Novel Split Intein-Mediated Enzymatic Channeling Accelerates the Multimeric Bioconversion Pathway of Ginsenoside.

Cascade reaction systems, such as protein fusion and synthetic protein scaffold systems, can both spatially control the metabolic flux and boost the productivity of multistep enzymatic reactions. Despite many efforts to generate fusion proteins, this task remains challenging due to the limited expression of complex enzymes. Therefore, we developed a novel fusion system that bypasses the limited expression of complex enzymes via a post-translational linkage. Here, we report a split intein-mediated cascade system wherein orthogonal split inteins serve as adapters for protein ligation. A genetically programmable, self-assembled, and traceless split intein was utilized to generate a biocatalytic cascade to produce the ginsenoside compound K (CK) with various pharmacological activities, including anticarcinogenic, anti-inflammatory, and antidiabetic effects. We used two types of split inteins, consensus atypical (Cat) and Rma DnaB, to form a covalent scaffold with the three enzymes involved in the CK conversion pathway. The multienzymatic complex with a size greater than 240 kDa was successfully assembled in a soluble form and exhibited specific activity toward ginsenoside conversion. Furthermore, our split intein cascade system significantly increased the CK conversion rate and reduced the production time by more than 2-fold. Our multienzymatic cascade system that uses split inteins can be utilized as a platform for regulating multimeric bioconversion pathways and boosting the production of various high-value substances.

Ginsenoside Rh2 Ameliorates Atopic Dermatitis in NC/Nga Mice by Suppressing NF-kappaB-Mediated Thymic Stromal Lymphopoietin Expression and T Helper Type 2 Differentiation.

Ginsenosides are known to have various highly pharmacological activities, such as anti-cancer and anti-inflammatory effects. However, the search for the most effective ginsenosides against the pathogenesis of atopic dermatitis (AD) and the study of the effects of ginsenosides on specific cytokines involved in AD remain unclear. In this study, ginsenoside Rh2 was shown to exert the most effective anti-inflammatory action on thymic stromal lymphopoietin (TSLP) and interleukin 8 in tumor necrosis factor-alpha and polyinosinic: polycytidylic acid induced normal human keratinocytes by inhibiting proinflammatory cytokines at both protein and transcriptional levels. Concomitantly, Rh2 also efficiently alleviated 2,4-dinitrochlorobenzene-induced AD-like skin symptoms when applied topically, including suppression of immune cell infiltration, cytokine expression, and serum immunoglobulin E levels in NC/Nga mice. In line with the in vitro results, Rh2 inhibited TSLP levels in AD mice via regulation of an underlying mechanism involving the nuclear factor κB pathways. In addition, in regard to immune cells, we showed that Rh2 suppressed not only the expression of TSLP but the differentiation of naïve CD4+ T-cells into T helper type 2 cells and their effector function in vitro. Collectively, our results indicated that Rh2 might be considered as a good therapeutic candidate for the alternative treatment of AD.

Ginsenoside Rh2 Ameliorates Doxorubicin-Induced Senescence Bystander Effect in Breast Carcinoma Cell MDA-MB-231 and Normal Epithelial Cell MCF-10A.

The anthracycline antibiotic doxorubicin is commonly used antineoplastic drug in breast cancer treatment. Like most chemotherapy, doxorubicin does not selectively target tumorigenic cells with high proliferation rate and often causes serve side effects. In the present study, we demonstrated the cellular senescence and senescence associated secretory phenotype (SASP) of both breast tumor cell MDA-MB-231 and normal epithelial cell MCF-10A induced by clinical dose of doxorubicin (100 nM). Senescence was confirmed by flattened morphology, increased level of beta galactose, accumulating contents of lysosome and mitochondrial, and elevated expression of p16 and p21 proteins. Similarly, SASP was identified by highly secreted proteins IL-6, IL-8, GRO, GM-CSF, MCP-1, and MMP1 by antibody array assay. Reciprocal experiments, determined by cell proliferation and apoptosis assays and cell migration and cell invasion, indicated that SASP of MDA-MB-231 cell induces growth arrest of MCF-10A, whereas SASP of MCF-10A significantly stimulates the proliferation of MDA-MB-231. Interestingly, SASP from both cells powerfully promotes the cell migration and cell invasion of MDA-MB-231 cells. Treatment with the natural product ginsenoside Rh2 does not prevent cellular senescence or exert senolytic. However, SASP from senescent cells treated with Rh2 greatly attenuated the above-mentioned bystander effect. Altogether, Rh2 is a potential candidate to ameliorate this unwanted chemotherapy-induced senescence bystander effect.

Enhanced Production of Gypenoside LXXV Using a Novel Ginsenoside-Transforming ß-Glucosidase from Ginseng-Cultivating Soil Bacteria and Its Anti-Cancer Property.

Minor ginsenosides, such as compound K, Rg3(S), which can be produced by deglycosylation of ginsenosides Rb1, showed strong anti-cancer effects. However, the anticancer effects of gypenoside LXXV, which is one of the deglycosylated shapes of ginsenoside Rb1, is still unknown due to the rarity of its content in plants. Here, we cloned and characterized a novel ginsenoside-transforming ß-glucosidase (BglG167b) derived from Microbacterium sp. Gsoil 167 which can efficiently hydrolyze gypenoside XVII into gypenoside LXXV, and applied it to the production of gypenoside LXXV at the gram-scale with high specificity. In addition, the anti-cancer activity of gypenoside LXXV was investigated against three cancer cell lines (HeLa, B16, and MDA-MB231) in vitro. Gypenoside LXXV significantly reduced cell viability, displaying an enhanced anti-cancer effect compared to gypenoside XVII and Rb1. Taken together, this enzymatic method would be useful in the preparation of gypenoside LXXV for the functional food and pharmaceutical industries.

Comparative analysis of the expression level of recombinant ginsenoside-transforming ß-glucosidase in GRAS hosts and mass production of the ginsenoside Rh2-Mix.

The ginsenoside Rh2, a pharmaceutically active component of ginseng, is known to have anticancer and antitumor effects. However, white ginseng and red ginseng have extremely low concentrations of Rh2 or Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, and Rh3]. To enhance the production of food-grade ginsenoside Rh2, an edible enzymatic bioconversion technique was developed adopting GRAS host strains. A ß-glucosidase (BglPm), which has ginsenoside conversion ability, was expressed in three GRAS host strains (Corynebacterium glutamicum, Saccharomyces cerevisiae and Lactococus lactis) by using a different vector system. Enzyme activity in these three GRAS hosts were 75.4%, 11.5%, and 9.3%, respectively, compared to that in the E. coli pGEX 4T-1 expression system. The highly expressed BglPm_C in C. glutamicum can effectively transform the ginsenoside Rg3-Mix [20(S)-Rg3, 20(R)-Rg3, Rk1, Rg5] to Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, Rh3] using a scaled-up biotransformation reaction, which was performed in a 10-L jar fermenter at pH 6.5/7.0 and 37°C for 24 h. To our knowledge, this is the first report in which 50 g of PPD-Mix (Rb1, Rb2, Rb3, Rc, and Rd) as a starting substrate was converted to ginsenoside Rg3-Mix by acid heat treatment and then 24.5-g Rh2-Mix was obtained by enzymatic transformation of Rg3-Mix through by BglPm_C. Utilization of this enzymatic method adopting a GRAS host could be usefully exploited in the preparation of ginsenoside Rh2-Mix in cosmetics, functional food, and pharmaceutical industries, thereby replacing the E. coli expression system.

Identification and characterization of a Mucilaginibacter sp. strain QM49 ß-glucosidase and its use in the production of the pharmaceutically active minor ginsenosides (S)-Rh1 and (S)-Rg2.

Here, we isolated and characterized a new ginsenoside-transforming ß-glucosidase (BglQM) from Mucilaginibacter sp. strain QM49 that shows biotransformation activity for various major ginsenosides. The gene responsible for this activity, bglQM, consists of 2,346 bp and is predicted to encode 781 amino acid residues. This enzyme has a molecular mass of 85.6 kDa. Sequence analysis of BglQM revealed that it could be classified into glycoside hydrolase family 3. The enzyme was overexpressed in Escherichia coli BL21(DE3) using a maltose binding protein (MBP)-fused pMAL-c2x vector system containing the tobacco etch virus (TEV) proteolytic cleavage site. Overexpressed recombinant BglQM could efficiently transform the protopanaxatriol-type ginsenosides Re and Rg1 into (S)-Rg2 and (S)-Rh1, respectively, by hydrolyzing one glucose moiety attached to the C-20 position at pH 8.0 and 30°C. The Km values for p-nitrophenyl-ß-d-glucopyranoside, Re, and Rg1 were 37.0 ± 0.4 µM and 3.22 ± 0.15 and 1.48 ± 0.09 mM, respectively, and the Vmax values were 33.4 ± 0.6 µmol min(-1) mg(-1) of protein and 19.2 ± 0.2 and 28.8 ± 0.27 nmol min(-1) mg(-1) of protein, respectively. A crude protopanaxatriol-type ginsenoside mixture (PPTGM) was treated with BglQM, followed by silica column purification, to produce (S)-Rh1 and (S)-Rg2 at chromatographic purities of 98% ± 0.5% and 97% ± 1.2%, respectively. This is the first report of gram-scale production of (S)-Rh1 and (S)-Rg2 from PPTGM using a novel ginsenoside-transforming ß-glucosidase of glycoside hydrolase family 3.

Mass production of the ginsenoside Rg3(S) through the combinative use of two glycoside hydrolases.

The ginsenoside Rg3(S), which is one of the exceptional components of Korean red ginseng extract, has been known to have anti-cancer, anti-metastatic, and anti-obesity effects. An enzymatic bioconversion method was developed to obtain the ginsenoside Rg3(S) with a high specificity, yield, and purity. Two glycoside hydrolases (BglBX10 and Abf22-3) were employed to produce Rg3(S) as a 100g unit. The conversion reaction transformed ginsenoside Rc to Rd using Abf22-3, followed by Rb1 and Rd to Rg3(S), using BglBX10. It was performed in a 10L jar fermenter at pH 6.0 and 37°C for 24h, with a high concentration of 50mg/ml of purified ginsenoside mixture obtained from ginseng roots. Finally, 144g of Rg3(S) was produced from 250g of root extract with 78±1.2% chromatographic purity. These results suggest that this enzymatic method would be useful in the preparation of ginsenoside Rg3(S) for the functional food and pharmaceutical industries.

Mucilaginibacter composti sp. nov., with ginsenoside converting activity, isolated from compost.

The Gram-negative, strictly aerobic, non-motile, non-spore-forming, rod shaped bacterial strain designated TR6-03(T) was isolated from compost, and its taxonomic position was investigated by using a polyphasic approach. Strain TR6-03(T) grew at 4-42°C and at pH 6.0-8.0 on R2A and nutrient agar without NaCl supplement. Strain TR6-03(T) had ß-glucosidase activity, which was responsible for its ability to transform ginsenoside Re (one of the dominant active components of ginseng) to Rg(2). On the basis of 16S rRNA gene sequence similarity, strain TR6-03(T) was shown to belong to the family Sphingobacteriaceae and to be related to Mucilaginibacter lappiensis ANJLI2(T) (96.3% sequence similarity), M. dorajii FR-f4(T) (96.1%), and M. rigui WPCB133(T) (94.1%). The G+C content of the genomic DNA was 45.6%. The predominant respiratory quinone was MK-7 and the major fatty acids were summed feature 3 (comprising C(16:1) ω7c and/or iso-C(15:0) 20H), iso-C(16:0) and iso-C(17:0) 3OH. DNA and chemotaxonomic data supported the affiliation of strain TR6-03(T) to the genus Mucilaginibacter. Strain TR6-03(T) could be differentiated genotypically and phenotypically from the recognized species of the genus Mucilaginibacter. The isolate therefore represents a novel species, for which the name Mucilaginibacter composti sp. nov. is proposed, with the type strain TR6-03(T) (=KACC 14956(T) = KCTC 12642(T) =LMG 23497(T)).

'Working' cardiomyocytes exhibiting plateau action potentials from human placenta-derived extraembryonic mesodermal cells.

The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac therapy.

Menstrual blood-derived cells confer human dystrophin expression in the murine model of Duchenne muscular dystrophy via cell fusion and myogenic transdifferentiation.

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood-derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood-derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood-derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood-derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo.

Activation of epidermal growth factor receptor via CCR3 in bronchial epithelial cells.

We have previously found that bronchial epithelial cells express CCR3 whose signaling elicits mitogen-activated protein (MAP) kinase activation and cytokine production. Several investigators have focused on the signaling crosstalk between G protein-coupled receptors (GPCRs) and epidermal growth factor receptor (EGFR) in cancer cells. In this study, we investigated the role of EGFR in CCR3 signaling in the bronchial epithelial cell line NCI-H292. Eotaxin (1-100 nM) induced dose-dependent tyrosine phosphorylation of EGFR in NCI-H292 cells. Pretreatment of the cells with the EGFR inhibitor (AG1478) significantly inhibited the MAP kinase phosphorylation induced by eotaxin. Eotaxin stimulated IL-8 production, which was inhibited by AG1478. The transactivation of EGFR through CCR3 is a critical pathway that elicits MAP kinase activation and cytokine production in bronchial epithelial cells. The delineation of the signaling pathway of chemokines will help to develop a new therapeutic strategy to allergic diseases including bronchial asthma.

The role of mitogen-activated protein kinases in eotaxin-induced cytokine production from bronchial epithelial cells.

Eotaxin is a critical chemokine eliciting migration of eosinophils and basophils in the pathogenesis of bronchial asthma. Recent studies have shown that the specific receptor for eotaxin, CCR3, is expressed in bronchial epithelial cells. Although mitogen-activated protein (MAP) kinases are involved in diverse cell functions of bronchial epithelial cells, their role in eotaxin signaling is unknown. In this study, we studied the activation and functional relevance of MAP kinases in bronchial epithelial cells stimulated with eotaxin. Eotaxin (1-100 nM) induced tyrosine/threonine phosphorylation and activation of extracellular regulated kinase (ERK) 1/2 and p38 in NCI-H(292) cells and normal human bronchial epithelial cells. The phosphorylation of these MAP kinases was detectable after 30 s, and peaked at 5 min. Eotaxin stimulated production of interleukin-8 and granulocyte macrophage colony-stimulating factor. Pretreatment of Compound X (a specific CCR3 antagonist), pertussis toxin, genistein, and wortmannin reduced the MAP kinase phosphorylation and cytokine production. The eotaxin-induced cytokine production was inhibited by specific inhibitors for MAP/ERK kinase (PD98059) and p38 MAP kinase (SB202190). These results suggest that both ERK1/2 and p38 MAP kinase activated by eotaxin have a critical role in the pathogenesis of asthma.