血影膜及脂质体的融合及其与膜流动性的关系

本文采用Tb/DPA荧光方法研究了血影膜及磷脂酰丝氨酸脂质体在不同Ca~(2 )浓度诱导下的融合动力学过程,并通过改变温度研究了血影膜、PS脂质体的融合与膜流动性的关系.血影膜、PS脂质体的最大融合程度在一定范围内随膜流动性增加而增大,超出此范围则随膜流动增加而减小.对此结果进行了讨论.     

Use of Staphylococcus aureus 6-P-beta-galactosidase and GFP as fusion partners for lactose-specific IIC domain from Staphylococcus aureus

The hydrophilic part of membrane proteins plays an important role in the formation of 3D crystals. The construction of fusion proteins using well crystallizing proteins as fusion partners is a possibility to increase the hydrophilic part of membrane proteins lacking large hydrophilic domains. These fusion proteins might be easier to crystallize. Two bifunctional fusion proteins containing the membrane-bound, lactose-specific enzyme IIC domain of the lactose transporter (IICB(lac)) from S. aureus as N-terminal fusion partner were constructed by gene fusion. The C-terminal fusion partners were S. aureus 6-P-beta-Galactosidase and GFP, respectively. Both proteins were overexpressed in E. coli, purified to homogeneity and kinetically characterized: In the presence of the components of the lactose phosphotransferase system of S. aureus, the hybrid proteins phosphorylated their substrates, indicating that the fusion partners are sufficiently flexibly linked to allow the interaction of the IIC(lac) domain with the IIB(lac) domain of the lactose transporter. The activity of the 6-P-beta-Galactosidase as well as the fluorescence of GFP were preserved in the fusion proteins. The Vmax values determined for the IIC domain in the fusion proteins were dramatically reduced compared with the values determined for the separate IIC(lac) domain and the complete lactose transporter (IICB(lac)). The Km values were only slightly increased indicating that the Vmax values are much more influenced by the fusion than the substrate affinities. The substrate affinity and the Vmax value determined for the GFP-fused IIC(lac) domain are higher than for the 6-P-beta-Galactosidase-fused IIC(lac). The results suggest that the fusion with GFP enables a better interaction with the IIB(lac) domain than the fusion with 6-P-beta-Galactosidase. Moreover, the GFP-fused IIC(lac) domain proved to be more stable than the 6-P-beta-Galactosidase fusion protein.     

Electron microscopic analysis of protoplast fusion in Streptomyces hygroscopicus and consideration on structural alterations in fusing membranes

Protoplasts of Streptomyces hygroscopicus were treated with polyethylene glycol and prepared for electron microscopic investigation as ultrathin sections. About 5% binary fusion products and 0.9% multicellular fusion products have been obtained in the sections. Three main types may be differentiated among binary fusion products, characterized by a successive loss of the bispherical shape and of continuous membrane structures in fusion zones.Analysing the membrane alterations a contact zone characterized by intact cytoplasmic membranes in both protoplasts, a fusion zone with a trilaminar fusion membrane of about 13–17 nm in thickness, and a fusion zone without continuous membrane structure can be distinguished. The different fusion areas are considered as stages in the fusion process. The data will be discussed in conjunction with a model for membrane alterations during fusion at the molecular level.     

Quantitative measurement of paramyxovirus fusion: differences in requirements of glycoproteins between simian virus 5 and human parainfluenza virus 3 or Newcastle disease virus.

To compare the requirements for paramyxovirus-mediated cell fusion, the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of simian virus 5 (SV5), human parainfluenza virus 3 (HPIV-3), and Newcastle disease virus (NDV) were expressed individually or coexpressed in either homologous or heterologous combinations in CV-1 or HeLa-T4 cells, using the vaccinia virus-T7 polymerase transient expression system. The contribution of individual glycoproteins in virus-induced membrane fusion was examined by using a quantitative assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecyl rhodamine (R18) and a quantitative assay for content mixing based on the cytoplasmic activation of a reporter gene, beta-galactosidase. In these assays, expression of the individual F glycoproteins did not induce significant levels of cell fusion and no cell fusion was observed in experiments when cells individually expressing homologous F or HN proteins were mixed. However, coexpression of homologous F and HN glycoproteins resulted in extensive cell fusion. The kinetics of fusion were found to be very similar for all three paramyxoviruses studied. With NDV and HPIV-3, no cell fusion was detected when F proteins were coexpressed with heterologous HN proteins or influenza virus hemagglutinin (HA). In contrast, SV5 F protein exhibited a considerable degree of fusion activity when coexpressed with either NDV or HPIV-3 HN or with influenza virus HA, although the kinetics of fusion were two- to threefold higher when the homologous SV5 F and HN proteins were coexpressed. Thus, these data indicate that among the paramyxoviruses tested, SV5 has different requirements for cell fusion.     

Phagosomal acidification in Paramecium: effects on lysosomal fusion

Phagosomes of paramecia and amoeba and endosomes of fibroblasts and other mammalian cells are acidified prior to lysosomal fusion. The question, whether the phagosomal acidification process in paramecia is required for phagosome-lysosome fusion, was studied using ionophores, weak bases, and cytochalasin B (CB) in combination with monoclonal antibodies, acid phosphatase (AcPase) cytochemistry, and lysosome morphometry. Digestive vacuoles (DVs) of known ages were treated and examined. In untreated cells, lysosome binding to the membrane of the acidified DV increased linearly with age and reached a maximum before lysosome-DV fusion. When the fusion of the acidosomes with the very young DVs was prevented by CB, causing a block in the normal vacuole-pH drop, lysosome binding to the DVs as well as the rate and extent of lysosome-DV fusion were all greatly reduced. These effects of CB were reversible. When present prior to acidification, three ionophores and two weak bases did not inhibit the acidosome-DV fusion but raised the phagosomal pH and reduced both the rates of DV acidification and of lysosome-DV fusion. However, when added after acidification but prior to lysosome-DV fusion, five of the six perturbants studied did not inhibit this fusion but prolonged the period when DVs remained AcPase positive. Lastly, lysosome-DV fusion rates were found to be related to acidification rates. We conclude that an inhibition in acidosome-DV fusion or a reduction in both the acidification rate and vacuolar-pH drop would inhibit lysosome-DV fusion.     

Effective donor cell fusion conditions for production of cloned dogs by somatic cell nuclear transfer

As shown by the birth of the first cloned dog ‘Snuppy', a protocol to produce viable cloned dogs has been reported. In order to evaluate optimum fusion conditions for improving dog cloning efficiency, in vivo matured oocytes were reconstructed with adult somatic cells from a female Pekingese using different fusion conditions. Fusion with needle vs chamber methods, and with low vs high pulse strength was compared by evaluating fusion rate and in vivo development of canine cloned embryos. The fusion rates in the high voltage groups were significantly higher than in the low voltage groups regardless of fusion method (83.5 vs 66.1% for the needle fusion method, 67.4 vs 37.9% for the fusion chamber method). After embryo transfer, one each pregnancy was detected after using the needle fusion method with high and low voltage and in the chamber fusion method with high voltage, whereas no pregnancy was detected using the chamber method with low voltage. However, only the pregnancy from the needle fusion method with high voltage was maintained to term and one healthy puppy was delivered. The results of the present study demonstrated that two DC pulses of 3.8 to 4.0 kV/cm for 15 μsec using the needle fusion method were the most effective method for the production of cloned dogs under the conditions of this experiment.     

Separate epitopes in the envelope of visna virus are responsible for fusion and neutralization: biological implications for anti-fusion antibodies in limiting virus replication.

Visna virus is a lentivirus which causes fusion of infected cells in vitro. Two types of fusion occur. Fusion from without requires no viral replication and a relatively high multiplicity of infection; fusion from within results from the replication of virus in cells. By using fusion from without as an assay, the mechanism of fusion by visna virus was investigated. Immune sera which contained both anti-fusion and neutralizing antibodies interacted with the virus with rapid kinetics in blocking fusion but relatively slow kinetics in the virus neutralization assay. By using visna virus and an antigenic variant, the epitopes responsible for fusion and virus neutralization were shown to be different. Antigenic variation of visna virus resulted in alteration of the neutralization epitope and conservation of the fusion epitope. This suggested that there were two populations of antibodies and that the viral epitopes for fusion and neutralization were separate. These data suggest that visna virus is capable of infecting cells via two pathways: one via the fusion site and the other via the viral epitope which mediates neutralization.     

FUS1 regulates the opening and expansion of fusion pores between mating yeast

Mating yeast cells provide a genetically accessible system for the study of cell fusion. The dynamics of fusion pores between yeast cells were analyzed by following the exchange of fluorescent markers between fusion partners. Upon plasma membrane fusion, cytoplasmic GFP and DsRed diffuse between cells at rates proportional to the size of the fusion pore. GFP permeance measurements reveal that a typical fusion pore opens with a burst and then gradually expands. In some mating pairs, a sudden increase in GFP permeance was found, consistent with the opening of a second pore. In contrast, other fusion pores closed after permitting a limited amount of cytoplasmic exchange. Deletion of FUS1 from both mating partners caused a >10-fold reduction in the initial permeance and expansion rate of the fusion pore. Although fus1 mating pairs also have a defect in degrading the cell wall that separates mating partners before plasma membrane fusion, other cell fusion mutants with cell wall remodeling defects had more modest effects on fusion pore permeance. Karyogamy is delayed by >1 h in fus1 mating pairs, possibly as a consequence of retarded fusion pore expansion.     

Interaction of Influenza Virus Fusion Peptide with Lipid Membranes: Effect of Lysolipid

The effect of lysophosphatidylcholine (LPC) on lipid vesicle fusion and leakage induced by influenza virus fusion peptides and the peptide interaction with lipid membranes were studied by using fluorescence spectroscopy and monolayer surface tension measurements. It was confirmed that the wild-type fusion peptide-induced vesicle fusion rate increased several-fold between pH 7 and 5, unlike a mutated peptide, in which valine residues were substituted for glutamic acid residues at positions 11 and 15. This mutated peptide exhibited a much greater ability to induce lipid vesicle fusion and leakage but in a less pH-dependent manner compared to the wild-type fusion peptide. The peptide-induced vesicle fusion and leakage were well correlated with the degree of interaction of these peptides with lipid membranes, as deduced from the rotational correlation time obtained for the peptide tryptophan fluorescence. Both vesicle fusion and leakage induced by the peptides were suppressed by LPC incorporated into lipid vesicle membranes in a concentration-dependent manner. The rotational correlation time associated with the peptide’s tryptophan residue, which interacts with lipid membranes containing up to 25 mole % LPC, was virtually the same compared to lipid membranes without LPC, indicating that LPC-incorporated membrane did not affect the peptide interaction with the membrane. The adsorption of peptide onto a lipid monolayer also showed that the presence of LPC did not affect peptide adsorption.     

The Transmembrane Domain and Acidic Lipid Flip-Flop Regulates Voltage-Dependent Fusion Mediated by Class II and III Viral Proteins

Voltage dependence of fusion induced by class II and class III viral fusion proteins was investigated. Class II proteins from Ross River and Sindbus virus and a mutant class III protein from Epstein Barr virus were found to induce cell-cell fusion that is voltage dependent. Combined with previous studies, in all, four class II and two class III protein have now been shown to exhibit voltage-dependent fusion, demonstrating that this is probably a general phenomenon for these two classes of viral fusion proteins. In the present study, monitoring fusion of pseudovirus expressing Vesicular Stomatitis virus (VSV) G within endosomes shows that here, too, fusion is voltage dependent. This supports the claim that voltage dependence of fusion is biologically relevant and that cell-cell fusion reliably models the voltage dependence. Fusion induced by class I viral proteins is independent of voltage; chimeras expressing the ectodomain of a class I fusion protein and the transmembrane domain of VSV G could therefore be used to explore the location within the protein responsible for voltage dependence. Results showed that the transmembrane domain is the region associated with voltage dependence. Experiments in which cells were enriched with acidic lipids led to the conclusion that it is the flip-flop of acidic lipids that carries the charge responsible for the observed voltage dependence of fusion. This flip-flop occurred downstream of hemifusion, in accord with previous findings that the voltage dependent steps of fusion occur at a stage subsequent to hemifusion.     

Fusion of short telomeres in human cells is characterized by extensive deletion and microhomology,and can result in complex rearrangements

Telomere fusion is an important mutational event that has the potential to lead to large-scale genomic rearrangements of the types frequently observed in cancer. We have developed single-molecule approaches to detect, isolate and characterize the DNA sequence of telomere fusion events in human cells. Using these assays, we have detected complex fusion events that include fusion with interstitial loci adjacent to fragile sites, intra-molecular rearrangements, and fusion events involving the telomeres of both arms of the same chromosome consistent with ring chromosome formation. All fusion events were characterized by the deletion of at least one of the telomeres extending into the sub-telomeric DNA up to 5.6 kb; close to the limit of our assays. The deletion profile indicates that deletion may extend further into the chromosome. Short patches of DNA sequence homology with a G:C bias were observed at the fusion point in 60% of events. The distinct profile that accompanies telomere fusion may be a characteristic of the end-joining processes involved in the fusion event.     

Murine immune responses to recombinant Toxoplasma gondii antigens

Recombinant proteins of the RH strain of Toxoplasma gondii were produced by expression in Escherichia coli as glutathione S-transferase (GST) fusion proteins. Enzyme-linked immunosorbent assays were established using 2 of these fusion proteins termed H4/GST and H11/GST. The assays were able to detect antibodies in the sera of mice orally infected with either the cyst or oocyst stage of a pork isolate of T. gondii. In addition, the sera from mice infected with 1 of 4 different T. gondii isolates were investigated for their binding to these fusion proteins. Antibodies in the sera of all mice bound to H11/GST, but not all sera recognized H4/GST. Delayed-type hypersensitivity (DTH) responses to the fusion proteins were found when the mice were sensitized intradermally with H4/GST and H11/GST and challenged with the homologous fusion protein. However, no DTH response was recorded when mice were challenged with homologous fusion proteins after infection with T. gondii, or after immunization with a sonicate of the RH strain of the parasite. In addition, cellular responses were not stimulated against either of the fusion proteins in in vitro assays. These 2 fusion proteins were recognized by anti-T. gondii antibodies in experimental murine infections, and they are therefore potential candidates as antigens in assays for the diagnosis of human toxoplasmosis.     

Local electrostatic interactions determine the diameter of fusion pores

In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least 2 mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore membrane composition.     

Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved.     

Membrane fusion machines of paramyxoviruses: capture of intermediates of fusion

Peptides derived from heptad repeat regions adjacent to the fusion peptide and transmembrane domains of many viral fusion proteins form stable helical bundles and inhibit fusion specifically. Paramyxovirus SV5 fusion (F) protein-mediated fusion and its inhibition by the peptides N-1 and C-1 were analyzed. The temperature dependence of fusion by F suggests that thermal energy, destabilizing proline residues and receptor binding by the hemagglutinin-neuraminidase (HN) protein collectively contribute to F activation from a metastable native state. F-mediated fusion was reversibly arrested by low temperature or membrane-incorporated lipids, and the resulting F intermediates were characterized. N-1 inhibited an earlier F intermediate than C-1. Co-expression of HN with F lowered the temperature required to attain the N-1-inhibited intermediate, consistent with HN binding to its receptor stimulating a conformational change in F. C-1 bound and inhibited an intermediate of F that could be detected until a point directly preceding membrane merger. The data are consistent with C-1 binding a pre-hairpin intermediate of F and with helical bundle formation being coupled directly to membrane fusion.     

斑玉蕈育种中漆酶转化体系建立的初步研究

漆酶具有降解木质素,氧化降解酚类物质,抑制杂菌,改善出菇品质等作用。采用酶学与原生质体融合相结合的技术,建立斑玉蕈育种中漆酶转化体系,将漆酶活性较高、生长速度较快的凤尾菇原生质体经高温灭活与斑玉蕈原生质体融合,通过RB-PDA平板显色技术筛选出具有漆酶活性较高的两融合菌株Ⅲ18C、Ⅲ2A,并对其进行了拮抗试验、RAPD分子标记、漆酶基因扩增等研究。结果表明,筛选出的两融合菌株与两亲本具有明显的拮抗线,随即引物扩增的条带与两亲本有明显的差异,并扩增出漆酶基因的一个片段。同时也表明利用漆酶转化体系筛选融合菌株具有目标明确、准确、快速的优点。     

Cell-to-cell fusion of lens fiber cells in situ: correlative light, scanning electron microscopic, and freeze-fracture studies

We have discovered cell-to-cell fusion between fiber cells of adult frog lenses in situ. Stereo scanning electron microscopy (SEM) revealed fusion between neighboring fiber cells in radial cell columns (RCCs) and in the same growth ring, respectively. Cell-to-cell fusion of fiber cells in the lens produced fusion zones that in cross-section were larger and of different polygonal shapes than unfused fiber cells. The shape and sizes of fiber cells surrounding fusion zones and the alignment of RCCs were also altered. Serial sectioning through fusion zones confirmed that they were areas of cell-to-cell continuity established by the union of neighboring fiber cells as seen by SEM. Fusion zones represent a previously unrecognized intercellular pathway in the adult frog lens. Although numerous fusion zones were seen throughout the lens cortex and nucleus, cell-to-cell fusion was rarely observed to have occurred between elongating fiber cells. Interestingly, communicating junctions with an unusual ultrastructure that closely resembles the appearance of membranes in the process of fusion demonstrated in other systems were frequently seen in the region of the superficial cortex where fusion zones were most numerous. The fact that such unusual communicating junctions were not found in any other region of the lens leads us to speculate that structural changes in fiber cell communicating junctions may herald the formation of fusion zones and that the initial site of cell-to-cell fusion between fiber cells may be within communicating junctional plaques.     

The transmembrane domain and cytoplasmic tail of herpes simplex virus type 1 glycoprotein H play a role in membrane fusion

Herpes simplex virus glycoprotein H (gH) is one of the four virion envelope proteins which are required for virus entry and for cell-cell fusion in a transient system. In this report, the role of the transmembrane and cytoplasmic tail domains of gH in membrane fusion was investigated by generating chimeric constructs in which these regions were replaced with analogous domains from other molecules and by introducing amino acid substitutions within the membrane-spanning sequence. gH molecules which lack the authentic transmembrane domain or cytoplasmic tail were unable to mediate cell-cell fusion when coexpressed with gB, gD, and gL and were unable to rescue the infectivity of a gH-null virus as efficiently as a wild-type gH molecule. Many amino acid substitutions of specific amino acid residues within the transmembrane domain also affected cell-cell fusion, in particular, those introduced at a conserved glycine residue. Some gH mutants that were impaired in cell-cell fusion were nevertheless able to rescue the infectivity of a gH-negative virus, but these pseudotyped virions entered cells more slowly than wild-type virions. These results indicate that the fusion event mediated by the coexpression of gHL, gB, and gD in cells shares common features with the fusion of the virus envelope with the plasma membrane, they point to a likely role for the membrane-spanning and cytoplasmic tail domains of gH in both processes, and they suggest that a conserved glycine residue in the membrane-spanning sequence is crucial for efficient fusion.     

Use of carboxypeptidase Y propeptide as a fusion partner for expression of small polypeptides in Escherichia coli

The carboxypeptidase Y (CPY) propeptide from Saccharomyces cerevisiae was developed as a fusion partner for the efficient expression of small polypeptides in Escherichia coli. Six consecutive histidine residues (6xHis) were fused to the N-terminus of the CPY propeptide for the facilitated purification of fusion proteins using immobilized metal ion affinity chromatography. In addition, a methionine or the pentapeptide (Asp)(4)-Lys linker was inserted at the junction between the CPY propeptide and the target polypeptide to release the target polypeptide by digestion with cyanogen bromide or enterokinase. Therapeutically valuable peptide hormones, such as salmon calcitonin precursor (sCAL-Gly), a fragment of human parathyroid hormone (hPTH(1-34)), and human glucagon were successfully expressed in E. coli as fusion polypeptides with the fusion partner. SDS-PAGE analyses showed that the majority of the expressed fusion sCAL-Gly and fusion hPTH(1-34) were present in the form of inclusion bodies, whereas about 66% of the expressed human glucagon was in a soluble form. Almost complete cleavage of the fusion polypeptides was obtained by digestion with enterokinase. Reverse-phase HPLC analyses showed that the target polypeptides released from the fusion proteins were identical to their native forms.     

重组单链抗体-低分子质量尿激酶融合蛋白在 CHO 细胞中的抗降解研究

抗人纤维蛋白单链抗体-低分子质量尿激酶(Ⅱn-UK)融合蛋白,兼有单链抗体对纤维蛋白的亲和性和尿激酶的溶栓活性,有望开发成为新型导向溶栓药物.但基于通用连接肽(G4S)3的Ⅱn-linker-UK融合蛋白在CHO细胞中表达时出现明显的降解.为了解决此问题,利用分子生物学方法,对Hn-UK融合蛋白进行了分子改造,包括置换连接肽,改变两个半分子(moiety)的相对位置,以及对连接肽附近明确的蛋白酶位点进行突变等方法,并分别研究了改造后的11种Ⅱn-1inker-UK或UK-linker-Ⅱn突变体在CHO细胞中分泌性表达时的稳定性,最终筛选到一种抗降解的突变体.