Degradative enzymes of oral streptococci

Members of the Streptococcus sanguis group (SSG) and Streptococcus milleri group (SMG) were screened for their ability to produce glycosidase, arylamidase (peptidase), protease, dextranase and glycosyltransferase activities. Species within each group produced unique patterns of activity. The most commonly produced glycosidases were β-D-glucosidase, β-D- galactosidase, N-acetyl-β-D-glucosaminidase and N-acetyl-β-D-galactosaminidase and the least commonly produced glycosidase activity was β-fucosidase with Streptococcus intermedius (SMG) being the only species capable of producing the activity. For arylamidase activity, the most commonly produced type was lysine-arylamidase. Glycosidase and arylamidase activities were localized to particular sub-cellular fractions. α-galactosidase was found only in culture supernatant fluids whereas N-acetyl-β-D-glucosaminidase was found in all fractions; the culture supernatant, cell wall, cell membrane and cytoplasm. No arylamidase activity was seen in culture supernatants. Phe-arg-arylamidase was found only in cytoplasmic fractions whereas val-pro-arg-arylamidase was found in cell walls, cell membranes and cytoplasmic fraction. Protease activity was measured as the degradation of bovine serum albumin (BSA) and casein. Casein was degraded by a number of strains whereas no species/strains were able to degrade BSA. Streptococcus intermedius, Streptococcus constellates (SMG), Streptococcus mitior and Streptococcus defectivus (SSG) were the only species that produced hyaluronidase and no species produced chondroitin sulphatase. The groups were also examined for their abilities to produce glycosyltransferase and dextranase. Strep. sanguis, Streptococcus gordonii, Streptococcus mitis and Streptococcus oralis produced glucosyltransferase and, with the exception of the latter species, fructosyl-transferase. No species within the SMG was capable of producing either glycosyltransferase. No species within the SSG or SMG was able to produce dextranase activity. The ability of species to produce different types of enzymes was related to their taxonomy, allowing the differentiation of several new taxonomic types within the SSG and may be related to pathogenesis.     

The structure of POMGNT2 provides new insights into the mechanism to determine the functional O-mannosylation site on α-dystroglycan

Defects in the O-mannosyl glycan of α-dystroglycan (α-DG) are associated with α-dystroglycanopathy, a group of congenital muscular dystrophies. While α-DG has many O-mannosylation sites, only the specific positions can be modified with the functional O-mannosyl glycan, namely, core M3-type glycan. POMGNT2 is a glycosyltransferase which adds β1,4-linked GlcNAc to the O-mannose (Man) residue to acquire core M3-type glycan. Although it is assumed that POMGNT2 extends the specific O-Man residues around particular amino acid sequences, the details are not well understood. Here, we determined a series of crystal structures of POMGNT2 with and without the acceptor O-mannosyl peptides and identified the critical interactions between POMGNT2 and the acceptor peptide. POMGNT2 has an N-terminal catalytic domain and a C-terminal fibronectin type III (FnIII) domain and forms a dimer. The acceptor peptide is sandwiched between the two protomers. The catalytic domain of one protomer recognizes the O-mannosylation site (TPT motif), and the FnIII domain of the other protomer recognizes the C-terminal region of the peptide. Structure-based mutational studies confirmed that amino acid residues of the catalytic domain interacting with mannose or the TPT motif are essential for POMGNT2 enzymatic activity. In addition, the FnIII domain is also essential for the activity and it interacts with the peptide mainly by hydrophobic interaction. Our study provides the first atomic-resolution insights into specific acceptor recognition by the FnIII domain of POMGNT2. The catalytic mechanism of POMGNT2 is proposed based on the structure.     

An enzymatic approach to configurationally rare trans‐androsteronyl‐α‐glucoside and Its potential anticancer application

Enzymatic glycosylation of sterols/steroids with glycosyltransferase HP0421 shows protein plasticity on generation of configurationally rare steryl‐α‐glucosides. Investigation of trans‐androsteronyl‐α‐glucoside on tamoxifen‐treated MCF‐7 breast cancer cells shows dose‐dependent depression of cell viability and enhanced drug effectiveness, illustrating a new avenue for the production of novel steryl‐α‐glucosides with useful biological activities.     

Enzymatic glycosylation of oleanane-type triterpenoids

Abstract

Glycosylation is an effective approach to improve the druggability of natural products by increasing their water solubility. In this work, we report the glycosylation of oleanane-type triterpenoids by a recombinant microbial glycosyltransferase YjiC1. A preliminary screening test indicated YjiC1 exhibited robust capabilities for O-glycosylation of triterpenoids, based on LC/MS analysis. Among the products, two new compounds (2a and 3a), together with a known one (1a), were isolated and characterized. These products exhibited improved water solubility, and 3a showed moderate anti-HIV activities at 100 μM. This reaction provides a facile and efficient approach to synthesize the glucosides of triterpenoids.     

Ceramides are associated with inflammatory processes in human mediastinal adipose tissue

Background and AimCeramides are poorly characterized in human adipose tissue. The aim of this study was to investigate concentrations of different ceramide species in human subcutaneous and visceral adipose tissue depots and to determine associations between ceramides and global gene expression profiles.Methods and ResultsConcentrations of six ceramide species were determined in plasma and in subcutaneous and mediastinal adipose tissue from 10 overweight subjects (BMI 29.4 ± 4.9 kg/m²). In the adipose tissue biopsies gene expression arrays were performed and relationships between ceramides and gene expression analyzed. Immunostaining of the two adipose tissue depots was performed in an independent group of 10 patients. Mediastinal adipose tissue contained significantly higher concentrations (p < 0.05) of all six ceramide species than the subcutaneous depot. Of the six ceramides in plasma, concentrations of only two (Cer d18:1/18:0 and Cer d18:1/22:0) correlated significantly (p < 0.05) with the corresponding species in mediastinal adipose tissue, but there were no significant correlations between ceramides in plasma and subcutaneous adipose tissue. Multivariate analysis identified significant correlations between the total ceramide concentration and global gene expression within mediastinal, but not subcutaneous adipose tissue, according to cross-validation. Gene ontology analysis of genes related to ceramides in the mediastinal depot revealed that genes positively correlated with ceramides were associated mainly with immune and inflammatory categories, while genes negatively correlated with ceramides were associated mainly with lipid and carbohydrate metabolism.ConclusionsCeramides in human mediastinal adipose tissue may be involved in inflammation and lipid and carbohydrate metabolism.     

A surprising sweetener from enteropathogenic Escherichia coli

Infections with enteropathogenic Escherichia coli (EPEC) are remarkably devoid of gut inflammation and necrotic damage compared to infections caused by invasive pathogens such as Salmonella and Shigella. Recently, we observed that EPEC blocks cell death using the type III secretion system (T3SS) effector NleB. NleB mediated post-translational modification of death domain containing adaptor proteins by the covalent attachment of N-acetylglucosamine (GlcNAc) to a conserved arginine in the death domain.  N-linked glycosylation of arginine has not previously been reported in mammalian cell biology and the precise biochemistry of this modification is not yet defined. Although the addition of a single GlcNAc to arginine is a seemingly slight alteration, the impact of NleB is considerable as arginine in this location is critical for death domain interactions and death receptor induced apoptosis. Hence, by blocking cell death, NleB promotes enterocyte survival and thereby prolongs EPEC attachment to the gut epithelium.     

Suppression of lung metastasis of mouse Lewis lung cancer P29 with transfection of the ganglioside GM2/GD2 synthase gene

Ganglioside functions in tumor metastasis were analyzed by carbohydrate remodeling of a mouse Lewis lung cancer (subline P29) by introducing beta1,4GalNAc-T cDNA. Although P29 was originally a low-metastatic subline in the s.c. injection system, it showed high potential in lung metastasis when i.v.-injected via the tail vein. Two lines of GM(2)(+) transfectants showed markedly reduced metastatic potential to the lung compared to 2 control lines. However, cell proliferation rates and expression levels of various cell adhesion molecules, e.g., integrin family members, SLe(x) and CD44, were essentially unchanged after transfection of the cDNA. Then, cell adhesion to fibronectin-coated dishes was examined, showing that GM(2) (+) transfectants attached to the plates much more slowly than controls, suggesting functional modulation of integrins with newly expressed GM(2). Phosphorylation of the FAK located at downstream of integrin molecules was markedly reduced in GM(2)(+) transfectants, suggesting that GM(2) suppressed cell adhesion signals via fibronectin-integrin interaction.     

Galactolipid synthesis in chloroplast inner envelope is essential for proper thylakoid biogenesis, photosynthesis, and embryogenesis

The biogenesis of thylakoid membranes, an indispensable event for the photoautotrophic growth of plants, requires a significant increase in the level of the unique thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG), which constitutes the bulk of membrane lipids in chloroplasts. The final step in MGDG biosynthesis occurs in the plastid envelope and is catalyzed by MGDG synthase. Here we report the identification and characterization of an Arabidopsis mutant showing a complete defect in MGDG synthase 1. The mutant seeds germinated as small albinos only in the presence of sucrose. The seedlings lacked galactolipids and had disrupted photosynthetic membranes, leading to the complete impairment of photosynthetic ability and photoautotrophic growth. Moreover, invagination of the inner envelope, which is not seen in mature WT chloroplasts, was observed in the mutant, supporting an old hypothesis that envelope invagination is a major event in early chloroplast biogenesis. In addition to the defective seedling phenotype, embryo development was arrested in the mutant, although seeds with impaired embryos could germinate heterotrophically. These results demonstrate the importance of galactolipids not only in photosynthetic growth but also in embryogenesis.     

T-cell activation results in microheterogeneous changes in glycosylation of CD45

During T-cell development and activation, dramatic changes occur in glycan structures that decorate cell-surface glycoproteins. These changes have been considered to be general cellular events that affect many glycans on many glycoproteins. For example, loss of sialic acid from core 1 O-glycans on T-cell surface glycoproteins CD45, CD43 and CD8, detected with peanut agglutinin (PNA), is a hallmark of immature thymocytes and activated peripheral T cells. Loss of cell-surface sialic acid during T-cell activation has been proposed to enhance TCR reactivity with antigen. However, CD4 T-cell activation also results in increased binding of the CZ-1 antibody that recognizes a sialic acid-containing epitope on CD45RB. This indicates that increased sialylation of the CZ-1 epitope occurs during CD4 T cell activation, and that loss of cell surface sialic acid during T-cell activation is a selective event rather than affecting all cell surface glycans. As specific glycans on specific glycoprotein backbones control critical events in T-cell maturation and survival, understanding mechanisms of selective glycoprotein glycosylation is important for regulating T-cell development and function. We define the sialylated O-glycan epitope recognized by CZ-1, and find that, paradoxically, CZ-1 and PNA binding are simultaneously increased on activated CD4(+) T cells, demonstrating site-specific changes in CD45 sialylation. Moreover, we identify ST3Gal I as the sialyltransferase responsible for creating the CZ-1 epitope. Thus, changes in glycan structure during T-cell activation are microheterogeneous and unique to individual glycans on specific glycoproteins, implying that these glycans have precise functions in T-cell biology.