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1.
Adv Exp Med Biol ; 1148: 1-24, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482492

RESUMO

The use of therapeutic enzymes embraces currently a vast array of applications, abridging from diggestive disorders to cancer therapy, cardiovascular and lysosomal storage diseases. Enzyme drugs bind and act on their targets with great affinity and specificity, converting substrates to desired products in a reduced time frame with minimal side reactions. These characteristics have resulted in the development of a multitude of enzyme biopharmaceuticals for a wide range of human disorders.The advances in genetic engineering and DNA recombination techniques facilitated the production of therapeutical human-like enzymes, using different cells as host organisms. The selection of hosts generally privileges those that secrete the enzyme into the culture medium, as this eases the purification process, and those that are able to express complex glycoproteins, with glycosylation patterns and other post-translational modifications close to human proteins. Moreover, engineering approaches such as pegylation, encapsulation in micro- and nanocarriers, and mutation of amino acid residues of the native enzyme molecule to yield variants with improved therapeutic activity, half-life and/or stability, have been also addressed. Engineered enzyme products have been designed to display enhanced delivery to target sites and reduced adverse side-effects (e.g., immunogenicity) upon continuous drug administration.Irrespectively of the production method, the final formulation of therapeutic enzymes must display high purity and specificity, and they are often marketed as lyophilized pure preparations with biocompatible buffering salts and diluents to prepare the reconstituted aqueous solution before treatment.


Assuntos
Enzimas/biossíntese , Enzimas/isolamento & purificação , Enzimas/farmacologia , Produtos Biológicos , Meios de Cultura , Engenharia Genética , Glicosilação , Humanos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes
2.
J Agric Food Chem ; 67(35): 9950-9957, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31403788

RESUMO

Protein glycosylation is a ubiquitous posttranslational modification that modulates protein properties, thereby influencing bioactivities within a system. Duck egg white (DEW) proteins exhibit diverse biological properties compared with their chicken egg white (CEW) counterparts, which might be related to glycosylation. N-Glycoproteome analysis of DEW was conducted, and a total of 231 N-glycosites from 68 N-glycoproteins were identified. Gene ontology analysis was used to elucidate the biofunctions of DEW N-glycoproteins and compare them with those of CEW, which showed that the differences mostly involved molecular functions and biological processes. The biological functions of DEW N-glycoproteins were illuminated through bioinformatics analysis and comparison with CEW orthologues, which showed different allergenicities and antibacterial abilities. These divergences might be initiated by specific alterations in glycosylation, which can enhance the proteolysis resistance and protein steric hindrance. These results provide new insights for discovering the effects of N-glycosylation on biofunctions during the divergence of homologous proteins.


Assuntos
Galinhas/genética , Patos/genética , Proteínas do Ovo/química , Glicoproteínas/química , Animais , Evolução Biológica , Galinhas/metabolismo , Patos/metabolismo , Proteínas do Ovo/genética , Proteínas do Ovo/metabolismo , Clara de Ovo/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Proteômica
3.
J Agric Food Chem ; 67(33): 9411-9422, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31393126

RESUMO

Royal jelly (RJ) is secreted by young worker bees, and it plays key roles in the development and physiological function in honeybees and can improve human health. Although there have been analyses on the glycosylation modification of RJ proteins, none of these methods have been conducted on a site-specific analysis of glycosylation from these glycoproteins. Here, a combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity of RJ glycoproteins. First, global characterization of the N-glycome of RJ was performed using a direct infusion ion trap-sequential mass spectrometry (IT-MSn) method. Second, tryptic glycopeptides were enriched and separated by hydrophilic interaction liquid chromatography-ion trap-sequential mass spectrometry (HILIC-IT-MSn). A total of 50 N-glycopeptides and 30 N-glycans have been site-specific glycosylation profiled in major royal jelly protein 1 (MRJP1) and MRJP2 of RJ for the first time. Eighteen of the identified N-glycans have been structurally characterized by IT-MSn, including oligosaccharide composition, sequence, branching, and linkage. Two N-glycosylation sites (N177 and N394), 3 sites (N145, N178, and N92), and 1 site of N183 were identified in MRJP1, MRJP2, and MRJP3, respectively. There were 18, 17, and 2 N-glycans attached to MRJP1, MRJP2, and MRJP3, respectively. The diversity of N-glycans attached to each single glycosylation site of these glycoproteins confirmed that MRJP1 and MRJP2 heterogeneity was mostly associated with their glycoform populations. Understanding the properties of the site-specific glycosylation heterogeneity of the RJ glycoproteins can be potentially useful for producing a glycoprotein with desirable pharmacokinetic and biological activity.


Assuntos
Ácidos Graxos/química , Glicoproteínas/química , Proteínas de Insetos/química , Motivos de Aminoácidos , Animais , Abelhas , Cromatografia Líquida , Glicômica , Glicosilação , Espectrometria de Massas em Tandem
4.
J Agric Food Chem ; 67(28): 8020-8028, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31259548

RESUMO

In this study, a monoglucosyl rebaudioside A product was isolated from the mixture of glucosylated rebaudioside A obtained from the most reported and industrial used cyclodextrin glycosyl transferase, Toruzyme 3.0 L (CGTase, Toruzyme 3.0 L). The molecular structure of the monoglucosyl rebaudioside A was characterized using LC-MS/MS and methylation analysis combined with 1D and 2D NMR, indicating that it is 13-[(2-O-(3-α-O-D-glucopyranosyl)-ß-D-glucopyranosyl-3-O-ß-D-glucopyranosyl-ß-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid ß-D-glucopyranosyl ester (also known as RQ3, which naturally exists in Stevia extract as an isomer of rebaudioside D). This study may help to further understand the reaction mechanism of glucosylation of steviol glycoside assisted by Toruzyme 3.0 L in the aspect of molecule linkage pattern, and also benefit the application of the glucosylated rebaudiosides.


Assuntos
Diterpenos de Caurano/química , Glucosiltransferases/química , Glicosídeos/química , Biocatálise , Glicosilação , Isomerismo , Estrutura Molecular , Espectrometria de Massas em Tandem
5.
J Agric Food Chem ; 67(28): 7821-7831, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31260293

RESUMO

The mechanism of inhibition of advanced glycation end product (AGE) formation by protocatechuic acid and 3,4-dihydroxyphenylacetic acid (DHPA) has been studied using a widespread applied in vitro model system composed of bovine serum albumin (BSA) and supraphysiological glucose concentrations. Protocatechuic acid and DHPA inhibited the formation of Amadori compounds, fluorescent AGEs (IC50 = 62.1 ± 1.4 and 155.4 ± 1.1 µmol/L, respectively), and Nε-(carboxymethyl)lysine (IC50 = 535.3 ± 1.1 and 751.2 ± 1.0 µmol/L, respectively). BSA was pretreated with the two phenolic acids, and the formation of BSA-phenolic acid adducts was estimated by nanoflow liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry. Results showed that the tested phenolic acids bound key sites of glycation in BSA through a metal-catalyzed oxidative mechanism. The antiglycative activity mechanism involved the formation of BSA-phenolic acid adducts, and it is unlikely that this occurs in vivo. These results raise the problem to design in vitro models closer to physiological conditions to reach biologically sound conclusions.


Assuntos
Ácido 3,4-Di-Hidroxifenilacético/química , Hidroxibenzoatos/química , Lisina/química , Metais/química , Soroalbumina Bovina/química , Animais , Catálise , Bovinos , Cromatografia Líquida , Glicosilação , Oxirredução , Espectrometria de Massas por Ionização por Electrospray
6.
J Agric Food Chem ; 67(28): 7961-7967, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31260294

RESUMO

Food-derived glycated phospholipids is potentially hazardous to human health. However, there are few studies on the effects of lipids on the formation of glycated phospholipids. In this work, two model systems were established: (1) a model system including 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (PE), glucose, and Fenton reagent and (2) a model system including PE, glucose, and five kind of vegetable oils. The contents of carboxymethyl-PE, carboxyethyl-PE, Amadori-PE, hydroxyl radical (OH•), glyoxal, and methylglyoxal were determined with high-performance liquid chromatography mass spectrometry. The results of the first model system showed that OH• oxidized glucose to produce glyoxal and methylglyoxal, which then reacted with PE to form carboxymethyl-PE and carboxyethyl-PE. OH• also oxidized Amadori-PE to form carboxymethyl-PE. The results of the second model system showed that vegetable oils with higher number of moles of carbon-carbon unsaturated double bond in vegetable oil per kilogram could produce more OH•, which promote the formation of carboxymethyl-PE and carboxyethyl-PE by oxidizing glucose and oil. We elucidated the effects of oils on the formation of glycated phospholipids in terms of OH• and intermediates. This work will contribute to better understanding the formation mechanism of glycated phospholipids with oil.


Assuntos
Radical Hidroxila/química , Lipídeos/química , Fosfolipídeos/química , Cromatografia Líquida de Alta Pressão , Glucose/química , Glicosilação , Glioxal/química , Reação de Maillard , Espectrometria de Massas , Modelos Químicos , Oxirredução , Aldeído Pirúvico/química
7.
J Agric Food Chem ; 67(29): 8138-8148, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31294563

RESUMO

The aim of the present study was to compare various glycated ovalbumin (OVA)-monosaccharides, including OVA-mannose (Man), -glucose, -ribose, and -fructose, in the attenuation of OVA-induced allergic response in a BALB/C mouse model and the potential mechanisms of immunological modulation. The glycated OVA forms were prepared by Maillard reactions. OVA-Man significantly reduced the frequency of allergic signs. Mouse mast cell protease enzyme concentration was significantly reduced in the OVA-Man group (549.80 ± 84.67 ng/mL, p < 0.05). The OVA-Man group also had a lower histamine concentration (30.96 ± 1.12 ng/mL) as compared with the positive control OVA group (44.43 ± 0.71 ng/mL, p < 0.05). Both specific IgG and IgE were significantly reduced in the OVA-Man-treated group (p < 0.05). The OVA-Man group exhibited decreased concentrations of IL-4 (67.98 ± 3.11 pg/mL) and IL-17 (67.98 ± 3.11 pg/mL) and an increased concentration of IL-12 (336.70 ± 18.69 pg/mL, p < 0.05) compared with the positive control. Mannosylation played a vital role in allergen recognition, implicating deleterious downstream Th2 cell activation, cytokine secretion, and IgE production. This result indicates that different glycans target specific DC receptors, and differential DC processing, antigen presentation, and T cell response leads to altered variation in allergic response. OVA-Man exhibited minimal DC internalization, DC processing, MHC antigen presentation, and antigen-specific T cell activation, resulting in an attenuated allergic response and validating its efficacy as a potential immunotherapeutic candidate to treat egg allergy.


Assuntos
Hipersensibilidade a Ovo/imunologia , Monossacarídeos/química , Ovalbumina/química , Ovalbumina/imunologia , Animais , Citocinas/imunologia , Células Dendríticas/imunologia , Modelos Animais de Doenças , Hipersensibilidade a Ovo/etiologia , Feminino , Glicosilação , Humanos , Imunoglobulina E/imunologia , Reação de Maillard , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/efeitos adversos , Linfócitos T/imunologia
8.
Nat Commun ; 10(1): 2898, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263112

RESUMO

The HIV-1 envelope (Env) is the target for neutralizing antibodies and exists on the surface of virions in open or closed conformations. Difficult-to-neutralize viruses (tier 2) express Env in a closed conformation antigenic for broadly neutralizing antibodies (bnAbs) but not for third variable region (V3) antibodies. Here we show that select V3 macaque antibodies elicited by Env vaccination can neutralize 26% of otherwise tier 2 HIV-1 isolates in standardized virus panels. The V3 antibodies only bound to Env in its open conformation. Thus, Envs on tier 2 viruses sample a state where the V3 loop is not in its closed conformation position. Envelope second variable region length, glycosylation sites and V3 amino acids were signatures of neutralization sensitivity. This study determined that open conformations of Env with V3 exposed are present on a subset of otherwise neutralization-resistant virions, therefore neutralization of tier 2 HIV-1 does not always indicate bnAb induction.


Assuntos
Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Motivos de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Glicosilação , Infecções por HIV/virologia , HIV-1/química , HIV-1/genética , Humanos , Macaca mulatta , Testes de Neutralização , Conformação Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/química
9.
Adv Exp Med Biol ; 1140: 199-224, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347049

RESUMO

There are only 30,000 human genes, which, according to the central dogma from biology, it means that there should be 30,000 mRNA and 30,000 proteins. However, there are at least 1-2 million protein entities that are expressed in a cell at a given time. This is primarily due to alternative splicing in different cells and tissues, which may lead to expression of different protein isoforms within one cell, but also different protein isoforms in different tissues. A new level of complexity of proteins and protein isoforms is then given by posttranslational modifications (PTMs) of proteins. Here, we discuss the PTMs in proteins and how they are identified by mass spectrometry and proteomics, with specific examples on identification of acetylation, phosphorylation, glycosylation, alkylation, hydroxinonenal-modification or assignment of intramolecular and intermolecular disulfide bridges.


Assuntos
Espectrometria de Massas , Processamento de Proteína Pós-Traducional , Proteômica , Acetilação , Alquilação , Glicosilação , Humanos , Fosforilação
10.
Adv Exp Med Biol ; 1140: 299-316, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347055

RESUMO

The pharmaceutical and clinical industries are imperative for the maintenance of global health and welfare and require accurate, reproducible, and high throughput analyses. Technological advancements, such as the development and implementation of liquid chromatography-tandem mass spectrometry (LC-MS), have allowed for improvements in these areas, however there is still room for development. One way in which current analyses may be improved is by the implementation of ion mobility technology. Ion mobility has the capability to produce much more comprehensive data sets, by providing separation of isomers, as well as improving throughput, with separations being performed as fast as 60 ms. Here we will discuss the potential for ion mobility to assist in the two specific areas of glycosylation monitoring of biological drugs, and vitamin D analysis, as representatives of ion mobility's potential in both the pharmaceutical and clinical industries, respectively, as well as the current hurdles of ion mobility adoption in both fields.


Assuntos
Química Farmacêutica/métodos , Cromatografia Líquida , Espectrometria de Massas em Tandem , Glicosilação , Isomerismo , Preparações Farmacêuticas/análise , Vitamina D/análise
11.
J Agric Food Chem ; 67(31): 8573-8580, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31293156

RESUMO

Glycosylation endows both natural and synthetic small molecules with modulated physicochemical and biological properties. Plant and bacterial glycosyltransferases capable of decorating various privileged scaffolds have been extensively studied, but those from kingdom Fungi still remain underexploited. Here, we use a combination of genome mining and heterologous expression techniques to identify four novel glycosyltransferase-methyltransferase (GT-MT) functional modules from Hypocreales fungi. These GT-MT modules display decent substrate promiscuity and regiospecificity, methylglucosylating a panel of natural products such as flavonoids, stilbenoids, anthraquinones, and benzenediol lactones. Native GT-MT modules can be split up and regrouped into hybrid modules with similar or even improved efficacy as compared with native pairs. Methylglucosylation of kaempferol considerably improves its insecticidal activity against the larvae of oriental armyworm Mythimna separata (Walker). Our work provides a set of efficient biocatalysts for the combinatorial biosynthesis of small molecule glycosides that may have significant importance to the pharmaceutical, agricultural, and food industries.


Assuntos
Proteínas Fúngicas/química , Glicosiltransferases/química , Hypocreales/enzimologia , Metiltransferases/química , Fenóis/química , Animais , Biocatálise , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Hypocreales/genética , Inseticidas/química , Inseticidas/farmacologia , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Mariposas/efeitos dos fármacos , Fenóis/farmacologia , Especificidade por Substrato
12.
Biochemistry (Mosc) ; 84(Suppl 1): S124-S143, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31213199

RESUMO

Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia accompanied by the disruption of carbohydrate, lipid, and proteins metabolism and development of long-term microvascular, macrovascular, and neuropathic changes. This review presents the results of spectroscopic studies on the glycation of tissues and cell proteins in organisms with naturally developing and model diabetes and in vitro glycated samples in a wide range of electromagnetic waves, from visible light to terahertz radiation. Experiments on the refractometric measurements of glycated and oxygenated hemoglobin in broad wavelength and temperature ranges using digital holographic microscopy and diffraction tomography are discussed, as well as possible application of these methods in the diabetes diagnostics. It is shown that the development and implementation of multimodal approaches based on a combination of phase diagnostics with other methods is another promising direction in the diabetes diagnostics. The possibilities of using optical clearing agents for monitoring the diffusion of substances in the glycated tissues and blood flow dynamics in the pancreas of animals with induced diabetes have also been analyzed.


Assuntos
Diabetes Mellitus/diagnóstico por imagem , Hemoglobina A Glicada/ultraestrutura , Animais , Velocidade do Fluxo Sanguíneo , Glicosilação , Holografia/métodos , Humanos , Microscopia/métodos , Espectrofotometria Infravermelho/métodos , Espectroscopia Terahertz/métodos , Tomografia/métodos
13.
BMC Plant Biol ; 19(1): 276, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234799

RESUMO

BACKGROUND: Aspartic protease (AP) is one of four large proteolytic enzyme families that are involved in plant growth and development. Little is known about the AP gene family in tree species, although it has been characterized in Arabidopsis, rice and grape. The AP genes that are involved in tree wood formation remain to be determined. RESULTS: A total of 67 AP genes were identified in Populus trichocarpa (PtAP) and classified into three categories (A, B and C). Chromosome mapping analysis revealed that two-thirds of the PtAP genes were located in genome duplication blocks, indicating the expansion of the AP family by segmental duplications in Populus. The microarray data from the Populus eFP browser demonstrated that PtAP genes had diversified tissue expression patterns. Semi-qRT-PCR analysis further determined that more than 10 PtAPs were highly or preferentially expressed in the developing xylem. When the involvement of the PtAPs in wood formation became the focus, many SCW-related cis-elements were found in the promoters of these PtAPs. Based on PtAPpromoter::GUS techniques, the activities of PtAP66 promoters were observed only in fiber cells, not in the vessels of stems as the xylem and leaf veins developed in the transgenic Populus tree, and strong GUS signals were detected in interfascicular fiber cells, roots, anthers and sepals of PtAP17promoter::GUS transgenic plants. Intensive GUS activities in various secondary tissues implied that PtAP66 and PtAP17 could function in wood formation. In addition, most of the PtAP proteins were predicted to contain N- and (or) O-glycosylation sites, and the integration of PNGase F digestion and western blotting revealed that the PtAP17 and PtAP66 proteins were N-glycosylated in Populus. CONCLUSIONS: Comprehensive characterization of the PtAP genes suggests their functional diversity during Populus growth and development. Our findings provide an overall understanding of the AP gene family in trees and establish a better foundation to further describe the roles of PtAPs in wood formation.


Assuntos
Ácido Aspártico Proteases/genética , Genes de Plantas , Família Multigênica , Proteínas de Plantas/genética , Populus/genética , Madeira/crescimento & desenvolvimento , Parede Celular/genética , Sequência Conservada , Duplicação Gênica , Perfilação da Expressão Gênica , Glicosilação , Filogenia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Populus/enzimologia , Populus/crescimento & desenvolvimento , Regiões Promotoras Genéticas
14.
Chem Soc Rev ; 48(15): 4006-4018, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31169838

RESUMO

In the pursuit of developing potent drug molecules, more efficient and straightforward procedures are in high demand. The evergrowing interest in carbohydrate-based therapeutics and vaccines particularly calls for such reliable and universal approaches that assemble oligosaccharides rapidly and stereoselectively. Hereby, we compiled remarkable efforts made in exploring the possibilities of protection-less glycosylation strategies. Pioneering works using organotin reagents or catalysts were introduced first, followed by the organoboron successors that were deemed less toxic and more versatile alternatives. In the meantime, more species such as copper or caesium were also included and supported by a mechanistic rationale. Lastly, we hope to bring further insights into the synthesis of intricate carbohydrate derivatives, achieved with the aid of glycosylation methods discussed herein.


Assuntos
Produtos Biológicos/química , Polissacarídeos/química , Açúcares/química , Produtos Biológicos/síntese química , Glicosilação , Estrutura Molecular , Polissacarídeos/síntese química , Estereoisomerismo , Açúcares/síntese química
15.
Chemistry ; 25(44): 10505-10510, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31173420

RESUMO

Precision cell-selective surface glycan remodeling is of vital importance for modulation of cell surface dynamics, tissue-specific imaging, and immunotherapy, but remains an unsolved challenge. Herein, we report a switchable enzymatic accessibility (SEA) strategy for highly specific editing of carbohydrate moieties of interest on the target cell surface. We demonstrate the blocking of enzyme in the inaccessible state with a metal-organic framework (MOF) cage and instantaneous switching to the accessible state through disassembly of MOF. We further show that this level of SEA regulation enables initial guided enzyme delivery to the target cell surface for subsequent cell-specific glycan remodeling, thus providing a temporally and spatially controlled tool for tuning the glycosylation architectures. Terminal galactose/N-acetylgalactosamine (Gal/GalNAc) remodeling and terminal sialic acid (Sia) desialylation have been precisely achieved on target cells even with other cell lines in close spatial proximity. The SEA protocol features a modular and generically adaptable design, a very short protocol duration (ca. 30 min or shorter), and a very high spatial resolving power (ability to differentiate immediately neighboring cell lines).


Assuntos
Membrana Celular/enzimologia , Polissacarídeos/metabolismo , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Aptâmeros de Nucleotídeos/química , Biocatálise , Membrana Celular/química , Ativação Enzimática , Galactose/química , Galactose/metabolismo , Galactose Oxidase/antagonistas & inibidores , Galactose Oxidase/metabolismo , Glicosilação , Células Hep G2 , Humanos , Células MCF-7 , Estruturas Metalorgânicas/química , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Imagem Óptica/métodos , Polissacarídeos/química , Propriedades de Superfície
16.
Microb Cell Fact ; 18(1): 97, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151435

RESUMO

BACKGROUND: Transglycosylation represents one of the most promising approaches for obtaining novel glycosides, and plant phenols and polyphenols are emerging as one of the best targets for creating new molecules with enhanced capacities. These compounds can be found in diet and exhibit a wide range of bioactivities, such as antioxidant, antihypertensive, antitumor, neuroprotective and anti-inflammatory, and the eco-friendly synthesis of glycosides from these molecules can be a suitable alternative for increasing their health benefits. RESULTS: Transglycosylation experiments were carried out using different GH3 ß-glucosidases from the fungus Talaromyces amestolkiae. After a first screening with a wide variety of potential transglycosylation acceptors, mono-glucosylated derivatives of hydroxytyrosol, vanillin alcohol, 4-hydroxybenzyl alcohol, and hydroquinone were detected. The reaction products were analyzed by thin-layer chromatography, high-pressure liquid chromatography, and mass spectrometry. Hydroxytyrosol and vanillyl alcohol were selected as the best options for transglycosylation optimization, with a final conversion yield of 13.8 and 19% of hydroxytyrosol and vanillin glucosides, respectively. NMR analysis confirmed the structures of these compounds. The evaluation of the biological effect of these glucosides using models of breast cancer cells, showed an enhancement in the anti-proliferative capacity of the vanillin derivative, and an improved safety profile of both glucosides. CONCLUSIONS: GH3 ß-glucosidases from T. amestolkiae expressed in P. pastoris were able to transglycosylate a wide variety of acceptors. Between them, phenolic molecules like hydroxytyrosol, vanillin alcohol, 4-hydroxybenzyl alcohol, and hydroquinone were the most suitable for its interesting biological properties. The glycosides of hydroxytyrosol and vanillin were tested, and they improved the biological activities of the original aglycons on breast cancer cells.


Assuntos
Neoplasias da Mama , Celulases/metabolismo , Glicosídeos/farmacologia , Talaromyces/enzimologia , Benzaldeídos/metabolismo , Álcoois Benzílicos/metabolismo , Celulases/química , Celulases/isolamento & purificação , Glicosídeos/química , Glicosídeos/isolamento & purificação , Glicosilação , Humanos , Hidroquinonas/metabolismo , Células MCF-7 , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/metabolismo , Especificidade por Substrato
17.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 605-615, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205022

RESUMO

The discovery of new glycoside hydrolases that can be utilized in the chemoenzymatic synthesis of carbohydrates has emerged as a promising approach for various biotechnological processes. In this study, recombinant Ps_Cel5A from Pseudomonas stutzeri A1501, a novel member of the GH5_5 subfamily, was expressed, purified and crystallized. Preliminary experiments confirmed the ability of Ps_Cel5A to catalyze transglycosylation with cellotriose as a substrate. The crystal structure revealed several structural determinants in and around the positive subsites, providing a molecular basis for a better understanding of the mechanisms that promote and favour synthesis rather than hydrolysis. In the positive subsites, two nonconserved positively charged residues (Arg178 and Lys216) were found to interact with cellobiose. This adaptation has also been reported for transglycosylating ß-mannanases of the GH5_7 subfamily.


Assuntos
Proteínas de Bactérias/química , Celulase/química , Celulose/química , Pseudomonas stutzeri/enzimologia , Trioses/química , Celulose/metabolismo , Cristalização , Cristalografia por Raios X/métodos , Escherichia coli , Glicosilação , Especificidade por Substrato , Trioses/metabolismo
18.
Plant Physiol Biochem ; 141: 183-192, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31174035

RESUMO

The present study is focused on the characterization of yacon [Smallanthus sonchifolius (Poepp. et Endl.) H. Robinson] accessions from different geographic origins (Bolivia, Ecuador, and Peru) by iPBS markers and metabolomic fingerprinting. The results showed that the number of amplified polymorphic fragment levels ranged from 20 up to 27 with a level of polymorphism ranging from 80 to 100%. Five of the iPBS primers used in this study provided no specific banding pattern able to discriminate between the different yacon accessions. However, two iPBS primer pairs were able to separate Peru accessions from those of Ecuador and Bolivia. The UPLC-HRMS/MS-based metabolomic fingerprinting showed highly similar metabolomic fingerprints characterized by the accumulation of high quantities of sesquiterpene lactones and diterpenes, but no apparent geographic clustering. The present study demonstrates that yacon accessions from different geographical origins maintained ex situ (in the Czech Republic) present a rather low chemical and genetic diversity.


Assuntos
Antioxidantes/química , Asteraceae/química , Diterpenos/química , Lactonas/química , Extratos Vegetais/química , Sesquiterpenos/química , Asteraceae/genética , Bolívia , Análise por Conglomerados , República Tcheca , Equador , Variação Genética , Geografia , Glicosilação , Espectrometria de Massas , Metabolômica , Análise Multivariada , Mapeamento de Peptídeos , Peru , Raízes de Plantas/química , Retroelementos
19.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1122-1123: 64-72, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31154130

RESUMO

Glycosylation plays an important role in the maintenance of the structure and function of glycoproteins, while aberrant protein glycosylation is correlated with various diseases. Human immunoglobulin G (IgG), which is composed of four subclasses (IgG1, IgG2, IgG3 and IgG4), is one of the most dominant and significant glycoprotein in human serum. The glycosylation on IgG-Fc moiety is known to be alternated with various physiological and pathological states. We herein report an integrated approach for comprehensive profiling and quantitation of IgG-Fc glycopeptides. Firstly, IgG N-glycans were profiled by using mAb-Glyco chip quadrupole time-of-flight mass spectrometry (Q-TOF-MS), resulting characterization of 87 N­glycans originating from 29 different oligosaccharide compositions. Secondly, subclass-specific glycopeptides were identified on the basis of high-resolution MS and MS/MS data by using ultra high performance liquid chromatography (UHPLC) coupled to Q-TOF-MS. As a result, 83 IgG-Fc glycopeptides from human serum, including 17 sialylated glycopeptides, were identified. In addition, a quantitation method with high sensitivity and repeatability was established by using UHPLC triple quadrupole (QQQ) MS. We applied this approach to carry out quantitative analysis of IgG glycosylation in RA patients. Finally, 36 potential glycopeptide biomarkers, including 13 species from IgG1, 12 species from IgG2/3 and 11 species from IgG4 were identified.


Assuntos
Artrite Reumatoide/sangue , Cromatografia Líquida de Alta Pressão/métodos , Glicopeptídeos/sangue , Imunoglobulina G/química , Espectrometria de Massas em Tandem/métodos , Idoso , Artrite Reumatoide/metabolismo , Estudos de Casos e Controles , Feminino , Glicopeptídeos/química , Glicosilação , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes
20.
Chemistry ; 25(45): 10585-10589, 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31215694

RESUMO

Late-stage C-H glycosylations of structurally complex amino acids and peptides were accomplished by means of racemization-free manganese(I)-catalyzed C-H activation. Thus, glycosylative modifications proved to be viable by a linch-pin approach, featuring chemo- and site-selective C-H transformations. The peptide-saccharide conjugation provided modular access to structurally complex glycopeptides, likewise enabling the assembly of fluorescent-labelled glycopeptides.


Assuntos
Carboidratos/química , Glicopeptídeos/química , Manganês/química , Peptídeos/química , Carbono/química , Catálise , Glicopeptídeos/síntese química , Glicosilação , Hidrogênio/química
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