Engineering of Chimeric Protein Based on E Protein Domain III of Tick- Borne Encephalitis Virus and OmpF Porin of Yersinia pseudotuberculosis.

BACKGROUND: Tick-borne encephalitis poses a serious public health threat in the endemic regions. The disease treatment is restricted to symptomatic therapy, so great expectations are in the development of the prophylactic and therapeutic vaccines. The domain III of E protein of the tickborne encephalitis virus is the main antigenic domain which includes virus-specific epitopes recognized by neutralizing antibodies. OBJECTIVES: The main objective of this study was to design, express, isolate and characterize the chimeric protein based on the fusion of domain III of E protein of the tick-borne encephalitis virus and bacterial porin OmpF from Yersinia pseudotuberculosis. METHODS: The chimeric gene was obtained by the PCR based fusion method from two fragments containing overlapping linker sequences. Resulting plasmids were transformed into BL21(DE3) pLysS electrocompetent cells for subsequent heterologous protein expression. All recombinant proteins were purified using immobilized metal affinity chromatography under denaturing conditions. The identity of the chimeric protein was confirmed by MALDI-TOF mass spectrometry and immunoblot analysis. The content of antibodies against the EIII protein was estimated in mice blood serum by ELISA. RESULTS: The bacterial partner protein was used for decreasing toxicity and increasing immunogenicity of antigen. The chimeric protein was successfully expressed by the Escherichia coli cells. The purified protein was recognized with immunoblots by anti-E protein of tick-borne encephalitis virus monoclonal antibodies. Furthermore, the protein was able to elicit antibody response against domain III of E protein in immunized mice. CONCLUSION: The newly obtained chimeric antigen could be valuable for the development of the preventing tick-borne encephalitis subunit vaccines.

Peculiarities of thermal denaturation of OmpF porin from Yersinia ruckeri.

Irreversible denaturation of membrane proteins in detergent solutions is similar to unfolding of water-soluble multidomain proteins and represents a complex, multistage process. Pore-forming proteins of Gram-negative bacteria are heat-modifiable proteins, i.e., proteins altering their molecular forms (trimers or monomers), and accordingly, their electrophoretic mobilities depending upon denaturation conditions. There are still some contradictory data on the peculiarities of the conformational changes in the porin structure with temperature. Some authors demonstrated the loss of the porin trimeric structure only after unfolding of monomer subunits. Other researchers initially observed the dissociation of porin oligomers into the folded monomers. Using SDS-PAGE, spectroscopic methods and differential scanning calorimetry, a detailed study of thermally induced changes in the spatial structure of OmpF porin from the fish pathogen Yersinia ruckeri (Yr-OmpF) was carried out. The data obtained allowed us to conclude unambiguously that changes in the spatial structure of the monomers of Yr-OmpF precede the dissociation of the porin trimer.

Opposite Effects of Lysophosphatidylethanolamines on Conformation of OmpF-like Porin from Yersinia pseudotuberculosis.

Lysophosphatidyletnolamine (LPE) is one of enigmatic lipids of bacteria. It is generated from major membrane lipid - phosphatidylethanolamine at severe changes of the bacterial growth conditions. Accumulation of this phospholipid in cells of Gram-negative enterobacterium Yersinia pseudotuberculosis results in the enhanced thermostability of OmpF-like porin (YOmpF) from the same bacteria. The respective integral conformational rearrangements may disturb the channel permeability of protein under stress conditions. However, role of fatty acid composition of LPE in this effect remained unclear. Present work demonstrated that the level of unsaturated LPE is 3.5 times higher than saturated one in total LPE of bacterial cells exposed to stress (phenol treatment). Unsaturated 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (MOPE) and saturated LPE 1-palmitoyl-2- hydroxy-sn-glycero-3-phosphoethanolamine (MPPE) oppositely affect the conformation of YOmpF. MOPE increases the protein thermal stability due to more dense packing of monomers in porin and preserves its trimeric form at elevated temperature, while MPPE weakens the contact between monomers and promotes dissociation of the protein.

Cucumarioside E from the Far Eastern Sea Cucumber Cucumaria japonica (Cucumariidae, Dendrochirotida), New Minor Monosulfated Holostane Triterpene Pentaoside with Glucose as the Second Monosaccharide Residue.

New minor triterpene glycoside, cucumarioside E (1) has been isolated from the Far Eastern sea cucumber Cucumaria japonica. The structure of the glycoside was elucidated by 2D-NMR specroscopy and mass-spectrometry. The glycoside has glucose instead of quinovose as the second monosaccharide residue and xylose as third monosaccharide residue that is unique structural feature for triterpene glycosides carbohydrate chains from sea cucumbers belonging to the genus Cucumaria.

The influence of monogalactosyldiacylglycerols from different marine macrophytes on immunogenicity and conformation of protein antigen of tubular immunostimulating complex.

The tubular immunostimulating complex (TI-complex) is a novel nanoparticulate antigen delivery system consisting of cholesterol, triterpene glycoside cucumarioside A(2)-2, and glycolipid monogalactosyldiacylglycerol (MGDG) isolated from marine macrophytes. MGDG is crucial for the formation of a lipid matrix for the protein antigen incorporated in TI-complexes. Fatty acid composition and the physical state of this glycolipid depend on the taxonomic position of marine macrophytes. Therefore, the aim of the present work was to study the capacity of MGDGs, isolated from five species of marine macrophytes, to influence conformation and to enhance immunogenicity of porin from Yersinia pseudotuberculosis (YOmpF) as a model antigen of subunit vaccine based on TI-complexes. The trimeric porin was chosen for these experiments, because it was approximately two times more immunogenic than monomeric porin incorporated in TI-complexes. Immunization of mice with YOmpF within TI-complexes, comprised of different MGDGs, revealed a dependence of the immunostimulating effect of TI-complexes on the microvicosity of this glycolipid. TI-complexes comprising MGDGs from Sargassum pallidum and Ulva fenestrata with medium microviscosity induced maximal levels of anti-porin antibodies (four times higher when compared with those induced by pure porin). The adjuvant effect of TI-complexes based on other MGDGs varied by 2.8, 2.3 and 1.3 times for TI-complexes comprised of MGDGs from Zostera marina, Ahnfeltia tobuchiensis, and Laminaria japonica, respectively. MGDGs are also able to influence cytokine mechanisms of immunological regulation. DSC and spectroscopic studies showed that maximal immunostimulating effect of TI-complexes correlated with a moderate stabilizing influence of MGDGs from S. pallidum and U. fenestrata on the conformation of porin. The results obtained suggest lipid "nanofluidics" as a novel strategy for optimizing the immune response to protein antigens within lipid particulate systems.

Tubular immunostimulating complex based on cucumarioside A2-2 and monogalactosyldiacylglycerol from marine macrophytes.

BACKGROUND: There is an urgent need to develop safe and effective adjuvants for the new generation of subunit vaccines. We developed the tubular immunostimulating complex (TI-complex) as a new nanoparticulate antigen delivery system. The morphology and composition of TI-complexes principally differ from the known vesicular immunostimulating complexes (ISCOMs). However, methodology for the preparation of TI-complexes has suffered a number of shortcomings. The aim of the present work was to obtain an antigen carrier consisting of triterpene glycosides from Cucumaria japonica, cholesterol, and monogalactosyldiacylglycerol from marine macrophytes with reproducible properties and high adjuvant activity. RESULTS: The cucumarioside A2-2 - cholesterol - MGalDG ratio of 6:2:4 (by weight) was found to provide the most effective formation of TI-complexes and the minimum hemolytic activity in vitro. Tubules of TI-complexes have an outer diameter of about 16 nm, an inner diameter of 6 nm, and a length of 500 nm. A significant dilution by the buffer gradually destroyed the tubular nanoparticles. The TI-complex was able to increase the immunogenicity of the protein antigens from Yersinia pseudotuberculosis by three to four times. CONCLUSIONS: We propose an optimized methodology for the preparation of homogeneous TI-complexes containing only tubular particles, which would achieve reproducible immunization results. We suggest that the elaborated TI-complexes apply as a universal delivery system for different subunit antigens within anti-infectious vaccines and enhance their economic efficacy and safety.

Seasonal changes of fatty acid composition and thermotropic behavior of polar lipids from marine macrophytes.

Major glyco- and phospholipids as well as betaine lipid 1,2-diacylglycero-O-4'-(N,N,N-tri-methyl)-homoserine (DGTS) were isolated from five species of marine macrophytes harvested in the Sea of Japan in summer and winter at seawater temperatures of 20-23 and 3 degrees C, respectively. GC and DSC analysis of lipids revealed a common increase of ratio between n-3 and n-6 polyunsaturated fatty acids (PUFAs) of polar lipids from summer to winter despite their chemotaxonomically different fatty acid (FA) composition. Especially, high level of different n-3 PUFAs was observed in galactolipids in winter. However, the rise in FA unsaturation did not result in the lowering of peak maximum temperature of phase transition of photosynthetic lipids (glycolipids and phosphatidylglycerol (PG)) in contrast to non-photosynthetic ones [phosphatidylcholine (PC) and phosphatidylethanolamine (PE)]. Different thermotropic behavior of these lipid groups was accompanied by higher content of n-6 PUFAs from the sum of n-6 and n-3 PUFAs in PC and PE compared with glycolipids and PG in both seasons. Seasonal changes of DSC transitions and FA composition of DGTS studied for the first time were similar to PC and PE. Thermograms of all polar lipids were characterized by complex profiles and located in a wide temperature range between -130 and 80 degrees C, while the most evident phase separation occurred in PGs in both seasons. Polarizing microscopy combined with DSC has shown that the liquid crystal - isotropic melt transitions of polar lipids from marine macrophytes began from 10 to 30 degrees C mostly, which can cause the thermal sensitivity of plants to superoptimal temperatures in their environment.

Morphological and immunological characterization of immunostimulatory complexes based on glycoglycerolipids from Laminaria japonica.

Some physicochemical properties of glycoglycerolipids (monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol) from the sea algae Laminaria japonica, as well as their ability to become incorporate into immunostimulating complexes (ISCOMs), used as a delivery system of microbial and tumor antigens in vesicular form, were studied. These glycolipids were found to differ essentially in fatty acid composition, unsaturation index and thermotropic behavior. The possibility of ISCOM modification by embedding the glycolipids studied instead of a phospholipid component in vesicles was shown. A preliminary research of the immunogenicity of the pore-forming protein from Yersinia pseudotuberculosis in modified (by monogalactosyldiacylglycerol) and typical (egg phosphatidylcholine) ISCOMs did not reveal a significant enhancement of immune response in comparison with that of isolated protein.

Fatty acid composition of individual polar lipid classes from marine macrophytes.

Major glycolipids [monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)) and phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG)] as well as betaine lipid 1,2-diacylglycero-O-4'-(N,N,N-tri-methyl)-homoserine (DGTS) were isolated from Anfeltia tobuchiensis (Rhodophyta), Laminaria japonica, Sargassum pallidum (Phaeophyta), Ulva fenestrata (Chlorophyta) and Zostera marina (Embriophyta), harvested in the Sea of Japan. GC analysis of their fatty acid (FA) composition revealed that the n-6 polyunsaturated FAs (PUFAs) shared the most part of the sum of n-6 and n-3 PUFAs in PC and PE compared with glycolipids and PG. In algae, it was related to the prevalence of 20:4n-6 over 20:5n-3 in non-photosynthetic lipids. Percentage of n-6 PUFAs as well as the sum of n-3 and n-6 PUFAs decreased in the following sequence: PC-->PE-->PG. The saturation increased in the lines of MGDG-->DGDG-->SQDG and PC-->PE-->PG. PG was close to SQDG by the level of saturation. Distribution of C(18) and C(20) PUFAs in polar lipids depended on taxonomic position of macrophytes. Balance between C(18) and C(20) PUFAs was preferably shifted to the side of C(20) PUFAs in PC and PE that was observed in contrast to glycolipids and PG from L. japonica containing both series of FAs. The set of major FAs of polar lipid classes can essentially differ from each other and from total lipids of macrophytes. For example, MGDG was found to accumulate characteristic fatty acids 16:4n-3, 16:3n-3, 18:3n-6 and 18:4n-3, 20:3n-6 in U. fenestrata, Z. marina, L. japonica and S. pallidum, respectively.

Thermotropic behavior of major phospholipids from marine invertebrates: changes with warm-acclimation and seasonal acclimatization.

The crystal-liquid crystal-isotropic melt phase transitions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from muscle tissue of five species (actinia Metridium senile fimbriatum, mussel Crenomytilus grayanus, sea-urchin Strongylocentrotus intermedius, starfish Distolasterias nipon and the ascidian Halocynthia aurantium) of marine invertebrates, collected in winter at 0 degrees C and then acclimated to 18.5 degrees C for 5 days, were studied by differential scanning calorimetry and polarising microscopy. To elevate temperature from 0 to 18.5 degrees C, we used the rate of 4.5 degrees C/h. Although phase transitions of both phospholipids from animals collected in summer occurred already at temperatures below -1.7 oC (minimal temperature of seawater in winter), compensatory mechanisms resulted in a decrease by 29-43 oC in the phase transition temperature of PE in winter. Thermotropic behavior of PCs changed in various trends. However, the total heat of their phase transitions always decreased in winter compared with summer. For all species, except the mussel, the time of warm-acclimation was insufficient to adjust the thermotropic behavior of either phospholipid. Nevertheless, the unsaturation index decreased to achieve summer values, due primarily to decreased proportions of eicosapentaenate and docosahexaenate. The accumulation of arachidonate, during warm-acclimation, might be connected to the signalling properties of n-6 eicosanoids. Absence of effective homeoviscous mechanisms suggests that most of the studied marine invertebrates have very limited capacity to survive an acute temperature elevation, e.g. at the appearance of thermal currents.