Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum

N-glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteins and N-glycans in microalgae. Here, N-glycoproteomic and N-glycomic approaches were used to unveil the N-glycoproteins and N-glycans in the model diatom Phaeodactylum tricornutum. In total, 863 different N-glycopeptides corresponding to 639 N-glycoproteins were identified from P. tricornutum. These N-glycoproteins participated in a variety of important metabolic pathways in P. tricornutum. Twelve proteins participating in the N-glycosylation pathway were identified as N-glycoproteins, indicating that the N-glycosylation of these proteins might be important for the protein N-glycosylation pathway. Subsequently, 69 N-glycans corresponding to 59 N-glycoproteins were identified and classified into high mannose and hybrid type N-glycans. High mannose type N-glycans contained four different classes, such as Man-5, Man-7, Man-9, and Man-10 with a terminal glucose residue. Hybrid type N-glycan harbored Man-4 with a terminal GlcNAc residue. The identification of N-glycosylation on nascent proteins expanded our understanding of this modification at a N-glycoproteomic scale, the analysis of N-glycan structures updated the N-glycan database in microalgae. The results obtained from this study facilitate the elucidation of the precise function of these N-glycoproteins and are beneficial for future designing the microalga to produce the functional humanized biopharmaceutical N-glycoproteins for the clinical therapeutics.

Novel Perbutyrylated Glucose Derivatives of (–)-Epigallocatechin-3-Gallate Inhibit Cancer Cells Proliferation by Decreasing Phosphorylation of the EGFR: Synthesis, Cytotoxicity, and Molecular Docking

Lung cancer is one of the most commonly occurring cancer mortality worldwide. The epidermal growth factor receptor (EGFR) plays an important role in cellular functions and has become the new promising target. Natural products and their derivatives with various structures, unique biological activities, and specific selectivity have served as lead compounds for EGFR. D-glucose and EGCG were used as starting materials. A series of glucoside derivatives of EGCG (7–12) were synthesized and evaluated for their in vitro anticancer activity against five human cancer cell lines, including HL-60, SMMC-7721, A-549, MCF-7, and SW480. In addition, we investigated the structure-activity relationship and physicochemical property–activity relationship of EGCG derivatives. Compounds 11 and 12 showed better growth inhibition than others in four cancer cell lines (HL-60, SMMC-7721, A-549, and MCF), with IC50 values in the range of 22.90–37.87 μM. Compounds 11 and 12 decreased phosphorylation of EGFR and downstream signaling protein, which also have more hydrophobic interactions than EGCG by docking study. The most active compounds 11 and 12, both having perbutyrylated glucose residue, we found that perbutyrylation of the glucose residue leads to increased cytotoxic activity and suggested that their potential as anticancer agents for further development.

The Possibility of obtaining inulin from the tubers of Helianthus tuberosus L and Inula helenium L

Inulin is a natural polysaccharide abundant in plants. In the latest decades, in Mongolia, have been cultivated some plants which used in traditional medicine. Inulin polysaccharides were isolated from the roots of the Helianthus tuberosus L. and Inula helenium L. by ultrasound-assisted, microwave extraction, and conventional extraction methods. This polysaccharide is light yellow, tasteless, powder. The polysaccharide structure was confirmed by infrared spectroscopy (FT-IR). The poly fructose content of the Helianthus tuberosus L. ranged from 69 to 84%; in comparison, Inula helenium L ranged from 13 to 51%. The IR-FT spectra revealed typical inulin structure - 820, 876, and 937 cm-1 with terminated α-D-glucose residue. Булцуут цэцэг (Helianthus tuberosus l.) Өндөр зоосон цэцэг (Inula helenium l.) -ийн үндэснээс инулин гарган авах боломж Инулин нь ургамалд элбэг байдаг байгалийн полисахарид бөгөөд манай оронд инулинаар баялаг зарим төрлийн ургамлыг амжилттай нутагшуулан тарималжуулж байгаа билээ. Манай оронд нутагшсан Булцуут цэцэг (Helianthus tuberosus L.) болон Өндөр зоосон цэцэг (Inula helenium L.) ургамлын үндэснээс инулин полисахаридыг хэт авиа (ХА), богино долгион (БД) болон уламжлалт хандлах аргын тусламжтайгаар ялган авлаа. Энэ полисахарид нь ямар нэгэн амтгүй, цайвар шаргал өнгөтэй, нунтаг хэлбэртэй. Уг полисахаридын бүтцийг нил улаан туяаны спектроскопийн (FT-IR) аргаар баталгаажуулсан ба инулины агууламжийг нийт фруктозод шилжүүлэн тооцов. Булцуут цэцгийн полифруктаны агууламж 69-84%, Өндөр зоосон цэцгийнх 13-51% хооронд хэлбэлзэж байсан бөгөөд богино долгионы тусламжтай ялган авсан инулины полифруктаны агууламж хамгийн өндөр байв. НУТ-ны спектрийн шингээлтээр инулины бүтцийн гол зурвасууд нь 820, 876 саяны хэсэгт болон фураноз хэлбэртэй b-D-фруктоз, харин 937 см-1 саяны хэсэгт пираноз хэлбэртэй a-D-глюкозын үлдэгдэл байгааг харуулж байна. Түлхүүр үг: булцуут цэцэг, инулин, НУТ спектроскоп, өндөр зоосон цэцэг, полифруктан

Syntheses and Anticancer Activities of Novel Glucosylated (-)-Epigallocatechin-3-Gallate Derivatives Linked via Triazole Rings

Novel glucosylated (-)-epigallocatechin-3-gallate derivatives 10 – 13 having the EGCG analogues conjugated to the D-glucosyl azide were synthesized by carrying out the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, and were evaluated for their cytotoxicities against a panel of five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480) using MTT assays. Compounds 10 and 11 showed the highest levels of cytotoxicity against the HL-60 cells with IC50 values of 4.57 μM and 3.78 μM, respectively, and showed moderate selectivity towards cancer cell lines. Compound 11 was also shown to induce apoptosis in HL-60 cells. Most notably, inclusion of the perbutyrylated glucose residue in an EGCG derivative was concluded to lead to increased anticancer activity.

Kurilosides A1, A2, C1, D, E and F—Triterpene Glycosides from the Far Eastern Sea Cucumber Thyonidium (= Duasmodactyla) kurilensis (Levin): Structures with Unusual Non-Holostane Aglycones and Cytotoxicities

Six new monosulfated triterpene tetra-, penta- and hexaosides, namely, the kurilosides A1 (1), A2 (2), C1 (3), D (4), E (5) and F (6), as well as the known earlier kuriloside A (7), having unusual non-holostane aglycones without lactone, have been isolated from the sea cucumber Thyonidium (= Duasmodactyla) kurilensis (Levin) (Cucumariidae, Dendrochirotida), collected in the Sea of Okhotsk near Onekotan Island from a depth of 100 m. Structures of the glycosides were established by 2D NMR spectroscopy and HR-ESI mass spectrometry. Kurilosides of the groups A and E contain carbohydrate moieties with a rare architecture (a pentasaccharide branched by C(4) Xyl1), differing from each other in the second monosaccharide residue (quinovose or glucose, correspondingly); kurilosides of the group C are characterized by a unique tetrasaccharide branched by a C(4) Xyl1 sugar chain; and kurilosides of the groups D and F are hexaosides differing from each other in the presence of an O-methyl group in the fourth (terminal) sugar unit. All these glycosides contain a sulfate group at C-6 of the glucose residue attached to C-4 Xyl1 and the non-holostane aglycones have a 9(11) double bond and lack γ-lactone. The cytotoxic activities of compounds 1–7 against mouse neuroblastoma Neuro 2a, normal epithelial JB-6 cells and erythrocytes were studied. Kuriloside A1 (1) was the most active compound in the series, demonstrating strong cytotoxicity against the erythrocytes and JB-6 cells and a moderate effect against Neuro 2a cells.

Glucocerebrosidases catalyze a transgalactosylation reaction that yields a newly-identified brain sterol metabolite, galactosylated cholesterol

β-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form β-cholesterylglucoside (β-GlcChol) in vitro. β-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate β-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (β-GalChol), in addition to β-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for β-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for β-GalChol formation. Liquid chromatography–tandem MS revealed that β-GlcChol and β-GalChol are present throughout development from embryo to adult in the mouse brain. We found that β-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of β-GalChol biosynthesis appeared to be during myelination. We also found that β-GlcChol and β-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form β-GalChol. This is the first report of the existence of β-GalChol in vertebrates and how β-GlcChol and β-GalChol are formed in the brain.

Characterization of β-Glycosidase from Caldicellulosiruptor owensensis and Its Application in the Production of Platycodin D from Balloon Flower Leaf

Platycodin D has diverse pharmacological activities. An efficient and economical mechanism for obtaining platycosides (platycodin D in particular) would be very useful. Balloon flower leaf extract (BFLE) was obtained by recycling leaves discarded from Platycodi radix production, as they have a high platycoside E content. A recombinant β-glycosidase from Caldicellulosiruptor owensensis was characterized and applied to BFLE for platycoside bioconversion. The enzyme specifically hydrolyzed the glucose residue at the C-3 position in platycosides and was suitable for platycodin D production. Under optimized reaction conditions, β-glycosidase from C. owensensis completely converted platycoside E from BFLE into platycodin D with the highest concentration and productivity reported so far. These results greatly improve the production process for deglycosylated platycosides.

Structures and Bioactivities of Psolusosides B1, B2, J, K, L, M, N, O, P, and Q from the Sea Cucumber Psolus fabricii. The First Finding of Tetrasulfated Marine Low Molecular Weight Metabolites

Ten new di-, tri- and tetrasulfated triterpene glycosides, psolusosides B1 (1), B2 (2), J (3), K (4), L (5), M (6), N (7), O (8), P (9), and Q (10), were isolated from the sea cucumber Psolus fabricii collected in the Sea of Okhotsk near the Kurile Islands. Structures of these glycosides were established by two-dimensional (2D) NMR spectroscopy and HR-ESI mass-spectrometry. It is particularly interesting that highly polar compounds 9 and 10 contain four sulfate groups in their carbohydrate moieties, including two sulfates in the same terminal glucose residue. Glycoside 2 has an unusual non-holostane aglycone with 18(16)-lactone and a unique 7,8-epoxy fragment. Cytotoxic activities of compounds 1–10 against several mouse cell lines such as Ehrlich ascites carcinoma cells, neuroblastoma Neuro 2A, normal epithelial JB-6 cells, and erythrocytes were quite different depending both on structural peculiarities of these glycosides and the type of cells subjected to their actions. Psolusoside L (5), pentaoside, with three sulfate groups at C-6 of two glucose and one 3-O-methylglucose residue and holostane aglycone, is the most active compound in the series. The presence of a sulfate group at C-2 of the terminal glucose residue attached to C-4 of the first (xylose) residue significantly decreases activities of the corresponding glycosides. Psolusosides of group B (1, 2, and known psolusoside B) are inactive in all tests due to the presence of non-holostane aglycones and tetrasaccharide-branched sugar chains sulfated by C-2 of Glc4.

Converting Galactose into the Rare Sugar Talose with Cellobiose 2-Epimerase as Biocatalyst

Cellobiose 2-epimerase from Rhodothermus marinus (RmCE) reversibly converts a glucose residue to a mannose residue at the reducing end of β-1,4-linked oligosaccharides. In this study, the monosaccharide specificity of RmCE has been mapped and the synthesis of d-talose from d-galactose was discovered, a reaction not yet known to occur in nature. Moreover, the conversion is industrially relevant, as talose and its derivatives have been reported to possess important antimicrobial and anti-inflammatory properties. As the enzyme also catalyzes the keto-aldo isomerization of galactose to tagatose as a minor side reaction, the purity of talose was found to decrease over time. After process optimization, 23 g/L of talose could be obtained with a product purity of 86% and a yield of 8.5% (starting from 4 g (24 mmol) of galactose). However, higher purities and concentrations can be reached by decreasing and increasing the reaction time, respectively. In addition, two engineering attempts have also been performed. First, a mutant library of RmCE was created to try and increase the activity on monosaccharide substrates. Next, two residues from RmCE were introduced in the cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) (S99M/Q371F), increasing the kcat twofold.

Structural insights into the substrate specificity of a glycoside hydrolase family 5 lichenase from Caldicellulosiruptor sp. F32

Glycoside hydrolase (GH) family 5 is one of the largest GH families with various GH activities including lichenase, but the structural basis of the GH5 lichenase activity is still unknown. A novel thermostable lichenase F32EG5 belonging to GH5 was identified from an extremely thermophilic bacterium Caldicellulosiruptor sp. F32. F32EG5 is a bi-functional cellulose and a lichenan-degrading enzyme, and exhibited a high activity on β-1,3-1,4-glucan but side activity on cellulose. Thin-layer chromatography and NMR analyses indicated that F32EG5 cleaved the β-1,4 linkage or the β-1,3 linkage while a 4-O-substitued glucose residue linked to a glucose residue through a β-1,3 linkage, which is completely different from extensively studied GH16 lichenase that catalyses strict endo-hydrolysis of the β-1,4-glycosidic linkage adjacent to a 3-O-substitued glucose residue in the mixed-linked β-glucans. The crystal structure of F32EG5 was determined to 2.8 Å resolution, and the crystal structure of the complex of F32EG5 E193Q mutant and cellotetraose was determined to 1.7 Å resolution, which revealed that the exit subsites of substrate-binding sites contribute to both thermostability and substrate specificity of F32EG5. The sugar chain showed a sharp bend in the complex structure, suggesting that a substrate cleft fitting to the bent sugar chains in lichenan is a common feature of GH5 lichenases. The mechanism of thermostability and substrate selectivity of F32EG5 was further demonstrated by molecular dynamics simulation and site-directed mutagenesis. These results provide biochemical and structural insights into thermostability and substrate selectivity of GH5 lichenases, which have potential in industrial processes.