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1.
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
2.
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
3.
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
4.
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
5.
Mol Cell ; 78(5): 824-834.e15, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32325029

RESUMO

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.


Assuntos
Glicosiltransferases/metabolismo , Polissacarídeos/metabolismo , Engenharia de Proteínas/métodos , Vias Biossintéticas , Membrana Celular/metabolismo , Glicosilação , Glicosiltransferases/química , Glicosiltransferases/fisiologia , Células HEK293 , Células Hep G2 , Humanos , Células K562 , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/metabolismo , N-Acetilgalactosaminiltransferases/fisiologia , Polissacarídeos/química , Proteínas/metabolismo
6.
Nat Chem Biol ; 16(4): 450-457, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32152541

RESUMO

Lipopolysaccharide O-antigen is an attractive candidate for immunotherapeutic strategies targeting antibiotic-resistant Klebsiella pneumoniae. Several K. pneumoniae O-serotypes are based on a shared O2a-antigen backbone repeating unit: (→ 3)-α-Galp-(1 → 3)-ß-Galf-(1 →). O2a antigen is synthesized on undecaprenol diphosphate in a pathway involving the O2a polymerase, WbbM, before its export by an ATP-binding cassette transporter. This dual domain polymerase possesses a C-terminal galactopyranosyltransferase resembling known GT8 family enzymes, and an N-terminal DUF4422 domain identified here as a galactofuranosyltransferase defining a previously unrecognized family (GT111). Functional assignment of DUF4422 explains how galactofuranose is incorporated into various polysaccharides of importance in vaccine production and the food industry. In the 2.1-Å resolution structure, three WbbM protomers associate to form a flattened triangular prism connected to a central stalk that orients the active sites toward the membrane. The biochemical, structural and topological properties of WbbM offer broader insight into the mechanisms of assembly of bacterial cell-surface glycans.


Assuntos
Glicosiltransferases/metabolismo , Antígenos O/metabolismo , Antígenos O/ultraestrutura , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Glicosiltransferases/fisiologia , Hexosiltransferases , Klebsiella pneumoniae/metabolismo , Lipopolissacarídeos/química , Polissacarídeos Bacterianos/química
7.
Nature ; 579(7799): 443-447, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32103179

RESUMO

In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins1. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates2. The responsible enzymes-ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10-are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate3,4. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0 Å resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9 Å resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms.


Assuntos
Microscopia Crioeletrônica , Retículo Endoplasmático/enzimologia , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Biocatálise , Domínio Catalítico , Sequência Conservada , Dolicol Monofosfato Manose/metabolismo , Fosfatos de Dolicol/metabolismo , Glucose/análogos & derivados , Glucose/metabolismo , Glicosiltransferases/deficiência , Técnicas In Vitro , Lipídeos , Proteínas de Membrana/deficiência , Modelos Moleculares , Mutação , Monossacarídeos de Poli-Isoprenil Fosfato/química , Monossacarídeos de Poli-Isoprenil Fosfato/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Especificidade por Substrato
8.
Enzyme Microb Technol ; 134: 109479, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32044026

RESUMO

Mangiferin, a major constituent of Mangifera indica L., has attracted substantial attention due to its anti-oxidant, anti-diabetic, anti-inflammatory, and anti-microbial activities. However, its poor solubility in water limits its use in food and pharmaceutical industries. In this study, novel mangiferin-(1→6)-α-d-glucopyranoside (Mg-G1) was enzymatically synthesized from mangiferin and sucrose using glucansucrase from Leuconostoc mesenteroides B-512F/KM, and optimized using response surface methodology. The water solubility of Mg-G1 was found to be 824.7 mM, which is more than 2300-fold higher than that of mangiferin. Mg-G1 also showed DPPH radical scavenging activity and superoxide dismutase (SOD)-like scavenging activity, which were 4.77- and 3.71-fold higher than that of mangiferin, respectively. Mg-G1 displayed inhibitory activity against human intestinal maltase and COX-2. Thus, the novel glucosylated mangiferin may be used as an ingredient in functional food and pharmaceutical application.


Assuntos
Glucosídeos/biossíntese , Glicosiltransferases/metabolismo , Leuconostoc mesenteroides/enzimologia , Mangifera/química , Xantonas/metabolismo , Antioxidantes/metabolismo , Inibidores de Ciclo-Oxigenase 2/metabolismo , Humanos , Solubilidade , Sacarose/metabolismo , Superóxido Dismutase/metabolismo , alfa-Glucosidases/metabolismo
9.
Food Chem ; 312: 126124, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31926461

RESUMO

Apple is rich in flavonol glycosides, which are believed to contribute to putative health benefits associated with apple consumption. Glycosylation, catalyzed by uridine diphospho-glycosyltransferases (UGTs), is the last step in flavonol biosynthesis, which confers molecular stability and solubility to the flavonol. In the present study, the involvement of two UGTs, MdUGT75B1 and MdUGT71B1, in flavonol biosynthesis in apple was investigated. The major flavonols are quercetin 3-O-glycosides, and UV-B and blue light treatment significantly enhanced the accumulation of quercetin 3-O-galactoside, quercetin 3-O-glucoside, and kaempferol 3-O-galactoside. Transcript levels of MdUGT75B1 and MdUGT71B1 in fruit subjected to different treatments were correlated well with flavonol accumulation. MdUGT75B1 showed flavonol-specific activity with a preference for UDP-galactose as the sugar donor, while MdUGT71B1 using UDP-glucose exhibited a wider substrate acceptance. Thus, MdUGT75B1 and MdUGT71B1 are key UGTs involved in flavonol biosynthesis and may have important roles in regulating accumulation of these health-promoting bioactive compounds in apple.


Assuntos
Galactosídeos/biossíntese , Glucosídeos/biossíntese , Glicosiltransferases/metabolismo , Quempferóis/biossíntese , Malus/química , Quercetina/análogos & derivados , Frutas/química , Frutas/metabolismo , Malus/metabolismo , Quercetina/biossíntese , Uridina/metabolismo
10.
PLoS One ; 15(1): e0219413, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31899920

RESUMO

Seed dormancy and germination are the two important traits related to plant survival, reproduction and crop yield. To understand the regulatory mechanisms of these traits, it is crucial to clarify which genes or pathways participate in the regulation of these processes. However, little information is available on seed dormancy and germination in peanut. In this study, seeds of the variety Luhua No.14, which undergoes nondeep dormancy, were selected, and their transcriptional changes at three different developmental stages, the freshly harvested seed (FS), the after-ripening seed (DS) and the newly germinated seed (GS) stages, were investigated by comparative transcriptomic analysis. The results showed that genes with increased transcription in the DS vs FS comparison were overrepresented for oxidative phosphorylation, the glycolysis pathway and the tricarboxylic acid (TCA) cycle, suggesting that after a period of dry storage, the intermediates stored in the dry seeds were rapidly mobilized by glycolysis, the TCA cycle, the glyoxylate cycle, etc.; the electron transport chain accompanied by respiration was reactivated to provide ATP for the mobilization of other reserves and for seed germination. In the GS vs DS pairwise comparison, dozens of the upregulated genes were related to plant hormone biosynthesis and signal transduction, including the majority of components involved in the auxin signal pathway, brassinosteroid biosynthesis and signal transduction as well as some GA and ABA signal transduction genes. During seed germination, the expression of some EXPANSIN and XYLOGLUCAN ENDOTRANSGLYCOSYLASE genes was also significantly enhanced. To investigate the effects of different hormones during seed germination, the contents and differential distribution of ABA, GAs, BRs and IAA in the cotyledons, hypocotyls and radicles, and plumules of three seed sections at different developmental stages were also investigated. Combined with previous data in other species, it was suggested that the coordination of multiple hormone signal transduction nets plays a key role in radicle protrusion and seed germination.


Assuntos
Arachis/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Germinação/genética , Proteínas de Plantas/genética , Sementes/genética , Transcriptoma , Ácido Abscísico/metabolismo , Trifosfato de Adenosina/biossíntese , Arachis/crescimento & desenvolvimento , Arachis/metabolismo , Brassinosteroides/metabolismo , Ciclo do Ácido Cítrico/genética , Ontologia Genética , Redes Reguladoras de Genes , Glicólise/genética , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Ácidos Indolacéticos/metabolismo , Anotação de Sequência Molecular , Fosforilação Oxidativa , Dormência de Plantas , Proteínas de Plantas/metabolismo , Característica Quantitativa Herdável , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Transdução de Sinais
11.
Plant Mol Biol ; 102(4-5): 389-401, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31894456

RESUMO

KEY MESSAGE: This study revealed that the Arabidopsis UGT75B1 plays an important role in modulating ABA activity by glycosylation when confronting stress environments. The cellular ABA content and activity can be tightly controlled in several ways, one of which is glycosylation by family 1 UDP-glycosyltransferases (UGTs). Previous analysis has shown UGT75B1 activity towards ABA in vitro. However, the biological role of UGT75B1 remains to be elucidated. Here, we characterized the function of UGT75B1 in abiotic stress responses via ABA glycosylation. GUS assay and qRT-PCR indicated that UGT75B1 is significantly upregulated by adverse conditions, such as osmotic stress, salinity and ABA. Overexpression of UGT75B1 in Arabidopsis leads to higher seed germination rates and seedling greening rates upon exposure to salt and osmotic stresses. In contrast, the big UGT75B1 overexpression plants are more sensitive under salt and osmotic stresses. Additionally, the UGT75B1 overexpression plants showed larger stomatal aperture and more water loss under drought condition, which can be explained by lower ABA levels examined in UGT75B1 OE plants in response to water deficit conditions. Consistently, UGT75B1 ectopic expression leads to downregulation of many ABA-responsive genes under stress conditions, including ABI3, ABI5 newly germinated seedlings and RD29A, KIN1, AIL1 in big plants. In summary, our results revealed that the Arabidopsis UGT75B1 plays an important role in coping with abiotic stresses via glycosylation of ABA.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/fisiologia , Glicosiltransferases/metabolismo , Estresse Fisiológico , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Catálise , Secas , Genes de Plantas , Germinação , Glucosiltransferases/genética , Glicosilação , Glicosiltransferases/genética , Pressão Osmótica , Plantas Geneticamente Modificadas/genética , Salinidade , Plântula/genética , Plântula/fisiologia , Cloreto de Sódio , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
12.
Int J Mol Sci ; 21(2)2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936666

RESUMO

Glycosylation is the most ubiquitous post-translational modification in eukaryotes. N-glycan is attached to nascent glycoproteins and is processed and matured by various glycosidases and glycosyltransferases during protein transport. Genetic and biochemical studies have demonstrated that alternations of the N-glycan structure play crucial roles in various physiological and pathological events including progression of cancer, diabetes, and Alzheimer's disease. In particular, the formation of N-glycan branches regulates the functions of target glycoprotein, which are catalyzed by specific N-acetylglucosaminyltransferases (GnTs) such as GnT-III, GnT-IVs, GnT-V, and GnT-IX, and a fucosyltransferase, FUT8s. Although the 3D structures of all enzymes have not been solved to date, recent progress in structural analysis of these glycosyltransferases has provided insights into substrate recognition and catalytic reaction mechanisms. In this review, we discuss the biological significance and structure-function relationships of these enzymes.


Assuntos
Glicosiltransferases/química , Glicosiltransferases/metabolismo , Modelos Moleculares , Polissacarídeos/metabolismo , Animais , Cristalografia por Raios X , Humanos , Polissacarídeos/química , Relação Estrutura-Atividade
13.
mSphere ; 5(1)2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941812

RESUMO

The pathogenic fungus Aspergillus fumigatus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise α-(1→2)-/α-(1→6)-mannan and ß-(1→5)-/ß-(1→6)-galactofuranosyl chains. We previously revealed that GfsA is a ß-galactofuranoside ß-(1→5)-galactofuranosyltransferase involved in the biosynthesis of ß-(1→5)-galactofuranosyl chains. In this study, we clarified the biosynthesis of ß-(1→5)-galactofuranosyl chains in A. fumigatus Two paralogs exist within A. fumigatus: GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are ß-galactofuranoside ß-(1→5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize ß-(1→5)-galactofuranosyl oligomers at up to lengths of 7, 3, and 5 galactofuranoses within an established in vitro highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from ΔgfsB, ΔgfsC, ΔgfsAC, and ΔgfsABC strains revealed that GfsA and GfsC synthesized all ß-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans and that GfsB exhibited limited function in A. fumigatus The loss of ß-(1→5)-galactofuranosyl residues decreased the hyphal growth rate and conidium formation ability and increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immunocompromised mice.IMPORTANCE ß-(1→5)-Galactofuranosyl residues are widely distributed in the subphylum Pezizomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of ß-(1→5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for understanding them. In this study, we showed that GfsA and GfsC are responsible for the biosynthesis of all ß-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans. The data presented here indicate that ß-(1→5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of ß-(1→5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the members of the subphylum Pezizomycotina.


Assuntos
Aspergillus fumigatus/enzimologia , Mananas/biossíntese , Mananas/química , Manose/química , Animais , Aspergillus fumigatus/genética , Parede Celular/química , Parede Celular/metabolismo , Glicosiltransferases/metabolismo , Hifas , Manose/biossíntese , Camundongos , Virulência
14.
J Biol Chem ; 295(5): 1411-1425, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31882545

RESUMO

The importance of the microbiome in health and its disruption in disease is continuing to be elucidated. However, the multitude of host and environmental factors that influence the microbiome are still largely unknown. Here, we examined UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 (Galnt3)-deficient mice, which serve as a model for the disease hyperphosphatemic familial tumoral calcinosis (HFTC). In HFTC, loss of GALNT3 activity in the bone is thought to lead to altered glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), resulting in hyperphosphatemia and subdermal calcified tumors. However, GALNT3 is expressed in other tissues in addition to bone, suggesting that systemic loss could result in other pathologies. Using semiquantitative real-time PCR, we found that Galnt3 is the major O-glycosyltransferase expressed in the secretory cells of salivary glands. Additionally, 16S rRNA gene sequencing revealed that the loss of Galnt3 resulted in changes in the structure, composition, and stability of the oral microbiome. Moreover, we identified the major secreted salivary mucin, Muc10, as an in vivo substrate of Galnt3. Given that mucins and their O-glycans are known to interact with various microbes, our results suggest that loss of Galnt3 decreases glycosylation of Muc10, which alters the composition and stability of the oral microbiome. Considering that oral findings have been documented in HFTC patients, our study suggests that investigating GALNT3-mediated changes in the oral microbiome may be warranted.


Assuntos
Calcinose/metabolismo , Calcinose/microbiologia , Hiperostose Cortical Congênita/metabolismo , Hiperostose Cortical Congênita/microbiologia , Hiperfosfatemia/metabolismo , Hiperfosfatemia/microbiologia , Microbiota/genética , N-Acetilgalactosaminiltransferases/metabolismo , Glândulas Salivares/metabolismo , Animais , Calcinose/genética , Feminino , Glicosilação , Glicosiltransferases/metabolismo , Hiperostose Cortical Congênita/genética , Hiperfosfatemia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucinas/química , Mucinas/metabolismo , N-Acetilgalactosaminiltransferases/genética , Polissacarídeos/metabolismo , RNA Ribossômico 16S/genética
15.
Dev Growth Differ ; 62(1): 35-48, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31886522

RESUMO

Notch signaling is an evolutionarily conserved signaling pathway and is essential for cell-fate specification in metazoans. Dysregulation of Notch signaling results in various human diseases, including cardiovascular defects and cancer. In 2000, Fringe, a known regulator of Notch signaling, was discovered as a Notch-modifying glycosyltransferase. Since then, glycosylation-a post-translational modification involving literal sugars-on the Notch extracellular domain has been noted as a critical mechanism for the regulation of Notch signaling. Additionally, the presence of diverse O-glycans decorating Notch receptors has been revealed in the extracellular domain epidermal growth factor-like (EGF) repeats. Here, we concisely summarize the recent studies in the human diseases associated with aberrant Notch glycosylation.


Assuntos
Doenças Cardiovasculares , Proteínas de Neoplasias , Neoplasias , Receptores Notch , Animais , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Domínios Proteicos , Receptores Notch/genética , Receptores Notch/metabolismo , Sequências Repetitivas de Aminoácidos
16.
Biochim Biophys Acta Gen Subj ; 1864(3): 129509, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31884067

RESUMO

BACKGROUND: Brains express structurally unique glycans, including human natural killer-1 (HNK-1), which participate in development and high-order functions. However, the regulatory mechanisms of expression of these brain-specific glycans are largely unknown. We examined whether arginine methylation, another type of protein modification essential for neural development, impacts the expression of various glycans in the developing brain. METHODS: We analyzed several types of glycans, including the HNK-1 epitope, in the cerebellum and cerebral cortex from mice with nervous system-specific knockout of protein arginine methyltransferase 1 (PRMT1). We also analyzed the expression levels of glycosyltransferases responsible for HNK-1 and of HNK-1 carrier glycoproteins by quantitative RT-PCR and western blotting. RESULTS: Among several glycans, expression of HNK-1 glycan was strikingly upregulated in the PRMT1-deficient cerebellum. Furthermore, such upregulation was found in the cerebellum but not in the cerebral cortex. Regarding the mechanisms, we demonstrated that the mRNA level and activity of the responsible glycosyltransferase (B3gat1) were elevated in the knockout cerebellum. We also showed that the expression of HNK-1 carrier glycoproteins such as neural cell adhesion molecule (NCAM), L1 and AMPA receptor subunit GluA2 were also increased in the PRMT1-deficient cerebellum. CONCLUSIONS: Loss of arginine methylation leads to an increase in HNK-1 glycan in the developing cerebellum but not in the cerebral cortex via upregulation of the biosynthetic enzyme and carrier glycoproteins. GENERAL SIGNIFICANCE: PRMT1 is a novel regulator of HNK-1 glycan production in the cerebellum. Mechanisms involving crosstalk between glycosylation and arginine methylation are suggested to occur.


Assuntos
Antígenos CD57/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Encéfalo , Cerebelo , Córtex Cerebral , Epitopos/genética , Feminino , Glucuronosiltransferase/metabolismo , Glicosilação , Glicosiltransferases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polissacarídeos/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Receptores de AMPA/genética , Proteínas Repressoras/genética , Ativação Transcricional
17.
Acta Neurobiol Exp (Wars) ; 79(4): 338-351, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31885391

RESUMO

Over the last twenty years chondroitin sulfate (CS) has become a focus of interest of neuroscience due to its indubitable role in shaping axonal growth, synaptic plasticity and glial scar forming. Various patterns of sulfation give rise to various CS molecules with different properties that are capable of interactions with a plethora of molecules, including growth factors, receptors and guidance molecules. The involvement of CS chains has been implicated in visual critical period regulation, memory formation, spinal cord regeneration. As part of proteoglycan molecules, they are widely expressed in the central nervous system, however, little is known about the enzymatic machinery responsible for CS synthesis and degradation. In this review we attempt to extract and collect the available information concerning the expression and function of enzymes of CS metabolism in the brain.


Assuntos
Encéfalo/metabolismo , Sulfatos de Condroitina/metabolismo , Animais , Encéfalo/enzimologia , Configuração de Carboidratos , Retículo Endoplasmático/metabolismo , Matriz Extracelular/metabolismo , Glicosiltransferases/metabolismo , Humanos , Redes e Vias Metabólicas , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal , Proteoglicanas/metabolismo , Xilose/metabolismo
18.
Nat Commun ; 10(1): 5404, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776339

RESUMO

Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by an N-glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications.


Assuntos
Sistema Livre de Células/metabolismo , Engenharia de Proteínas/métodos , Proteínas/metabolismo , Antígenos CD/metabolismo , Escherichia coli/genética , Glicoproteínas/biossíntese , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Humanos , Redes e Vias Metabólicas , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Proteínas/genética , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismo
19.
J Agric Food Chem ; 67(46): 12806-12815, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31650841

RESUMO

Lactic acid bacteria are known to produce extracellular polysaccharides such as α-glucan, levan, and inulin, which are promising for applications in food systems because of their prebiotic properties. In this work, a novel glucansucrase (GS) Gtf-DSM from Lactobacillus ingluviei DSM 14792 was recombinantly expressed and biochemically characterized as a dextransucrase capable of producing a dextran polysaccharide with four types of linkages, including 69% (α1 → 6), 24% (α1 → 3), 6% (α1 → 4), and 1% (α1 → 2). Intriguingly, the dextransucrase Gtf-DSM had a sequence identity of 99.3% with the reuteransucrase GtfO producing a reuteran with 21% (α1 → 6) and 79% (α1 → 4) linkages. Thus, the dextransucrase Gtf-DSM is a unique target for understanding the linkage specificity of GSs and the investigation of site-directed mutagenesis using Gtf-DSM and GtfO as templates is underway.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Lactobacillus/enzimologia , Proteínas de Bactérias/genética , Dextranos/química , Dextranos/metabolismo , Glucosiltransferases/genética , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Lactobacillus/química , Lactobacillus/genética , Lactobacillus/metabolismo , Mutagênese Sítio-Dirigida , Especificidade por Substrato
20.
Int J Mol Sci ; 20(20)2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31635144

RESUMO

Strain GA A07 was identified as an intestinal Bacillus bacterium of zebrafish, which has high efficiency to biotransform the triterpenoid, ganoderic acid A (GAA), into GAA-15-O-ß-glucoside. To date, only two known enzymes (BsUGT398 and BsUGT489) of Bacillus subtilis ATCC 6633 strain can biotransform GAA. It is thus worthwhile to identify the responsible genes of strain GA A07 by whole genome sequencing. A complete genome of strain GA A07 was successfully assembled. A phylogenomic analysis revealed the species of the GA A07 strain to be Bacillus thuringiensis. Forty glycosyltransferase (GT) family genes were identified from the complete genome, among which three genes (FQZ25_16345, FQZ25_19840, and FQZ25_19010) were closely related to BsUGT398 and BsUGT489. Two of the three candidate genes, FQZ25_16345 and FQZ25_19010, were successfully cloned and expressed in a soluble form in Escherichia coli, and the corresponding proteins, BtGT_16345 and BtGT_19010, were purified for a biotransformation activity assay. An ultra-performance liquid chromatographic analysis further confirmed that only the purified BtGT_16345 had the key biotransformation activity of catalyzing GAA into GAA-15-O-ß-glucoside. The suitable conditions for this enzyme activity were pH 7.5, 10 mM of magnesium ions, and 30 °C. In addition, BtGT_16345 showed glycosylation activity toward seven flavonoids (apigenein, quercetein, naringenein, resveratrol, genistein, daidzein, and 8-hydroxydaidzein) and two triterpenoids (GAA and antcin K). A kinetic study showed that the catalytic efficiency (kcat/KM) of BtGT_16345 was not significantly different compared with either BsUGT398 or BsUGT489. In short, this study identified BtGT_16345 from B. thuringiensis GA A07 is the catalytic enzyme responsible for the 15-O-glycosylation of GAA and it was also regioselective toward triterpenoid substrates.


Assuntos
Bacillus thuringiensis/enzimologia , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Glicosiltransferases/metabolismo , Ácidos Heptanoicos/química , Ácidos Heptanoicos/metabolismo , Lanosterol/análogos & derivados , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Biotransformação , Catálise , Glicosilação , Glicosiltransferases/genética , Lanosterol/química , Lanosterol/metabolismo , Filogenia , Especificidade por Substrato , Sequenciamento Completo do Genoma
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