An engineered eukaryotic protein glycosylation pathway in Escherichia coli

We performed bottom-up engineering of a synthetic pathway in Escherichia coli for the production of eukaryotic trimannosyl chitobiose glycans and the transfer of these glycans to specific asparagine residues in target proteins. The glycan biosynthesis was enabled by four eukaryotic glycosyltransferases, including the yeast uridine diphosphate-N-acetylglucosamine transferases Alg13 and Alg14 and the mannosyltransferases Alg1 and Alg2. By including the bacterial oligosaccharyltransferase PglB from Campylobacter jejuni, we successfully transferred glycans to eukaryotic proteins.     

Burkholderia mallei expresses a unique lipopolysaccharide mixture that is a potent activator of human Toll-like receptor 4 complexes

Burkholderia mallei, the aetiologic agent of glanders, causes a variety of illnesses in animals and humans ranging from occult infections to acute fulminating septicaemias. To better understand the role of lipopolysaccharide (LPS) in the pathogenesis of these diseases, studies were initiated to characterize the structural and biological properties of lipid A moieties expressed by this organism. Using a combination of chemical analyses and MALDI-TOF mass spectrometry, B. mallei was shown to express a heterogeneous mixture of tetra- and penta-acylated lipid A species that were non-stoichiometrically substituted with 4-amino-4-deoxy-arabinose residues. The major penta-acylated species consisted of bisphosphorylated d-glucosamine disaccharide backbones possessing two amide linked 3-hydroxyhexadecanoic acids, two ester linked 3-hydroxytetradecanoic acids [C14:0(3-OH)] and an acyloxyacyl linked tetradecanoic acid, whereas, the major tetra-acylated species possessed all but the 3'-linked C14:0(3-OH) residues. In addition, although devoid of hexa-acylated species, B. mallei LPS was shown to be a potent activator of human Toll-like receptor 4 complexes and stimulated human macrophage-like cells (THP-1 and U-937), monocyte-derived macrophages and dendritic cells to produce high levels of TNF-alpha, IL-6 and RANTES. Based upon these results, it appears that B. mallei LPS is likely to play a significant role in the pathogenesis of human disease.     

Glycosylation Variants of a β-Glucosidase Secreted by a Taiwanese Fungus,Chaetomella raphigera,Exhibit Variant-Specific Catalytic and Biochemical Properties

Cellulosic biomass is an abundant and promising energy source. To make cellulosic biofuels competitive against conventional fuels, conversion of rigid plant materials into sugars must become efficient and cost-effective. During cellulose degradation, cellulolytic enzymes generate cellobiose (β-(1→4)-glucose dimer) molecules, which in turn inhibit such enzymes by negative feedback. β-Glucosidases (BGLs) cleave cellobiose into glucose monomers, assisting overall cellulolytic activities. Therefore, BGLs are essential for efficient conversion of cellulosic biomass into biofuels, and it is important to characterize newly isolated BGLs for useful traits. Here, we report our discovery that the indigenous Taiwanese fungus Chaetomella raphigera strain D2 produces two molecular weight variants of a single BGL, D2-BGL (shortened to “D2”), which differ in O-glycosylation. The more extensively O-glycosylated form of native D2 (nD2L) has increased activity toward the natural substrate, cellobiose, compared to the less O-glycosylated form (nD2S). nD2L is more stable at 60°C, in acidic pH, and in the presence of the ionic detergent sodium dodecyl sulfate than nD2S. Furthermore, unlike nD2S, nD2L does not display substrate inhibition by an artificial substrate p-nitrophenyl glucopyranoside (pNPG), and the glucose feedback inhibition kinetics of nD2L is competitive (while it is non-competitive for nD2S), suggesting that these two glycovariants of D2 bind substrates differently. Interestingly, D2 produced in a heterologous system, Pichia pastoris, closely mimics properties of nD2S. Our studies suggest that O-glycosylation of D2 is important in determining its catalytic and biochemical properties.     

Glycyrrhizin production by in vitro cultured <Emphasis Type="Italic">Glycyrrhiza glabra</Emphasis> elicited by methyl Jasmonate and salicylic acid

Licorice (Glycyrrhiza glabra L. var. glabra, Fabaceae) is considered as a model plant synthesizing triterpenoid secondary compounds. It is known that glycyrrhizin is accumulated in thickened intact licorice roots. The effects of methyl jasmonate (MeJa) and salicylic acid (SA) on plant growth and production of glycyrrhizin in the roots of in vitro cultured 65-day-old plants were studied. Increasing amounts of glycyrrhizin in the roots treated with MeJa inhibited root growth, while SA increased the amount of glycyrrhizin without negative effects on growth. Treatment of plantlets with 0.1–2 mM MeJa and 0.1 and 1 mM SA enhanced the production of glycyrrhizin by 3.8 and 4.1 times, respectively, as compared to the controls. Results support the hypothesis that production of glycyrrhizin is related to a defense response system of the licorice.     

Design and Synthesis of Lipopolysaccharide-Binding Antimicrobial Peptides Based on Truncated Rabbit and Human CAP18 Peptides and Evaluation of Their Action Mechanism

Lipopolysaccharide (LPS) is a toxic and immunogenic agent for human. Additionally, LPS is a good target for some antimicrobial compounds, including antimicrobial peptides (AMPs). LPS-binding peptides (LBPs) can recognize and neutralize LPS. Rabbit and human cathelicidins are AMPs with LPS-binding activity. In this study, we designed and synthesized two new truncated LBPs from rabbit and human CAP18 peptides by in silico methods. After synthesis of peptides, the antimicrobial properties and LPS-binding activity of these peptides were evaluated. The parental rabbit and human CAP18 peptides were selected as positive controls. Next, the changes in the secondary structure of these peptides before and after treatment with LPS were measured by circular dichroism (CD). Human cytotoxicity of the peptides was evaluated by MTT and red blood cells (RBCs) hemolysis assays. Finally, field emission scanning electron microscopy (FE-SEM), confocal microscopy, and flow cytometry were performed to study the action mechanism of these peptides. Results indicated that the hCap18 and rCap18 had antibacterial activity (at a MIC of 4–128 μg/mL). The results of the quantitative LAL test demonstrated that LPS-binding activity of hCap18 peptide was better than rCap18, while rCap18 peptide had better antimicrobial properties. Furthermore, rCap18 had less cytotoxicity than hCap18. However, both peptides were nontoxic for normal human skin fibroblast cell in MIC range. In conclusion, rCap18 has good antibacterial properties, while hCap18 can be tested as a diagnostic molecule in our future studies.

     

Structure of Compositionally Simple Lipopolysaccharide from Marine Synechococcus

Lipopolysaccharide (LPS) is the first defense against changing environmental factors for many bacteria. Here, we report the first structure of the LPS from cyanobacteria based on two strains of marine Synechococcus, WH8102 and CC9311. While enteric LPS contains some of the most complex carbohydrate residues in nature, the full-length versions of these cyanobacterial LPSs have neither heptose nor 3-deoxy-d-manno-octulosonic acid (Kdo) but instead 4-linked glucose as their main saccharide component, with low levels of glucosamine and galacturonic acid also present. Matrix-assisted laser desorption ionization mass spectrometry of the intact minimal core LPS reveals triacylated and tetraacylated structures having a heterogeneous mix of both hydroxylated and nonhydroxylated fatty acids connected to the diglucosamine backbone and a predominantly glucose outer core-like region for both strains. WH8102 incorporated rhamnose in this region as well, contributing to differences in sugar composition and possibly nutritional differences between the strains. In contrast to enteric lipid A, which can be liberated from LPS by mild acid hydrolysis, lipid A from these organisms could be produced by only two novel procedures: triethylamine-assisted periodate oxidation and acetolysis. The lipid A contains odd-chain hydroxylated fatty acids, lacks phosphate, and contains a single galacturonic acid. The LPS lacks any limulus amoebocyte lysate gelation activity. The highly simplified nature of LPSs from these organisms leads us to believe that they may represent either a primordial structure or an adaptation to the relatively higher salt and potentially growth-limiting phosphate levels in marine environments.Lipopolysaccharide (LPS) in the outer membrane layer is known to be the first line of defense against environmental factors in many gram-negative organisms, preventing lysis by complement, antimicrobial peptides and detergents (17, 21, 47). In proteobacteria, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and phosphate are key parts of the conserved inner core of the LPS which connects the less-well-conserved outer core and sometimes an attached polysaccharide to the lipid A anchor. Why heptose is so well conserved is a mystery, but the prevalence of Kdo and phosphate may be related to the charge which they impart to the outer membrane and to their ability to bind divalent cations. The Kdo-phosphate metal binding center is capable of binding calcium with a dissociation constant (K_d) of 12 to 13 μM (28). This high-affinity binding of divalent cations is known to be necessary for the low permeability of LPS bilayers to some antibiotics (32), and it has been hypothesized that divalent cation cross-bridges may link LPS molecules on the bacterial cell surfaces of enterobacteria into a giant complex with very low membrane permeability (16).Though the LPSs of many proteobacteria are well characterized, the LPSs from cyanobacteria are much less studied. The cell envelopes of cyanobacteria resemble those of gram-negative bacteria structurally, consisting of a cytoplasmic membrane, a peptidoglycan layer, an outer membrane containing LPS, and sometimes additional structures (9, 14). Previous chemical analyses have shown the LPS of some cyanobacteria to be devoid of phosphate, Kdo, and heptose (11, 12, 42, 43). Given the lack of Kdo in these organisms as well as the fact that the lability of the Kdo-glucosamine ketosidic linkage allows for the mild acid hydrolysis of LPS to lipid A, it is perhaps not surprising that many attempts at hydrolysis of cyanobacterial LPS to lipid A have failed (for an example, see reference 29).Within the cyanobacteria, the genus Synechococcus represents a polyphyletic group of unicellular morphotypes. Synechococcus cells are found in both freshwater and marine environments. Organisms from group A Synechococcus and its sister taxon Prochlorococcus are extremely important primary producers in marine environments, with multiple “clades” similar to “species” described for other bacteria, dominating in different environments (3, 22). Unlike enterobacteria, which must frequently contend with an onslaught of host factors, members of the Synechococcus face grazing by protists and bacteriophages as their primary survival challenges.The genome of Synechococcus sp. strain CC9311 has been shown to be devoid of the genes for Kdo biosynthesis, while strain WH8102 has several putative genes for Kdo biosynthesis (18, 20). This suggests that the LPS of cyanobacteria could be significantly different from that of enteric bacteria and could show species/strain variation as well. A comparison of the structures of LPS from cyanobacteria and enterobacteria would afford a unique opportunity to understand which elements of LPS structure are essential to bacterial survival and which are adaptations to the environment in which the bacteria live. To further this understanding, we present here an analysis of the LPS structure from two strains of marine Synechococcus: an open-ocean-dwelling strain having the putative genes for Kdo biosynthesis (strain WH8102; clade III) and a coastal strain lacking these genes (strain CC9311; clade I). We further present two novel methods for producing lipid A from bacteria lacking the labile Kdo ketosidic linkage.     

The Lewis x epitope is a major non-reducing structure in the sulphated N-glycans attached to Asn-65 of bovine pro-opiomelanocortin

The N-terminal glycopeptide of pro-opiomelanocortin (POMC),designated as the 16K fragment, is highly conserved throughoutvertebrates from amphibians to mammals and is likely thereforeto have an important functional role. In this paper, we reportthe first structural characterization of N-glycans attachedto asparagine-65 of a 16K glycopeptide. The 16K fragment wasisolated from bovine pituitaries and the N-glycans were analysedusing fast atom bombardment mass spectrometry together withsugar and linkage analysis. Sulphated-N-acetylgalactosamine-cappedantennae, typical of the pituitary glycohormones, were presentin the major acidic components. The POMC oligo-saccharides aredistinct from those of the pituitary glycohormones because thesulphate is exclusively located on the 3-arm of biantennarystructures and, in addition, a significant proportion of themolecules carry the Lewis x epitope. It is probable that thesedifferences reflect the absence of a tripeptide motif in POMCwhich fully conforms to the criteria previously defined forthe recognition sequence for the N-acetylgalactosamine transferasethat is specific for the pituitary glycohormones [Smith andBaenziger (1992) Proc. Natl. Acad. Sci. USA, 89, 329–333].It remains to be seen whether the Lewis x epitope is involvedin selectin-mediated events, but previous studies suggest thatthe sulphated moieties are unlikely to play a major role inclearance. The Lewis x epitope is also present in the neutralN-linked oligosaccharides, together with a variety of otherantennae including a rarely found fucosylated GalNAc-GlcNAcstructure. FAB-MS glycoprotein Lewis x POMC sulphated-GalNAc     

Identification of a Novel Triterpenoid Saponin from Pisum sativum as a Specific Inhibitor of the Diguanylate Cyclase of Acetobacter xylinum

A specific and highly potent inhibitor of diguanylate cyclase,the key regulatory enzyme of the cellulose synthesizing apparatusin the bacterium Acetobacter xylinum, was isolated from extractsof etiolated pea shoots (Pisum sativum). The inhibitor has beenpurified by a multistep procedure, and sufficient amounts ofhighly purified compound (3-8 mg) for spectral analysis wereobtained. The structure of this compound was established as3-O-a-L-rhamnopyranosyl-(l