Cloning, expression and characterization of a new endo-β-N-acetylglucosaminidase from Streptomyces alfalfae / 生物工程学报

Endo-β-N-acetylglucosaminidase is used widely in the glycobiology studies and industries. In this study, a new endo-β-N-acetylglucosaminidase, designated as Endo SA, was cloned from Streptomyces alfalfae ACCC 40021 and expressed in Escherichia coli BL21 (DE3). The purified recombinant Endo SA exhibited the maximum activity at 35 ºC and pH 6.0, good thermo/pH stability and high specific activity (1.0×10⁶ U/mg). It displayed deglycosylation activity towards different protein substrates. These good properties make EndoSA a potential tool enzyme and industrial biocatalyst.

Advances in metabolism of natural flavonoid glycosides / 中草药

Flavonoid glycosides (FGs) are secondary metabolites of many plants widely found in nature, and exhibit significant biological activities, such as anticancer, antioxidant and antimicrobial. According to the glycosidic bonds, FGs are divided into flavonoid O-glycosides and flavonoid C-glycosides. The main metabolic processes of FGs in vivo were specific hydrolysis in the gastrointestinal tract and glucuronidation in liver. Glucose, xylose, rhamnose and other glycosyl groups were hydrolyzed to produce secondary glycosides or aglycones in the gastrointestinal tract that were absorbed into blood, and then further glucuronidation and methylation metabolites are mainly produced by phase II metabolism in liver. This article reviews the metabolism in vivo and biotransformation in vitro of some typical natural flavonoid glycosides exited in Chinese materia medica (CMMs), such as flavonoid O-glycosides in Epimedii Folium, Glycyrrhizae Radix et Rhizoma, Scutellariae Radix, Citri Reticulatae Pericarpium, and Cirsii Japonici Herba, and flavonoid C-glycosides in Anemarrhenae Rhizoma and Puerariae Lobatae Radix. The investigation of the metabolisms of FGs in vivo is helpful for the clarification of the effective ingredients in CMMs, which will provide the basis for new drugs development based on metabolites in vivo.

Changes and mechanisms of terrestroside B and terrestrosin K in stir-frying Tribuli Fructus / 中国中药杂志

The contents of terrestroside B and terrestrosin K in Tribuli Fructus with different degree of stir-frying were determined by high performance liquid chromatography with evaporative light-scattering detector( HPLC-ELSD). The results showed that the contents of terrestroside B and terrestrosin K were increased at first and then decreased,and both of them had the highest content at the best time of heating. The results of simulated processing of Tribulus Terrestris saponins showed that when the processing time kept constant,the contents of terrestroside B and terrestrosin K were decreased gradually with the increase of processing temperature from 180 ℃ to240 ℃. At a certain temperature,the content of terrestrosin K was increased first and then decreased with the prolongation of processing time,and reached the highest level at 5 min. However,the content of terrestroside B was increased first and then decreased with the increase of processing time only at 180 ℃,and reached the highest level at 10 min. When the processing temperature was controlled at200,220 and 240 ℃ respectively,the content of terrestroside B was decreased gradually with the increase of processing time. The simulated processing products of tribuluside A,terrestroside B and terrestrosin K were qualitatively characterized by ultra-performance liquid chromatography-time of flight mass spectrometry( UPLC-TOF/MS). It was proved that tribuluside A and terrestrosin Ⅰ containing C-22-OH were dehydroxylated in the processing of Tribuli Fructus and transformed respectively into terrestroside B and terrestrosin K containing C-20-C-22 double bond. As a result,the contents of terrestroside B and terrestrosin K were increased. The sugar chains at C-3 and C-26 positions of terrestroside B and terrestrosin K could be deglycosylated and converted into monosaccharide chain saponins and short sugar chain saponins,so the contents of terrestroside B and terrestrosin K were reduced. The study provides reference for further revealing the processing principle of Tribuli Fructus.

Study on transformation rules of terrestrosin D in course of Tribuli Fructus stir-frying based on simulated processing technology / 中国中药杂志

The contents of terrestrosin D and hecogenin from Tribuli Fructus were determined before and after stir-frying. The results showed that the content of terrestrosin D was decreased significantly,and the content of hecogenin was increased significantly after such processing. In order to verify the inference that terrestrosin D was converted to hecogenin by stir-frying,the quantitative variation rules of terrestrosin D and hecogenin were studied by simulated processing technology,and the simulated processing product of terrestrosin D was qualitatively characterized by ultra performance liquid chromatography/time of flight mass spectrometry( UPLC-TOF/MS) to clarify its transformation process during stir-frying. The results showed that the content of terrestrosin D was decreased significantly at first and then a platform stage appeared with the prolongation of processing time at a certain temperature. Raising the stir-frying temperature could further decrease the content of terrestrosin D and delay the time that the platform stage appeared. When the processing was simulated at higher temperatures( 220 ℃ and 240 ℃),the content of hecogenin was increased gradually with the increase of processing temperature and the prolongation of processing time. In the process of stir-frying,the deglycosylation reaction of terrestrosin D to hecogenin was not completed in one step. The deglycosylation reaction occurred first at the end of the sugar chain,and then other glycosyl units in the sugar chain were sequentially removed from the outside to the inside to finally form the hecogenin. This study provides a basis for further revealing the detoxification mechanism of stir-fried Tribuli Fructus.

Metabolism of six saponins by rat intestinal bacteria in vitro / 中国中药杂志

To investigate the metabolism of six saponins by rat intestinal bacteria in vitro.Six saponins, including notoginsenoside R₁, ginsenoside Rg₁, ginsenoside Rg₂, ginsenoside Re, ginsenoside Rd and ginsenoside Rb₁, were incubated for 8 and 24 h with rat intestinal bacteria under anaerobic environment, respectively. After the samples were precipitated by acetonitrile and extracted with ethyl acetate, LC-Q-TOF-MS/MS was applied for the qualitative analysis of the metabolites. The potential metabolites in rat feces were analyzed by comparing the total ion current of the test samples and blank samples and analyzing the quasi-molecular ion and fragment ion of all chromatograms. The results showed that six saponins could be easily metabolized by rat intestinal bacteria. Notoginsenoside R₁ was mainly metabolized into five metabolites, and it's metabolic pathway was notoginsenoside R₁→ginsenoside Rg₁→ginsenoside Rh₁ and ginsenoside F₁→protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Rg₁ was mainly metabolized into four metabolites, and it's metabolic pathway was ginsenoside Rg₁→ginsenoside Rh₁ and ginsenoside F₁→protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Rg₂ was mainly metabolized into two metabolites, and it's metabolic pathway was ginsenoside Rg₂→ protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Re was mainly metabolized into four metabolites, and it's metabolic pathway was ginsenoside Re→ginsenoside Rg₂→ginsenoside F₁→protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Rd was mainly metabolized into four metabolites, and it's metabolic pathway was ginsenoside Rd→ginsenoside Rg₃ and ginsenoside F₂→ginsenoside Rh₂→protopanaxadiol. Ginsenoside Rb1 was mainly metabolized into five metabolites, and it's metabolic pathway was ginsenoside Rb₁→ginsenoside Rd→ginsenoside Rg₃ and ginsenoside F₂→ginsenoside Rh₂→protopanaxadiol. In summary, six saponins could be quickly metabolized by rat intestinal bacteria in vitro. Their major metabolic pathways were deglycosylation and dehydrogenation.

Comparative study on contents and pharmacokinetics of raw and steamed Notoginseng Radix et Rhizoma and its anti-platelet aggregation activity / 中草药

Objective: To compare the relationship of ingredients and pharmacokinetics of steamed Notoginseng Radix et Rhizoma (the roots of Panax notoginseng) with different time (0, 2, 4, and 8 h), and accompany with its anti-platelet aggregation activity. Methods: The components with different steaming duration were determined by HPLC method. Concentration of saponins in Notoginseng Radix et Rhizoma before and after steaming at different time points were detected by UPLC-MS/MS. Pharmacokinetic parameters of each compound were calculated using DAS 3.2.6 software. The anti-platelet aggregation activity was measured by platelet aggregation/clotting analyzer. Results: The results showed that the steaming process reduced the contents of certain bioactive substances (NG-R1, Rg1, Rd, Rb1, and Re) and produced some new components (Rh1, Rg3, Rk3, and Rh4). Ginsenoside Rg3, deglycosylated metabolites of ginsenoside Rb1 possessed lower tmax than ginsenoside Rb1 that indicated the course of deglycosylation made faster absorption. Steamed Notoginseng Radix et Rhizoma had stronger antiplatelet activity, following higher antiplatelet and anticoagulant activities with increasing steaming durations. Conclusion: The results inspire us that saponins may become more active ingredients after deglycosylation, saponins with deglycosylated in vitro become more and more active ingredients into the blood, which could make stronger anticoagulant activity.

Analysis on in vivo metabolites of α-hederin in rats by UPLC-MS/MS / 中草药

Objective: To investigate the metabolites of α-hederin in feces of rats after ig administration. Methods: An UPLC-Q/Trap-MS method was used for the identification of metabolites of α-hederin in feces of rats after ig administration of α-hederin (150 mg/kg). Results: Seven metabolites were detected in rat feces, including demethylation of α-hederin (M1), 3-O-α-L-arabinopyranoside hederagenin (M2), hederagenin (M3), glucuronideconjugate of α-hederin (M4), and double bond bonus of α-hederin (M5, M6-1, and M6-2). Conclusion: α-Hederin experiences a variety of metabolic reactions in rat gastrointestinal tract, mainly including deglycosylation, glucuronidation, demethylation, etc.

Binding Specificity of Philyra pisum Lectin to Pathogen-Associated Molecular Patterns, and Its Secondary Structure

We recently reported a Philyra pisum lectin (PPL) that exerts mitogenic effects on human lymphocytes, and its molecular characterization. The present study provides a more detailed characterization of PPL based on the results from a monosaccharide analysis indicating that PPL is a glycoprotein, and circular dichroism spectra revealing its estimated alpha-helix, beta-sheet, beta-turn, and random coil contents to be 14.0%, 39.6%, 15.8%, and 30.6%, respectively. These contents are quite similar to those of deglycosylated PPL, indicating that glycans do not affect its intact structure. The binding properties to different pathogen-associated molecular patterns were investigated with hemagglutination inhibition assays using lipoteichoic acid from Gram-positive bacteria, lipopolysaccharide from Gram-negative bacteria, and both mannan and beta-1,3-glucan from fungi. PPL binds to lipoteichoic acids and mannan, but not to lipopolysaccharides or beta-1,3-glucan. PPL exerted no significant antiproliferative effects against human breast or bladder cancer cells. These results indicate that PPL is a glycoprotein with a lipoteichoic acid or mannan-binding specificity and which contains low and high proportions of alpha-helix and beta-structures, respectively. These properties are inherent to the innate immune system of P. pisum and indicate that PPL could be involved in signal transmission into Gram-positive bacteria or fungi.

The impact of site-directed deglycosylation of the HIV-1 envelope (Env) on its immunogenicity and assembly of functional pseudovirus / 中华微生物学和免疫学杂志

Objective To study the influence of site-directed deglycosylation of the HIV-1 envelope (Env) on its immunogenicity and assembly of functional pseudovirus. Methods Site-directed deglycosylation were performed using cycling mutagenesis and selection of mutants with DpnⅠ. Single-cycle infection assay was employed to analyze the effect of the mutations on the ability of functional pseudovirus assembly. The influence of deglycosylations on the immunodeficiency of Env was evaluated using pseudovirusbased neutralization assay and ELISPOT assay. Results Mutant N197Q induced higher neutralization activities for both pseudoviruses, but lower Env-specific T-cell response. And N197Q rendered the Env to lose the ability of functional pseudovirus assembly. Mutant G2 induced higher neutralization activities for pseudovirus 74-2 but lower for pseudovirus Wt, and had almost no influence on Env-specific T-cell response and functional pseudovirus forming. Conclusion The site-directed deglycosylation of the HIV-1 Env affects the pseudovirus forming and its immunogenicity.

Relationship between the integrity of SEA module and the proteolytic digestion within C-terminal domain of rodent Muc3 / 解放军医学杂志

Objective It has been known that the C-terminal domain of rodent Muc3 underwent proteolytic digestion.G/S within the motif of cleavage,LSKGSIVV,was one of the important pivots for digestion.The present investigation was aiming at exploring the unknown relationship between the integrity of SEA module and the proteolytic digestion.Method Truncated rodent Muc3 C-terminal domains(p20t and p20SEA) were produced by site-directed mutagenesis to insert a stop code in the required place.Proteins were detected by SDS/PAGE and Western blotting.Deglycosylation of the expressed protein was performed by digestion using N-glycosidase F.Results Muc3 C-terminal domain was posttranslationally cleaved to produce a V5-tagged 30kDa extracellular glycopeptide and a Myc-tagged 49kDa membrane-associated glycopeptide.The 30kDa N-terminal fragment shifted to 22kDa after deglycosylation.The truncated rodent Muc3 C-terminal domain containing complete SEA module,but without the following residues after SEA module,was 30kDa Mw as detected with anti-V5 antibody,and it was shifted to 22kDa after deglycosylation.But the truncated rodent Muc3 C-terminal domain containing incomplete SEA module(p20t) of 26-30kDa Mw was shifted to 26kDa after deglycosylation.Conclusion There was proteolytic digestion in both complete rodent C-terminal domain and complete SEA module without residues after SEA module.But proteolytic digestion does not occur in the incomplete SEA module of rodent Muc3.So it may be concluded that the integrity of SEA module of rodent Muc3 was also a crucial condition for its proteolytic digestion.