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1.
Henk Engel AnneMarie van Leeuwen Arnoud Dijkstra Wolfgang Keck 《Applied microbiology and biotechnology》1992,37(6):772-783
Summary In order to produce biologically active 1,6-anhydro-muropeptides in large amounts by enzymatic degradation of isolated bacterial murein polymer highly specific periplasmic murein-metabolizing enzymes from Escherichia coli are made available. The genes slt, dacB, and mepA, encoding the soluble lytic transglycosylase (Slt), the penicillin-sensitive DD-endopeptidase (PBP4), and the penicillin-insensitive murein endopeptidase A (MepA), were independently fused to the N-terminal encoding sequence of staphylococcal protein A (SpA) under control of the temperature-inducible phage p
R promoter. The SpA fusion proteins were stably over-produced at high levels in E. coli upon temperature induction at 42°C and account for 3% (5 mg SpASlt/l culture), 3% (5 mg SpAPBP4/l culture), and 0.3% (0.5 mg SpAMepA/l culture) of total protein. The SpA fusion proteins, immobilized on IgG Sepharose, are proteolytically sensitive, in vitro, resulting in complete degradation of the SpA portion of the fusion proteins and release of the murein hydrolases in intact and enzymatically active form into the supernatant. Proteolytic degradation could be prevented by p-hydroxymercuribenzoic acid (PHMB) or ethylenediaminetetraacetate (EDTA) suggesting the involvement of the periplasmic protease Pi from E. coli. The immobilized fusion proteins were enzymatically active and could be used for the batch production of biologically active 1,6-anhydro-muropeptides, which were successively separated on HPLC. Isolated murein polymer was degraded quantitatively to monomeric 1,6-anhydro-muropeptides when immunoglobulin G (IgG)-SpASlt was used in combination with IgG-SpAMepA. A combination of IgG-SpASlt with IgG-SpAPBP4 left the 1,6-anhydro-dimers and oligomers being cross-linked via an LD-peptide bond (m-DAP-m-DAP) uncleaved.
Correspondence to: W. Keck 相似文献
2.
Martina H. Stiasny Michael Sswat Felix H. Mittermayer Inger‐Britt Falk‐Petersen Nalani K. Schnell Velmurugu Puvanendran Atle Mortensen Thorsten B. H. Reusch Catriona Clemmesen 《Global Change Biology》2019,25(3):839-849
In order to understand the effect of global change on marine fishes, it is imperative to quantify the effects on fundamental parameters such as survival and growth. Larval survival and recruitment of the Atlantic cod (Gadus morhua) were found to be heavily impaired by end‐of‐century levels of ocean acidification. Here, we analysed larval growth among 35–36 days old surviving larvae, along with organ development and ossification of the skeleton. We combined CO2 treatments (ambient: 503 µatm, elevated: 1,179 µatm) with food availability in order to evaluate the effect of energy limitation in addition to the ocean acidification stressor. As expected, larval size (as a proxy for growth) and skeletogenesis were positively affected by high food availability. We found significant interactions between acidification and food availability. Larvae fed ad libitum showed little difference in growth and skeletogenesis due to the CO2 treatment. Larvae under energy limitation were significantly larger and had further developed skeletal structures in the elevated CO2 treatment compared to the ambient CO2 treatment. However, the elevated CO2 group revealed impairments in critically important organs, such as the liver, and had comparatively smaller functional gills indicating a mismatch between size and function. It is therefore likely that individual larvae that had survived acidification treatments will suffer from impairments later during ontogeny. Our study highlights important allocation trade‐off between growth and organ development, which is critically important to interpret acidification effects on early life stages of fish. 相似文献
3.
A DNA immunization model study with constructs expressing the tick-borne encephalitis virus envelope protein E in different physical forms 总被引:6,自引:0,他引:6
Aberle JH Aberle SW Allison SL Stiasny K Ecker M Mandl CW Berger R Heinz FX 《Journal of immunology (Baltimore, Md. : 1950)》1999,163(12):6756-6761
We have conducted a DNA immunization study to evaluate how the immune response is influenced by the physical structure and secretion of the expressed Ag. For this purpose, we used a series of plasmid constructs encoding different forms of the envelope glycoprotein E of the flavivirus tick-borne encephalitis virus. These included a secreted recombinant subviral particle, a secreted carboxyl-terminally truncated soluble homodimer, a nonsecreted full-length form, and an inefficiently secreted truncated form. Mice were immunized using both i.m. injection and Gene Gun-mediated application of plasmids. The functional immune response was evaluated by determining specific neutralizing and hemagglutination-inhibiting Ab activities and by challenging the mice with a lethal dose of the virus. As a measure for the induction of a Th1 and/or Th2 response, we determined specific IgG subclasses and examined IFN-gamma, Il-4, and Il-5 induction. The plasmid construct encoding a secreted subviral particle, which carries multiple copies of the protective Ag on its surface, was superior to the other constructs in terms of extent and functionality of the Ab response as well as protection against virus challenge. As expected, the type of Th response was largely dependent on the mode of application (i.m. vs Gene Gun), but our data show that it was also strongly influenced by the properties of the Ag. Most significantly, the plasmid encoding the particulate form was able to partially overcome the Th2 bias imposed by the Gene Gun, resulting in a balanced Th1/Th2 response. 相似文献
4.
Flavivirus membrane fusion is triggered by acidic pH and mediated by the major envelope protein E. A structurally very similar fusion protein is found in alphaviruses, and these molecules are designated class II viral fusion proteins. In contrast to that of flaviviruses, however, alphavirus fusion has been shown to be absolutely dependent on the presence of cholesterol and sphingomyelin in the target membrane, suggesting significant differences in the fusion protein-membrane interactions that lead to fusion. With the flavivirus tick-borne encephalitis virus (TBEV), we have therefore conducted a study on the lipid requirements of viral fusion with liposomes and on the processes preceding fusion, specifically, the membrane-binding step and the fusion-associated oligomeric switch from E protein dimers to trimers. As with alphaviruses, cholesterol had a strong promoting effect on membrane binding and trimerization of the fusion protein, and-as shown by the use of cholesterol analogs-the underlying interactions involve the 3beta-hydroxyl group at C-3 in both viral systems. In contrast to alphaviruses, however, these effects are much less pronounced with respect to the overall fusion of TBEV and can only be demonstrated when fusion is slowed down by lowering the temperature. The data presented thus suggest the existence of structurally related interactions of the flavivirus and alphavirus fusion proteins with cholesterol in the molecular processes required for fusion but, at the same time, point to significant differences between the class II fusion machineries of these viruses. 相似文献
5.
Characterization of a membrane-associated trimeric low-pH-induced Form of the class II viral fusion protein E from tick-borne encephalitis virus and its crystallization
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The interaction of a dimeric membrane anchor-free form of the envelope protein E (sE dimer) from tick-borne encephalitis virus with liposomes at acidic pH levels leads to its conversion into membrane-inserted sE trimers. Electron microscopy shows that these trimers have their long dimensions along the threefold molecular axis, which is oriented perpendicularly to the plane of the membrane, where the protein inserts via the internal fusion peptide. Liposomes containing sE at their surface display paracrystalline arrays of protein in a closely packing arrangement in which each trimer is surrounded by six others, suggesting cooperativity in the insertion process. sE trimers, solubilized with nonionic detergents, yielded three-dimensional crystals suitable for X-ray diffraction analysis. 相似文献
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8.
Impact of Quaternary Organization on the Antigenic Structure of the Tick-Borne Encephalitis Virus Envelope Glycoprotein E
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The envelope protein E of flaviviruses mediates both receptor-binding and membrane fusion. At the virion surface, 180 copies of E are tightly packed and organized in a herringbone-like icosahedral structure, whereas in noninfectious subviral particles, 60 copies are arranged in a T=1 icosahedral symmetry. In both cases, the basic building block is an E dimer which exposes the binding sites for neutralizing antibodies at its surface. It was the objective of our study to assess the dependence of the antigenic structure of E on its quaternary arrangement, i.e., as part of virions, recombinant subviral particles, or soluble dimers. For this purpose, we used a panel of 11 E protein-specific neutralizing monoclonal antibodies, mapped to distinct epitopes in each of the three E protein domains, and studied their reactivity with the different soluble and particulate forms of tick-borne encephalitis virus E protein under nondenaturing immunoassay conditions. Significant differences in the reactivities with these forms were observed that could be related to (i) limited access of certain epitopes at the virion surface; (ii) limited occupancy of epitopes in virions due to steric hindrance between antibodies; (iii) differences in the avidity to soluble forms compared to the virion, presumably related to the flexibility of E at its domain junctions; and (iv) modulations of the external E protein surface through interactions with its stem-anchor structure. We have thus identified several important factors that influence the antigenicity of the flavivirus E protein and have an impact on the interaction with neutralizing antibodies.Flaviviruses form a genus in the family Flaviviridae (52) and comprise a number of important human pathogens such as yellow fever, dengue, Japanese encephalitis, West Nile, and tick-borne encephalitis (TBE) viruses (30). They are small, enveloped viruses with only three structural proteins, designated C (capsid), M (membrane), and E (envelope). The E protein is oriented parallel to the viral membrane and forms a head-to-tail homodimeric complex (Fig. 1A and B). The structure of the E ectodomain (soluble E [sE])—consisting of about 400 amino acids and lacking the 100 C-terminal amino acids (including the so-called stem and two transmembrane helices)—has been determined by X-ray crystallography for several flaviviruses (Fig. (Fig.1A)1A) (25, 34, 36, 38, 44, 55). Both of the essential entry functions—receptor-binding and membrane fusion after uptake by receptor-mediated endocytosis—are mediated by E, which is therefore the primary target for virus-neutralizing antibodies (11, 42, 43, 45).Open in a separate windowFIG. 1.Structures and schematic representations of the TBE virus E protein, virions, and RSPs. In all panels, DI, DII, and DIII of the E protein are shown in red, yellow, and blue, respectively, and the fusion peptide (FP) is in orange. (A) Ribbon diagram of the sE dimer (top view). (B) Schematic of the full-length E dimer in a top view (upper panel) and side view (lower panel). The position of the two transmembrane helices of the membrane anchor and the two helices of the stem are based on Zhang et al. (54) and are shown in green and purple, respectively. (C) Pseudo-atomic structure of the virion based on cryo-EM reconstructions of dengue and West Nile viruses (27, 37, 54). One of the 30 rafts, each consisting of three parallel dimers, is highlighted. DIIIs of three monomers belonging to one icosahedral asymmetric unit are labeled by white stars. (D) Pseudo-atomic structure of RSP based on cryo-EM reconstructions (12).As revealed by cryo-electron microscopy (cryo-EM), mature infectious virions have smooth surfaces, comparable to a golf ball (27, 37). Their envelopes are icosahedrally symmetric and consist of a closed shell of 180 E monomers that are arranged in a herringbone-like pattern of 30 rafts of three dimers each (Fig. (Fig.1C)1C) (27). On the other hand, capsid-lacking subviral particles, which can be produced in recombinant form by the coexpression of prM and E, have a different symmetry, with 30 E dimers in a T=1 icosahedral structure (Fig. (Fig.1D)1D) (12, 49).The peculiar organization of E in virions is reminiscent of the tight packing of capsid proteins in nonenveloped viruses, for which it was shown that the native antigenic structure is strongly dependent on the intact capsid structure and not completely represented by isolated forms of capsid proteins (1, 41, 53). Such modulations of antigenic structure may be due to conformational changes in the course of packaging the capsid proteins into virions and/or to the fact that antibody binding sites at the virion surface are composed of residues that come together only through the juxtaposition of capsid proteins or neighboring protein subunits. Even in the case of spiky viral envelope proteins, the dependence of certain epitopes on the quaternary organization of the envelope glycoproteins has been described (8, 47).For flaviviruses, structural studies provide evidence for the considerable flexibility of E, especially at the junctions between the individual domains I, II, and III (DI, DII, and DIII) (7, 35, 55), suggesting that soluble forms may display differences in antigenic structure compared to those fixed in the closed envelope shell of whole virions. Furthermore, because of the tight packing of E at the virion surface, certain epitopes may be cryptic in the context of whole virus particles but accessible in soluble forms of E (40, 51).Studies on the antigenic structure of flaviviruses have used different antigen preparations including virions, recombinant subviral particles (RSPs), and soluble forms and subunits of E (10, 15-17, 32, 39, 40, 46, 49, 51), but so far no systematic comparative analysis of E in different physical forms and quaternary arrangements has been conducted. It was therefore the objective of our study, using TBE virus as a model, to investigate possible structural and/or antigenic differences between (i) soluble dimeric forms of E, including C-terminally truncated sE and detergent-solubilized full-length E (Fig. 1A and B); (ii) E in the context of whole virions (Fig. (Fig.1C);1C); and (iii) E in the context of RSPs (Fig. (Fig.1D).1D). For this purpose we used, and further characterized, a set of monoclonal antibodies (MAbs) directed to each of the three domains of E. All of these MAbs have neutralizing activity (17, 24) and therefore, by definition, react with infectious virions.Through these analyses, we demonstrate that the reactivity of several MAbs is significantly dependent on the quaternary arrangement of E and differs between virions, RSPs, and/or sE dimers. We thus provide evidence for previously unrecognized structural factors that have an impact on the antigenicity of the flavivirus E protein. 相似文献
9.
Obesity is associated with adipose tissue remodeling, characterized by macrophage accumulation, adipocyte hypertrophy, and apoptosis. We previously reported that macrophage-conditioned medium (MacCM) protects preadipocytes from apoptosis, due to serum withdrawal, in a platelet-derived growth factor (PDGF)-dependent manner. We have now investigated the role of intracellular signaling pathways, activated in response to MacCM versus PDGF, in promoting preadipocyte survival. Exposure of 3T3-L1 preadipocytes to J774A.1-MacCM or PDGF strongly stimulated Akt and ERK1/2 phosphorylation from initially undetectable levels. Inhibition of the upstream regulators of Akt or ERK1/2, i.e. phosphoinositide 3-kinase (PI3K; using wortmannin or LY294002) or MEK1/2 (using UO126 or PD98509), abrogated the respective phosphorylation responses, and significantly impaired pro-survival activity. J774A.1-MacCM increased reactive oxygen species (ROS) levels by 3.4-fold, and diphenyleneiodonium (DPI) or N-acetyl cysteine (NAC) significantly inhibited pro-survival signaling and preadipocyte survival in response to J774A.1-MacCM. Serum withdrawal itself also increased ROS levels (2.1-fold), and the associated cell death was attenuated by DPI or NAC. In summary, J774A.1-MacCM-dependent 3T3-L1 preadipocyte survival requires the Akt and ERK1/2 signaling pathways. Furthermore, ROS generation by J774A.1-MacCM is required for Akt and ERK1/2 signaling to promote 3T3-L1 preadipocyte survival. These data suggest potential mechanisms by which macrophages may alter preadipocyte fate. 相似文献
10.
The fusion of enveloped viruses with cellular membranes is mediated by proteins that are anchored in the lipid bilayer of the virus and capable of triggered conformational changes necessary for driving fusion. The flavivirus envelope protein E is the only known viral fusion protein with a double membrane anchor, consisting of two antiparallel transmembrane helices (TM1 and TM2). TM1 functions as a stop-transfer sequence and TM2 as an internal signal sequence for the first nonstructural protein during polyprotein processing. The possible role of this peculiar C-terminal helical hairpin in membrane fusion has not been investigated so far. We addressed this question by studying TM mutants of tick-borne encephalitis virus (TBEV) recombinant subviral particles (RSPs), an established model system for flavivirus membrane fusion. The engineered mutations included the deletion of TM2, the replacement of both TM domains (TMDs) by those of the related Japanese encephalitis virus (JEV), and the use of chimeric TBEV-JEV membrane anchors. Using these mutant RSPs, we provide evidence that TM2 is not just a remnant of polyprotein processing but, together with TM1, plays an active role in fusion. None of the TM mutations, including the deletion of TM2, affected early steps of the fusion process, but TM interactions apparently contribute to the stability of the postfusion E trimer and the completion of the merger of the membranes. Our data provide evidence for both intratrimer and intertrimer interactions mediated by the TMDs of E and thus extend the existing models of flavivirus membrane fusion. 相似文献