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1.
Nat Commun ; 12(1): 5656, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34580305

RESUMO

Glycosyltransferases (GTs) play fundamental roles in nearly all cellular processes through the biosynthesis of complex carbohydrates and glycosylation of diverse protein and small molecule substrates. The extensive structural and functional diversification of GTs presents a major challenge in mapping the relationships connecting sequence, structure, fold and function using traditional bioinformatics approaches. Here, we present a convolutional neural network with attention (CNN-attention) based deep learning model that leverages simple secondary structure representations generated from primary sequences to provide GT fold prediction with high accuracy. The model learns distinguishing secondary structure features free of primary sequence alignment constraints and is highly interpretable. It delineates sequence and structural features characteristic of individual fold types, while classifying them into distinct clusters that group evolutionarily divergent families based on shared secondary structural features. We further extend our model to classify GT families of unknown folds and variants of known folds. By identifying families that are likely to adopt novel folds such as GT91, GT96 and GT97, our studies expand the GT fold landscape and prioritize targets for future structural studies.


Assuntos
Aprendizado Profundo , Glicosiltransferases/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos/genética , Biologia Computacional/métodos , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Glicosilação , Glicosiltransferases/genética , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência
2.
Molecules ; 26(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34500611

RESUMO

Human natural killer-1 (HNK-1) is a sulfated glyco-epitope regulating cell adhesion and synaptic functions. HNK-1 and its non-sulfated forms, which are specifically expressed in the brain and the kidney, respectively, are distinctly biosynthesized by two homologous glycosyltransferases: GlcAT-P in the brain and GlcAT-S in the kidney. However, it is largely unclear how the activity of these isozymes is regulated in vivo. We recently found that bisecting GlcNAc, a branching sugar in N-glycan, suppresses both GlcAT-P activity and HNK-1 expression in the brain. Here, we observed that the expression of non-sulfated HNK-1 in the kidney is unexpectedly unaltered in mutant mice lacking bisecting GlcNAc. This suggests that the biosynthesis of HNK-1 in the brain and the kidney are differentially regulated by bisecting GlcNAc. Mechanistically, in vitro activity assays demonstrated that bisecting GlcNAc inhibits the activity of GlcAT-P but not that of GlcAT-S. Furthermore, molecular dynamics simulation showed that GlcAT-P binds poorly to bisected N-glycan substrates, whereas GlcAT-S binds similarly to bisected and non-bisected N-glycans. These findings revealed the difference of the highly homologous isozymes for HNK-1 synthesis, highlighting the novel mechanism of the tissue-specific regulation of HNK-1 synthesis by bisecting GlcNAc.


Assuntos
Antígenos CD57/biossíntese , Glucuronosiltransferase/metabolismo , Animais , Encéfalo/metabolismo , Células COS , Linhagem Celular , Chlorocebus aethiops , Epitopos/metabolismo , Glicosiltransferases/metabolismo , Células HEK293 , Humanos , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Polissacarídeos/metabolismo
4.
J Org Chem ; 86(15): 10819-10828, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34254798

RESUMO

A diversity-oriented chemoenzymatic approach for the collective preparation of sulfated core 2 O-GalNAc glycans and their nonsulfated counterparts was described. A sulfated trisaccharide and a nonsulfated trisaccharide were chemically synthesized by combining flexible protected group manipulations and sequential one-pot glycosylations. The divergent enzymatic extension of these two trisaccharides, using a panel of robust glycosyltransferases that can recognize sulfated substrates and differentiating the branches with specifically designed glycosylation sequences to achieve regioselective sialylation, provided 36 structurally well-defined O-GalNAc glycans.


Assuntos
Polissacarídeos , Sulfatos , Glicosilação , Glicosiltransferases/metabolismo , Trissacarídeos
5.
Molecules ; 26(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34299397

RESUMO

We followed a comparative approach to investigate how heavy vacuum gas oil (HVGO) affects the expression of genes involved in biosurfactants biosynthesis and the composition of the rhamnolipid congeners in Pseudomonas sp. AK6U. HVGO stimulated biosurfactants production as indicated by the lower surface tension (26 mN/m) and higher yield (7.8 g/L) compared to a glucose culture (49.7 mN/m, 0.305 g/L). Quantitative real-time PCR showed that the biosurfactants production genes rhlA and rhlB were strongly upregulated in the HVGO culture during the early and late exponential growth phases. To the contrary, the rhamnose biosynthesis genes algC, rmlA and rmlC were downregulated in the HVGO culture. Genes of the quorum sensing systems which regulate biosurfactants biosynthesis exhibited a hierarchical expression profile. The lasI gene was strongly upregulated (20-fold) in the HVGO culture during the early log phase, whereas both rhlI and pqsE were upregulated during the late log phase. Rhamnolipid congener analysis using high-performance liquid chromatography-mass spectrometry revealed a much higher proportion (up to 69%) of the high-molecularweight homologue Rha-Rha-C10-C10 in the HVGO culture. The results shed light on the temporal and carbon source-mediated shifts in rhamonlipids' composition and regulation of biosynthesis which can be potentially exploited to produce different rhamnolipid formulations tailored for specific applications.


Assuntos
Proteínas de Bactérias/metabolismo , Gases/farmacologia , Glicolipídeos/biossíntese , Glicosiltransferases/metabolismo , Óleos Voláteis/farmacologia , Pseudomonas/metabolismo , Percepção de Quorum , Pseudomonas/efeitos dos fármacos , Pseudomonas/crescimento & desenvolvimento , Ramnose/metabolismo , Tensoativos/farmacologia , Volatilização
6.
Molecules ; 26(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299465

RESUMO

Lytic transglycosylases such as Slt35 from E. coli are enzymes involved in bacterial cell wall remodelling and recycling, which represent potential targets for novel antibacterial agents. Here, we investigated a series of known glycosidase inhibitors for their ability to inhibit Slt35. While glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, thiamet G and miglitol had no effect, the phenothiazinium dye thionine acetate was found to be a weak inhibitor. IC50 values and binding constants for thionine acetate were similar for Slt35 and the hen egg white lysozyme. Molecular docking simulations suggest that thionine binds to the active site of both Slt35 and lysozyme, although it does not make direct interactions with the side-chain of the catalytic Asp and Glu residues as might be expected based on other inhibitors. Thionine acetate also increased the potency of the beta-lactam antibiotic ampicillin against a laboratory strain of E. coli.


Assuntos
Glicosiltransferases/metabolismo , Fenotiazinas/farmacologia , Acetatos/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/química , Sítios de Ligação/genética , Domínio Catalítico/genética , Parede Celular/metabolismo , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Glicosiltransferases/antagonistas & inibidores , Glicosiltransferases/efeitos dos fármacos , Modelos Moleculares , Simulação de Acoplamento Molecular , Muramidase/antagonistas & inibidores , Muramidase/metabolismo , Peptidoglicano/metabolismo , Fenotiazinas/metabolismo , Conformação Proteica/efeitos dos fármacos
7.
New Phytol ; 232(2): 642-654, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34289137

RESUMO

The levels of the important plant growth regulator indole-3-acetic acid (IAA) are tightly controlled within plant tissues to spatiotemporally orchestrate concentration gradients that drive plant growth and development. Metabolic inactivation of bioactive IAA is known to participate in the modulation of IAA maxima and minima. IAA can be irreversibly inactivated by oxidation and conjugation to aspartate and glutamate. Usually overlooked because of its reversible nature, the most abundant inactive IAA form is the IAA-glucose (IAA-glc) conjugate. Glycosylation of IAA in Arabidopsis thaliana is reported to be carried out by UDP-glycosyltransferase 84B1 (UGT84B1), while UGT74D1 has been implicated in the glycosylation of the irreversibly formed IAA catabolite oxIAA. Here we demonstrated that both UGT84B1 and UGT74D1 modulate IAA levels throughout plant development by dual IAA and oxIAA glycosylation. Moreover, we identified a novel UGT subfamily whose members redundantly mediate the glycosylation of oxIAA and modulate skotomorphogenic growth.


Assuntos
Proteínas de Arabidopsis , Glicosiltransferases , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Homeostase , Ácidos Indolacéticos , Desenvolvimento Vegetal , Difosfato de Uridina
8.
Molecules ; 26(11)2021 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-34204142

RESUMO

Broussonetia papyrifera is a multifunctional deciduous tree that is both a food and a source of traditional Chinese medicine for both humans and animals. Further analysis of the UGT gene family is of great significance to the utilization of B. papyrifera. The substrates of plant UGT genes include highly diverse and complex chemicals, such as flavonoids and terpenes. In order to deepen our understanding of this family, a comprehensive analysis was performed. Phylogenetic analysis showed that 155 BpUGTs were divided into 15 subgroups. A conserved motif analysis showed that BpUGT proteins in the same subgroups possessed similar motif structures. Tandem duplication was the primary driving force for the expansion of the BpUGT gene family. The global promoter analysis indicated that they were associated with complex hormone regulatory networks and the stress response, as well as the synthesis of secondary metabolites. The expression pattern analysis showed that the expression level of BpUGTs in leaves and roots was higher than that in fruits and stems. Next, we determined the composition and content of flavonoids, the main products of the BpUGT reaction. A total of 19 compounds were isolated and analyzed by UPLC-ESI-MS/MS in 3 species of Broussonetia including B. kazinoki, B. papyrifera, and B. kazinoki × B. papyrifera, and the number of compounds was different in these 3 species. The total flavonoid content and antioxidant capacities of the three species were analyzed respectively. All assays exhibited the same trend: the hybrid paper mulberry showed a higher total flavonoid content, a higher total phenol content and higher antioxidant activity than the other two species. Overall, our study provides valuable information for understanding the function of BpUGTs in the biosynthesis of flavonoids.


Assuntos
Broussonetia/química , Flavonoides/isolamento & purificação , Glicosiltransferases/genética , Broussonetia/genética , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Glicosiltransferases/classificação , Glicosiltransferases/metabolismo , Família Multigênica , Filogenia , Folhas de Planta/química , Folhas de Planta/genética , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/química , Raízes de Plantas/genética , Distribuição Tecidual
9.
PLoS Pathog ; 17(6): e1009658, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34133469

RESUMO

During infection, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) directly manipulate various aspects of host cell function through the translocation of type III secretion system (T3SS) effector proteins directly into the host cell. Many T3SS effector proteins are enzymes that mediate post-translational modifications of host proteins, such as the glycosyltransferase NleB1, which transfers a single N-acetylglucosamine (GlcNAc) to arginine residues, creating an Arg-GlcNAc linkage. NleB1 glycosylates death-domain containing proteins including FADD, TRADD and RIPK1 to block host cell death. The NleB1 paralogue, NleB2, is found in many EPEC and EHEC strains but to date its enzymatic activity has not been described. Using in vitro glycosylation assays combined with mass spectrometry, we found that NleB2 can utilize multiple sugar donors including UDP-glucose, UDP-GlcNAc and UDP-galactose during glycosylation of the death domain protein, RIPK1. Sugar donor competition assays demonstrated that UDP-glucose was the preferred substrate of NleB2 and peptide sequencing identified the glycosylation site within RIPK1 as Arg603, indicating that NleB2 catalyses arginine glucosylation. We also confirmed that NleB2 catalysed arginine-hexose modification of Flag-RIPK1 during infection of HEK293T cells with EPEC E2348/69. Using site-directed mutagenesis and in vitro glycosylation assays, we identified that residue Ser252 in NleB2 contributes to the specificity of this distinct catalytic activity. Substitution of Ser252 in NleB2 to Gly, or substitution of the corresponding Gly255 in NleB1 to Ser switches sugar donor preference between UDP-GlcNAc and UDP-glucose. However, this switch did not affect the ability of the NleB variants to inhibit inflammatory or cell death signalling during HeLa cell transfection or EPEC infection. NleB2 is thus the first identified bacterial Arg-glucose transferase that, similar to the NleB1 Arg-GlcNAc transferase, inhibits host protein function by arginine glycosylation.


Assuntos
Arginina/metabolismo , Escherichia coli Enteropatogênica/metabolismo , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Glicosiltransferases/metabolismo , Fatores de Virulência/metabolismo , Linhagem Celular , Humanos
10.
ACS Chem Biol ; 16(6): 1059-1069, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34080843

RESUMO

C-Glycosylation in the biosynthesis of bioactive natural products is quite unique, which has not been studied well. Medermycin, as an antitumor agent in the family of pyranonaphthoquinone antibiotics, is featured with unique C-glycosylation. Here, a new C-glycosyltransferase (C-GT) Med-8 was identified to be essential for the biosynthesis of medermycin, as the first example of C-GT to recognize a rare deoxyaminosugar (angolosamine). med-8 and six genes (med-14, -15, -16, -17, -18, and -20 located in the medermycin biosynthetic gene cluster) predicted for the biosynthesis of angolosamine were proved to be functional and sufficient for C-glycosylation. A C-glycosylation cassette composed of these seven genes could convert a proposed substrate into a C-glycosylated product. In conclusion, these genes involved in the C-glycosylation of medermycin were functionally identified and biosynthetically engineered, and they provided the possibility of producing new C-glycosylated compounds.


Assuntos
Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Glicosiltransferases/metabolismo , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Genes Bacterianos , Glicosiltransferases/genética , Modelos Moleculares , Família Multigênica , Naftoquinonas/metabolismo , Filogenia , Streptomyces/genética
11.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073255

RESUMO

Whole genome sequences of two Acinetobacter baumannii clinical isolates, 48-1789 and MAR24, revealed that they carry the KL106 and KL112 capsular polysaccharide (CPS) biosynthesis gene clusters, respectively, at the chromosomal K locus. The KL106 and KL112 gene clusters are related to the previously described KL11 and KL83 gene clusters, sharing genes for the synthesis of l-rhamnose (l-Rhap) and 6-deoxy-l-talose (l-6dTalp). CPS material isolated from 48-1789 and MAR24 was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy. The structures of K106 and K112 oligosaccharide repeats (K units) l-6dTalp-(1→3)-D-GlcpNAc tetrasaccharide fragment share the responsible genes in the respective gene clusters. The K106 and K83 CPSs also have the same linkage between K units. The KL112 cluster includes an additional glycosyltransferase gene, Gtr183, and the K112 unit includes α l-Rhap side chain that is not found in the K106 structure. K112 further differs in the linkage between K units formed by the Wzy polymerase, and a different wzy gene is found in KL112. However, though both KL106 and KL112 share the atr8 acetyltransferase gene with KL83, only K83 is acetylated.


Assuntos
Acinetobacter baumannii , Desoxiaçúcares , Hexoses , Polissacarídeos Bacterianos , Acinetobacter baumannii/química , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Desoxiaçúcares/química , Desoxiaçúcares/genética , Desoxiaçúcares/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Hexoses/química , Hexoses/genética , Hexoses/metabolismo , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Especificidade da Espécie
12.
Commun Biol ; 4(1): 678, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083706

RESUMO

Toxicity mechanisms of metal oxide nanoparticles towards bacteria and underlying roles of membrane composition are still debated. Herein, the response of lipopolysaccharide-truncated Escherichia coli K12 mutants to TiO2 nanoparticles (TiO2NPs, exposure in dark) is addressed at the molecular, single cell, and population levels by transcriptomics, fluorescence assays, cell nanomechanics and electrohydrodynamics. We show that outer core-free lipopolysaccharides featuring intact inner core increase cell sensitivity to TiO2NPs. TiO2NPs operate as membrane strippers, which induce osmotic stress, inactivate cell osmoregulation and initiate lipid peroxidation, which ultimately leads to genesis of membrane vesicles. In itself, truncation of lipopolysaccharide inner core triggers membrane permeabilization/depolarization, lipid peroxidation and hypervesiculation. In turn, it favors the regulation of TiO2NP-mediated changes in cell Turgor stress and leads to efficient vesicle-facilitated release of damaged membrane components. Remarkably, vesicles further act as electrostatic baits for TiO2NPs, thereby mitigating TiO2NPs toxicity. Altogether, we highlight antagonistic lipopolysaccharide-dependent bacterial responses to nanoparticles and we show that the destabilized membrane can generate unexpected resistance phenotype.


Assuntos
Vesículas Citoplasmáticas/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Pressão Osmótica/efeitos dos fármacos , Titânio/toxicidade , Vesículas Citoplasmáticas/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Microscopia de Força Atômica/métodos , Mutação
13.
Int J Biol Macromol ; 184: 1014-1025, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34171260

RESUMO

There are many reports on exopolysaccharides of lactic acid bacteria (LAB EPS) such as isolation, production and applications. The LAB EPS have been proved to exhibit significantly improved texture and rheological properties in order to prevent syneresis of fermented foods. Furthermore, they are known to have many biological properties such as mouthwatering flavors, antioxidant activity, cholesterol lowering and antimicrobial activities. Considering their GRAS status, LAB EPS need to be explored for better titre and improved biological properties, where strain improvement by genetic engineering has a major role for making tailor-made EPS. The genetic overview of the EPS production by LAB is an auxiliary area of interest as the process and the biosynthetic pathway involves numerous genes and their proteins. Among them Glycosyltransferases (gtfs) are the key enzymes involved in EPS biosynthesis. Current knowledge of gtfs of LAB and its manipulation is limited. The present review spotlights the importance of glycosyltransferases and their specific role on the biosynthesis of LAB EPS and addresses the functionality and applicability of these enzymes and their products. It enfold the available literature including some patents in recent past to underline the fact that glycosyltransferases are un-reluctantly the key proteins involved in the EPS biosynthesis.


Assuntos
Genômica/métodos , Glicosiltransferases/genética , Lactobacillales/enzimologia , Polissacarídeos Bacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fermentação , Microbiologia de Alimentos , Glicosiltransferases/metabolismo , Lactobacillales/genética , Probióticos , Reologia
14.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064863

RESUMO

At the plasma membrane, gangliosides, a group of glycosphingolipids, are expressed along with glycosphingolipids, phospholipids, and cholesterol in so-called lipid rafts that interact with signaling receptors and related molecules. Most cancers present abnormalities in the intracellular signal transduction system involved in tumor growth, invasion, and metastasis. To date, the roles of gangliosides as regulators of signal transduction have been reported in several cancer types. Gangliosides can be expressed by the exogenous ganglioside addition, with their endogenous expression regulated at the enzymatic level by targeting specific glycosyltransferases. Accordingly, the relationship between changes in the composition of cell surface gangliosides and signal transduction has been investigated by controlling ganglioside expression. In cancer cells, several types of signaling molecules are positively or negatively regulated by ganglioside expression levels, promoting malignant properties. Moreover, antibodies against gangliosides have been shown to possess cytotoxic effects on ganglioside-expressing cancer cells. In the present review, we highlight the involvement of gangliosides in the regulation of cancer cell signaling, and we explore possible therapies targeting ganglioside-expressing cancer.


Assuntos
Gangliosídeos/metabolismo , Glicosiltransferases/metabolismo , Neoplasias/patologia , Animais , Humanos , Neoplasias/metabolismo , Transdução de Sinais
15.
Plant Cell ; 33(3): 735-749, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33955489

RESUMO

The tradeoff between growth and defense is a critical aspect of plant immunity. Therefore, the plant immune response needs to be tightly regulated. Salicylic acid (SA) is an important plant hormone regulating defense against biotrophic pathogens. Recently, N-hydroxy-pipecolic acid (NHP) was identified as another regulator for plant innate immunity and systemic acquired resistance (SAR). Although the biosynthetic pathway leading to NHP formation is already been identified, how NHP is further metabolized is unclear. Here, we present UGT76B1 as a uridine diphosphate-dependent glycosyltransferase (UGT) that modifies NHP by catalyzing the formation of 1-O-glucosyl-pipecolic acid in Arabidopsis thaliana. Analysis of T-DNA and clustered regularly interspaced short palindromic repeats (CRISPR) knock-out mutant lines of UGT76B1 by targeted and nontargeted ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) underlined NHP and SA as endogenous substrates of this enzyme in response to Pseudomonas infection and UV treatment. ugt76b1 mutant plants have a dwarf phenotype and constitutive defense response which can be suppressed by loss of function of the NHP biosynthetic enzyme FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1). This suggests that elevated accumulation of NHP contributes to the enhanced disease resistance in ugt76b1. Externally applied NHP can move to distal tissue in ugt76b1 mutant plants. Although glycosylation is not required for the long-distance movement of NHP during SAR, it is crucial to balance growth and defense.


Assuntos
Proteínas de Arabidopsis/metabolismo , Glicosiltransferases/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Regulação da Expressão Gênica de Plantas , Glicosiltransferases/genética , Ácidos Pipecólicos/metabolismo , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Pseudomonas syringae/patogenicidade , Ácido Salicílico/metabolismo
16.
Rev Physiol Biochem Pharmacol ; 180: 85-117, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34031738

RESUMO

Among neoplasia-associated epigenetic alterations, changes in cellular glycosylation have recently received attention as a key component of hematological malignancy progression. Alterations in glycosylation appear to not only directly impact cell growth and survival, but also alter the adhesion of tumor cells and their interactions with the microenvironment, facilitating cancer-induced immunomodulation and eventual metastasis. Changes in glycosylation arise from altered expression of glycosyltransferases, enzymes that catalyze the transfer of saccharide moieties to a wide range of acceptor substrates, such as proteins, lipids, and other saccharides in the endoplasmic reticulum (ER) and Golgi apparatus. Novel glycan structures in hematological malignancies represent new targets for the diagnosis and treatment of blood diseases. This review summarizes studies of the aberrant expression of glycans commonly found in hematological malignancies and their potential mechanisms and defines the specific roles of glycans as drivers or passengers in the development of hematological malignancies.


Assuntos
Glicosiltransferases , Neoplasias Hematológicas , Células Sanguíneas/metabolismo , Glicosilação , Glicosiltransferases/metabolismo , Humanos , Polissacarídeos , Microambiente Tumoral
17.
J Agric Food Chem ; 69(19): 5491-5499, 2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-33973475

RESUMO

Cinmethylin is a well-known benzyl-ether derivative of the natural terpene 1,4-cineole that is used industrially as a pre-emergence herbicide in grass weed control for crop protection. Cinmethylin detoxification in plants has not been reported, but in animals, it prominently involves hydroxylation at the benzylic C15 methyl group. Here, we show enzymatic ß-glycosylation of synthetic 15-hydroxy-cinmethylin to prepare a putative phase II detoxification metabolite of the cinmethylin in plants. We examined eight Leloir glycosyltransferases for reactivity with 15-hydroxy cinmethylin and revealed the selective formation of 15-hydroxy cinmethylin ß-d-glucoside from uridine 5'-diphosphate (UDP)-glucose by the UGT71E5 from safflower (Carthamus tinctorius). The UGT71E5 showed a specific activity of 431 mU/mg, about 300-fold higher than that of apple (Malus domestica) UGT71A15 that also performed the desired 15-hydroxy cinmethylin mono-glycosylation. Bacterial glycosyltransferases (OleD from Streptomyces antibioticus, 2.9 mU/mg; GT1 from Bacillus cereus, 60 mU/mg) produced mixtures of 15-hydroxy cinmethylin mono- and disaccharide glycosides. Using UDP-glucose recycling with sucrose synthase, 15-hydroxy cinmethylin conversion with UGT71E5 efficiently provided the ß-mono-glucoside (≥95% yield; ∼9 mM) suitable for biological studies.


Assuntos
Herbicidas , Malus , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Malus/metabolismo , Uridina Difosfato Glucose/metabolismo
18.
J Exp Bot ; 72(11): 4053-4067, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33948638

RESUMO

This review commemorates the 50th anniversary of the Nobel Prize in Chemistry awarded to Luis F. Leloir 'for his discovery of sugar-nucleotides and their role in the biosynthesis of carbohydrates'. He and his co-workers discovered that activated forms of simple sugars, such as UDP-glucose and UDP-galactose, are essential intermediates in the interconversion of sugars. They elucidated the biosynthetic pathways for sucrose and starch, which are the major end-products of photosynthesis, and for trehalose. Trehalose 6-phosphate, the intermediate of trehalose biosynthesis that they discovered, is now a molecule of great interest due to its function as a sugar signalling metabolite that regulates many aspects of plant metabolism and development. The work of the Leloir group also opened the doors to an understanding of the biosynthesis of cellulose and other structural cell wall polysaccharides (hemicelluloses and pectins), and ascorbic acid (vitamin C). Nucleotide-sugars also serve as sugar donors for a myriad of glycosyltransferases that conjugate sugars to other molecules, including lipids, phytohormones, secondary metabolites, and proteins, thereby modifying their biological activity. In this review, we highlight the diversity of nucleotide-sugars and their functions in plants, in recognition of Leloir's rich and enduring legacy to plant science.


Assuntos
Parede Celular , Plantas , Metabolismo dos Carboidratos , Parede Celular/metabolismo , Glicosiltransferases/metabolismo , Uridina Difosfato Glucose/metabolismo
19.
Plant Cell ; 33(3): 714-734, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33955482

RESUMO

Glucosylation modulates the biological activity of small molecules and frequently leads to their inactivation. The Arabidopsis thaliana glucosyltransferase UGT76B1 is involved in conjugating the stress hormone salicylic acid (SA) as well as isoleucic acid (ILA). Here, we show that UGT76B1 also glucosylates N-hydroxypipecolic acid (NHP), which is synthesized by FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1) and activates systemic acquired resistance (SAR). Upon pathogen attack, Arabidopsis leaves generate two distinct NHP hexose conjugates, NHP-O-ß-glucoside and NHP glucose ester, whereupon only NHP-O-ß-glucoside formation requires a functional SA pathway. The ugt76b1 mutants specifically fail to generate the NHP-O-ß-glucoside, and recombinant UGT76B1 synthesizes NHP-O-ß-glucoside in vitro in competition with SA and ILA. The loss of UGT76B1 elevates the endogenous levels of NHP, SA, and ILA and establishes a constitutive SAR-like immune status. Introgression of the fmo1 mutant lacking NHP biosynthesis into the ugt76b1 background abolishes this SAR-like resistance. Moreover, overexpression of UGT76B1 in Arabidopsis shifts the NHP and SA pools toward O-ß-glucoside formation and abrogates pathogen-induced SAR. Our results further indicate that NHP-triggered immunity is SA-dependent and relies on UGT76B1 as a common metabolic hub. Thereby, UGT76B1-mediated glucosylation controls the levels of active NHP, SA, and ILA in concert to balance the plant immune status.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glicosiltransferases/metabolismo , Ácidos Pipecólicos/metabolismo , Imunidade Vegetal/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Glicosiltransferases/genética , Imunidade Vegetal/genética
20.
Appl Environ Microbiol ; 87(14): e0032621, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33931419

RESUMO

In the marine environment, phosphorus availability significantly affects the lipid composition in many cosmopolitan marine heterotrophic bacteria, including members of the SAR11 clade and the Roseobacter clade. Under phosphorus stress conditions, nonphosphorus sugar-containing glycoglycerolipids are substitutes for phospholipids in these bacteria. Although these glycoglycerolipids play an important role as surrogates for phospholipids under phosphate deprivation, glycoglycerolipid synthases in marine microbes are poorly studied. In the present study, we biochemically characterized a glycolipid glycosyltransferase (GTcp) from the marine bacterium "Candidatus Pelagibacter sp." strain HTCC7211, a member of the SAR11 clade. Our results showed that GTcp is able to act as a multifunctional enzyme by synthesizing different glycoglycerolipids with UDP-glucose, UDP-galactose, or UDP-glucuronic acid as sugar donors and diacylglycerol (DAG) as the acceptor. Analyses of enzyme kinetic parameters demonstrated that Mg2+ notably changes the enzyme's affinity for UDP-glucose, which improves its catalytic efficiency. Homology modeling and mutational analyses revealed binding sites for the sugar donor and the diacylglycerol lipid acceptor, which provided insights into the retaining mechanism of GTcp with its GT-B fold. A phylogenetic analysis showed that GTcp and its homologs form a group in the GT4 glycosyltransferase family. These results not only provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodeling but also describe an efficient enzymatic tool for the future synthesis of bioactive molecules. IMPORTANCE The bilayer formed by membrane lipids serves as the containment unit for living microbial cells. In the marine environment, it has been firmly established that phytoplankton and heterotrophic bacteria can replace phospholipids with nonphosphorus sugar-containing glycoglycerolipids in response to phosphorus limitation. However, little is known about how these glycoglycerolipids are synthesized. Here, we determined the biochemical characteristics of a glycolipid glycosyltransferase (GTcp) from the marine bacterium "Candidatus Pelagibacter sp." strain HTCC7211. GTcp and its homologs form a group in the GT4 glycosyltransferase family and can synthesize neutral glycolipids (monoglucosyl-1,2-diacyl-sn-glycerol [MGlc-DAG] and monogalactosyl [MGal]-DAG) and monoglucuronic acid diacylglycerol (MGlcA-DAG). We also uncovered the key residues for DAG binding through molecular docking, site-direct mutagenesis, and subsequent enzyme activity assays. Our data provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodeling.


Assuntos
Alphaproteobacteria/enzimologia , Glicolipídeos/química , Glicosiltransferases/química , Alphaproteobacteria/genética , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Metais Pesados/química , Especificidade por Substrato
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