Self-Assembly of Proteinaceous Multishell Structures Mediated by a Supercharged Protein

Engineered variants of the capsid-forming enzyme lumazine synthase can exploit electrostatic interactions to encapsulate complementarily charged guest macromolecules. Here we investigate the effect of ionic strength and cargo molecules on assembly of AaLS-13, a negatively supercharged lumazine synthase protein cage, and we show that multishell structures are produced upon mixing the capsid core with free capsomers and a positively supercharged variant of the green fluorescent protein GFP­(+36). The assembly process is mediated by favorable electrostatic interactions between the negatively charged capsid shells/capsomers and GFP­(+36) molecules, and it is therefore strongly dependent on ionic strength. The mechanism of formation of these assemblages is likely similar to the assembly of multishell structures of some virus-like particles, where outer shells organize as nonicosahedral structures with larger radii of curvature than the templating inner shell. In contrast to the viral multishell structures, the positively charged mediator was found to be essential for the assembly of multilayered structures of different shapes and sizes constituted of AaLS-13 capsomers. This mediator-bridging approach may be widely applicable to create protein-based hierarchical nanostructures for various nanotechnology applications such as drug delivery and bioimaging

Mechanistic Studies of the Biosynthesis of 2-Thiosugar: Evidence for the Formation of an Enzyme-Bound 2-Ketohexose Intermediate in BexX-Catalyzed Reaction

The first mechanistic insight into 2-thiosugar production in an angucycline-type antibiotic, BE-7585A, is reported. d-Glucose 6-phosphate was identified as the substrate for the putative thiosugar biosynthetic protein, BexX, by trapping the covalently bonded enzyme−substrate intermediate. The site-specific modification at K110 residue was determined by mutagenesis studies and LC−MS/MS analysis. A key intermediate carrying a keto functionality was confirmed to exist in the enzyme−substrate complex. These results suggest that the sulfur insertion mechanism in 2-thiosugar biosynthesis shares similarities with that for thiamin biosynthesis

A Biosynthetic Pathway for BE-7585A, a 2-Thiosugar-Containing Angucycline-Type Natural Product

Sulfur is an essential element found ubiquitously in living systems. However, there exist only a few sulfur-containing sugars in nature and their biosyntheses have not been studied. BE-7585A produced by Amycolatopsis orientalis subsp. vinearia BA-07585 has a 2-thiosugar and is a member of the angucycline class of compounds. We report herein the results of our initial efforts to study the biosynthesis of BE-7585A. Spectroscopic analyses verified the structure of BE-7585A, which is closely related to rhodonocardin A. Feeding experiments using 13C-labeled acetate were carried out to confirm that the angucycline core is indeed polyketide-derived. The results indicated an unusual manner of angular tetracyclic ring construction, perhaps via a Baeyer−Villiger type rearrangement. Subsequent cloning and sequencing led to the identification of the bex gene cluster spanning ∼30 kbp. A total of 28 open reading frames, which are likely involved in BE-7585A formation, were identified in the cluster. In view of the presence of a homologue of a thiazole synthase gene (thiG), bexX, in the bex cluster, the mechanism of sulfur incorporation into the 2-thiosugar moiety could resemble that found in thiamin biosynthesis. A glycosyltransferase homologue, BexG2, was heterologously expressed in Escherichia coli. The purified enzyme successfully catalyzed the coupling of 2-thioglucose 6-phoshate and UDP-glucose to produce 2-thiotrehalose 6-phosphate, which is the precursor of the disaccharide unit in BE-7585A. On the basis of these genetic and biochemical experiments, a biosynthetic pathway for BE-7585A can now be proposed. The combined results set the stage for future biochemical studies of 2-thiosugar biosynthesis and BE-7585A assembly

Construction of the Octose 8‑Phosphate Intermediate in Lincomycin A Biosynthesis: Characterization of the Reactions Catalyzed by LmbR and LmbN

Lincomycin A is a potent antimicrobial agent noted for its unusual C1 methylmercapto-substituted 8-carbon sugar. Despite its long clinical history for the treatment of Gram-positive infections, the biosynthesis of the C8-sugar, methylthiolincosamide (MTL), is poorly understood. Here, we report our studies of the two initial enzymatic steps in the MTL biosynthetic pathway leading to the identification of d-erythro-d-gluco-octose 8-phosphate as a key intermediate. Our experiments demonstrate that this intermediate is formed via a transaldol reaction catalyzed by LmbR using d-fructose 6-phosphate or d-sedoheptulose 7-phosphate as the C3 donor and d-ribose 5-phosphate as the C5 acceptor. Subsequent 1,2-isomerization catalyzed by LmbN converts the resulting 2-keto C8-sugar (octulose 8-phosphate) to octose 8-phosphate. These results provide, for the first time, in vitro evidence for the biosynthetic origin of the C8 backbone of MTL

In Vitro Characterization of LmbK and LmbO: Identification of GDP‑d-<i>erythro</i>-α‑d-<i>gluco</i>-octose as a Key Intermediate in Lincomycin A Biosynthesis

Lincomycin A is a clinically useful antibiotic isolated from <i>Streptomyces lincolnensis</i>. It contains an unusual methyl­mercapto-substituted octose, methylthio­lincos­amide (MTL). While it has been demonstrated that the C₈ backbone of MTL moiety is derived from d-fructose 6-phosphate and d-ribose 5-phosphate via a transaldol reaction catalyzed by LmbR, the subsequent enzymatic transformations leading to the MTL moiety remain elusive. Here, we report the identification of GDP-d-<i>erythro</i>-α-d-<i>gluco</i>-octose (GDP-d-α-d-octose) as a key intermediate in the MTL biosynthetic pathway. Our data show that the octose 1,8-bisphosphate intermediate is first converted to octose 1-phosphate by a phosphatase, LmbK. The subsequent conversion of the octose 1-phosphate to GDP-d-α-d-octose is catalyzed by the octose 1-phosphate guanylyltransferase, LmbO. These results provide significant insight into the lincomycin biosynthetic pathway, because the activated octose likely serves as the acceptor for the installation of the C1 sulfur appendage of MTL

Gating strategies used for FACS analysis of eosinophils and neutrophils after vaccination with primary alveolar macrophages.

(A) Gating strategies used for the FACS analysis of eosinophils and neutrophils. (B) Gating strategies used for FACS analysis of primary alveolar macrophages and associated impurities. Relative to Fig 2. (TIF)</p

Alum increases MHC class II expression levels of APCs in an IL-33-dependent manner.

WT or Il33-/- mice were intranasally administered SV or SV plus alum. At 24 h after administration, the mice were euthanized, and the lungs or BALF were collected. CD103+ DCs, IMs, and CD11b+ DCs in the lung were gated as live (propidium iodide negative) CD45+SiglecF-CD103+CD11bmidCD11c+CD11c+CD24mid, CD45+SiglecF-CD11c+CD11b+CD24-, and CD45+SiglecF-CD11c+CD11b+CD24+, respectively. Their MHC class II expression levels were individually analyzed (A). The median fluorescence intensity (MFI) of MHC class II levels in each cell population was determined (B). The IL-33 concentrations in BALF were determined using ELISA (C). The data are from three independently performed experiments (A–C), and the error bars are presented as the mean (± SEM) of three mice per group (A–C). For more than three groups, significance was assessed using one-way ANOVA and Dunnett’s multiple-comparison test to determine differences between SA and other groups. The student’s t-test was used to compare two groups. **p ***p p p < 0.001.</p

Alum increases IL-5 and IL-13 expression in lung ILC2s but does not induce ILC2 accumulation in the lung.

WT or Il33-/- mice were intranasally administered SA or alum. At 6 h after administration, the mice were euthanized, and the lungs were collected. ILC2s in the lung were gated as live (propidium iodide negative) CD45+Lineage (B220/FcεRI/CD11b/CD3ε/SiglecF)-ICOS+ST2+ (A). The ILC2 percentage of CD45+ cells was determined for each group (A). Intracellular staining with anti-IL-5 Ab (B) or anti-IL-13 Ab (C) was performed to determine their expression levels in ILC2s. The rates of CD45+IL-5+ or CD45+IL-13+ cells were calculated. Intracellular staining experiments were performed using pooled single suspensions from three mice for each group. The data are from two independently performed experiments (A–C), and the error bars are presented as the mean (± SEM) of four mice per group (A–C). Each dot indicates the result of individual experiments (B and C). For more than three groups, significance was assessed using one-way ANOVA and Dunnett’s multiple-comparison test to determine differences between SA and other groups. The student’s t-test was used to compare two groups. p * p ***p < 0.001 compared with the DQ-OVA group (A) or SA group (B and C).</p

Alveolar epithelial cells lead to necroptosis-dependent IL-33 secretion.

Nontreated mice were euthanized, and the lungs were collected. Fresh frozen sections were prepared and stained with anti-IL-1α Ab, anti-F4/80 Ab, anti-Pro-SP-C Ab and DAPI (A), or anti-IL-33 Ab, anti-F4/80 Ab, anti- Pro-SP-C Ab, and DAPI (B). In both A and B, the top and bottom images are from the same section, however, there are different fluorescent combinations. Images were analyzed with an Olympus BX53 fluorescence microscope. Adobe Photoshop was used to adjust the brightness and contrast (changes were applied to the entirety of all images equally). To calculate cell populations, more than 100 IL-1α+ or IL-33+ cells were counted in specimens from each animal. Among them, F4/80+, Pro-SP-C+, and F4/80-Pro-SP-C- cells were counted, and then the ratios of each population were calculated. The data are presented as the mean ratio from three animals (±SD). (C) Freshly isolated alveolar epithelial cells were prepared from WT mice. The purities of the alveolar epithelial cells were determined using FACS analysis as shown in S3 Fig. The cells were stimulated with PBS, 50, or 100 μg alum or 10% HP-β-CD for 5 h. After stimulation, a cell-free medium was collected to determine the cell viability with a Cytotoxicity LDH Assay Kit-WST and the IL-33 concentration using ELISA. (D) To assess the involvement of necroptosis signaling, alum was coadministered with the RIP3K inhibitor GSK’872, RIP1K inhibitor necrostatin-1, or MLKL inhibitor necrosulfonamide. DMSO was added as a vehicle control. The data are from three independently performed experiments (C and D), and the error bars are presented as the mean (± SEM) of three independently performed experiments. For more than three groups, significance was assessed using one-way ANOVA and Dunnett’s multiple-comparison test to determine differences between SA and other groups. The student’s t-test was used to compare two groups. *p p ***p t-test was used to determine differences between the DMSO- and GSK’872-treated groups, and *p < 0.05 compared with the DMSO group (E).</p

Alum has strong adjuvant effects in nasal influenza vaccines, and IL-33 has a significant influence on the induction of Ag-specific IgA Ab production by alum.

(A) WT and IL-33-deficient (Il33-/-) mice were intranasally administered SA, 100 μg of alum, or 100 μg of NanoSiO2. At 1, 3, 6, and 24 h after administration, the mice were euthanized, and BALF was collected. The concentrations of DAMPs in BALF were determined using ELISA. Quantification of dsDNA was performed using the Quant-iT Picogreen dsDNA Assay Kit. (B) For dosing amount-dependent responses, 5, 10, 50, or 100 μg of alum was intranasally administered. BALF was collected 6 h and 24 h after alum intranasal administration to analyze IL-33 and IL-1α levels using ELISA. Nontreated (NT) mice were used as controls. (C) BALF Ag-specific IgA Ab and serum IgE Ab concentration were determined using ELISA 14 days after the second immunization with SV, SV combined with alum, SV combined with rIL-33, OVA, or OVA combined with PVNO (n = 4–6 per group). (D) Nonimmunized, SV-immunized, or SV plus alum-immunized WT or Il33-/- mice were challenged with a 10 LD50 dose of A/Osaka/129/2009 [A(H1N1)pdm09] influenza virus 14 days after the second alum-adjuvanted SV immunization. Their body weight changes and survival were monitored (n = 4–10 per group). (E) BALF was collected 6 h after SA or alum intranasal administration. IL-1α, IL-1β, and IL-6 concentrations in BALF were determined using ELISA, and quantification of dsDNA was performed using the Quant-iT Picogreen dsDNA Assay Kit (n = 4–6 per group). (F) The mice were immunized with SV alone, SV with 2 μg dsDNA, or SV with alum. In some groups, 600 IU of RNase-free recombinant DNase I was intranasally administered 3 h before and 18 h after each immunization. Another group was similarly administered PBS as a control for DNase I treatment. At 14 days after immunizations, the mice were euthanized, and BALF was collected to determine Ag-specific IgA Ab concentrations using ELISA (n = 4 per group). The data are from two (A, B, C, E, and F) or three (D) independently performed experiments and are presented as the mean (± SEM). Each dot indicates the result of an individual animal (A, C, and D). For more than three groups, significance was assessed using one-way ANOVA and Dunnett’s multiple-comparison test to determine differences between SA and other groups. The student’s t-test was used to compare two groups. *p $p 2 groups compared with the SA group, respectively. *p ***p p ***p < 0.001. N.S. means not significant.</p