Genetics and Biochemistry of Polyhydroxyalkanoate Granule Self-assembly: The Key Role of Polyester Synthases

PHAs (polyhydroxyalkanoates = biopolyester) composed of hydroxy fatty acids represent a rather complex class of storage polymers synthesized by various bacteria and archaea and are deposited as water-insoluble cytoplasmic nano-sized inclusions. These spherical particles are composed of a polyester core surrounded by phospholipids and proteins. The key enzymes of polyester biosynthesis and polyester particle formation are the polyester synthases, which catalyze the formation of polyesters. Various metabolic routes have been identified and established in bacteria to provide substrate for polyester synthases. Although not essential for particle formation, non-covalently attached proteins, the so-called phasins, can be found at the particle surface and are considered as structural proteins. Protein engineering of polyester synthases and phasins was used to shed light into the topology of these granule attached proteins. Biopolyesters and the respective micro-/nano-structures are currently considered as biocompatible and biodegradable biomaterials with numerous potential applications particularly in the medical field. Received 12 October 2005; Revisions requested 1 November 2005; Revisions received 25 November 2005; Accepted 25 November 2005     

Role of leucine zipper-like motifs in the oligomerization of Pseudomonas putida phasins

Background

Phasins are low molecular mass proteins that accumulate strongly in bacterial cells in response to the intracellular storage of polyhydroxyalkanoates (PHA). Although lacking catalytic activity, phasins are the major components of the surface of the PHA granules and could be potentially involved in the formation of a network-like protein layer surrounding the polyester inclusions. Structural models revealed phasins to possess coiled-coil regions that might be important in the establishment of protein-protein interactions. However, there is not experimental evidence of a coiled-coil mediated oligomerization in these proteins.

Characterization of the lipid and protein organization in HBsAg viral particles by steady-state and time-resolved fluorescence spectroscopy

Hepatitis B surface antigen (HBsAg) particles, produced in the yeast Hansenula polymorpha, are 20 nm particles, composed of S surface viral proteins and host-derived lipids. Since the detailed structure of these particles is still missing, we further characterized them by fluorescence techniques. Fluorescence correlation spectroscopy indicated that the particles are mainly monomeric, with about 70 S proteins per particle. The S proteins were characterized through the intrinsic fluorescence of their thirteen Trp residues. Fluorescence quenching and time-resolved fluorescence experiments suggest the presence of both low emissive embedded Trp residues and more emissive Trp residues at the surface of the HBsAg particles. The low emission of the embedded Trp residues is consistent with their close proximity in alpha-helices. Furthermore, S proteins exhibit restricted movement, as expected from their tight association with lipids. The lipid organization of the particles was studied using viscosity-sensitive DPH-based probes and environment sensitive 3-hydroxyflavone probes, and compared to lipid vesicles and low density lipoproteins (LDLs), taken as models. Like LDLs, the HBsAg particles were found to be composed of an ordered rigid lipid interface, probably organized as a phospholipid monolayer, and a more hydrophobic and fluid inner core, likely composed of triglycerides and free fatty acids. However, the lipid core of HBsAg particles was substantially more polar than the LDL one, probably due to its larger content in proteins and its lower content in sterols. Based on our data, we propose a structural model for HBsAg particles where the S proteins deeply penetrate into the lipid core.     

Ralstonia eutropha strain H16 as model organism for PHA metabolism and for biotechnological production of technically interesting biopolymers

The Gram-negative, facultative chemolithoautotrophic bacterium Ralstonia eutropha has been intensively investigated for almost 50 years. Today it is the best studied 'Knallgas' bacterium and producer of poly(3-hydroxybutyric acid). This polyester provides the basis for renewable resource-based biodegradable plastic materials and has attracted much biotechnological interest. The polymer is accumulated in large amounts in the cell and can be used for various applications ranging from replacement of fossil resource-based bulk plastics to high-value special purpose polymers. To further enhance productivity and to allow tailormade poly(hydroxyalkanoic acids) (PHA) with different monomer compositions by metabolic engineering, the knowledge of metabolic pathways and of the biochemical properties of the enzymes involved is essential. Furthermore, proteins covering the PHA granule surface, which are referred to as phasins, and fusions of these phasins to other proteins are promising candidates for various protein technologies. The recently published genome sequence of strain H16 allows researchers to take a closer look at the genetic potential of this versatile bacterium. R. eutropha is, however, not limited to PHAs and to PHA-related polymers like poly(mercaptoalkanoic acids) as it can also be employed for production of a range of other interesting polymers including polyamides like cyanophycin.     

Three envelope proteins of hepatitis B virus: large S, middle S, and major S proteins needed for the formation of Dane particles.

The infectious particles of hepatitis B virus are called Dane particles and consist of viral nucleic acid encapsulated within a core particle that is enveloped by virus-coded surface proteins. The major S protein constitutes a significant fraction of these surface proteins. In addition, there are two other related proteins (large S and middle S), but their role in envelope formation has not yet been elucidated. We modified the translation initiation codon ATG of each of the envelope proteins by site-directed mutagenesis and found that mutant genomes that did not produce one or two of these proteins were unable to form Dane particles. The particles released into the culture medium by such mutants did not carry DNA. Synthesis of virus-coded RNA still occurred normally, and core particles carrying DNA accumulated intracellularly. The DNA in such core particles was mostly in the double-stranded open circular form, in contrast to the normal situation in which the particles contain mostly RNA and its complementary single-stranded DNA or else contain linear DNA that is partially single stranded and otherwise duplex. The role of the large S and middle S proteins in the formation of Dane particles is discussed.     

Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.     

The role of phasins in the morphogenesis of poly(3-hydroxybutyrate) granules

Recent developments in the understanding of the structure of polyhydroxyalkanoate, PHA, granules in bacteria are documented in the literature and point to the role of structural proteins, phasins, in granule formation and stabilization. We have previously conceived a computer program which successfully simulates granule formation in vitro, in the absence of phasins. Now we are extending the computer model to a more complex system, including phasins, to quantify their anticipated effect on the granule properties. The simulation enabled us to propose real experiments to test the validity of the model and provide a framework for a better understanding of PHA granule formation in vivo.     

The specific delivery of proteins to human liver cells by engineered bio-nanocapsules

A bio-nanocapsule (BNC), composed of the surface antigen (sAg) of the hepatitis B virus, is an efficient nanomachine with which to accomplish the liver-specific delivery of genes and drugs. Approximately 110 molecules of sAg are associated to form a BNC particle with an average diameter of 130 nm. The L protein is an sAg peptide composed mainly of preS and S regions. The preS region, with specific affinity for human hepatocytes, is localized in the N-terminus. The S region following the preS has two transmembrane regions responsible for the formation of particles. In this study, the fusion of emerald green fluorescent protein (EGFP) at the C-terminus of the S region was designed to deliver proteins to human hepatocytes. Truncation of the C-terminus of the S region was required to obtain sufficient expression levels in Cos7 cells. The nanoparticles that were produced delivered EGFP to human hepatoma cells, displaying the EGFP moiety outside, or enclosing it inside. However, only a single orientation characterizes the particle, so that either type of L fusion particle could be effectively and independently separated by an antibody affinity column. The dual C-terminal topologies of the L fusion particles designed in this study could be applied to various proteins for the C-terminal moiety of the L fusion proteins, depending on the character of the proteins, such as cytoplasmic proteins, as well as cytokines or ligands to cell surface receptors. We suggest that this fusion design is the most efficient way to prepare a BNC that delivers proteins to specific cells or tissues.     

Studies on the Nucleocapsid Structure of a Group A Arbovirus

When Sindbis virus (273S) was treated with sodium desoxycholate, a nonhemagglutinating 136S particle was liberated from the virion, representing the viral nucleocapsid (core). Electron microscopically it appeared as a spherical particle 35 nm in diameter, showing ringlike morphological units 12 to 14 nm in diameter on its surface. When the one- and two-sided images of core particles were correlated, their structure could be demonstrated to have the T = 3 arrangement of 32 hexamer-pentamer morphological units within a symmetrical surface lattice. The core contained a further spherical structure (12 to 16 nm in diameter) which was designated as the central core component. Two proteins were found associated with the core, a third viral protein belonged to the hemagglutinating surface structures. The significance of these findings for virus classification is discussed.     

Binding of the major phasin, PhaP1, from Ralstonia eutropha H16 to poly(3-hydroxybutyrate) granules

The surface of polyhydroxybutyrate (PHB) storage granules in bacteria is covered mainly by proteins referred to as phasins. The layer of phasins stabilizes the granules and prevents coalescence of separated granules in the cytoplasm and nonspecific binding of other proteins to the hydrophobic surfaces of the granules. Phasin PhaP1(Reu) is the major surface protein of PHB granules in Ralstonia eutropha H16 and occurs along with three homologues (PhaP2, PhaP3, and PhaP4) that have the capacity to bind to PHB granules but are present at minor levels. All four phasins lack a highly conserved domain but share homologous hydrophobic regions. To identify the region of PhaP1(Reu) which is responsible for the binding of the protein to the granules, N-terminal and C-terminal fusions of enhanced green fluorescent protein with PhaP1(Reu) or various regions of PhaP1(Reu) were generated by recombinant techniques. The fusions were localized in the cells of various recombinant strains by fluorescence microscopy, and their presence in different subcellular protein fractions was determined by immunodetection of blotted proteins. The fusions were also analyzed to determine their capacities to bind to isolated PHB granules in vitro. The results of these studies indicated that unlike the phasin of Rhodococcus ruber, there is no discrete binding motif; instead, several regions of PhaP1(Reu) contribute to the binding of this protein to the surface of the granules. The conclusions are supported by the results of a small-angle X-ray scattering analysis of purified PhaP1(Reu), which revealed that PhaP1(Reu) is a planar, triangular protein that occurs as trimer. This study provides new insights into the structure of the PHB granule surface, and the results should also have an impact on potential biotechnological applications of phasin fusion proteins and PHB granules in nanobiotechnology.     

Mapping of amino acid side chains on the surface of hepatitis B virus capsids required for envelopment and virion formation

The crystal structure of recombinant hepatitis B virus (HBV) capsids formed by 240 core proteins has recently been published. We wanted to map sites on the surface of the icosahedral 35-nm particle that are important for nucleocapsid envelopment by HBV surface proteins during virion morphogenesis. For this purpose, we individually mutated 52 amino acids (aa) within the N-terminal 140 aa of the 185-aa long core protein displaying their side chains to the external surface of the capsid to alanine residues. The phenotype of the mutations with respect to virion formation was tested by transcomplementation of a core gene-negative HBV genome in transiently cotransfected cells, immunoprecipitation of nucleocapsids from cells and secreted virions from culture media, and detection of the particles by radioactive endogenous polymerase reactions. Thirteen point mutations impeded nucleocapsid detection by endogenous polymerase reactions. Twenty-seven mutations were compatible with virion formation. Among these were all capsid-forming mutations in the upper half of the spike protruding from the particle shell and two additional triple mutations at tip of the spike. Eleven mutations (S17, F18, L60, L95, K96, F122, I126, R127, N136, A137, and I139) allowed nucleocapsid formation but blocked particle envelopment and virion formation to undetectable levels. These mutations map to a ring-like groove around the base of the spike and to a small area at the capsid surface close to the pores in the capsid shell. These residues are candidate sites for the interaction with envelope proteins during virion morphogenesis.     

Hepatitis B virus DNA-negative dane particles lack core protein but contain a 22-kDa precore protein without C-terminal arginine-rich domain

DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles.     

Nanoscale topography of hepatitis B antigen particles by atomic force microscopy

Hepatitis B virus envelope is mainly composed of three forms of the same protein expressed from different start codons of the same open reading frame. The smaller form named S protein corresponds to the C-terminal common region and represents about 80% of the envelope proteins. It is mainly referred as hepatitis B virus surface antigen (HBsAg). Over expressed in the host cell, this protein can be produced as spherical and tubular self-organized particles. Highly immunogenic, these particles are used in licensed hepatitis B vaccines. In this study we have combined transmission electron microscopy and atomic force microscopy to determine the shape and size of HBsAg particles produced from the yeast Hansenula polymorpha. Tapping mode atomic force microscopy in liquid allows structural details of the surface to be delineated with a resolution in the nanometer range. Particles were decorated by closely packed spike-like structures protruding from particle surface. Protrusions appeared uniformly distributed at the surface and an average number of 75 protrusions per particle were calculated. Importantly, we demonstrated that proteins mainly contribute to the topography of the protrusions.     

Production of hepatitis B virus nucleocapsidlike core particles in Xenopus oocytes: assembly occurs mainly in the cytoplasm and does not require the nucleus.

The location of hepatitis B virus (HBV) nucleocapsid (core particle) assembly in infected cells remains controversial. Some lines of evidence implicate the nucleus; others favor the cytoplasm. Via injection of a synthetic mRNA encoding the HBV nucleocapsid protein (p21.5), we have expressed both unassembled p21.5 and nucleocapsidlike core particles in Xenopus oocytes. Subcellular fractionation reveals that approximately 91% of the unassembled p21.5 and 95% of the core particles are cytoplasmic, with only 9 and 5%, respectively, in the nucleus. We present evidence showing that unassembled p21.5 equilibrates between nucleus and cytoplasm by passive diffusion and that intact core particles do not enter the nucleus. To examine the role of the nucleus in core particle formation, we expressed p21.5 in surgically anucleate oocytes. We show that anucleate oocytes support efficient core particle formation, indicating that (i) the nucleus is not essential for assembly and (ii) the cytoplasm can assemble most core particles found in oocytes. On the basis of our data, we propose that in oocytes, most core particle assembly (up to 95%) occurs in the cytoplasm, but that at least approximately 5% of the cellular core particles are assembled in the nucleus and remain there. We discuss the implications of these findings for the formation of replication-competent core particles in infected cells.     

The duck hepatitis B virus core protein contains a highly phosphorylated C terminus that is essential for replication but not for RNA packaging.

In this report, we present biochemical and mutational analyses of the duck hepatitis B virus core protein (DHBcAg). The data show that duck hepatitis B virus core particles consist of at least four different proteins with sizes between 32 and 34 kilodaltons, all of which react with DHBcAg-specific antiserum. Most of the heterogeneity was found to be due to extensive phosphorylation of the DHBcAg C terminus. Bacterially synthesized DHBcAg was not phosphorylated, and mutations within the viral P gene did not influence phosphorylation, suggesting that the kinase activity is not encoded by the viral C or P gene. Removal of the last 12 C-terminal DHBcAg amino acids, which are at least in part located on the core particle surface, had only a minor effect on DHBcAg phosphorylation and did not interfere with packaging of the capsids into viral envelopes or with genome replication. However, an attempt to infect ducklings with this mutant failed. Removal of the last 36 C-terminal DHBcAg amino acids abolished core protein heterogeneity but did not prevent particle formation. Interestingly, these particles were defective in genome replication, although they could still package viral pregenomic RNA.     

Reconsideration of hydrophobic lipid distributions in lipoprotein particles

Lipoprotein particles are commonly known as micellar aggregates with hydrophobic lipids located within the core and amphipathic molecules in the surface. Using a new structural model for optimizing the distribution of hydrophobic lipids, namely triglyceride (TG) and cholesterol ester (CE) molecules, we reveal that particle size-dependent proportion of these 'core lipids' may locate in the surface of lipoprotein particles. The composition of the particles also strongly influences the actual molecular content of the surface. For example, in high-density lipoprotein (HDL) particles the percentage of CEs of all surface lipids is between 13% and 27% due to the high tendency of CEs to locate in the surface and the high concentration of CEs in the particles. Conversely, although the percentage of TG molecules in the surface of HDL particles is also high, approximately 60% as for CE, the percentage of TGs of all surface lipids is low, only up to 5%, because HDL particles have a low-TG concentration. These structural models provide an intuitive and coherent structural rationale for various metabolic cascades in lipoprotein metabolism with the catalytic enzyme action and molecular binding for transport proteins taking place at the surface of the particles.     

Genetic Determinants of Rous Sarcoma Virus Particle Size

The Gag proteins of retroviruses are the only viral products required for the release of membrane-enclosed particles by budding from the host cell. Particles released when these proteins are expressed alone are identical to authentic virions in their rates of budding, proteolytic processing, and core morphology, as well as density and size. We have previously mapped three very small, modular regions of the Rous sarcoma virus (RSV) Gag protein that are necessary for budding. These assembly domains constitute only 20% of RSV Gag, and alterations within them block or severely impair particle formation. Regions outside of these domains can be deleted without any effect on the density of the particles that are released. However, since density and size are independent parameters for retroviral particles, we employed rate-zonal gradients and electron microscopy in an exhaustive study of mutants lacking the various dispensable segments of Gag to determine which regions would be required to constrain or define the particle dimensions. The only sequence found to be absolutely critical for determining particle size was that of the initial capsid cleavage product, CA-SP, which contains all of the CA sequence plus the spacer peptides located between CA and NC. Some regions of CA-SP appear to be more important than others. In particular, the major homology region does not contribute to defining particle size. Further evidence for interactions among CA-SP domains was obtained from genetic complementation experiments using mutant ΔNC, which lacks the RNA interaction domains in the NC sequence but retains a complete CA-SP sequence. This mutant produces low-density particles heterogeneous in size. It was rescued into particles of normal size and density, but only when the complementing Gag molecules contained the complete CA-SP sequence. We conclude that CA-SP functions during budding in a manner that is independent of the other assembly domains.     

Sponge aggregation factor: identification of the specific collagen-binding site by means of a monoclonal antibody

The aggregation factor (AF) from the sponge Geodia cydonium is known to be a complex proteinaceous particle, composed of a series of different (glyco)proteins (Mr lower than 150,000) around a 90S sunburst-like core structure. One of the low-Mr proteins is the 47-KD cell binding fragment. We describe a new monoclonal antibody (mAb), III1E6, raised against purified AF particles, which recognizes in tissue slices structures present both on the plasma membrane and in a network-like manner in the extracellular space. By applying immunoelectron microscopical, immunoblotting, and immunoaffinity chromatographical techniques, the mAb III1E6 was shown to recognize the core structure of the AF particle. Cell adhesion studies revealed that the mAb does not inhibit AF mediated cell-cell adhesion but abolishes AF-caused attachment of cells to collagen. Electron microscopic data show that III1E6 prevents association of AF particles with collagen fibrils. By applying the techniques of immunoblotting and of protein-protein recognition on the solid phase in vitro, we could formulate the following series of events: the AF particle recognizes, with its 47-KD cell binding fragment, the aggregation receptor protein in the plasma membrane and with its core structure the collagen fibrils. These fibrils interact optionally, either via the same route or via the collagen assembly factor, with an adjacent cell surface. These findings demonstrate that the AF particle is not only the key molecule for cell-cell adhesion but also a component of cell-matrix interactions.     

Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope.

Hepatitis delta virus (HDV)-associated particles were purified from the serum of an experimentally infected chimpanzee by size chromatography and by density centrifugation. Hepatitis delta antigen (HDAg) was detected after mild detergent treatment at a column elution volume corresponding to 36-nm particles and banded at a density of 1.25 g/ml. The serum had an estimated titer of 10(9) to 10(10) HDV-associated particles and had only a 10-fold excess of hepatitis B surface antigen (HBsAg) not associated with HDAg. Therefore, HDV appears to be much more efficiently packed and secreted than is its helper virus, hepatitis B virus (HBV), which is usually accompanied by a 1,000-fold excess of HBsAg. The protein compositions of the HDAg-containing particles were analyzed by immunoblotting with HDAg-, HBsAg-, and hepatitis B core antigen-specific antisera and monoclonal antibodies to HBV surface gene products. The HBsAg envelope of HDAg contained approximately 95% P24/GP27s, 5% GP33/36s, and 1% P39/GP42s proteins. This protein composition was more similar to that of the 22-nm particles of HBsAg than to that of complete HBV. The significant amount of GP33/36s suggests that the HBsAg component of the HDV-associated particle carries the albumin receptor. Two proteins of 27 and 29 kilodaltons which specifically bound antibody to HDAg but not HBV-specific antibodies were detected in the interior of the 36-nm particle. Since these proteins were structural components of HDAg and were most likely coded for by HDV, they were designated P27d and P29d.