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1.
Int J Mol Sci ; 24(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36614218

RESUMO

The xyloglucan endotransglucosylase/hydrolase (XET/XEH, also named XTH) family is a multigene family, the function of which plays a significant role in cell-wall rebuilding and stress tolerance in plants. However, the specific traits of the XTH gene family members and their expression pattern in different tissues and under stress have not been carried out in sweet potato. Thirty-six XTH genes were identified in I. batatas, all of which had conserved structures (Glyco_hydro_16). Based on Neighbor-Joining phylogenetic analysis the IbXTHs can be divided into three subfamilies-the I/II, IIIA, and IIIB subfamilies, which were unevenly distributed on 13 chromosomes, with the exception of Chr9 and Chr15. Multiple cis-acting regions related to growth and development, as well as stress responses, may be found in the IbXTH gene promoters. The segmental duplication occurrences greatly aided the evolution of IbXTHs. The results of a collinearity analysis showed that the XTH genes of sweet potato shared evolutionary history with three additional species, including A. thaliana, G. max, and O. sativa. Additionally, based on the transcriptome sequencing data, the results revealed that the IbXTHs have different expression patterns in leaves, stems, the root body (RB), the distal end (DE), the root stock (RS), the proximal end (PE), the initiative storage root (ISR), and the fibrous root (FR), and many of them are well expressed in the roots. Differentially expressed gene (DEG) analysis of FRs after hormone treatment of the roots indicated that IbXTH28 and IbXTH30 are up-regulated under salicylic acid (SA) treatment but down-regulated under methyl jasmonate (MeJA) treatment. Attentionally, there were only two genes showing down-regulation under the cold and drought treatment. Collectively, all of the findings suggested that genes from the XTH family are crucial for root specificity. This study could provide a theoretical basis for further research on the molecular function of sweet potato XTH genes.


Assuntos
Ipomoea batatas , Ipomoea batatas/genética , Ipomoea batatas/metabolismo , Filogenia , Glicosiltransferases/metabolismo , Hidrolases/metabolismo , Regulação da Expressão Gênica de Plantas
2.
Biomed Res Int ; 2023: 6082635, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36685667

RESUMO

Glycosylation is the most common posttranslational modification of proteins. Glycosyltransferase gene differential expression dictates the glycosylation model and is epigenetically regulating glioma progression and immunity. This study is aimed at identifying the glycosyltransferase gene signature to predict the prognosis and immune characteristics of glioma. The glycosyltransferase gene signature of glioma was identified in the TCGA database and validated in the CGGA database. Glioma patients were then divided into high- and low-risk groups based on risk scores to compare survival differences and predictive capacity. Subsequently, validation of glycosyltransferase gene signature merits by comparing with other signatures and utility in clinical judgment. The immune cell infiltration, immune pathways, and immune checkpoint expression level were also analyzed and compared in the high- and low-risk groups. Finally, the signature and its gene function were tested in our cohort and in vitro experiments. Eight glycosyltransferase genes were identified to establish the glycosyltransferase signature to predict the prognosis of glioma patients. The survival time was shorter in the high-risk group compared to the low-risk group based on glycosyltransferase signature and was confirmed in an independent external cohort. The glycosyltransferase signature displayed outstanding predictive capacity than other signatures in the TCGA and CGGA database cohorts. Furthermore, patients in the high-risk group were positively correlated with TAM infiltration, immune checkpoint expression level, and protumor immune pathways in TCGA cohorts. Validation of clinical tissue specimens revealed that the high-risk group was closely associated with infiltration of M2 TAMs. High-risk genes in the signature promote glioma proliferation, invasion, and macrophage recruitment in an in vitro validation of U87 and U251 cell lines. This carefully constructed that glycosyltransferase signature can predict the prognosis and immune profile of gliomas and help us evaluate subsequent macrophage-targeted therapies as well as other immune microenvironment modulation therapeutic strategies.


Assuntos
Glioma , Glicosiltransferases , Humanos , Glicosiltransferases/genética , Prognóstico , Glicosilação , Fenótipo , Glioma/genética , Microambiente Tumoral/genética
3.
Int J Mol Sci ; 24(2)2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36674551

RESUMO

The biosynthesis of subunits of rhizobial exopolysaccharides is dependent on glycosyltransferases, which are usually encoded by large gene clusters. PssA is a member of a large family of phosphoglycosyl transferases catalyzing the transfer of a phosphosugar moiety to polyprenol phosphate; thus, it can be considered as priming glycosyltransferase commencing synthesis of the EPS repeating units in Rhizobium leguminosarum. The comprehensive analysis of PssA protein features performed in this work confirmed its specificity for UDP-glucose and provided evidence that PssA is a monotopic inner membrane protein with a reentrant membrane helix rather than a transmembrane segment. The bacterial two-hybrid system screening revealed interactions of PssA with some GTs involved in the EPS octasaccharide synthesis. The distribution of differentially expressed genes in the transcriptome of the ΔpssA mutant into various functional categories indicated complexity of cell response to the deletion, which can mostly be attributed to the lack of exopolysaccharide and downstream effects caused by such deficiency. The block in the EPS biosynthesis at the pssA step, potentially leading to an increased pool of UDP-glucose, is likely to be filtered through to other pathways, and thus the absence of EPS may indirectly affect the expression of proteins involved in these pathways.


Assuntos
Rhizobium leguminosarum , Transferases , Transferases/metabolismo , Rhizobium leguminosarum/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Fenótipo , Glucose/metabolismo , Difosfato de Uridina/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas de Bactérias/metabolismo
4.
Planta ; 257(2): 43, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36689015

RESUMO

MAIN CONCLUSION: We have demonstrated that the Arabidopsis FRA9 (fragile fiber 9) gene is specifically expressed in secondary wall-forming cells and essential for the synthesis of the unique xylan reducing end sequence. Xylan is made of a linear chain of ß-1,4-linked xylosyl (Xyl) residues that are often substituted with (methyl)glucuronic acid [(Me)GlcA] side chains and may be acetylated at O-2 and/or O-3. The reducing end of xylan from gymnosperms and dicots contains a unique tetrasaccharide sequence consisting of ß-D-Xylp-(1 → 3)-α-L-Rhap-(1 → 2)-α-D-GalpA-(1 → 4)-D-Xylp, the synthesis of which requires four different glycosyltransferase activities. Genetic analysis in Arabidopsis thaliana has so far implicated three glycosyltransferase genes, FRA8 (fragile fiber 8), IRX8 (irregular xylem 8) and PARVUS, in the synthesis of this unique xylan reducing end sequence. Here, we report the essential role of FRA9, a member of glycosyltransferase family 106 (GT106), in the synthesis of this sequence. The expression of the FRA9 gene was shown to be induced by secondary wall master transcriptional regulators and specifically associated with secondary wall-forming cells, including xylem and fiber cells. T-DNA knockout mutation of the FRA9 gene caused impaired secondary cell wall thickening in leaf veins and a severe arrest of plant growth. RNA interference (RNAi) downregulation of FRA9 led to a significant reduction in secondary wall thickening of fibers, a deformation of xylem vessels and a decrease in xylan content. Structural analysis of xylanase-released xylooligomers revealed that RNAi downregulation of FRA9 resulted in a diminishment of the unique xylan reducing end sequence and complete methylation of xylan GlcA side chains, chemotypes reminiscent of those of the fra8, irx8 and parvus mutants. Furthermore, two FRA9 close homologs from Populus trichocarpa were found to be wood-associated functional orthologs of FRA9. Together, our findings uncover a member of the GT106 family as a new player involved in the synthesis of the unique reducing end sequence of xylan.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Glicosiltransferases/genética , Proteínas de Arabidopsis/metabolismo , Xilanos/metabolismo , Mutação , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas
5.
Carbohydr Res ; 523: 108737, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36657220

RESUMO

Steviol glycosides have attracted great interest because of their high levels of sweetness and safety, and absence of calories. Improvement of their sensory qualities via glycosylation modification by glycosyltransferase is a research hotspot. In this study, YjiC, a uridine diphosphate-dependent glycosyltransferase from Bacillus subtilis 168, was found with the ability to glycosylate rebaudioside A (Reb A) to produce a novel mono ß-1, 6-glycosylated Reb A derivative rebaudioside L2 (Reb L2). It has an improved sweetness compared with Reb A. Next, a cascade reaction was established by combining YjiC with sucrose synthase AtSuSy from Arabidopsis thaliana for scale-up preparation of Reb L2. It shows that Reb L2 (30.94 mg/mL) could be efficiently synthesized with an excellent yield of 91.34% within 12 h. Therefore, this study provides a potential approach for the production and application of new steviol glycoside Reb L2, expanding the scope of steviol glycosides.


Assuntos
Diterpenos do Tipo Caurano , Stevia , Glicosiltransferases , Glucosídeos , Catálise
6.
J Ovarian Res ; 16(1): 5, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36611197

RESUMO

BACKGROUND: Ovarian cancer (OC) is the most fatal gynaecological malignancy and has a poor prognosis. Glycosylation, the biosynthetic process that depends on specific glycosyltransferases (GTs), has recently attracted increasing importance due to the vital role it plays in cancer. In this study, we aimed to determine whether OC patients could be stratified by glycosyltransferase gene profiles to better predict the prognosis and efficiency of immune checkpoint blockade therapies (ICBs). METHODS: We retrieved transcriptome data across 420 OC and 88 normal tissue samples using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases, respectively. An external validation dataset containing 185 OC samples was downloaded from the Gene Expression Omnibus (GEO) database. Knockdown and pathway prediction of B4GALT5 were conducted to investigate the function and mechanism of B4GALT5 in OC proliferation, migration and invasion. RESULTS: A total of 50 differentially expressed GT genes were identified between OC and normal ovarian tissues. Two clusters were stratified by operating consensus clustering, but no significant prognostic value was observed. By applying the least absolute shrinkage and selection operator (LASSO) Cox regression method, a 6-gene signature was built that classified OC patients in the TCGA cohort into a low- or high-risk group. Patients with high scores had a worse prognosis than those with low scores. This risk signature was further validated in an external GEO dataset. Furthermore, the risk score was an independent risk predictor, and a nomogram was created to improve the accuracy of prognostic classification. Notably, the low-risk OC patients exhibited a higher degree of antitumor immune cell infiltration and a superior response to ICBs. B4GALT5, one of six hub genes, was identified as a regulator of proliferation, migration and invasion in OC. CONCLUSION: Taken together, we established a reliable GT-gene-based signature to predict prognosis, immune status and identify OC patients who would benefit from ICBs. GT genes might be a promising biomarker for OC progression and a potential therapeutic target for OC.


Assuntos
Neoplasias dos Genitais Femininos , Neoplasias Ovarianas , Feminino , Humanos , Imunoterapia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Prognóstico , Glicosiltransferases/metabolismo
7.
J Agric Food Chem ; 71(1): 963-973, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36548634

RESUMO

Ginsenosides, the main bioactive ingredients of the Panax genus, are dammarane or oleanane triterpenoids with glycosylated modifications at C3/C6/C20 hydroxyls or C28 carboxyl, and their diverse glycosylation pattern has attracted great attention. However, the biosynthesis of some important saponins is still unclear. In this study, six UGTs were characterized, two of which were novel. PnUGT71A3 catalyzes not only the C6 hydroxyl glycosylation of protopanaxatriol (PPT) and F1 to form Rh1 and Rg1, respectively, but also the C20 hydroxyl glycosylation of protopanaxadiol (PPD)-type Rg3 to generate Rd. Especially, PnUGT94M1 is UDP-ß-l-rhamnose (UDP-Rha)-dependent, regioselectively catalyzing the C2' hydroxyl rhamnosylation of C6 glucose of the PPT-type ginsenosides Rg1 and Rh1 to generate ginsenosides Re and Rg2, respectively. Site-directed mutagenesis showed that His21, Asp120, Ser363, and Pro372 are key residues, and the triple mutant (G344S/G345S/L346T) highly improved the activity toward Rg1 and Rh1. The findings in this study, perfect main ginsenosides biosynthetic pathways in the Panax genus, expand the biocatalyst toolbox for ginsenoside production and show that the PSPG motif is one of the options to modify UGTs to improve their activities.


Assuntos
Ginsenosídeos , Panax notoginseng , Panax , Glicosiltransferases/metabolismo , Panax notoginseng/metabolismo , Vias Biossintéticas , Glicosilação , Panax/química
8.
Carbohydr Res ; 523: 108727, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36521208

RESUMO

Nucleotide sugars play an elementary role in nature as building blocks of glycans, polysaccharides, and glycoconjugates used in the pharmaceutical, cosmetics, and food industries. As substrates of Leloir-glycosyltransferases, nucleotide sugars are essential for chemoenzymatic in vitro syntheses. However, high costs and the limited availability of nucleotide sugars prevent applications of biocatalytic cascades on a large industrial scale. Therefore, the focus is increasingly on nucleotide sugar synthesis strategies to make significant application processes feasible. The chemical synthesis of nucleotide sugars and their derivatives is well established, but the yields of these processes are usually low. Enzyme catalysis offers a suitable alternative here, and in the last 30 years, many synthesis routes for nucleotide sugars have been discovered and used for production. However, many of the published procedures shy away from assessing the practicability of their processes. With this review, we give an insight into the development of the (chemo)enzymatic nucleotide sugar synthesis pathways of the last years and present an assessment of critical process parameters such as total turnover number (TTN), space-time yield (STY), and enzyme loading.


Assuntos
Nucleotídeos , Açúcares , Glicosiltransferases/metabolismo , Biocatálise , Catálise
9.
Methods Mol Biol ; 2557: 691-707, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36512245

RESUMO

The Golgi apparatus is one of the major sites of protein and lipid glycosylation and processing. Protein N-glycosylation plays critical roles in protein folding, transport, stability, and activity. Various glycosyltransferases and glycoside hydrolases are localized at each cisterna in the Golgi apparatus and synthesize a large variety of N-glycan structures. The biosynthetic pathways of N-glycans are complicated, which hiders the rational design of glycan metabolic pathways. In addition, the analysis of glycan structure requires specialized instruments for analyses such as mass spectrometry, high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy, which are not familiar to all laboratories. Here, we introduce relatively simple methods for N-glycan analysis, including disruption of genes encoding glycosyltransferases or glycoside hydrolases, glycan structural analysis using lectins and mass spectrometry, and visualization of glycan metabolic pathways in silico.


Assuntos
Glicosiltransferases , Lectinas , Espectrometria de Massas , Glicosiltransferases/metabolismo , Polissacarídeos/química , Glicosídeo Hidrolases/metabolismo , Coloração e Rotulagem
10.
Front Immunol ; 13: 974377, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36458010

RESUMO

Background: Cellular senescence plays an irreplaceable role in tumorigenesis, progression, and tumor microenvironment (TME) remodeling. However, to date, there is limited research delineating the landscape of cellular senescence in hepatocellular carcinoma (HCC), and an improved understanding on the interaction of tumor-associated cellular senescence with HCC prognosis, TME, and response to immunotherapy is warrant. Methods: Tumorigenic and immune infiltration-associated senescence genes were determined by weighted gene co-expression network analysis (WGCNA) and the Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) algorithm, and subsequently, a prognostic scoring model (named TIS) was constructed using multiple survival analysis algorithms to classify the senescence-related subtypes of HCC. Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were conducted to identify the distinct hallmark pathways between high- and low-risk subtypes. Additionally, we carried out correlation analyses for TIS and clinical traits, senescence-associated secretory phenotype (SASP), immune infiltration and evasion, immune checkpoint factors, drug response, and immunotherapeutic efficacy. External experimental validation was conducted to delineate the association of CPEP3 (a TIS gene) with HCC phenotypes through assays of proliferation, colony formation, and invasion. Results: A five-gene TIS, composed of NET1, ATP6V0B, MMP1, GTDC1, and CPEB3, was constructed and validated using TCGA and ICGC datasets, respectively, and showed a highly robust and plausible signature for overall survival (OS) prediction of HCC in both training and validation cohorts. Patients in the TIS-high group were accompanied by worse OS, activation of carcinogenetic pathways, infiltration of immunosuppressive cells, exclusion of effector killing cells, overexpression of immunomodulatory genes and SASP, and unsatisfied response to immunotherapy. In response to anticancer drugs, patients in the TIS-high group exhibited enhanced susceptibility to several conventional chemotherapeutic agents (5-fluorouracil, docetaxel, doxorubicin, gemcitabine, and etoposide), as well as several inhibitors of pathways involved in cellular senescence (cell-cycle inhibitors, bromodomain and extraterminal domain family (BET) inhibitors, PI3K-AKT pathway inhibitors, and multikinase inhibitors). Additionally, four putative drugs (palbociclib, JAK3 inhibitor VI, floxuridine, and lestaurtinib) were identified as potential compounds for patients in the TIS-high group. Notably, in vitro functional validation showed that CPEB3 knockdown boosted the phenotypes of proliferation, clonogenicity, and invasion in HCC cells, whereas CPEB3 overexpression attenuated these phenotypes. Conclusions: Our study provides comprehensive clues demonstrating the role of novel TIS in predicting HCC prognosis, immunotherapeutic response, and candidate drugs. This work highlights the significance of tumorigenesis- and immune infiltration-related cellular senescence in cancer therapy.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Fosfatidilinositol 3-Quinases , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Prognóstico , Imunoterapia , Carcinogênese , Fatores Imunológicos , Senescência Celular/genética , Microambiente Tumoral/genética , Proteínas de Ligação a RNA , Glicosiltransferases
11.
Food Res Int ; 162(Pt B): 112119, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36461352

RESUMO

The 1,4-α-glucan branching enzymes (GBEs, EC 2.4.1.18) catalyze the formation of new α-1,6 branching points in starch, playing an irreplaceable role in controlling the frequency and position of branch points in glucan chains, which present several potential applications in starch industry. Previously, a thermostable GBE that originates from Rhodothermus obamensis STB05 (RoGBE) is reported to be an excellent glycosyltransferase to modify the structures of starch. However, until now, how to control the length of the transferred glucan chains is still a challenge. Structural analysis of RoGBE shows that the residue at amino acid site 489 connects with the reducing end of the substrate, which may determine the chain length of binding oligosaccharides. The substitutions of Gln at this site with Arg, Glu and Gly result in alternations at catalytic activities and transglycosylation patterns of GBE. Specifically, the Q489E, and Q489R mutants had 5-10 % increases in catalytic activities, the Q489G shows that a slight decrease in activity. versus modified maltodextrin with wild-type RoGBE, maltodextrin incubated with Q489G, Q489E, and Q489R mutants show a 4.17 %-22.43 % increase in the ratio of glucan chains with DP < 13 in the oligosaccharide chains of modified maltodextrin. Crystallographic analyses and simulations were performed to reveal the structural alternations mediated by the introduced mutations. These results are important in the context of understanding the mechanism of action of the thermostable glycosyltransferase and can help develop more functional glycosyltransferases for controlling the glucan chains of polysaccharides.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Glucanos , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Domínio Catalítico , Polissacarídeos , Amido , Glicosiltransferases/genética
12.
Nat Commun ; 13(1): 7424, 2022 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-36456586

RESUMO

Arabidopsis glycosyltransferase family 41 (GT41) protein SPINDLY (SPY) plays pleiotropic roles in plant development. Despite the amino acid sequence is similar to human O-GlcNAc transferase, Arabidopsis SPY has been identified as a novel nucleocytoplasmic protein O-fucosyltransferase. SPY-like proteins extensively exist in diverse organisms, indicating that O-fucosylation by SPY is a common way to regulate intracellular protein functions. However, the details of how SPY recognizes and glycosylates substrates are unknown. Here, we present a crystal structure of Arabidopsis SPY/GDP complex at 2.85 Å resolution. SPY adopts a head-to-tail dimer. Strikingly, the conformation of a 'catalytic SPY'/GDP/'substrate SPY' complex formed by two symmetry-related SPY dimers is captured in the crystal lattice. The structure together with mutagenesis and enzymatic data demonstrate SPY can fucosylate itself and SPY's self-fucosylation region negatively regulates its enzyme activity, reveal SPY's substrate recognition and enzyme mechanism, and provide insights into the glycan donor substrate selection in GT41 proteins.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fucosiltransferases , Arabidopsis/enzimologia , Arabidopsis/genética , Fucosiltransferases/genética , Glicosiltransferases , Proteínas de Arabidopsis/genética
13.
Planta ; 257(1): 18, 2022 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-36538078

RESUMO

MAIN CONCLUSION: Transcriptome and biochemical analyses are applied to individual plant cell types to reveal potential players involved in the molecular machinery of cell wall formation in specialized cells such as collenchyma. Plant collenchyma is a mechanical tissue characterized by an irregular, thickened cell wall and the ability to support cell elongation. The composition of the collenchyma cell wall resembles that of the primary cell wall and includes cellulose, xyloglucan, and pectin; lignin is absent. Thus, the processes associated with the formation of the primary cell wall in the collenchyma can be more pronounced compared to other tissues due to its thickening. Primary cell walls intrinsic to different tissues may differ in structure and composition, which should be reflected at the transcriptomic level. For the first time, we conducted transcriptome profiling of collenchyma strands isolated from young celery petioles and compared them with other tissues, such as parenchyma and vascular bundles. Genes encoding proteins involved in the primary cell wall formation during cell elongation, such as xyloglucan endotransglucosylase/hydrolases, expansins, and leucine-rich repeat proteins, were significantly activated in the collenchyma. As the key players in the transcriptome orchestra of collenchyma, xyloglucan endotransglucosylase/hydrolase transcripts were characterized in more detail, including phylogeny and expression patterns. The comprehensive approach that included transcriptome and biochemical analyses allowed us to reveal peculiarities of collenchyma cell wall formation and modification, matching the abundance of upregulated transcripts and their potential substrates for revealed gene products. As a result, specific isoforms of multigene families were determined for further functional investigation.


Assuntos
Apium , Apium/genética , Celulose/metabolismo , Perfilação da Expressão Gênica , Plantas/genética , Glicosiltransferases/genética , Verduras/genética , Verduras/metabolismo , Parede Celular/metabolismo
14.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 30(6): 1851-1855, 2022 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-36476915

RESUMO

OBJECTIVE: Activities of ABO blood group glycosyltransferases in the plasma of blood donors with different blood groups were detected to discover their normal ranges. In addition, the influence of different plasma storage temperatures and time on the enzyme activity was studied, so as to establish a stable ABO blood group glycosyltransferase activity detection technology system for the auxiliary identification of ABO blood groups. METHODS: Detect the activities of glycosyltransferase A (GTA) in plasma of type A, AB and O blood donors, and glycosyltransferase B (GTB) in plasma of type B, AB and O blood donors, respectively, to determine the activity range of GTA and GTB in the plasma of normal blood group under this detection technique. RESULTS: The activities of GTA and GTB in plasma of the same ABO blood groups were relatively consistent, while significant difference was found among different ABO blood groups. The activity of GTA was around 27.9±0.3 in plasma of A blood group and 28.3±0.5 in plasma of AB blood group. The activity of GTB in plasma of B blood group was about 24.4±0.5, and that in plasma of AB blood group was about 25.6±0.5. The activities of GTA and GTB in plasma of O blood group were negative. The storage temperature and time of plasma would affect the activities of GTA and GTB. There were no significant changes of the activities of GTA and GTB when the plasma was stored at 4 ℃ for 7 days and -40℃ for 21 days. However, after 28 days of storage at -40 ℃, the activities of GTA and GTB were both decreased significantly. CONCLUSION: The preservation condition suitable for the detection of ABO glycosyltransferase activity in plasma samples contain short-term storage at 4 ℃ for one week, and cryopreserved at -40 ℃ for no more than three weeks.


Assuntos
Sistema ABO de Grupos Sanguíneos , Glicosiltransferases , Humanos
15.
PLoS One ; 17(12): e0278713, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36512577

RESUMO

LARGE1 is a bifunctional glycosyltransferase responsible for generating a long linear polysaccharide termed matriglycan that links the cytoskeleton and the extracellular matrix and is required for proper muscle function. This matriglycan polymer is made with an alternating pattern of xylose and glucuronic acid monomers. Mutations in the LARGE1 gene have been shown to cause life-threatening dystroglycanopathies through the inhibition of matriglycan synthesis. Despite its major role in muscle maintenance, the structure of the LARGE1 enzyme and how it assembles in the Golgi are unknown. Here we present the structure of LARGE1, obtained by a combination of X-ray crystallography and single-particle cryo-EM. We found that LARGE1 homo-dimerizes in a configuration that is dictated by its coiled-coil stem domain. The structure shows that this enzyme has two canonical GT-A folds within each of its catalytic domains. In the context of its dimeric structure, the two types of catalytic domains are brought into close proximity from opposing monomers to allow efficient shuttling of the substrates between the two domains. Together, with putative retention of matriglycan by electrostatic interactions, this dimeric organization offers a possible mechanism for the ability of LARGE1 to synthesize long matriglycan chains. The structural information further reveals the mechanisms in which disease-causing mutations disrupt the activity of LARGE1. Collectively, these data shed light on how matriglycan is synthesized alongside the functional significance of glycosyltransferase oligomerization.


Assuntos
Matriz Extracelular , Glicosiltransferases , Glicosiltransferases/metabolismo , Glicosilação , Domínios Proteicos , Matriz Extracelular/metabolismo , Polissacarídeos , Cristalografia por Raios X
16.
Cells ; 11(23)2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36496988

RESUMO

Cuproptosis is a newly defined programmed cell death pattern and is believed to play an important role in tumorigenesis and progression. In addition, many studies have shown that glycosylation modification is of vital importance in tumor progression. However, it remains unclear whether glycosyltransferases, the most critical enzymes involved in glycosylation modification, are associated with cuproptosis. In this study, we used bioinformatic methods to construct a signature of cuproptosis-related glycosyltransferases to predict the prognosis of colon adenocarcinoma patients. We found that cuproptosis was highly correlated with four glycosyltransferases in COAD, and our model predicted the prognosis of COAD patients. Further analysis of related functions revealed the possibility that cuproptosis-related glycosyltransferase Exostosin-like 2 (EXTL2) participated in tumor immunity.


Assuntos
Adenocarcinoma , Apoptose , Neoplasias do Colo , Glicosiltransferases , Humanos , Glicosilação , Prognóstico
17.
Org Lett ; 24(49): 9065-9070, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36475912

RESUMO

The actinopyrone biosynthetic gene cluster (atpn) lacks glycosyl- and methyltransferase genes, yet its product clearly calls for such enzymes. Using bioinformatics and biochemical methods, we confirmed that the mt3913 and gt723 genes, well beyond the atpn cluster boundaries, encode methyltransferase and glycosyltransferase, respectively. Moreover, homologous protein GT1507 enabled us to produce 14 non-natural actinopyrone analogues. PM050463 (3) was found to display potent anti-Helicobacter pylori activity and no signs of cytotoxicity.


Assuntos
Helicobacter pylori , Metiltransferases , Metiltransferases/metabolismo , Glicosiltransferases/genética , Família Multigênica , Helicobacter pylori/genética
18.
Int J Mol Sci ; 23(24)2022 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-36555445

RESUMO

Glycosylation, which consists of the enzymatic addition of sugars to proteins and lipids, is one of the most important post-co-synthetic modifications of these molecules, profoundly affecting their activity. Although the presence of carbohydrate chains is crucial for fine-tuning the interactions between cells and molecules, glycosylation is an intrinsically stochastic process regulated by the relative abundance of biosynthetic (glycosyltransferases) and catabolic (glycosidases) enzymes, as well as sugar carriers and other molecules. Non-coding RNAs, which include microRNAs, long non-coding RNAs and circRNAs, establish a complex network of reciprocally interacting molecules whose final goal is the regulation of mRNA expression. Likewise, these interactions are stochastically regulated by ncRNA abundance. Thus, while protein sequence is deterministically dictated by the DNA/RNA/protein axis, protein abundance and activity are regulated by two stochastic processes acting, respectively, before and after the biosynthesis of the protein axis. Consequently, the worlds of glycosylation and ncRNA are closely interconnected and mutually interacting. In this paper, we will extensively review the many faces of the ncRNA-glycosylation interplay in cancer and other physio-pathological conditions.


Assuntos
MicroRNAs , Neoplasias , RNA Longo não Codificante , Humanos , Glicosilação , Glicosiltransferases/metabolismo , MicroRNAs/genética , Carboidratos , Neoplasias/genética , RNA Longo não Codificante/genética
19.
Molecules ; 27(23)2022 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-36500465

RESUMO

Trapa bispinosa Roxb. is an economical crop for medicine and food. Its roots, stems, leaves, and pulp have medicinal applications, and its shell is rich in active ingredients and is considered to have a high medicinal value. One of the main functional components of the Trapa bispinosa Roxb. shell is 1-galloyl-beta-D-glucose (ßG), which can be used in medical treatment and is also an essential substrate for synthesizing the anticancer drug beta-penta-o-Galloyl-glucosen (PGG). Furthermore, gallate 1-beta-glucosyltransferase (EC 2.4.1.136) has been found to catalyze gallic acid (GA) and uridine diphosphate glucose (UDPG) to synthesize ßG. In our previous study, significant differences in ßG content were observed in different tissues of Trapa bispinosa Roxb. In this study, Trapa bispinosa Roxb. was used to clone 1500 bp of the UGGT gene, which was named TbUGGT, to encode 499 amino acids. According to the specificity of the endogenous expression of foreign genes in Escherichia coli, the adaptation codon of the cloned original genes was optimized for improved expression. Bioinformatic and phylogenetic tree analyses revealed the high homology of TbUGGT with squalene synthases from other plants. The TbUGGT gene was constructed into a PET-28a expression vector and then transferred into Escherichia coli Transsetta (DE3) for expression. The recombinant protein had a molecular weight of 55 kDa and was detected using SDS-PAGE. The proteins were purified using multiple fermentation cultures to simulate the intracellular environment, and a substrate was added for in vitro reaction. After the enzymatic reaction, the levels of ßG in the product were analyzed using HPLC and LC-MS, indicating the catalytic activity of TbUGGT. The cloning and functional analysis of TbUGGT may lay the foundation for further study on the complete synthesis of ßG in E. coli.


Assuntos
Escherichia coli , Glicosiltransferases , Glicosiltransferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Filogenia , Clonagem Molecular
20.
Sci Rep ; 12(1): 19288, 2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36369343

RESUMO

Pathogenic bacteria encode virulent glycosyltransferases that conjugate various glycans onto host crucial proteins, which allows adhesion to mammalian cells and modulates host cellular processes for pathogenesis. Escherichia coli NleB1, Citrobacter rodentium NleB, and Salmonella enterica SseK1/3 type III effectors fatally glycosyltransfer N-acetyl glucosamine (GlcNAc) from UDP-GlcNAc to arginine residues of death domain-containing proteins that regulate host inflammation, intra-bacterial proteins, and themselves, whose post-translational modification disrupts host immune functions and prolongs bacterial viability inside host cells. However, unlike the similar NleB1/SseK1/SseK3, E. coli NleB2 and S. enterica SseK2 show deficient GlcNAcylation and neither intra-bacterial glycosylation nor auto-glycosylation. Here, as the major factor in SseK2/NleB2 deficiency, we focused on the catalytic Asp-x-Asp (DxD) motif conserved throughout all O-/N-glycosyltransferases to coordinate Mn2+. All DxD motifs in apo-glycosyltransferases form Type-I-turns for binding Mn2+, similar to the ligand-bound DxD motif, whereas TcnA/SseK2/NleB2 DxD motifs form Asx-turns, which are unable to bind Mn2+. Interestingly, methionine of the NleB2 DMD motif forms triple Met-aromatic interactions, as found in age-associated diseases and tumor necrosis factor (TNF) ligand-receptor complexes. The NleB1 A222M mutation induces triple Met-aromatic interactions to steeply attenuate glycosylation activity to 3% of that in the wild type. Thus, the characteristic conformation of the DxD motif is essential for binding Mn2+, donors, and glycosylate targets. This explains why SseK2/NleB2 effectors with the DxD motif caged in the Asp-/Asn-turn (Asx-turn) and triple Met-aromatic interactions have lower glycosyltransferase activity than that of other fatal NleB1/SseK1/SseK3 toxins.


Assuntos
Citrobacter rodentium , Escherichia coli , Glicosiltransferases , Salmonella enterica , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Ligantes
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