Prenyl proteins in eukaryotic cells: a new type of membrane anchor

Recent studies have indicated that eukaryotic cells contain proteins that are post-translationally modified by long-chain, thioether-linked prenyl groups. These proteins include yeast mating factors, ras proteins and nuclear lamins. The modification occurs on a cysteine residue near the C terminus and appears to initiate a set of additional protein modification reactions that promote attachment of the proteins to specific membranes.     

Construction of an artificial symbiotic community using a Chlorella-symbiont association as a model.

Chlorella sorokiniana IAM C-212 produces a polysaccharide gel, termed a sheath, under photoautotrophic conditions. The C. sorokiniana sheath is a suitable habitat for several symbiotic microorganisms because it ensures close proximity between the C. sorokiniana and symbionts. In this study, we established a method for increasing the volume of the sheath produced by C. sorokiniana, and proposed a method for constructing artificial communities of Chlorella and symbiotic microorganisms. The C. sorokiniana sheath was increased by addition of calcium chloride solution. The sheath resulted in coflocculation of C. sorokiniana and the associated symbiotic bacteria, thus strengthening the bacterial-Chlorella symbiotic association. An application of this technique was demonstrated by constructing a complex of C. sorokiniana and a propionate-degrading bacterium (PDS1). Although propionate inhibited the growth of axenic C. sorokiniana, the C. sorokiniana-PDS1 complex showed good growth in a medium containing a high concentration of propionate.     

A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells

Glycosyl-phosphatidylinositol- (GPI) anchored proteins contain a large extracellular protein domain that is linked to the membrane via a glycosylated form of phosphatidylinositol. We recently reported the polarized apical distribution of all endogenous GPI-anchored proteins in the MDCK cell line (Lisanti, M. P., M. Sargiacomo, L. Graeve, A. R. Saltiel, and E. Rodriguez-Boulan. 1988. Proc. Natl. Acad. Sci. USA. 85:9557-9561). To study the role of this mechanism of membrane anchoring in targeting to the apical cell surface, we use here decay-accelerating factor (DAF) as a model GPI-anchored protein. Endogenous DAF was localized on the apical surface of two human intestinal cell lines (Caco-2 and SK-CO15). Recombinant DAF, expressed in MDCK cells, also assumed a polarized apical distribution. Transfer of the 37-amino acid DAF signal for GPI attachment to the ectodomain of herpes simplex glycoprotein D (a basolateral antigen) and to human growth hormone (a regulated secretory protein) by recombinant DNA methods resulted in delivery of the fusion proteins to the apical surface of transfected MDCK cells. These results are consistent with the notion that the GPI anchoring mechanism may convey apical targeting information.     

Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif

A novel bacterial cell-surface display system was developed in Escherichia coli using omp1, a hypothetical outer membrane protein of Zymomonas mobilis. By using this system, we successfully expressed β-amylase gene of sweet potato in E. coli. The display of enzyme on the membrane surface was also confirmed. The recombinant β-amylase showed to significantly increase hydrolytic activity toward soluble starch. Our results provide a basis for constructing an engineered Z. mobilis strain directly fermenting raw starch to produce ethanol.     

Construction of an artificial symbiotic community using a Chlorella–symbiont association as a model

Chlorella sorokiniana IAM C-212 produces a polysaccharide gel, termed a sheath, under photoautotrophic conditions. The C. sorokiniana sheath is a suitable habitat for several symbiotic microorganisms because it ensures close proximity between the C. sorokiniana and symbionts. In this study, we established a method for increasing the volume of the sheath produced by C. sorokiniana , and proposed a method for constructing artificial communities of Chlorella and symbiotic microorganisms. The C. sorokiniana sheath was increased by addition of calcium chloride solution. The sheath resulted in coflocculation of C. sorokiniana and the associated symbiotic bacteria, thus strengthening the bacterial– Chlorella symbiotic association. An application of this technique was demonstrated by constructing a complex of C. sorokiniana and a propionate-degrading bacterium (PDS1). Although propionate inhibited the growth of axenic C. sorokiniana , the C. sorokiniana –PDS1 complex showed good growth in a medium containing a high concentration of propionate.     

Incorporation of sulfonylurea into N-isopropylacrylamide as an extracellular matrix for an artificial pancreas.

High-molecular-weight N-isopropylacrylamide copolymers with small amounts of sulfonylurea (SU, typically 2-4 mol% in the feed) were synthesized by free radical polymerization in benzene. SU-incorporated polymer solutions (5, 6, 8, and 10% w/v) in a culture medium (pH 7.4, 0.15 M ionic strength) with islet cells were mixed and poured into Millicells which supported gel formation. In order to increase the gelation temperature, the SU-incorporated copolymer gel, p(NiPAAm-co-SU), was blended with the p(NiPAAm-co-AAc) polymer at a ratio of 4 to 96. Interaction between the islet cells and the synthetic matrix of SU-incorporated copolymer gel resulted in effective cell viability and such cell functions as insulin secretion. To verify the specific interaction between the SU (K+ channel closer)-incorporated copolymer and islet cells, the cells were pretreated with diazoxide, an agonist of the ATP-sensitive K+ channel (K+ channel opener), before interaction between the polymer and islet cells. This treatment suppressed the action of SU on the islet cells. The results from this study provide evidence that the SU-incorporated copolymer stimulated insulin secretion by specific interaction between SU moieties in the polymer and the islet cells.     

Fabrication of a cell-adhesive protein imprinting surface with an artificial cell membrane structure for cell capturing

We proposed a new molecular imprinting procedure based on molecular integration for the purpose of cell capture. We selected the cell-adhesive protein fibronectin (FN) as the imprinting protein for preparing templates and evaluated selective cell adhesion on the FN imprinting substrate. Silica beads with a diameter of 15 μm were used as the stamp matrix and FN molecules were adsorbed as a monolayer. The FN recognition sites were constructed by integrating a surfactant as the ligand and immobilizing it with new biocompatible photoreactive phospholipid polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units. As control substrates, imprinting procedures were carried out using albumin (BSA imprinting substrate) and without imprinting protein (non-imprinting substrate). The binding of FN from the cell culture medium with the fetal calf serum was achieved on the FN imprinting substrate, and induced the cell adhesion. On the other hand, on the non-imprinted and BSA imprinting substrates, the FN scarcely bound from the cell culture medium, and subsequent cell adhesion could not be observed on the substrate. These results indicate that the FN binding sites were well constructed by arranging the ligand surfactant to a suitable position and immobilized by the photoreactive MPC polymer. The MPC polymer prevented the nonspecific adsorption of proteins from the cell culture medium. We concluded that this procedure is convenient and can be potentially used for the preparation of surfaces for cell engineering devices.     

Entamoeba histolytica as a model for the primitive eukaryotic cell

Entamoeba histolytica is a structurally simple eukaryote lacking mitochondria, peroxisomes and a well-developed Golgi apparatus, also in its biochemistry, it deviates substantially from the more complex eukoryotes. These features have alternatively been interpreted as archaic, ie. the ancestor of Entamoeba branched off before the primitive eukaryotic cell obtained proto-mitochondria, or as regressive, ie. Entamoeba has lost its mitochondria in the course of its adaptation to a parasitic life style. Tilly Bakker-Grunwald and Claudia W?stmann favor the first interpretation and discuss in which respects E. histolytica may serve as a model for the primitive eukaryote.     

Characterization of a liposome-based artificial skin membrane for in vitro permeation studies using Franz diffusion cell device

A prerequisite for successful transdermal or dermal drug therapy is the drug ability to penetration through the skin, especially stratum corneum (SC). The most acceptable technique for measuring skin permeation in vitro is the application of both the Franz diffusion cell device and the skin model. In the skin model, a liposome-based artificial skin membrane (LASM) consisting of tight layers of liposomes immobilized on a filter was prepared and characterized. Using porcine ear skin, rat skin and Strat-M? artificial membrane as control, the LASM was then evaluated in permeation studies with five active compounds: ferulic acid, paeoniflorin, albiflorin, tetrahydrocolumbamine, and tetrahydropalmatine. The scanning electron microscope images demonstrated complete filling of the membrane pores with lipids and the formation of a continuous liposomal coating. The contents of egg phosphatidylcholine (EPC) and cholesterol in LASM were measured to be 12.08?±?0.18 and 4.41?±?0.04?mg/cm², respectively. Moreover, revealed by the measurement of electrical resistance, the LASM remains intact for at least 12?h with the incubation of 20% ethanol. The results of permeation studies demonstrated a good correlation (r^2?=?0.9743, r?=?0.9871) of P_app values between the drugs’ permeation through LASM and porcine ear skin. In addition, by ATR-FTIR analysis, a slighter shift of CH₂ stretching frequency between LASM and porcine ear skin was observed compared with the shift between Strat-M? membrane and porcine ear skin. In summary, for the first time, the LASM has been proved to be a valuable alternative to porcine ear skin in permeation studies using Franz diffusion cell device.     

The autolysin Ami contributes to the adhesion of Listeria monocytogenes to eukaryotic cells via its cell wall anchor

Adherence of pathogenic microorganisms to the cell surface is a key event during infection. We have previously reported the characterization of Listeria monocytogenes transposon mutants defective in adhesion to eukaryotic cells. One of these mutants had lost the ability to produce Ami, a 102 kDa autolytic amidase with an N-terminal catalytic domain and a C-terminal cell wall-anchoring domain made up of repeated modules containing the dipeptide GW ('GW modules'). We generated ami null mutations by plasmid insertion into L. monocytogenes strains lacking the invasion proteins InlA (EGDDeltainlA), InlB (EGDDeltainlB) or both (EGDDeltainlAB). These mutants were 5-10 times less adherent than their parental strains in various cell types. The adhesion capacity of the mutants was restored by complementation with a DNA fragment encoding the Ami cell wall-anchoring domain fused to the Ami signal peptide. The cell-binding activity of the Ami cell wall-anchoring domain was further demonstrated using the purified polypeptide. Growth of the ami null mutants constructed in EGD and EGDDeltainlAB backgrounds was attenuated in the livers of mice inoculated intravenously, indicating a role for Ami in L. monocytogenes virulence. Adhesive properties have recently been reported in the non-catalytic domain of two other autolysins, Staphylococcus epidermidis AtlE and Staphylococcus saprophyticus Aas. Interestingly, we found that these domains were also composed of repeated GW modules. Thus, certain autolysins appear to promote bacterial attachment by means of their GW repeat domains. These molecules may contribute to the colonization of host tissues by Gram-positive bacteria.     

Blood feeding of Ornithodoros turicata larvae using an artificial membrane system

An artificial membrane system was adapted to feed Ornithodoros turicata (Ixodida: Argasidae) larvae from a laboratory colony using defibrinated swine blood. Aspects related to larval feeding and moulting to the first nymphal instar were evaluated. A total of 55.6% of all larvae exposed to the artificial membrane in two experimental groups fed to repletion and 98.0% of all fed larvae moulted. Mortality rates of first instar nymphs differed significantly depending on the sorting tools used to handle engorged larvae (χ² = 35.578, P < 0.0001): engorged larvae handled with featherweight forceps showed significantly higher mortality (odds ratio = 4.441) than those handled with a camel‐hair brush. Differences in the physical properties of the forceps and camel‐hair brush may affect the viability of fragile soft tick larvae even when care and the same technique are used to sort them during experimental manipulations. The current results represent those of the first study to quantify successful feeding to repletion, moulting and post‐moulting mortality rates in O. turicata larvae using an artificial membrane feeding system. Applications of the artificial membrane feeding system to fill gaps in current knowledge of soft tick biology and the study of soft tick–pathogen interactions are discussed.     

A hyperthermostable bacterial histone-like protein as an efficient mediator for transfection of eukaryotic cells

Gene delivery has shown potential in a variety of applications, including basic research, therapies for inborn genetic defects, cancer, AIDS, tissue engineering, and vaccination. Most available systems have serious drawbacks, such as safety hazards, inefficiency under in vivo-like conditions, and expensive production. When using naked DNA, for instance, a large amount of ultrapure DNA has to be applied as a result of degradation by nucleases. Similarly, the use of eukaryotic histones, synthetic peptides, or peptide nucleic acids may be limited by high production costs. We have demonstrated a biotechnologically feasible and economical approach for gene delivery using the histone-like protein from the hyperthermostable eubacterium Thermotoga maritima, TmHU as an efficient gene transfer reagent. HU can be easily isolated from recombinant Escherichia coli, is extraordinarily stable, and protects dsDNA from thermal denaturation. This study demonstrates its use as an inexpensive tool for gene delivery.     

An artificial hydrophobic sequence functions as either an anchor or a signal sequence at only one of two positions within the Escherichia coli outer membrane protein OmpA

The 325-residue outer membrane protein, OmpA, of Escherichia coli, like most other outer membrane proteins with known sequence, contains no long stretch of hydrophobic amino acids. A synthetic oligonucleotide, encoding the sequence Leu-Ala-Leu-Val, was inserted four times between the codons for amino acid residues 153 and 154 and two, three, or four times between the codons for residues 228 and 229, resulting in the OmpA153-4, OmpA-228-2, -3, and -4 proteins, respectively. In the first case, the lipophilic sequence anchored the protein in the plasma membrane. In the OmpA228 proteins, 16 but not 12 or 8 lipophilic residues most likely also acted as an anchor. By removal of the NH2-terminal signal peptide, the function of the insert in OmpA153-4 was converted to that of a signal-anchor sequence. Possibly due to differences in amino acid sequences surrounding the insert, no signal function was observed with the insert in OmpA228-4. Production of the OmpA153-4 protein, with or without the NH2-terminal signal sequence, resulted in a block of export of chromosomally encoded OmpA. Clearly, long hydrophobic regions are not permitted within proteins destined for the bacterial outer membrane, and these proteins, therefore, have had to evolve another mechanism of membrane assembly.     

利用Flo1p锚定蛋白在酿酒酵母表面展示三种纤维素酶的纤维素乙醇发酵

为了简化纤维素乙醇生产工艺,实现纤维素利用与乙醇发酵的同步进行,通过酵母细胞表面展示技术,以酿酒酵母菌株Saccharomyces cerevisiae Y5为受体,通过絮凝素(Flo1p)锚定方式,将来自丝状真菌里氏木霉Trichoderma reesei的内切葡聚糖酶Ⅱ(EGII)、纤维二糖水解酶Ⅱ(CBHII)以及来自棘孢曲霉Aspergillus aculeatus的β-葡糖苷酶Ⅰ(BGLI)展示在细胞表面,构建同时表达3种纤维素酶的酵母菌群系统。经过免疫荧光验证展示酶的细胞蛋白定位,酶活测定,乙醇发酵性能验证,结果表明:展示表达的3种纤维素酶具有良好的稳定性和功能活性;在EGII、CBHII和BGLI协同作用下重组酵母菌株能够水解溶胀磷酸纤维素(Phosphoric acid swollen cellulose,简称PASC)并产生乙醇,乙醇浓度达到最大值0.77 g/L,乙醇产量为0.35 g/g,相当于理论值的68.6%。本研究成功构建了利用Flo1p作为锚定蛋白的絮凝素展示系统,初步实现了纤维素利用与乙醇发酵的同步进行,为利用酿酒酵母表面展示技术固定并表达纤维素酶提供了一定的理论依据。     

Construction of an infectious clone of canine herpesvirus genome as a bacterial artificial chromosome

Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals.     

Plasma membrane budding as an alternative release mechanism of the extracellular enveloped form of vaccinia virus from HeLa cells

In HeLa cells the assembly of modified vaccinia virus Ankara (MVA), an attenuated vaccinia virus (VV) strain, is blocked. No intracellular mature viruses (IMVs) are made and instead, immature viruses accumulate, some of which undergo condensation and are released from the cell. The condensed particles may undergo wrapping by membranes of the trans-Golgi network and fusion with the plasma membrane prior to their release (M. W. Carroll and B. Moss, Virology 238:198-211, 1997). The present study shows by electron microscopy (EM), however, that the dense particles made in HeLa cells are also released by a budding process at the plasma membrane. By labeling the plasma membrane with antibodies to B5R, a membrane protein of the extracellular enveloped virus, we show that budding occurs at sites that concentrate this protein. EM quantitation revealed that the cell surface around a budding profile was as strongly labeled with anti-B5R antibody as were the extracellular particles, whereas the remainder of the plasma membrane was significantly less labeled. To test whether budding was a characteristic of MVA infection, HeLa cells were infected with the replication competent VV strains Western Reserve strain (WR) and International Health Department strain-J (IHD-J) and also prepared for EM. EM analyses, surprisingly, revealed for both virus strains IMVs that evidently budded at the cell surface at sites that were significantly labeled with anti-B5R. EM also indicated that budding of MVA dense particles was more efficient than budding of IMVs from WR- or IHD-J-infected cells. This was confirmed by semipurifying [(35)S]methionine-labeled dense particles or extracellular enveloped virus (EEVs) from the culture supernatant of MVA- or IHD-J-infected HeLa cells, respectively, showing that threefold more labeled dense particles were secreted than EEVs. Finally, although the released MVA dense particles contain some DNA, they are not infectious, as assessed by plaque assays.