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1.
J Biol Chem ; 299(11): 105294, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37774972

RESUMO

The glycoside hydrolase family 55 (GH55) includes inverting exo-ß-1,3-glucosidases and endo-ß-1,3-glucanases, acting on laminarin, which is a ß1-3/1-6-glucan consisting of a ß1-3/1-6-linked main chain and ß1-6-linked branches. Despite their different modes of action toward laminarin, endo-ß-1,3-glucanases share with exo-ß-1,3-glucosidases conserved residues that form the dead-end structure of subsite -1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-ß-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-ß-1,3-glucanases, degraded internal ß-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. ß1-3-Glucans lacking ß1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal ß1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain ß1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21 s-1 mM-1) were much lower than to laminarin (5920 s-1 mM-1). These results indicate that ß-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites -1 and -2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-ß-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-ß-1,3-glucanases.


Assuntos
Glicosídeo Hidrolases , beta-Glucanas , Glucanos/metabolismo , Glucose , Glucosidases/metabolismo , Glicosídeo Hidrolases/metabolismo , Especificidade por Substrato
2.
J Am Chem Soc ; 146(26): 17738-17746, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38957137

RESUMO

Various Xanthomonas species cause well-known plant diseases. Among various pathogenic factors, the role of α-1,6-cyclized ß-1,2-glucohexadecaose (CßG16α) produced by Xanthomonas campestris pv. campestris was previously shown to be vital for infecting model organisms, Arabidopsis thaliana and Nicotiana benthamiana. However, enzymes responsible for biosynthesizing CßG16α are essentially unknown, which limits the generation of agrichemicals that inhibit CßG16α synthesis. In this study, we discovered that OpgD from X. campestris pv. campestris converts linear ß-1,2-glucan to CßG16α. Structural and functional analyses revealed OpgD from X. campestris pv. campestris possesses an anomer-inverting transglycosylation mechanism, which is unprecedented among glycoside hydrolase family enzymes.


Assuntos
Xanthomonas campestris , Xanthomonas campestris/enzimologia , Xanthomonas/enzimologia , Doenças das Plantas/microbiologia , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Modelos Moleculares
3.
Biosci Biotechnol Biochem ; 87(10): 1111-1121, 2023 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-37407435

RESUMO

ß1-3/1-6 Glucans, known for their diverse structures, comprise a ß1-3-linked main chain and ß1-6-linked short branches. Laminarin, a ß1-3/1-6 glucan extracted from brown seaweed, for instance, includes ß1-6 linkages even in the main chain. The diverse structures provide various beneficial functions for the glucan. To investigate the relationship between structure and functionality, and to enable the characterization of ß1-3/1-6 glucan-metabolizing enzymes, oligosaccharides containing the exact structures of ß1-3/1-6 glucans are required. We synthesized the monomeric units for the synthesis of ß1-3/1-6 mixed-linked glucooligosaccharides. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-4,6-O-benzylidene-ß-d-glucopyranoside served as an acceptor in the formation of ß1-3 linkages. Phenyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(tert-butyldiphenylsilyl)-1-thio-ß-d-glucopyranoside and phenyl 2,3-di-O-benzoyl-4,6-di-O-levulinyl-1-thio-ß-d-glucopyranoside acted as donors, synthesizing acceptors suitable for the formation of ß1-3- and ß1-6-linkages, respectively. These were used to synthesize a derivative of Glcß1-6Glcß1-3Glcß1-3Glc, demonstrating that the proposed route can be applied to synthesize the main chain of ß-glucan, with the inclusion of both ß1-3 and ß1-6 linkages.


Assuntos
Glucosídeos , beta-Glucanas , Sequência de Carboidratos , Oligossacarídeos/química , Glucanos
4.
Mar Drugs ; 21(10)2023 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-37888475

RESUMO

We previously examined the cellular uptake of six types of vitamin D in human intestinal Caco-2 cells. Since vitamins D5-D7 were commercially unavailable, we synthesized these compounds organically before studying them. This process led us to understand that new secosteroids could be generated as vitamin D candidates, depending on the sterol used as the starting material. We obtained two new secosteroids-compounds 3 and 4-from fucosterol in the current study. We investigated the intestinal absorption of these compounds using Caco-2 cells cultured in Transwells and compared the results with vitamin D3, a representative secosteroid. The intestinal absorption of compound 4 was comparable to that of vitamin D3. Compound 3 showed similar uptake levels but transported about half as much as vitamin D3. These compounds demonstrated intestinal absorption at the cellular level. Vitamin D is known for its diverse biological activities manifest after intestinal absorption. Using PASS online simulation, we estimated the biological activity of compound 3's activated form. In several items indicated by PASS, compound 3 exhibited stronger biological activity than vitamins D2-D7 and was also predicted to have unique biological activities.


Assuntos
Secoesteroides , Vitamina D , Humanos , Vitamina D/farmacologia , Células CACO-2 , Vitaminas , Absorção Intestinal , Técnicas de Química Sintética
5.
Soft Matter ; 17(7): 1773-1778, 2021 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-33393948

RESUMO

A sugar-based, low-molecular-weight gelator 16AG, can gelatinize primary alcohols by forming supramolecular fibers. We obtained non-helical, tape-like fibers in methanol and ethanol but helical fibers in alcohols with at least three carbons. The pitch of the helical fibers became shorter with increasing carbon number.

6.
J Biol Chem ; 294(19): 7942-7965, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30926603

RESUMO

endo-ß-1,2-Glucanase (SGL) is an enzyme that hydrolyzes ß-1,2-glucans, which play important physiological roles in some bacteria as a cyclic form. To date, no eukaryotic SGL has been identified. We purified an SGL from Talaromyces funiculosus (TfSGL), a soil fungus, to homogeneity and then cloned the complementary DNA encoding the enzyme. TfSGL shows no significant sequence similarity to any known glycoside hydrolase (GH) families, but shows significant similarity to certain eukaryotic proteins with unknown functions. The recombinant TfSGL (TfSGLr) specifically hydrolyzed linear and cyclic ß-1,2-glucans to sophorose (Glc-ß-1,2-Glc) as a main product. TfSGLr hydrolyzed reducing-end-modified ß-1,2-gluco-oligosaccharides to release a sophoroside with the modified moiety. These results indicate that TfSGL is an endo-type enzyme that preferably releases sophorose from the reducing end of substrates. Stereochemical analysis demonstrated that TfSGL is an inverting enzyme. The overall structure of TfSGLr includes an (α/α)6 toroid fold. The substrate-binding mode was revealed by the structure of a Michaelis complex of an inactive TfSGLr mutant with a ß-1,2-glucoheptasaccharide. Mutational analysis and action pattern analysis of ß-1,2-gluco-oligosaccharide derivatives revealed an unprecedented catalytic mechanism for substrate hydrolysis. Glu-262 (general acid) indirectly protonates the anomeric oxygen at subsite -1 via the 3-hydroxy group of the Glc moiety at subsite +2, and Asp-446 (general base) activates the nucleophilic water via another water. TfSGLr is apparently different from a GH144 SGL in the reaction and substrate recognition mechanism based on structural comparison. Overall, we propose that TfSGL and closely-related enzymes can be classified into a new family, GH162.


Assuntos
Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Microbiologia do Solo , Talaromyces/enzimologia , Relação Estrutura-Atividade , Especificidade por Substrato
7.
J Food Sci Technol ; 56(5): 2778-2786, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31168160

RESUMO

The γ-oryzanol contents and the composition of steryl ferulates distributed in Japanese pigmented rice varieties were investigated using the high-performance liquid chromatography-ultraviolet detection method for the purpose of expanding their utilisation. The average γ-oryzanol content in nine black-purple, four red, four green and three brown rice varieties was 54.2, 47.3, 44.3 and 43.3 mg γ-oryzanol equivalent/100 g dried weight, respectively. Among the nine varieties of black-purple rice, five varieties showed steryl ferulate composition similar to that of brown, red and green varieties. In contrast, the composition of steryl ferulates in other four black-purple rice varieties was partially specific and was characterised by a low amount of campesteryl ferulate and high of campestanyl ferulate and stigmastanyl ferulate. The latter two steryl ferulates have been recognised as minor components of γ-oryzanol in rice and as major components in wheat and corn. These results indicate that the compositions of steryl ferulates vary among Japanese black-purple rice varieties.

8.
Mar Drugs ; 16(8)2018 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-30082622

RESUMO

Fucoxanthin has an antiproliferative effect on cancer cells, but its detailed structure⁻activity correlation has not yet been elucidated. To elucidate this correlation, fucoxanthin was degraded by ozonolysis. The degraded compounds of fucoxanthin obtained by ozonolysis were purified by HPLC and analyzed by NMR. The polyene chain of fucoxanthin was cleaved by ozonolysis, and the fucoxanthin was divided into two types of cyclohexyl derivatives, one with a ß,γ-epoxy ketone group and the other with an allenic bond. In order to elucidate the structure⁻activity correlation, Caco-2 cells (human colorectal carcinoma) were treated with fucoxanthin degradation compounds. It was found that the entire structure of fucoxanthin is not essential for its antiproliferative effect and that even a partial structure exerts this effect.


Assuntos
Antineoplásicos/farmacologia , Alga Marinha/metabolismo , Undaria/metabolismo , Xantofilas/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Células CACO-2 , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Oxirredução , Ozônio/química , Polienos/química , Polienos/farmacologia , Relação Estrutura-Atividade , Xantofilas/química , Xantofilas/isolamento & purificação
9.
Biosci Biotechnol Biochem ; 81(3): 573-580, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27876437

RESUMO

Oryzanol contained in rice bran is a complex mixture of steryl ferulates (SFs) with many identified health benefits. Recently, SF has been shown to exist in other cereals such as wheat, rye, and corn. In this study, SFs in several wheats produced in Japan were analyzed. For instance, SF content of whole wheat grain, Yumekaori (Japan) was 15.2 ± 1.4 mg-oryzanol-equivalent/100 g grain, while that of the imported one, 1CW (Canada) was 11.4 ± 1.3 mg-oryzanol-equivalent/100 g grain. The main SF components in the examined wheats were campesteryl ferulate, campestanyl ferulate, and sitostanyl ferulate. SF distribution in whole wheat grain was investigated using 14 fractions produced by a conventional test milling machine. SF was intensively accumulated in the four bran fractions (24 - 95 mg-oryzanol-equivalent/100 g bran fraction). These results suggest that the wheat bran would be an important source of SF.


Assuntos
Ácidos Cumáricos/metabolismo , Fenilpropionatos/metabolismo , Triticum/química , Ácidos Cumáricos/análise , Fibras na Dieta/análise , Análise de Alimentos , Japão , Fenilpropionatos/análise , Sementes/química , Sementes/metabolismo , Triticum/metabolismo
10.
Biochem Biophys Res Commun ; 434(3): 594-9, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23583401

RESUMO

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is an endothelial scavenger receptor that is important for oxidized low-density lipoprotein uptake. LOX-1 functions as an oligomer; however, little is known about the oligomeric complex and ligand processing after recognition by LOX-1. Here, we found that LOX-1 recognized and internalized ligands through the caveolae/raft-dependent endocytosis pathway in human coronary artery endothelial cells. Furthermore, we demonstrated that LOX-1 was palmitoylated and that both cysteine 36 and cysteine 46 were necessary for the recruitment of LOX-1 into raft microdomains and for its ligand uptake ability.


Assuntos
Cavéolas/metabolismo , Endocitose , Receptores Depuradores Classe E/metabolismo , Células Cultivadas , Endocitose/efeitos dos fármacos , Genisteína/farmacologia , Humanos , Ligantes , Lipoilação , Nistatina/farmacologia
11.
Biochem J ; 442(1): 171-80, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22077443

RESUMO

LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is an endothelial scavenger receptor that is important for the uptake of OxLDL (oxidized low-density lipoprotein) and contributes to the pathogenesis of atherosclerosis. However, the precise structural motifs of OxLDL that are recognized by LOX-1 are unknown. In the present study, we have identified products of lipid peroxidation of OxLDL that serve as ligands for LOX-1. We used CHO (Chinese-hamster ovary) cells that stably express LOX-1 to evaluate the ability of BSA modified by lipid peroxidation to compete with AcLDL (acetylated low-density lipoprotein). We found that HNE (4-hydroxy-2-nonenal)-modified proteins most potently inhibited the uptake of AcLDL. On the basis of the findings that HNE-modified BSA and oxidation of LDL resulted in the formation of HNE-histidine Michael adducts, we examined whether the HNE-histidine adducts could serve as ligands for LOX-1. The authentic HNE-histidine adduct inhibited the uptake of AcLDL in a dose-dependent manner. Furthermore, we found the interaction of LOX-1 with the HNE-histidine adduct to have a dissociation constant of 1.22×10(-8) M using a surface plasmon resonance assay. Finally, we showed that the HNE-histidine adduct stimulated the formation of reactive oxygen species and activated extracellular-signal-regulated kinase 1/2 and NF-κB (nuclear factor κB) in HAECs (human aortic endothelial cells); these signals initiate endothelial dysfunction and lead to atherosclerosis. The present study provides intriguing insights into the molecular details of LOX-1 recognition of OxLDL.


Assuntos
Aldeídos/metabolismo , Histidina/análogos & derivados , Receptores Depuradores Classe E/metabolismo , Aldeídos/farmacologia , Animais , Aorta/metabolismo , Células CHO , Cricetinae , Endotélio Vascular/citologia , Histidina/metabolismo , Histidina/farmacologia , Humanos , Ligantes , Lipoproteínas LDL/metabolismo , Espécies Reativas de Oxigênio/metabolismo
12.
RSC Adv ; 13(14): 9316-9321, 2023 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-36959883

RESUMO

We previously developed low-molecular-weight organogelators that can gel various types of oils by introducing various fatty acids into 1,5-anhydro-d-glucitol (1,5-AG), a cyclic polyol derived from starch. These gelators are composed of ester bonds between naturally occurring materials. We have established a method to synthesize these gelators by heating powdered 1,5-AG and powdered fatty acids to 230 °C to liquefy the compounds without using organic solvents or activated fatty acids and then performing the esterification reaction without a catalyst or with a base catalyst. This has created the possibility of synthesizing these gelling agents at very low cost.

13.
ACS Omega ; 6(32): 20912-20923, 2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34423199

RESUMO

We determined that compounds in which γ-aminobutyric acid (GABA) and linear saturated fatty acids of various lengths are amide-bonded, as found in the human brain, have the ability to gelate organic solvents. We also synthesized compounds of these GABA derivatives attached to 1,5-anhydro-d-glucitol (1,5-AG) or d-glucopyranose (Glc) via ester linkages, and these compounds were also found to be able to gelate organic solvents. From the comparative experiments of gelation using various lengths of N-linear saturated fatty acyl-GABAs and their ester derivatives, it was determined that the compound of N-tetradecanoic acyl-GABA bonded to 1,5-AG via ester linkage (C14GABA-AG) had a particularly high gel hardness and could gelate various organic solvents. Furthermore, field-emission scanning electron microscopy observations revealed the formation of a fibrous structure, which encapsulates the organic solvent and forms a gel. A variable-temperature Fourier transform infrared spectroscopy analysis revealed that the alkyl chains of N-linear saturated fatty acyl-GABAs are packed with an all-trans conformation, whereas the alkyl chains of the ester compounds attached to 1,5-AG or Glc are slightly skewed from the all-trans conformation due to the intermolecular hydrogen bonding of the amide groups. Here, we report the synthesis and analysis of N-linear saturated fatty acyl-GABA derivatives and the gelation mechanism.

14.
Nutrients ; 13(4)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805560

RESUMO

Vitamins D have various biological activities, as well as intestinal calcium absorption. There has been recent concern about insufficient vitamin D intake. In addition to vitamins D2 and D3, there are lesser-known vitamins D4-D7. We synthesized vitamins D5-D7, which are not commercially available, and then evaluated and compared the mixed micelles-solubilized vitamins D uptake by Caco-2 cells. Except for vitamin D5, the uptake amounts of vitamins D4-D7 by differentiated Caco-2 cells were similar to those of vitamins D2 and D3. The facilitative diffusion rate in the ezetimibe inhibited pathway was approximately 20% for each vitamin D type, suggesting that they would pass through the pathway at a similar rate. Lysophosphatidylcholine enhanced each vitamin D uptake by approximately 2.5-fold. Lysophosphatidylcholine showed an enhancing effect on vitamin D uptake by reducing the intercellular barrier formation of Caco-2 cells by reducing cellular cholesterol, suggesting that increasing the uptakes of vitamins D and/or co-ingesting them with lysophosphatidylcholine, would improve vitamin D insufficiency. The various biological activities in the activated form of vitamins D4-D7 were estimated by Prediction of Activity Spectra for Substances (PASS) online simulation. These may have some biological activities, supporting the potential as nutritional components.


Assuntos
Lisofosfatidilcolinas/farmacologia , Micelas , Vitamina D/classificação , Vitamina D/metabolismo , Transporte Biológico , Células CACO-2 , Humanos , Estrutura Molecular , Vitamina D/administração & dosagem
15.
Chempluschem ; 85(4): 701-710, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32267103

RESUMO

We synthesized a series of novel alicyclic compounds by modifying 1,5-anhydro-d-glucitol with two to four palmitoyl chains, and we explored their self-assembly and gelation behaviors in paraffin. The obtained organogels were studied by field emission scanning electron microscopy, atomic force microscopy, variable-temperature Fourier transform IR spectroscopy, X-ray diffraction analysis, polarized optical microscopy, and transmission spectroscopy. While all the palmitoylated derivatives spontaneously formed fibrous networks and gelated the paraffin, an acetylated derivative of 1,5-anhydro-d-glucitol did not gelatinize the solvent, thus indicating the importance of aliphatic chains for gelation. Interestingly, α- and ß- d-glucopyranose with five palmitoyl chains neither gelatinized the solvent nor formed fibrous networks, thus suggesting that the absence of C-1 substitution in 1,5-anhydro-d-glucitol is important for gelation. Fourier transform IR spectroscopy suggested that the formation of weak hydrogen bonds between the carbonyl groups and the C-H groups was the driving force for formation of the supramolecular fibers and for gelation of the solvent.

16.
Biotechniques ; 46(3): 225-7, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19317666

RESUMO

Using a combination of silicone and urethane resin, we established a rapid technique for preparing living specimens for microscopy. One major advantage of this technique is that the coverslip is rigidly attached and does not detach during handling. As a result, it is possible to continuously observe living cells at high magnification and resolution using an oil immersion objective. Another advantage is that living cells are quickly confined to the space between the glass slide and coverslip, protecting them from environmental changes, which can cause serious effects on cell function and morphology. Moreover, high-resolution observations of real-time responses of cells are possible, using the combination of the mounting technique and a simple flow chamber.


Assuntos
Adesivos/química , Técnicas de Cultura de Células/instrumentação , Células Cultivadas/citologia , Microscopia/instrumentação , Silicones/química , Manejo de Espécimes/instrumentação , Uretana/química , Técnicas de Cultura de Células/métodos , Desenho de Equipamento , Análise de Falha de Equipamento
17.
J Appl Glycosci (1999) ; 66(3): 103-112, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-34429688

RESUMO

We have developed a novel low-molecular-mass oil-gelling agent that is electrically neutral, has no nitrogen atoms and consists only of cyclic sugar alcohols and saturated linear fatty acids. The cyclic sugar alcohols were 1,5-anhydro-D-glucitol (1,5-AG) and 1,5-anhydro-D-mannitol (1,5-AM) derived from starch via 1,5-anhydro-D-fructose. Various saturated linear fatty acids with 10 to 18 and 22 carbon atoms were introduced into all the hydroxy groups of 1,5-AG. Various saturated linear fatty acids with 13 to 18 and 22 carbon atoms were introduced into all the hydroxy groups of 1,5-AM. Initially, the gelling ability increased as the carbon number increased, but the gelling ability decreased as the carbon number increased beyond 17 carbons. This trend was similar for both 1,5-AG and 1,5-AM. A comparison of 1,5-AG and 1,5-AM derivatives revealed that 1,5-AG derivatives had greater gelling abilities for different kinds of oils at the same fatty acid length. Further, it was confirmed by SEM observations that a three-dimensional fibrous structure was formed, and this network structure formed the gel and held the oil. Here, we report the synthesis and characteristics of a novel low-molecular-weight gelling agent and its gelation mechanism.

18.
Metabolites ; 9(6)2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31174367

RESUMO

We succeeded in simultaneously synthesizing the vitamin D family, vitamins D2, D4, D5, D6, and D7, from ß-sitosterol, which is sold as a commercially available reagent from Tokyo Chemical Industry Co., Ltd. It is officially sold as a mixture of four phytosterols {ß-sitosterol (40-45%), campesterol (20-30%), stigmasterol, and brassicasterol}. Owing to this, we anticipated that, using this reagent, various vitamin D analogs could be synthesized simultaneously. We also synthesized vitamin D3 from pure cholesterol and analyzed and compared all vitamin D analogs (D2, D3, D4, D5, D6, and D7) by HSQC NMR. We succeeded in clearly demonstrating the difference in the NMR chemical shifts for each vitamin D analog.

19.
J Biochem ; 143(2): 229-36, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18032414

RESUMO

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in the development of diabetic complications. Using an Escherichia coli expression system, we have successfully expressed and purified the C-terminal biotinylated extracellular domain of human RAGE (hsRAGE), which consists of three immunoglobulin-like domains carrying three putative disulfide bonds. Over 90% of hsRAGE was expressed in soluble form in trxB and gor mutant E. coli strain Origami (DE3). Most hsRAGE was biotinylated with a C-terminal AviTag, and stably immobilized onto matrix via streptavidin without any treatment. Immobilized hsRAGE without glycosylation recognized its ligands, such as AGEs. Biotinylated hsRAGE was also able to apply in the detection of AGEs on microtitre wells like antibodies used in enzyme-linked immunoassay. SPR analysis demonstrated that the dissociation constant (K(d)) of RAGE for AGE-BSA was 23.1 nM with the two-state reaction model, and 13.5 nM with the 1:1 binding model, comparable to those of RAGEs on cell surface. These results indicate that biotinylated hsRAGE must be useful not only in analysing RAGE-ligand interactions but also detect AGEs.


Assuntos
Escherichia coli/genética , Produtos Finais de Glicação Avançada/metabolismo , Receptores Imunológicos/metabolismo , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
Carbohydr Res ; 468: 13-22, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30121414

RESUMO

Sophorose (Sop2) is known as a powerful inducer of cellulases in Trichoderma reesei, and in recent years 1,2-ß-D-oligoglucan phosphorylase (SOGP) has been found to use Sop2 in synthetic reactions. From the structure of the complex of SOGP with Sop2, it was predicted that both the 3-hydroxy group at the reducing end glucose moiety of Sop2 and the 3'-hydroxy group at the non-reducing end glucose moiety of Sop2 were important for substrate recognition. In this study, three kinds of 3- and/or 3'-deoxy-Sop2 derivatives were synthesized to evaluate this mechanism. The deoxygenation of the 3-hydroxy group of D-glucopyranose derivative was performed by radical reduction using a toluoyl group as a leaving group. The utilization of a toluoyl group that plays two roles (a leaving group for the deoxygenation and a protecting group for a hydroxy group) resulted in efficient syntheses of the three target compounds. The NMR spectra of the two final compounds (3-deoxy- and 3,3'-dideoxy-Sop2) suggested that the glucose moiety of the reducing end of Sop2 can easily take on a furanose structure (five-membered ring structure) by deoxygenation of the 3-hydroxy group of Sop2. In addition, the ratio of the five- and six-membered ring structures changed depending on the temperature. The SOGPs exhibited remarkably lower specific activity for 3'-deoxy- and 3,3'-dideoxy-Sop2, indicating that the 3'-hydroxy group of Sop2 is important for substrate recognition by SOGPs.


Assuntos
Glucanos/química , Glucanos/síntese química , Fosforilases/metabolismo , Sequência de Aminoácidos , Indução Enzimática/efeitos dos fármacos , Glucanos/farmacologia , Modelos Moleculares , Fosforilases/biossíntese , Fosforilases/química , Conformação Proteica , Estereoisomerismo , Trichoderma/enzimologia
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