Diversity of protein inclusion bodies and identification of mosquitocidal protein in Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis subsp. israelensis produces, during sporulation, protein inclusion bodies of wide ranging sizes, all of which are toxic to mosquitoes. Two proteins are present in the smallest protein bodies (less than 0.2 micron dia.), but the number of proteins increases with increasing size of protein bodies. The largest bodies (greater than 1.5 micron dia.) contain seven proteins. All of the proteins are synthesized at different times during sporulation and are added to developing protein bodies in a stepwise manner. The protein component responsible for mosquitocidal activity is a 65,000-dalton protein, that is present in all of the protein bodies.     

Calreticulin mRNA and protein are localized to protein bodies in storage maize callus cells

Maize callus cells possess numerous protein bodies which develop as sub-compartments of the endoplasmic reticulum. We localized maize calreticulin mRNAs and protein in maize callus cells using in situ hybridization and immunocytochemistry. Calreticulin mRNAs were selectively targeted to the endoplasmic reticulum (ER) subdomains surrounding protein bodies. Profilin mRNAs, used as a positive control for in situ hybridization experiments, showed distinct and rather diffuse localization pattern. Using both, immunofluorescence and immunogold electron microscopy localization techniques, calreticulin was found to be enriched around and within protein bodies in maize callus storage cells. As a positive control for reticuloplasmins, HDEL antibody revealed labelling of protein bodies and of the nuclear envelope. The identity of protein bodies was confirmed by specific binding of an α zein antibody. These data suggest that calreticulin mRNA is targeted towards protein body forming subdomains of the ER, and that calreticulin is localized and enriched in these protein bodies. The possibility that calreticulin plays an important role in zein retention within the ER and/or its assembly and packaging into protein bodies during protein body biogenesis in maize callus is discussed.     

Protein Bodies in Nature and Biotechnology

Protein bodies are natural structures containing protein aggregates that exist in many organisms ranging from bacteria to mammals and plants. In bacteria they are often a phenomenon associated to over-expression of heterologous proteins. In mammals the so called Russell bodies indicate an accumulation of mutated immune globulins. In plants the protein bodies play a major role as protein storage organelle in seeds. Besides these natural cases, protein bodies can also be artificially induced primarily using self-assembling peptides. Frequently plant derived proteins such as prolamins or their derivatives are used. In some cases the help of an endoplasmatic retention signal is needed to create artificial protein bodies. The biotechnological application of protein bodies offers novel solutions such as the simplification of downstream processing in protein manufacture, the utilisation as particle for immunisation as vaccines or as carrier free self immobilised enzyme particle for many industrial catalytic processes.     

Changes in the zein composition of protein bodies during maize endosperm development.

Zeins, the seed storage proteins of maize, are synthesized during endosperm development by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum, where they assemble into protein bodies. To better understand the distribution of the various zeins throughout the endosperm, and within protein bodies, we used immunolocalization techniques with light and electron microscopy to study endosperm tissue at 14 days and 18 days after pollination. Protein bodies increase in size with distance from the aleurone layer of the developing endosperm; this reflects a process of cell maturation. The protein bodies within the subaleurone cell layer are the smallest and contain little or no alpha-zein; beta-zein and gamma-zein are distributed throughout these small protein bodies. The protein bodies in cells farther away from the aleurone layer are progressively larger, and immunostaining for alpha-zein occurs over locules in the central region of these protein bodies. In the interior of the largest protein bodies, the locules of alpha-zein are fused. Concomitant with the appearance of alpha-zein in the central regions of the protein bodies, most of the beta- and gamma-zeins become peripheral. These observations are consistent with a model in which specific zeins interact to assemble the storage proteins into a protein body.     

Baculovirus expression vectors that incorporate the foreign protein into viral occlusion bodies

Current baculovirus expression systems typically produce soluble proteins that accumulate within the infected insect cell or are secreted into the growth medium. A system has now been developed for the incorporation of foreign proteins, along with the matrix protein, polyhedrin, into baculovirus occlusion bodies. Initial studies showed that a recombinant virus expressing a translational fusion between polyhedrin and GFP did not form occlusion bodies. However, a baculovirus coexpressing native polyhedrin and the polyhedrin-GFP fusion protein formed occlusion bodies that fluoresced under UV light, demonstrating that they included the polyhedrin-GFP fusion protein. This was confirmed by immunoblot analysis. Thus, incorporation of a foreign protein into occlusion bodies depends on an interaction between native polyhedrin and the polyhedrin fusion protein. Electron microscopy demonstrated that the occlusion bodies containing GFP also incorporated virions as expected. These ColorPol occlusion bodies were as infectious to insect larvae as occlusion bodies produced by wild-type virus. This new system expands the capabilities for foreign gene expression by baculoviruses, which has implications for biopesticide design, novel vaccine delivery systems, and fusion protein purification applications.     

Evidence for the presence of two different types of protein bodies in wheat endosperm

Storage proteins of wheat grains (Triticum L. em Thell) are deposited in protein bodies inside vacuoles. However, the subcellular sites and mechanisms of their aggregation into protein bodies are not clear. In the present report, we provide evidence for two different types of protein bodies, low- and high-density types that accumulate concurrently and independently in developing wheat endosperm cells. Gliadins were present in both types of protein bodies, whereas the high molecular weight glutenins were localized mainly in the dense ones. Pulse-chase experiments verified that the dense protein bodies were not formed by a gradual increase in density but, presumably, by a distinct, quick process of storage protein aggregation. Subcellular fractionation and electron microscopy studies revealed that the wheat homolog of immunoglobulin heavy-chain-binding protein, an endoplasmic reticulum-resident protein, was present within the dense protein bodies, implying that these were formed by aggregation of storage proteins within the endoplasmic reticulum. The present results suggest that a large part of wheat storage proteins aggregate into protein bodies within the rough endoplasmic reticulum. Because these protein bodies are too large to enter the Golgi, they are likely to be transported directly to vacuoles. This route may operate in concert with the known Golgi-mediated transport to vacuoles in which the storage proteins apparently condense into protein bodies at a postendoplasmic reticulum location. Our results further suggest that although gliadins are transported by either one of these routes, the high molecular weight glutenins use only the Golgi bypass route.     

Localization and secretory pathways of a 58K-like protein in multi-vesicular bodies in callus of Arabidopsis thaliana

Multi-vesicular bodies in endocytosis and protoplasts are special cellular structures that are consid-ered to be originated from invagination of plasma membranes. However, the genesis and function of multi-vesicular bodies, the relationship with Golgi bodies and cell walls, and their secretory pathways remain controversial and ambiguous. Using a monoclonal antibody against an animal 58K protein, we have detected, by Western blotting and confocal microscopy, that a 58K-like protein is present in the calli of Arabidopsis thaliana and Hypericum perforatum. The results of immuno-electron microscopy showed that the 58K-like protein was located in the cisternae of Golgi bodies, secretory vesicles, multi-vesicular bodies, cell walls and vacuoles in callus of Arabidopsis thaliana, suggesting that the multi-vesicular bodies may be originated from Golgi bodies and function as a transporter carrying substances synthesized in Golgi bodies to cell walls and vacuoles. It seems that multi-vesicular bodies have a close relationship with the development of the cell wall and vacuole. The possible secretory pathways of multi-vesicular bodies might be in exocytosis, in which multi-vesicular bodies carry sub-stances to the cell wall for its construction, and in endocytosis, in which multi-vesicular bodies carry substances to the vacuole for its development, depending on what they carry and where the materials are transported. We hence propose that there is more than one pathway for the secretion of multi-vesicular bodies. In addition, our results provided a paradigm that a plant molecule, such as the 58k-like protein in callus of Arabidopsis thaliana, can be detected using a cross-reactive monoclonal antibody induced by an animal protein, and illustrate the existence of analog molecules in both animal and plant kingdoms.     

Early Stages in Wheat Endosperm Formation and Protein Body Initiation

The early stages of endosperm formation and protein body initiationare described for hard red winter wheat using light and transmissionelectron microscopy. Two days after flowering (DAF) the endospermwas a thin layer of coenocytic cytoplasm lining the embryo sac.By 4 DAF the endosperm had cellularized and completely filledthe embryo sac. Enough differentiation had occurred by 6 DAFto distinguish cells destined to become the aleurone layer,sub-aleurone region and central endosperm. Protein bodies wereinitiated at about 6–7 DAF and were first found near theGolgi apparatus. Wheat was ready for combine harvest at 34 DAF.Enlargement of the small protein bodies near the Golgi apparatusoccurred by several mechanisms: (1) fusion with one or moreof the dense Golgi vesicles or fusion with other protein bodies,(2) fusion with small electron-lucent Golgi-derived vesicles,(3) pinocytosis of a portion of the adjacent cytoplasm intothe developing protein body and (4) fusion of large proteinbodies with one another at later stages of grain development.Of the four mechanisms described, the pinocytotic vesicles andfusion of protein bodies were the most frequent and consistentprocesses observed. Direct connections between rough endoplasmicreticulum (RER) and protein bodies were not observed. The resultssuggest a rôle for the Golgi apparatus in the initiationof protein bodies. Also, the lack of RER derived vesicles suggestsa soluble mode of secretion of storage proteins involved inthe enlargement of protein bodies. Triticum aestivum, wheat endosperm, protein bodies Golgi apparatus     

Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins—oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology.     

A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides

An expression/purification system was developed using artificial oil bodies (AOB) as carriers for producing recombinant proteins. A target protein, green fluorescent protein (GFP), was firstly expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a linker sequence susceptible to factor Xa cleavage. Artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble recombinant protein, oleosin-Xa-GFP. After centrifugation, the oleosin-fused GFP was exclusively found on the surface of artificial oil bodies presumably with correct folding to emit fluorescence under excitation. Proteolytic cleavage with factor Xa separated soluble GFP from oleosin embedded in the artificial oil bodies; thus after re-centrifugation, GFP of high yield and purity was harvested simply by concentrating the ultimate supernatant.     

Wheat (Triticum aestivum L.) [gamma]-Gliadin Accumulates in Dense Protein Bodies within the Endoplasmic Reticulum of Yeast

Following their sequestration into the endoplasmic reticulum (ER), wheat storage proteins may either be retained and packaged into protein bodies within this organelle or transported via the Golgi to vacuoles. We attempted to study the processes of transport and packaging of wheat storage proteins using the heterologous expression system of yeast. A wild-type wheat [gamma]-gliadin, expressed in the yeast cells, accumulated mostly within the ER and was deposited in protein bodies with similar density to natural protein bodies from wheat endosperm. This suggested that wheat storage proteins contain sufficient information to initiate the formation of protein bodies in the ER of a heterologous system. Only a small amount of the [gamma]-gliadin was transported to the yeast vacuoles. When a deletion mutant of the [gamma]-gliadin, lacking the entire N-terminal repetitive region, was expressed in the yeast cells, the mutant was unable to initiate the formation of protein bodies within the ER and was completely transported to the yeast vacuole. This strongly indicated that the information for packaging into dense protein bodies within the ER resides in the N-terminal repetitive region of the [gamma]-gliadin. The advantage of using yeast to identify the signals and mechanisms controlling the transport of wheat storage proteins and their deposition in protein bodies is discussed.     

Subcellular localization of glutelin-2 in maize (Zea mays L.) endosperm

Accumulation of the 28 KD protein of the glutelin-(G2) fraction was followed in developing maize endosperm, using sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and peak integration of scanned gels. 28 KD glutelin-2 could already be observed from 15 days after pollination and its accumulates reached a plateau during the second half of the development period. The process of biosynthesis of 28 KD glutelin-2 and zeins occurs in a parallel way. Subcellular fractions obtained from linear sucrose gradient centrifugation of developing maize endosperms were analyzed by SDS-PAGE and immunoblotting using a serum reacting against glutelin-2 and 14 KD Z2. Glutelin-2 was found to be present in the protein bodies when subcellular fractionation was carried out without dithiothreitol (DTT). The presence of a reducing agent causes the elution of glutelin-2 from protein bodies. Immunocytochemical labelling using the protein A-colloidal gold technique in protein bodies incubated with anti-G2 IgG revealed that G2 is located mainly in the periphery of protein bodies. These results are interpreted as indicating a structural role for glutelins in protein bodies.     

Protein bodies in seeds

Membrane bound protein bodies (aleurone grains) are thought to be the main subcellular location of protein and mineral storage in seeds. In addition to structurally homogeneous proteinaceous matrix, protein bodies may contain protein crystalloids, electron–dense globoid crystals, electron–transparent soft globoids, and crystals of calcium oxalate. Protein crystalloids vary in shape, size and number. For example, cotyledon mesophyll cell protein bodies in the Cucurbitaceae generally contain protein crystalloids whereas those of Compositae and Cruciferae do not. Globoid crystals, which are rich in phytin, vary greatly in size and number per protein body. Some species have numerous small globoid crystals per protein body whereas others have one or two large globoid crystals per protein body. Phosphorus and various cations (K, Mg, Ca, Fe, Ba, Mn) located in globoid crystals can be studied with an energy dispersive X–ray (EDX) analysis system mounted on a transmission electron microscope. In some cases, cations such as Ca, Mn and Fe are specifically localized in globoid crystals of certain tissues or embryo regions. Further investigations may allow elemental composition of globoid crystals to be used in studies of systematics. Biref–ringent crystals, sometimes in the form of single large crystals but frequently in the form of druses, are present in protein bodies of some species. At least some endosperm protein bodies of all Umbelliferous species examined contain druse crystals. While seed protein bodies of relatively few species have been studied with electron microscopy, there are indications that protein bodies could be a useful character for studies in plant systematics.     

ULTRASTRUCTURE OF THE MATURE UNGERMINATED RICE (ORYZA SATIVA) CARYOPSIS. THE STARCHY ENDOSPERM

The structure of the starchy endosperm of rice (Oryza sativa) was studied by using light and transmission electron microscopy coupled with proteolytic enzyme digestions. The starchy endosperm was divided into two regions, the subaleurone and central, based on the number and types of protein bodies observed. The subaleurone region contained three different types of membrane bounded protein bodies—large spherical, small spherical, and crystalline protein bodies. The small spherical protein bodies were most numerous and the large spherical ones were least numerous. The crystalline protein bodies displayed crystal lattice fringes and were a composite of smaller angular components. The central region lacked both the small spherical and crystalline protein bodies. The large spherical protein bodies of this region were located in pockets of densely stained proteinaceous material. In contrast to the relatively well preserved cytoplasm of the subaleurone region, the central endosperm zone consistently was poorly preserved.     

The Uni2 phosphoprotein is a cell cycle regulated component of the basal body maturation pathway in Chlamydomonas reinhardtii

Mutations in the UNI2 locus in Chlamydomonas reinhardtii result in a "uniflagellar" phenotype in which flagellar assembly occurs preferentially from the older basal body and ultrastructural defects reside in the transition zones. The UNI2 gene encodes a protein of 134 kDa that shares 20.5% homology with a human protein. Immunofluorescence microscopy localized the protein on both basal bodies and probasal bodies. The protein is present as at least two molecular-weight variants that can be converted to a single form with phosphatase treatment. Synthesis of Uni2 protein is induced during cell division cycles; accumulation of the phosphorylated form coincides with assembly of transition zones and flagella at the end of the division cycle. Using the Uni2 protein as a cell cycle marker of basal bodies, we observed migration of basal bodies before flagellar resorption in some cells, indicating that flagellar resorption is not required for mitotic progression. We observed the sequential assembly of new probasal bodies beginning at prophase. The uni2 mutants may be defective in the pathways leading to flagellar assembly and to basal body maturation.     

Bld10/Cep135 stabilizes basal bodies to resist cilia-generated forces

Basal bodies nucleate, anchor, and organize cilia. As the anchor for motile cilia, basal bodies must be resistant to the forces directed toward the cell as a consequence of ciliary beating. The molecules and generalized mechanisms that contribute to the maintenance of basal bodies remain to be discovered. Bld10/Cep135 is a basal body outer cartwheel domain protein that has established roles in the assembly of nascent basal bodies. We find that Bld10 protein first incorporates stably at basal bodies early during new assembly. Bld10 protein continues to accumulate at basal bodies after assembly, and we hypothesize that the full complement of Bld10 is required to stabilize basal bodies. We identify a novel mechanism for Bld10/Cep135 in basal body maintenance so that basal bodies can withstand the forces produced by motile cilia. Bld10 stabilizes basal bodies by promoting the stability of the A- and C-tubules of the basal body triplet microtubules and by properly positioning the triplet microtubule blades. The forces generated by ciliary beating promote basal body disassembly in bld10Δ cells. Thus Bld10/Cep135 acts to maintain the structural integrity of basal bodies against the forces of ciliary beating in addition to its separable role in basal body assembly.     

Physicochemical characteristics of LR3-IGF1 protein inclusion bodies: electrophoretic mobility studies

A knowledge of the physicochemical properties of inclusion bodies is important for the rational design of potential recovery processes such as flotation and precipitation. In this study, measurement of the size and electrophoretic mobility of protein inclusion bodies and cell debris was undertaken. SDS-PAGE analysis of protein inclusion bodies subjected to different cleaning regimes suggested that electrophoretic mobility provides a qualitative measure of protein inclusion body purity. Electrophoretic mobility as a function of electrolyte type and ionic strength was investigated. The presence of divalent ions produced a stronger effect on electrophoretic mobility compared with monovalent ions. The isoelectric point of cell debris was significantly lower than that for the inclusion bodies. Hence, the contaminating cell debris may be separated from inclusion bodies using flotation by exploiting this difference in isoelectric points. Separation by this method is simple, convenient, and a possible alternative to the conventional route of centrifugation.     

Ultrastructure and Lipase Activity of Cotyledon cell During Pod Development in Peanut

A series of significant changes of the ultrastructure and lipase activity of cotyledon cell were found in peanut (Arachis hypogaea) during pod development. In he initial stage of cotyledon development there were many plastids which kept producing starch grain and there were low lipase activity and very few lipid and protein bodies in the cell. In the middle stage of cotyledon development, a great number of larger lipid bodies were seen in the cell and a lot of protein bodies formed in the vacuoles and continued to increase in size. Lipase activity increased in the cytoplasm, endoplasmic reticulum, protein bodies, plasmalemma and intercellular space. In the later stage of cotyledon development, the lipid bodies did not increase in number but became slightly larger. The protein bodies continued to increase both in number and in size. Lipase acttvity was even hegher in the cytoplasm. In the final stage the protein bodies became irregular in shape and some of them tended to disintegrate with their content entered into the space around the lipid bodies. The lipase activity in the cell declined. The results indicated that the lipid body originated in the cytoplasm and the protein body originated in the vacuole; that the accumulation of oil and protein in peanut cotyledon resulted from the formation and development of lipid and protein bodies in the cell, and that the changes of plasmid and lipase activity in the cell played a role in the development of lipid body during the development of cotyledon.     

Ultrastructure, Origin, and Composition of the Protein Bodies in the Ligule of Isoetes lacustris L.

The basal cells in the ligule of Isoetes lacustris contain numerousprotein bodies, the contents of which can be digested enzymicallyby pronase and are stained red by treatment with ninhydrin Schiff'sreagent. Two types of protein bodies can be distinguished ultrastructurally:spherically-shaped bodies with granular contents and spindle-likebodies with fibrillar contents. Both are ensheathed by singlemembranes and do not show any solid inclusions within theirmatrix. The protein bodies probably arise from dilatation of the endoplasmicreticulum (ER) cisternae. This conclusion is based upon threeobservations: (a) The protein bodies occasionally show membranecontinuity with the ER; (b) ribosomes and polysomes are frequentlyattached to the protein-body membranes; (c) the contents ofthe protein bodies and of the dilated ER cisternae show similarultrastructural features. The dilatation of the ER cisternae is assumed to be a resultof protein accumulation in the intracisternal space. Based upon the results of polyacrylamide gel electrophoresis,it is likely that the spherically-shaped protein bodies storepredominately two proteins with molecular weights of 51300 and55800 D, while the spindle-like bodies store two proteins withmolecular weights of 92000 and 98000 D. The results presented do not permit a definite conclusion regardingthe function of the ligule of Isoetes lacustris but it is suggestedthat it may have a nutritive role. Isoetes lacustris L., ligule, protein bodies, endoplasmic reticulum, ultrastructure     

Formation of Hirano bodies induced by expression of an actin cross-linking protein with a gain-of-function mutation

Hirano bodies are paracrystalline actin filament-containing structures reported to be associated with a variety of neurodegenerative diseases. However, the biological function of Hirano bodies remains poorly understood, since nearly all prior studies of these structures were done with postmortem samples of tissue. In the present study, we generated a full-length form of a Dictyostelium 34-kDa actin cross-linking protein with point mutations in the first putative EF hand, termed 34-kDa ΔEF1. The 34-kDa ΔEF1 protein binds calcium normally but has activated actin binding that is unregulated by calcium. The expression of the 34-kDa ΔEF1 protein in Dictyostelium induces the formation of Hirano bodies, as assessed by both fluorescence microscopy and transmission electron microscopy. Dictyostelium cells bearing Hirano bodies grow normally, indicating that Hirano bodies are not associated with cell death and are not deleterious to cell growth. Moreover, the expression of the 34-kDa ΔEF1 protein rescues the phenotypes of cells lacking the 34-kDa protein and cells lacking both the 34-kDa protein and α-actinin. Finally, the expression of the 34-kDa ΔEF1 protein also initiates the formation of Hirano bodies in cultured mouse fibroblasts. These results show that the failure to regulate the activity and/or affinity of an actin cross-linking protein can provide a signal for the formation of Hirano bodies. More generally, the formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton.