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1.
Nat Commun ; 11(1): 4874, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978395

RESUMO

Organic synthesis methodology enables the synthesis of complex molecules and materials used in all fields of science and technology and represents a vast body of accumulated knowledge optimally suited for deep learning. While most organic reactions involve distinct functional groups and can readily be learned by deep learning models and chemists alike, regio- and stereoselective transformations are more challenging because their outcome also depends on functional group surroundings. Here, we challenge the Molecular Transformer model to predict reactions on carbohydrates where regio- and stereoselectivity are notoriously difficult to predict. We show that transfer learning of the general patent reaction model with a small set of carbohydrate reactions produces a specialized model returning predictions for carbohydrate reactions with remarkable accuracy. We validate these predictions experimentally with the synthesis of a lipid-linked oligosaccharide involving regioselective protections and stereoselective glycosylations. The transfer learning approach should be applicable to any reaction class of interest.


Assuntos
Carboidratos/química , Aprendizado de Máquina , Oligossacarídeos/química , Metabolismo dos Carboidratos , Técnicas de Química Sintética , Glicosilação , Estrutura Molecular , Oligossacarídeos/metabolismo
2.
Nat Commun ; 11(1): 3285, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620774

RESUMO

The early life human gut microbiota exerts life-long health effects on the host, but the mechanisms underpinning its assembly remain elusive. Particularly, the early colonization of Clostridiales from the Roseburia-Eubacterium group, associated with protection from colorectal cancer, immune- and metabolic disorders is enigmatic. Here, we describe catabolic pathways that support the growth of Roseburia and Eubacterium members on distinct human milk oligosaccharides (HMOs). The HMO pathways, which include enzymes with a previously unknown structural fold and specificity, were upregulated together with additional glycan-utilization loci during growth on selected HMOs and in co-cultures with Akkermansia muciniphila on mucin, suggesting an additional role in enabling cross-feeding and access to mucin O-glycans. Analyses of 4599 Roseburia genomes underscored the preponderance and diversity of the HMO utilization loci within the genus. The catabolism of HMOs by butyrate-producing Clostridiales may contribute to the competitiveness of this group during the weaning-triggered maturation of the microbiota.


Assuntos
Butiratos/metabolismo , Clostridiales/metabolismo , Leite Humano/metabolismo , Mucinas/metabolismo , Oligossacarídeos/metabolismo , Bifidobacterium/metabolismo , Clostridiales/genética , Colo/microbiologia , Eubacterium/metabolismo , Microbioma Gastrointestinal/fisiologia , Humanos , Lactente , Recém-Nascido , Metabolismo/fisiologia , Leite Humano/química , Polissacarídeos/metabolismo , Verrucomicrobia/metabolismo , Desmame
3.
Food Chem ; 332: 127437, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32645670

RESUMO

In 'Crimson Seedless' grapes, the appearance of senescence caused by abnormal dark red color, the loss of crisp taste caused by the decrease in firmness, and the fading of sweetness caused by the decrease in total soluble sugar (TSS) are the main problems affecting its edible qualities after storage. In the mesocarp, burdock fructooligosaccharide (BFO) restricted sucrose export; therefore, more carbohydrates were retained directly leading to higher TSS and sweetness, and cell osmotic pressure and firmness were retained indirectly. In the exocarp, BFO restricted sucrose import; therefore, the signal molecule sucrose was reduced and the senescence-related processes were inhibited. The downregulation of SUC12 and SUC27 by BFO may play an important role in restricting sucrose transportation. The opposing effects exhibited by exogenous sucrose treatments compared to those of BFO further verified these mechanisms. Based on the above mechanisms, sucrose transportation mediates the fresh-keeping effects of BFO in 'Crimson Seedless' grapes.


Assuntos
Oligossacarídeos/metabolismo , Sacarose/metabolismo , Vitis/metabolismo , Transporte Biológico , Frutas/química , Frutas/metabolismo , Oligossacarídeos/análise , Sacarose/análise , Vitis/química
4.
Food Chem ; 332: 127438, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32645671

RESUMO

ß-N-acetylhexosaminidases have attracted much attention in recent years due to their potential application in oligosaccharide production, in particular lacto-N-triose II (LNT2) and lacto-N-neotetraose (LNnT) synthesis, which can be further used as backbone precursors for human milk oligosaccharides. A novel ß-N-acetylhexosaminidase gene from Tyzzerella nexilis (TnHex189) was heterologously expressed in Bacillus subtilis. The highest ß-N-acetylhexosaminidase activity of 14.5 U mL-1 was obtained in a 5-L fermentor by fed-batch fermentation for 27 h. TnHex189 was optimally active at pH 5.0 and 45 °C. It efficiently synthesized LNT2 with a conversion ratio of 57.2% (4.7 g L-1). The synthesized LNT2 was further converted to LNnT by a reported ß-galactosidase (BgaD-D) in 8 h, with a conversion ratio of 17.3% (6.1 g L-1). These unique synthesis activities may make this enzyme a good candidate for the food industry.


Assuntos
Proteínas de Bactérias/metabolismo , Clostridiales/enzimologia , Trissacarídeos/biossíntese , beta-N-Acetil-Hexosaminidases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clostridiales/genética , Estabilidade Enzimática , Fermentação , Expressão Gênica , Concentração de Íons de Hidrogênio , Oligossacarídeos/metabolismo , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/genética
5.
PLoS One ; 15(6): e0235120, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584851

RESUMO

Two low-phytate soybean (Glycine max (L.) Merr.) mutant lines- V99-5089 (mips mutation on chromosome 11) and CX-1834 (mrp-l and mrp-n mutations on chromosomes 19 and 3, respectively) have proven to be valuable resources for breeding of low-phytate, high-sucrose, and low-raffinosaccharide soybeans, traits that are highly desirable from a nutritional and environmental standpoint. A recombinant inbred population derived from the cross CX1834 x V99-5089 provides an opportunity to study the effect of different combinations of these three mutations on soybean phytate and oligosaccharides levels. Of the 173 recombinant inbred lines tested, 163 lines were homozygous for various combinations of MIPS and two MRP loci alleles. These individuals were grouped into eight genotypic classes based on the combination of SNP alleles at the three mutant loci. The two genotypic classes that were homozygous mrp-l/mrp-n and either homozygous wild-type or mutant at the mips locus (MIPS/mrp-l/mrp-n or mips/mrp-l/mrp-n) displayed relatively similar ~55% reductions in seed phytate, 6.94 mg g -1 and 6.70 mg g-1 respectively, as compared with 15.2 mg g-1 in the wild-type MIPS/MRP-L/MRP-N seed. Therefore, in the presence of the double mutant mrp-l/mrp-n, the mips mutation did not cause a substantially greater decrease in seed phytate level. However, the nutritionally-desirable high-sucrose/low-stachyose/low-raffinose seed phenotype originally observed in soybeans homozygous for the mips allele was reversed in the presence of mrp-l/mrp-n mutations: homozygous mips/mrp-l/mrp-n seed displayed low-sucrose (7.70%), high-stachyose (4.18%), and the highest observed raffinose (0.94%) contents per gram of dry seed. Perhaps the block in phytic acid transport from its cytoplasmic synthesis site to its storage site, conditioned by mrp-l/mrp-n, alters myo-inositol flux in mips seeds in a way that restores to wild-type levels the mips conditioned reductions in raffinosaccharides. Overall this study determined the combinatorial effects of three low phytic acid causing mutations on regulation of seed phytate and oligosaccharides in soybean.


Assuntos
Loci Gênicos , Mutação , Oligossacarídeos , Ácido Fítico/metabolismo , Sementes , Soja , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Sementes/genética , Sementes/metabolismo , Soja/genética , Soja/metabolismo
6.
Nat Chem Biol ; 16(7): 766-775, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483376

RESUMO

Cell surfaces are glycosylated in various ways with high heterogeneity, which usually leads to ambiguous conclusions about glycan-involved biological functions. Here, we describe a two-step chemoenzymatic approach for N-glycan-subtype-selective editing on the surface of living cells that consists of a first 'delete' step to remove heterogeneous N-glycoforms of a certain subclass and a second 'insert' step to assemble a well-defined N-glycan back onto the pretreated glyco-sites. Such glyco-edited cells, carrying more homogeneous oligosaccharide structures, could enable precise understanding of carbohydrate-mediated functions. In particular, N-glycan-subtype-selective remodeling and imaging with different monosaccharide motifs at the non-reducing end were successfully achieved. Using a combination of the expression system of the Lec4 CHO cell line and this two-step glycan-editing approach, opioid receptor delta 1 (OPRD1) was investigated to correlate its glycostructures with the biological functions of receptor dimerization, agonist-induced signaling and internalization.


Assuntos
Membrana Celular/química , Células Epiteliais/química , Glicoconjugados/química , Oligossacarídeos/química , Receptores Opioides delta/química , Animais , Células CHO , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colforsina/farmacologia , Cricetulus , Encefalina Leucina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica , Glicoconjugados/metabolismo , Glicosilação , Células HEK293 , Humanos , Camundongos , Oligossacarídeos/metabolismo , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Transgenes
7.
Proc Natl Acad Sci U S A ; 117(22): 11931-11939, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32424105

RESUMO

Cell surfaces are often decorated with glycoconjugates that contain linear and more complex symmetrically and asymmetrically branched carbohydrates essential for cellular recognition and communication processes. Mannose is one of the fundamental building blocks of glycans in many biological membranes. Moreover, oligomannoses are commonly found on the surface of pathogens such as bacteria and viruses as both glycolipids and glycoproteins. However, their mechanism of action is not well understood, even though this is of great potential interest for translational medicine. Sequence-defined amphiphilic Janus glycodendrimers containing simple mono- and disaccharides that mimic glycolipids are known to self-assemble into glycodendrimersomes, which in turn resemble the surface of a cell by encoding carbohydrate activity via supramolecular multivalency. The synthetic challenge of preparing Janus glycodendrimers containing more complex linear and branched glycans has so far prevented access to more realistic cell mimics. However, the present work reports the use of an isothiocyanate-amine "click"-like reaction between isothiocyanate-containing sequence-defined amphiphilic Janus dendrimers and either linear or branched oligosaccharides containing up to six monosaccharide units attached to a hydrophobic amino-pentyl linker, a construct not expected to assemble into glycodendrimersomes. Unexpectedly, these oligoMan-containing dendrimers, which have their hydrophobic linker connected via a thiourea group to the amphiphilic part of Janus glycodendrimers, self-organize into nanoscale glycodendrimersomes. Specifically, the mannose-binding lectins that best agglutinate glycodendrimersomes are those displaying hexamannose. Lamellar "raft-like" nanomorphologies on the surface of glycodendrimersomes, self-organized from these sequence-defined glycans, endow these membrane mimics with high biological activity.


Assuntos
Biomimética/métodos , Dendrímeros/síntese química , Glicoconjugados/síntese química , Nanopartículas/química , Membrana Celular/química , Glicolipídeos/química , Interações Hidrofóbicas e Hidrofílicas , Isotiocianatos/metabolismo , Lectinas/metabolismo , Manose/metabolismo , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Pesquisa Médica Translacional/métodos
8.
Am J Clin Nutr ; 112(1): 106-112, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32401307

RESUMO

BACKGROUND: Oligosaccharides are the third most abundant component in human milk. They are a potential protective agent against neonatal sepsis. OBJECTIVES: We aimed to explore the association between human milk oligosaccharides (HMOs) and late-onset sepsis in very-low-birth-weight infants, and to describe the composition and characteristics of HMOs in Peruvian mothers of these infants. METHODS: This is a secondary data analysis of a randomized clinical trial. We conducted a retrospective cohort study of mothers and their very-low-birth-weight (<1500 g) infants with ≥1 milk sample and follow-up data for >30 d. HMOs were measured by high performance liquid chromatography (HPLC). We used factor analysis and the Mantel-Cox test to explore the association between HMOs and late-onset neonatal sepsis. RESULTS: We included 153 mother-infant pairs and 208 milk samples. Overall, the frequency of the secretor phenotype was 93%. Secretors and nonsecretors were defined by the presence and near-absence of α1-2-fucosylated HMOs, respectively. The most abundant oligosaccharides were 2'-fucosyllactose, lacto-N-fucopentaose (LNFP) I, and difucosyllacto-N-tetraose in secretors and lacto-N-tetraose and LNFP II in nonsecretors. Secretors had higher amounts of total oligosaccharides than nonsecretors (11.45 g/L; IQR: 0.773 g/L compared with 8.04 g/L; IQR: 0.449 g/L). Mature milk samples were more diverse in terms of HMOs than colostrum (Simpson's Reciprocal Diversity Index). We found an association of factor 3 in colostrum with a reduced risk of late-onset sepsis (HR: 0.63; 95% CI: 0.41, 0.97). Fucosyl-disialyllacto-N-hexose (FDSLNH) was the only oligosaccharide correlated to factor 3. CONCLUSIONS: These findings suggest that concentrations of different HMOs vary from one individual to another according to their lactation period and secretor status. We also found that FDSLNH might protect infants with very low birth weight from late-onset neonatal sepsis. Confirming this association could prove 1 more mechanism by which human milk protects infants against infections and open the door to clinical applications of HMOs.This trial was registered at clinicaltrials.gov as NCT01525316.


Assuntos
Recém-Nascido de muito Baixo Peso/metabolismo , Leite Humano/química , Leite Humano/metabolismo , Sepse Neonatal/metabolismo , Oligossacarídeos/metabolismo , Adulto , Idade de Início , Colostro/química , Colostro/metabolismo , Feminino , Humanos , Lactente , Masculino , Oligossacarídeos/análise , Peru , Estudos Retrospectivos , Adulto Jovem
9.
Plant Mol Biol ; 103(4-5): 581-596, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32409993

RESUMO

KEY MESSAGE: N-glycans play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects. Asparagine-linked (Asn/N-) glycosylation is one of the most prevalent and complex protein modifications and the associated N-glycans play crucial roles on protein folding and secretion. The studies have shown that many glycoproteins hold multiple N-glycans, yet little is known about the redundancy of N-glycans on a protein. In this study, we used BRI1 to decipher the roles of N-glycans on protein secretion and function. We found that all 14 potential N-glycosylation sites on BRI1 were occupied with oligosaccharides. The elimination of single N-glycan had no obvious effect on BRI1 secretion or function except N154-glycan, which resulted in the retention of BRI1 in the endoplasmic reticulum (ER), similar to the loss of multiple highly conserved N-glycans. To misfolded bri1, the absence of N-glycans next to local structural defects enhanced the ER retention and the artificial addition of N-glycan could help the misfolded bri1-GFPs exiting from the ER, indicating that the N-glycans might serve as steric hindrance to protect the structure defects from ER recognition. We also found that the retention of misfolded bri1-9 by lectins and chaperones in the ER relied on the presence of multiple N-glycans distal to the local defects. Our findings revealed that the N-glycans might play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Polissacarídeos/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologia , Alcaloides/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Glicoproteínas/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação , Modelos Moleculares , Oligossacarídeos/metabolismo , Plantas Geneticamente Modificadas , Conformação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Plântula , Sementes/citologia , Sementes/metabolismo , Transdução de Sinais/genética
10.
PLoS Pathog ; 16(5): e1008501, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32369532

RESUMO

Plant-parasitic nematodes cause huge agricultural economic losses. Two major families of Bacillus thuringiensis crystal proteins, Cry5 and Cry6, show nematicidal activity. Previous work showed that binding to midgut receptors is a limiting step in Cry toxin mode of action. In the case of Cry5Ba, certain Caenorhabditis elegans glycolipids were identified as receptors of this toxin. However, the receptors for Cry6 toxin remain unknown. In this study, the C. elegans CUB-like-domain containing protein RBT-1, released by phosphatidylinositol-specific phospholipase C (PI-PLC), was identified as a Cry6Aa binding protein by affinity chromatography. RBT-1 contained a predicted glycosylphosphatidylinositol (GPI) anchor site and was shown to locate in lipid rafts in the surface of the midgut cells. Western ligand blot assays and ELISA binding analysis confirmed the binding interaction between Cry6Aa and RBT-1 showing high affinity and specificity. In addition, the mutation of rbt-1 gene decreased the susceptibility of C. elegans to Cry6Aa but not that of Cry5Ba. Furthermore, RBT-1 mediated the uptake of Cry6Aa into C. elegans gut cells, and was shown to be involved in triggering pore-formation activity, indicating that RBT-1 is required for the interaction of Cry6Aa with the nematode midgut cells. These results support that RBT-1 is a functional receptor for Cry6Aa.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/genética , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Mutação , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Receptores de Superfície Celular/genética
11.
Adv Exp Med Biol ; 1221: 61-69, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274706

RESUMO

Heparanase was discovered during a study of the heparin proteoglycan (serglycin) in mast cells. Newly synthesized polysaccharide chains, kDa 60-100 x 103, were rapidly degraded to fragments similar in size to commercially available heparin (averaging 15 x 103). Analysis of the degradation products identified reducing-terminal glucuronic acid residues, shown by studies of heparin biosynthesis to be of ßD-configuration in the intact polymer. Heparanase, thus identified as an endo-ßD-glucuronidase, was subsequently identified in a variety of tissues and cells. The enzyme was subsequently implicated with a variety of pathophysiological processes, including in particular cancer, inflammatory diseases, and amyloidosis, as detailed in subsequent chapters of this volume. The target for enzyme action in these settings is primarily extracellular heparan sulfate proteoglycans; furthermore, intracellular cleavage initiates degradation of heparan sulfate chains by exolytic hydrolases and sulfatases, as part of normal turnover of the polysaccharide. More unexpectedly, heparanase also influences heparan sulfate biosynthesis, such that overexpression of the enzyme results in generation of highly sulfated, heparin-like oligosaccharides. The mechanism behind this effect remains unclear - along with the overall design of the molecular machinery in control of proteoglycan biosynthesis.


Assuntos
Glucuronidase/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Oligossacarídeos/metabolismo , Especificidade por Substrato
12.
PLoS One ; 15(4): e0230472, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315306

RESUMO

BACKGROUND: Numerous studies have shown that specific components of breast milk, considered separately, are associated with disease status in the mother or the child using univariate analyses. However, very few studies have considered multivariate analysis approaches to evaluate the relationship between multiple breast milk components simultaneously. AIM: Here we aimed at visualizing breast milk component complex interactions in the context of the allergy status of the mother or the child. METHODS: Milk samples were collected from lactating mothers participating in the Leipziger Forschungszentrum für Zivilisationskrankheiten (LIFE) Child cohort in Leipzig, Germany. A total of 156 breast milk samples, collected at 3 months after birth from mother/infant pairs, were analyzed for 51 breast milk components. Correlation, principal component analysis (PCA) and graphical discovery analysis were used. RESULT: Correlations ranging from 0.40 to 0.96 were observed between breast milk fatty acid and breast milk phospholipids levels and correlations ranging from 0 to 0.76 between specific human milk oligosaccharides (HMO) were observed. No separation of the data based on the risk of allergy in the infants was identified using PCA. When graphical discovery analysis was used, dependencies between maternal plasma immunoglobulin E (IgE) level and the breast milk immune marker transforming growth factor-beta 2 (TGF-ß2), between TGF-ß2, breast milk immunoglobulin A (IgA) and TGF-ß1 as well as between breast milk total protein and birth weight were observed. Graphical discovery analysis also exemplifies a possible competition for the fucosyl group between 2'FL, LNFP-I and 3'FL in the HMO group. Additionally, dependencies between immune component IgA and specific HMO (6'SL and blood group A antigen tetraose type 5 or PI-HMO) were identified. CONCLUSION: Graphical discovery analysis applied to complex matrices such as breast milk composition can aid in understanding the complexity of interactions between breast milk components and possible relations to health parameters in the mother or the infant. This approach can lead to novel discoveries in the context of health and diseases such as allergy. Our study thus represents the first attempt to visualize the complexity and the inter-dependency of breast milk components.


Assuntos
Imunoglobulina A/metabolismo , Hipersensibilidade a Leite/etiologia , Leite Humano/química , Oligossacarídeos/metabolismo , Adulto , Aleitamento Materno , Criança , Estudos de Coortes , Dermatite Alérgica de Contato , Feminino , Humanos , Imunoglobulina E/sangue , Lactente , Lactação , Metacrilatos/efeitos adversos , Análise de Componente Principal/métodos , Fatores de Crescimento Transformadores/metabolismo
13.
J Biosci Bioeng ; 130(2): 128-136, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32265131

RESUMO

The N- and O-linked oligosaccharides from fission yeast Schizosaccharomyces pombe not only contain large amounts of d-mannose (Man) but also contain large amounts of d-galactose (Gal). Although the galactomannans of S. pombe are mainly composed of α1,2- or α1,3-linked Gals, some of the terminal α1,2-linked Gals are found to be linked to pyruvylated ß1,3-linked galactose (PvGal). We have determined the structural characteristics of the N-glycans and O-glycans in three Schizosaccharomyces species (S. japonicus, S. octosporus, and S. cryophilus) using lectin blot, 1H NMR spectroscopy, and size-fractionation high performance liquid chromatography (HPLC), and found that the galactosylation of oligosaccharides was a common feature in fission yeasts. In addition, each of the terminal Galα1,2-, Galß1,3- and non-substituted Man residues exhibited distinct characteristics. A BLAST search of gene databases in Schizosaccharomyces identified genes homologous to pvg1 encoding pyruvyltransferase of S. pombe. These genes, when expressed in an S. pombe pvg1Δ strains, led to the pyruvylation of non-reducing terminal ß-linked Gal, suggesting the biosynthetic pathway of PvGal-containing oligosaccharides is highly conserved in fission yeasts.


Assuntos
Oligossacarídeos/química , Polissacarídeos/química , Schizosaccharomyces/química , Lectinas/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Oligossacarídeos/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
14.
J Nutr ; 150(5): 1058-1067, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32133503

RESUMO

BACKGROUND: The use of biomarkers of food intake (BFIs) in blood and urine has shown great promise for assessing dietary intake and complementing traditional dietary assessment tools whose use is prone to misreporting. OBJECTIVE: Untargeted LC-MS metabolomics was applied to identify candidate BFIs for assessing the intake of milk and cheese and to explore the metabolic response to the ingestion of these foods. METHODS: A randomized controlled crossover study was conducted in healthy adults [5 women, 6 men; age: 23.6 ± 5.0 y; BMI (kg/m2): 22.1 ± 1.7].  After a single isocaloric intake of milk (600 mL), cheese (100 g), or soy-based drink (600 mL), serum and urine samples were collected postprandially up to 6 h and after fasting after 24 h. Untargeted metabolomics was conducted using LC-MS. Discriminant metabolites were selected in serum by multivariate statistical analysis, and their mass distribution and postprandial kinetics were compared. RESULTS: Serum metabolites discriminant for cheese intake had a significantly lower mass distribution than metabolites characterizing milk intake (P = 4.1 × 10-4). Candidate BFIs for milk or cheese included saccharides, a hydroxy acid, amino acids, amino acid derivatives, and dipeptides. Two serum oligosaccharides, blood group H disaccharide (BGH) and Lewis A trisaccharide (LeA), specifically reflected milk intake but with high interindividual variability. The 2 oligosaccharides showed related but opposing trends: subjects showing an increase in either oligosaccharide did not show any increase in the other oligosaccharide. This result was confirmed in urine. CONCLUSIONS: New candidate BFIs for milk or cheese could be identified in healthy adults, most of which were related to protein metabolism. The increase in serum of LeA and BGH after cow-milk intake in adults calls for further investigations considering the beneficial health effects on newborns of such oligosaccharides in maternal milk. The trial is registered at clinicaltrials.gov as NCT02705560.


Assuntos
Queijo , Dieta , Leite , Oligossacarídeos/sangue , Oligossacarídeos/metabolismo , Adolescente , Adulto , Animais , Biomarcadores/sangue , Estudos Cross-Over , Feminino , Humanos , Masculino , Oligossacarídeos/química , Adulto Jovem
15.
Enzyme Microb Technol ; 135: 109489, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32146932

RESUMO

The successful enzymatic synthesis of various ganglioside-related oligosaccharides requires many available glycan-processing enzymes. However, the number of available glycan-processing enzymes remains limited. In this study, the full-length CgtA43456 (ß-(1→4)-N-acetylgalactosaminyltransferase) and CgtB11168 (ß-(1→3)-galactosyltransferase) were successfully produced from Escherichia coli through the optimization of E. coli-preferable codon usage, selection of E. coli strain, and use of the molecular chaperone GroEL-GroES (GroEL/ES). The CgtA43456 enzyme was produced as a soluble form in E. coli C41(DE3) co-expressed with codon-optimized CgtA43456 and GroEL/ES. However, soluble CgtB11168 was well expressed in E. coli C41(DE3) with only the codon-optimized CgtB11168. Rather, when co-expressed with GroEL/ES, total production of CgtB11168 was reduced. Using immobilized-metal affinity chromatography, the CgtA43456 and CgtB11168 proteins were obtained with approximately 75-78 % purity. The purified CgtA43456 showed a specific activity of 21 mU/mg using UDP-N-acetylgalactosamine and GM3 trisaccharide as donor and acceptor, respectively. The purified CgtB11168 catalyzed the transfer of galactose from UDP-Gal to GM2 tetrasaccharide with a specific activity of 16 mU/mg. We propose that they could be used as catalysts for enzymatic synthesis of GM1 ganglioside-related oligosaccharides.


Assuntos
Proteínas de Bactérias/genética , Campylobacter jejuni/enzimologia , Galactosiltransferases/genética , Galactosiltransferases/isolamento & purificação , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/química , Campylobacter jejuni/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosiltransferases/química , Galactosiltransferases/metabolismo , Expressão Gênica , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Especificidade por Substrato
16.
Food Microbiol ; 89: 103430, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32138988

RESUMO

This study evaluates the influence of prebiotic carbohydrates, namely fructooligosaccharides (FOS) and galactooligosaccharides (GOS), on the protein expression of Enterococcus durans LAB18S. The strain was cultivated in 10 g L-1 FOS, GOS or glucose (control) and cellular proteins were extracted for mass spectrometry analysis. A total of 771 proteins were identified and 135 E. durans proteins were validated by the Scaffold algorithm. The proteins were functionally categorized according to Gene Ontology terms. Both FOS and GOS were used as carbon source by E. durans LAB18S, upregulating the production of proteins that may be associated with intestinal mucosa adhesion, carbohydrate and nitrogen metabolism, and stress response. Cells grown with GOS showed an increased expression of the cell division protein divIVA, EF-Tu and glyceraldehyde 3-phosphate dehydrogenase that have been associated with epithelial cell adhesion. The use of FOS stimulated the production of proteins related to amino acid metabolism and energy conversion, and ClpX protein, which plays an important role in protein turnover. The results of this study indicate that FOS and GOS can be metabolized by E. durans and stimulate the microorganism to produce proteins related to some desirable characteristics for a probiotic strain.


Assuntos
Enterococcus/crescimento & desenvolvimento , Oligossacarídeos/metabolismo , Prebióticos/microbiologia , Brasil , Proteômica
17.
J Med Chem ; 63(8): 4227-4255, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32216347

RESUMO

Heparanase cleaves polymeric heparan sulfate (HS) molecules into smaller oligosaccharides, allowing for release of angiogenic growth factors promoting tumor development and autoreactive immune cells to reach the insulin-producing ß cells. Interaction of heparanase with HS chains is regulated by specific substrate sulfation sequences. We have synthesized 11 trisaccharides that are highly tunable in structure and sulfation pattern, allowing us to determine how heparanase recognizes HS substrate and selects a favorable cleavage site. Our study shows that (1) N-SO3- at +1 subsite and 6-O-SO3- at -2 subsite of trisaccharides are critical for heparanase recognition, (2) addition of 2-O-SO3- at the -1 subsite and of 3-O-SO3- to GlcN unit is not advantageous, and (3) the anomeric configuration (α or ß) at the reducing end is crucial in controlling heparanase activity. Our study also illustrates that the α-trisaccharide having N- and 6-O-SO3- at -2 and +1 subsites inhibited heparanase and was resistant toward hydrolysis.


Assuntos
Ativação Enzimática/fisiologia , Glucuronidase/metabolismo , Glicosídeos/metabolismo , Heparitina Sulfato/metabolismo , Oligossacarídeos/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Glicosídeos/síntese química , Heparina/farmacologia , Heparitina Sulfato/antagonistas & inibidores , Humanos , Camundongos , Simulação de Acoplamento Molecular/métodos , Oligossacarídeos/síntese química
18.
J Agric Food Chem ; 68(10): 3184-3194, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32105462

RESUMO

Enzymatic hydrolysis of xylan represents a promising way to produce xylooligosaccharide (XOS), which is a novel ingredient in functional food. However, the recalcitrance of xylan in natural lignocellulosic biomass entails effective and robust xylanases. In the present study, we reported the isolation of a thermophilic Streptomyces sp. B6 from mushroom compost producing high xylanase activity. Two xylanases of Streptomyces sp. B6 belonging to GH10 (XynST10) and GH11 (XynST11) families were thus identified and biochemically characterized to be robust enzymes with high alkaline- and thermostability. Direct hydrolysis of neutralized viscose fiber production waste using XynST10 and XynST11 showed that while XynST10 produced 23.22 g/L XOS with a degree of polymerization (DP) of 2-4 and 9.27 g/L xylose, XynST11 produced much less xylose (1.19 g/L) and a higher amounts of XOS with a DP = 2-4 (28.29 g/L). Thus, XynST11 holds great potential for the production of XOS from agricultural and industrial waste.


Assuntos
Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/química , Glucuronatos/química , Oligossacarídeos/química , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Glucuronatos/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Resíduos Industriais/análise , Oligossacarídeos/metabolismo , Streptomyces/química , Streptomyces/genética , Xilose/química , Xilose/metabolismo
19.
Biochemistry (Mosc) ; 85(2): 234-240, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32093599

RESUMO

Helicobacter pylori is an important human pathogen that causes gastritis, gastric and duodenal ulcers, and gastric cancer. O-polysaccharides of H. pylori lipopolysaccharide (LPS) are composed of (ß1→3)-poly(N-acetyllactosamine) (polyLacNAc) decorated with multiple α-L-fucose residues. In many strains, their terminal LacNAc units are mono- or di-fucosylated to mimic Lewis X (Lex) and/or Lewis Y (Ley) oligosaccharides. The studies in rhesus macaques as a model of human infection by H. pylori showed that this bacterium adapts to the host during colonization by expressing host Lewis antigens. Here, we characterized LPS from H. pylori strains used in the previous study, including the parental J166 strain and the three derivatives (98-149, 98-169, and 98-181) isolated from rhesus macaques after long-term colonization. Chemical and NMR spectroscopic analyses of the LPS showed that the parent strain expressed Lex, Ley, and H type 1 terminal oligosaccharide units. The daughter strains were similar to the parental one in the presence of the same LPS core and fucosylated polyLacNAc chain of the same length but differed in the terminal oligosaccharide units. These were Lex in the isolates 98-149 and 98-169, which corresponded to the Lea phenotype of the host animals, and Ley was found in the 98-181 isolate from the macaque characterized by the Leb phenotype. As Lea and Leb are isomers of Lex and Ley, respectively, the observed correlation confirmed adaptation of the expression of terminal oligosaccharide units in H. pylori strains to the properties of the host gastric mucosa. The 98-181 strain also acquired glucosylation of the polyLacNAc chain and was distinguished by a lower expression of fucosylated internal LacNAc units (internal Lex) as a result of decoration of polyLacNAc with ß-glucopyranose, which may also play a role in the bacterial adaptation.


Assuntos
Helicobacter pylori/química , Lipopolissacarídeos/química , Macaca mulatta/microbiologia , Oligossacarídeos/genética , Polissacarídeos/metabolismo , Animais , Glicosilação , Helicobacter pylori/metabolismo , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/metabolismo , Oligossacarídeos/análise , Oligossacarídeos/metabolismo , Fenótipo , Polissacarídeos/química
20.
Am J Clin Nutr ; 111(4): 769-778, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32068776

RESUMO

BACKGROUND: Breastfeeding modulates infant growth and protects against the development of obesity. However, whether or not maternal variation in human milk components, such as human milk oligosaccharides (HMOs), is associated with programming of child growth remains unknown. OBJECTIVE: Our objective was to determine the association between maternal HMO composition and child growth during the first 5 y of life. In addition, the association between maternal prepregnancy BMI and HMO composition was assessed. METHODS: Human milk samples from 802 mothers were obtained from a prospective population-based birth cohort study, Steps to healthy development of Children (STEPS), conducted in Turku, Finland. HMO composition in these milk samples was analyzed by HPLC. Child growth data from 3 mo to 5 y were collected from municipal well-baby clinics and linked to maternal HMO composition data to test for associations. RESULTS: Maternal HMO composition 3 mo after delivery was associated with height and weight during the first 5 y of life in children of secretor mothers. Specifically, HMO diversity and the concentration of lacto-N-neo-tetraose (LNnT) were inversely associated and that of 2'-fucosyllactose (2'FL) was directly associated with child height and weight z scores in a model adjusted for maternal prepregnancy BMI, mode of delivery, birthweight z score, sex, and time. Maternal prepregnancy BMI was associated with HMO composition. CONCLUSIONS: The association between maternal HMO composition and childhood growth may imply a causal relation, which warrants additional testing in preclinical and clinical studies, especially since 2'FL and LNnT are among the HMOs now being added to infant formula. Furthermore, altered HMO composition may mediate the impact of maternal prepregnancy BMI on childhood obesity, which warrants further investigation to establish the cause-and-effect relation.


Assuntos
Desenvolvimento Infantil , Leite Humano/metabolismo , Adulto , Estatura , Peso Corporal , Aleitamento Materno , Pré-Escolar , Feminino , Finlândia , Humanos , Lactente , Estudos Longitudinais , Masculino , Leite Humano/química , Oligossacarídeos/análise , Oligossacarídeos/metabolismo , Estudos Prospectivos , Adulto Jovem
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