , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/isolamento & purificação , Humanos , Masculino , Camundongos , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transportador 2 de Glucose-Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Especificidade por Substrato
14.
PLoS One ; 15(7): e0235344, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628728

RESUMO

A Glycine max (soybean) hemicellulose modifying gene, xyloglucan endotransglycoslase/hydrolase (XTH43), has been identified as being expressed within a nurse cell known as a syncytium developing within the soybean root undergoing the process of defense to infection by the parasitic nematode, Heterodera glycines. The highly effective nature of XTH43 overexpression in suppressing H. glycines parasitism in soybean has led to experiments examining whether the heterologous expression of XTH43 in Gossypium hirsutum (upland cotton) could impair the parasitism of Meloidogyne incognita, that form a different type of nurse cell called a giant cell that is enclosed within a swollen root structure called a gall. The heterologous transgenic expression of XTH43 in cotton resulted in an 18% decrease in the number of galls, 70% decrease in egg masses, 64% decrease in egg production and a 97% decrease in second stage juvenile (J2) production as compared to transgenic controls. The heterologous XTH43 expression does not significantly affect root mass. The results demonstrate XTH43 expression functions effectively in impairing the development of M. incognita at numerous life cycle stages occurring within the cotton root. The experiments reveal that there are highly conserved aspects of the defense response of G. max that can function effectively in G. hirsutum to impair M. incognita having a different method of parasitism.


Assuntos
Glicosiltransferases/genética , Gossypium/parasitologia , Doenças das Plantas/prevenção & controle , Proteínas de Soja/genética , Soja/enzimologia , Tylenchoidea , Animais , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glicosiltransferases/metabolismo , Gossypium/genética , Doenças das Plantas/parasitologia , Raízes de Plantas/parasitologia , Plantas Geneticamente Modificadas , Proteínas de Soja/metabolismo , Soja/genética
15.
Mol Cell ; 78(4): 683-699.e11, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32386575

RESUMO

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides-arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate-binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4- and 3.5-Å structures of a mutant with impaired ACP binding reveal a conformational change, suggesting that ACP may regulate AftD function. Mutagenesis experiments using a conditional knockout constructed in M. smegmatis confirm the essentiality of the putative active site and the ACP binding for AftD function.


Assuntos
Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica/métodos , Glicosiltransferases/metabolismo , Mycobacterium smegmatis/enzimologia , Proteína de Transporte de Acila/genética , Proteínas de Bactérias/genética , Domínio Catalítico , Parede Celular/metabolismo , Galactanos/metabolismo , Glicosiltransferases/genética , Lipopolissacarídeos/metabolismo , Mutação , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Filogenia , Conformação Proteica , Especificidade por Substrato
16.
Nat Chem Biol ; 16(7): 740-748, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32424305

RESUMO

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.


Assuntos
Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Glicosiltransferases/genética , Proteínas de Plantas/genética , Saponinas/biossíntese , Spinacia oleracea/metabolismo , Terpenos/metabolismo , Beta vulgaris/genética , Beta vulgaris/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Celulose/metabolismo , Retículo Endoplasmático/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Glucosiltransferases/metabolismo , Ácido Glucurônico/metabolismo , Glicosilação , Glicosiltransferases/metabolismo , Glycyrrhiza/genética , Glycyrrhiza/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Spinacia oleracea/genética
17.
Nature ; 580(7801): 124-129, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32238941

RESUMO

Pluripotent stem cells are increasingly used to model different aspects of embryogenesis and organ formation1. Despite recent advances in in vitro induction of major mesodermal lineages and cell types2,3, experimental model systems that can recapitulate more complex features of human mesoderm development and patterning are largely missing. Here we used induced pluripotent stem cells for the stepwise in vitro induction of presomitic mesoderm and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modelling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We observed oscillatory expression of core segmentation clock genes, including HES7 and DKK1, determined the period of the human segmentation clock to be around five hours, and demonstrated the presence of dynamic travelling-wave-like gene expression in in vitro-induced human presomitic mesoderm. Furthermore, we identified and compared oscillatory genes in human and mouse presomitic mesoderm derived from pluripotent stem cells, which revealed species-specific and shared molecular components and pathways associated with the putative mouse and human segmentation clocks. Using CRISPR-Cas9-based genome editing technology, we then targeted genes for which mutations in patients with segmentation defects of the vertebrae, such as spondylocostal dysostosis, have been reported (HES7, LFNG, DLL3 and MESP2). Subsequent analysis of patient-like and patient-derived induced pluripotent stem cells revealed gene-specific alterations in oscillation, synchronization or differentiation properties. Our findings provide insights into the human segmentation clock as well as diseases associated with human axial skeletogenesis.


Assuntos
Relógios Biológicos/fisiologia , Desenvolvimento Embrionário/fisiologia , Células-Tronco Pluripotentes/citologia , Somitos/citologia , Somitos/crescimento & desenvolvimento , Anormalidades Múltiplas/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Relógios Biológicos/genética , Desenvolvimento Embrionário/genética , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento/genética , Glicosiltransferases/deficiência , Glicosiltransferases/genética , Hérnia Diafragmática/genética , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Fenótipo , Somitos/metabolismo , Fatores de Tempo
18.
Exp Anim ; 69(3): 261-268, 2020 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-32281559

RESUMO

Carbohydrate chains are attached to various proteins and lipids and modify their functions. The complex structures of carbohydrate chains, which have various biological functions, are involved not only in regulating protein conformation, transport, and stability but also in cell-cell and cell-matrix interactions. These functional carbohydrate structures are designated as "glyco-codes." Carbohydrate chains are constructed through complex reactions of glycosyltransferases, glycosidases, nucleotide sugars, and protein and lipid substrates in a cell. To elucidate the functions of carbohydrate chains, I and my colleagues generated and characterized knockout (KO) mice of galactosyltransferase family genes. In this review, I introduce our studies about galactosyltransferase family genes together with related studies performed by other researchers, which I presented in my award lecture for the Ando-Tajima Prize of the Japanese Association for Laboratory Animal Science (JALAS) in 2019.


Assuntos
Carboidratos/fisiologia , Glicosiltransferases/deficiência , Animais , Carboidratos/química , Comunicação Celular , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Camundongos Knockout , Transporte Proteico
19.
Sci Rep ; 10(1): 3773, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111874

RESUMO

Stevia rebaudiana (Bertoni) is one of a very few plant species that produce zero calorie, sweet compounds known as steviol glycosides (SG). SGs differ in their sweetness and organoleptic properties depending on the number and positioning of sugar groups on the core steviol backbone. There is great interest of modulating the SG profiles of the Stevia plant to enhance the flavor profile for a given application in the food and beverage industries. Here, we report a highly efficient Agrobacterium-mediated stable transformation system using axillary shoots as the initial explant. Using this system, we generated over 200 transgenic Stevia plants overexpressing a specific isoform of UGT76G1. By comparing the SG profiles among independent transgenic events, we demonstrated that altering UGT76G1 expression can change the ratios of specific SG species. Furthermore, using recombinant proteins produced in E. coli, we show that two closely related UGT76G1 isoforms differ in their substrate specificities, providing new insights into mechanisms underlying the diversity of SG profiles that are observed across Stevia germplasm. Finally, we found evidence suggesting that alternative and/or aberrant splicing may serve to influence the ability of the plant to produce functional UGT76G1 transcripts, and possibly produce enzyme variants within the plant.


Assuntos
Processamento Alternativo , Glicosiltransferases , Proteínas de Plantas , Plantas Geneticamente Modificadas , Stevia , Transformação Genética , Glicosiltransferases/biossíntese , Glicosiltransferases/genética , Isoenzimas/biossíntese , Isoenzimas/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Stevia/enzimologia , Stevia/genética
20.
Sci China Life Sci ; 63(7): 1037-1052, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32112268

RESUMO

Although natural variations in rice flavonoids exist, and biochemical characterization of a few flavonoid glycosyltransferases has been reported, few studies focused on natural variations in tricin-lignan-glycosides and their underlying genetic basis. In this study, we carried out metabolic profiling of tricin-lignan-glycosides and identified a major quantitative gene annotated as a UDP-dependent glycosyltransferase OsUGT706C2 by metabolite-based genome-wide association analysis. The putative flavonoid glycosyltransferase OsUGT706C2 was characterized as a flavonoid 7-O-glycosyltransferas in vitro and in vivo. Although the in vitro enzyme activity of OsUGT706C2 was similar to that of OsUGT706D1, the expression pattern and induced expression profile of OsUGT706C2 were very different from those of OsUGT706D1. Besides, OsUGT706C2 was specifically induced by UV-B. Constitutive expression of OsUGT706C2 in rice may modulate phenylpropanoid metabolism at both the transcript and metabolite levels. Furthermore, overexpressing OsUGT706C2 can enhance UV-B tolerance by promoting ROS scavenging in rice. Our findings might make it possible to use the glycosyltransferase OsUGT706C2 for crop improvement with respect to UV-B adaptation and/or flavonoid accumulation, which may contribute to stable yield.


Assuntos
Flavonoides/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/efeitos da radiação , Oryza/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/efeitos da radiação , Adaptação Fisiológica , Vias Biossintéticas , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Genes de Plantas/efeitos da radiação , Estudo de Associação Genômica Ampla , Glicosídeos/metabolismo , Lignanas/metabolismo , Metaboloma/efeitos da radiação , Mutação
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