分子筛层析法分析重组乙肝表面抗原聚集物的形成

分子筛层析作为分析蛋白质颗粒聚集物的一种有力工具,被用于研究重组乙肝表面抗原聚集物的形成。已去除聚集物的表面抗原放置在不同的理化条件下或经过不同的纯化方法处理后,应用HPLC分析其聚集物的形成。为研究发酵过程中是否形成表面抗原聚集物,酵母细胞破碎后立即用Sepharose 4 FF层析柱分离为不同的组分,并分别进行HPLC分析。结果发现,在纯化过程和酵母发酵阶段都有表面抗原聚集物的产生。     

Characterization of the large size aggregation of Hepatitis B virus surface antigen (HBsAg) formed in ultrafiltration process

This investigation focused on the structure change of Hepatitis B virus surface antigen (HBsAg) in the process of ultrafiltration (UF). Based on the assay of high performance size exclusion chromatography combining with on-line multi-angle laser light scattering (HPSEC-MALLS) and enzyme-linked immunosorbent assay (ELISA), the HBsAg assemblies were found to aggregate into large-size HBsAg aggregation with only about 20% HBsAg activity of the normal HBsAg assembly. The secondary structure of large size HBsAg aggregation was monitored by circular dichroism spectroscopy (CD) and demonstrated that the content of α-helix in HBsAg decreased from 48.2% to 34.4% and the content of γ-turn increased from 29.6% to 38.7% due to aggregation. The lipid structure of large size HBsAg aggregation was also changed markedly by the assay of infrared spectroscopy (IR) at the wavenumber 1750 cm⁻¹ which is corresponding to ester acyl.     

溶剂环境对乙肝表面抗原(HBsAg)结构的影响

HBsAg作为乙肝疫苗的主要成分,是一种病毒样颗粒,由蛋白质和脂类通过非共价键作用形成。HBsAg保持完整结构对其功能非常重要,而目前未见对其在溶液中结构变化的研究。考察了不同溶剂环境(温度、pH值、离子类型和盐浓度)对HBsAg结构的影响。实验发现,HBsAg在常温下比较稳定,但在温度超过60℃时稳定性明显下降;pH值小于4.0时引起不可逆聚集,但在pH5.0时的聚集部分可逆;不同离子对HBsAg的影响基本符合Hofmeister序列,不同之处是SO42-比F-更易引起HBsAg颗粒的聚集,在所考察的盐中,(NH4)2SO4对HBsAg有着较大的影响,0.4mol/L时就会引起HBsAg聚集,随着浓度增加,聚集现象更加严重,所以在HBsAg的疏水层析中要谨慎使用(NH4)2SO4。     

Insertions in the hepatitis B surface antigen. Effect on assembly and secretion of 22-nm particles from mammalian cells

The envelope protein of hepatitis B virus carrying the surface antigen, HBsAg, has the unique property of mobilizing cellular lipids into spherical or elongated particles, about 22 nm in diameter, which are secreted from mammalian cells. We have created mutant envelope proteins by insertion of various sequences of different lengths into two regions of the S gene encoding the major envelope protein. S genes carrying inserts in phase with HBsAg were expressed in mouse L cells from the simian virus 40 early promoter. Various single or double inserts in the two major hydrophilic domains of HBsAg were compatible with secretion of 22-nm particles. In all mutant envelope proteins studied, the HBsAg domains required for intracellular aggregation appeared to be intact. However, assembly into particles was not sufficient to assure transport into the extracellular space. The 22-nm HBsAg particle may be a useful vehicle for the export and presentation of foreign peptide sequences.     

Adenosine diphosphate-induced aggregation of human platelets in flow through tubes. I. Measurement of concentration and size of single platelets and aggregates.

A double infusion flow system and particle sizing technique were developed to study the effect of time and shear rate on adenosine diphosphate-induced platelet aggregation in Poiseuille flow. Citrated platelet-rich plasma, PRP, and 2 microM ADP were simultaneously infused into a 40-microliters cylindrical mixing chamber at a fixed flow ratio, PRP/ADP = 9:1. After rapid mixing by a rotating magnetic stirbar, the platelet suspension flowed through 1.19 or 0.76 mm i.d. polyethylene tubing for mean transit times, t, from 0.1 to 86 s, over a range of mean tube shear rate, G, from 41.9 to 1,000 s-1. Known volumes of suspension were collected into 0.5% buffered glutaraldehyde, and all particles in the volume range 1-10(5) microns 3 were counted and sized using a model ZM particle counter (Coulter Electronics Inc., Hialeah, FL) and a logarithmic amplifier. The decrease in the single platelet concentration served as an overall index of aggregation. The decrease in the total particle concentration was used to calculate the collision capture efficiency during the early stages of aggregation, and aggregate growth was followed by changes in the volume fraction of particles of successively increasing size. Preliminary results demonstrate that both collision efficiency and particle volume fraction reveal important aspects of the aggregation process not indicated by changes in the single platelet concentration alone.     

Effect of the N-terminal hydrophobic sequence of hepatitis B virus surface antigen on the folding and assembly of hybrid beta-galactosidase in Escherichia coli

To investigate the mechanism of inclusion body formation and the effect of a hydrophobic sequence on the in vivo polypeptide folding, the aggregation caused by recombinant fusion beta-galactosidase in Escherichia coli was examined. Two plasmids were constructed: pTBG(H-) carried only the preS2 sequence of the hepatitis B virus surface antigen (HBsAg) in front of the beta-galactosidase gene (lacZ) while pTBG(H+) carried an additional sequence encoding the amino-terminal hydrophobic sequence of the S region of HBsAg between preS2 and lacZ. Unlike cells expressing the fusion protein not containing the hydrophobic sequence, E. coli JM109/pTBG(H+) exhibited temperature-sensitive production of beta-galactosidase. As the culture temperature increased the activity decreased dramatically. This decrease in activity was not due to a decrease in fusion polypeptide production, but rather the fusion polypeptides containing the hydrophobic sequence aggregated within the cells at high temperature. However once the fusion polypeptides folded into proper conformation at low temperature, they maintained the activity even at high temperature. The results indicate that aggregation is a consequence of incorrect folding and assembly of the polypeptides, and is not derived from the native structure. The aggregates of the pTBG(H+)-encoded fusion polypeptides did not revert to active form when the culture temperature was lowered.     

Hepatitis B virus surface antigen assembly function persists when entire transmembrane domains 1 and 3 are replaced by a heterologous transmembrane sequence

Native hepatitis B surface antigen (HBsAg) spontaneously assembles into 22-nm subviral particles. The particles are lipoprotein micelles, in which HBsAg is believed to span the lipid layer four times. The first two transmembrane domains, TM1 and TM2, are required for particle assembly. We have probed the requirements for particle assembly by replacing the entire first or third TM domain of HBsAg with the transmembrane domain of HIV gp41. We found that either TM domain of HBsAg could be replaced, resulting in HBsAg-gp41 chimeras that formed particles efficiently. HBsAg formed particles even when both TM1 and TM3 were replaced with the gp41 domain. The results indicate remarkable flexibility in HBsAg particle formation and provide a novel way to express heterologous membrane proteins that are anchored to a lipid surface by their own membrane-spanning domain. The membrane-proximal exposed region (MPER) of gp41 is an important target of broadly reactive neutralizing antibodies against HIV-1, and HBsAg-MPER particles may provide a good platform for future vaccine development.     

Diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii

Aggregation of algae, mainly diatoms, is an important process in marine systems leading to the settling of particulate organic carbon predominantly in the form of marine snow. Exudation products of phytoplankton form transparent exopolymer particles (TEP), which acts as the glue for particle aggregation. Heterotrophic bacteria interacting with phytoplankton may influence TEP formation and phytoplankton aggregation. This bacterial impact has not been explored in detail. We hypothesized that bacteria attaching to Thalassiosira weissflogii might interact in a yet-to-be determined manner, which could impact TEP formation and aggregate abundance. The role of individual T. weissflogii-attaching and free-living new bacterial isolates for TEP production and diatom aggregation was investigated in vitro. T. weissflogii did not aggregate in axenic culture, and striking differences in aggregation dynamics and TEP abundance were observed when diatom cultures were inoculated with either diatom-attaching or free-living bacteria. The data indicated that free-living bacteria might not influence aggregation whereas bacteria attaching to diatom cells may increase aggregate formation. Interestingly, photosynthetically inactivated T. weissflogii cells did not aggregate regardless of the presence of bacteria. Comparison of aggregate formation, TEP production, aggregate sinking velocity and solid hydrated density revealed remarkable differences. Both, photosynthetically active T. weissflogii and specific diatom-attaching bacteria were required for aggregation. It was concluded that interactions between heterotrophic bacteria and diatoms increased aggregate formation and particle sinking and thus may enhance the efficiency of the biological pump.     

Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain

The mechanism of fibronectin (FN) assembly and the self-association sites are still unclear and contradictory, although the N-terminal 70-kDa region ((I)1-9) is commonly accepted as one of the assembly sites. We previously found that (I)1-9 binds to superfibronectin, which is an artificial FN aggregate induced by anastellin. In the present study, we found that (I)1-9 bound to the aggregate formed by anastellin and a small FN fragment, (III)1-2. An engineered disulfide bond in (III)2, which stabilizes folding, inhibited aggregation, but a disulfide bond in (III)1 did not. A gelatin precipitation assay showed that (I)1-9 did not interact with anastellin, (III)1, (III)2, (III)1-2, or several (III)1-2 mutants including (III)1-2KADA. (In contrast to previous studies, we found that the (III)1-2KADA mutant was identical in conformation to wild-type (III)1-2.) Because (I)1-9 only bound to the aggregate and the unfolding of (III)2 played a role in aggregation, we generated a (III)2 domain that was destabilized by deletion of the G strand. This mutant bound (I)1-9 as shown by the gelatin precipitation assay and fluorescence resonance energy transfer analysis, and it inhibited FN matrix assembly when added to cell culture. Next, we introduced disulfide mutations into full-length FN. Three disulfide locks in (III)2, (III)3, and (III)11 were required to dramatically reduce anastellin-induced aggregation. When we tested the disulfide mutants in cell culture, only the disulfide bond in (III)2 reduced the FN matrix. These results suggest that the unfolding of (III)2 is one of the key factors for FN aggregation and assembly.     

Analysis of shear-induced platelet aggregation with population balance mathematics.

Suspensions of blood platelets aggregate and degranulate when subjected to a shearing flow of sufficient intensity. This work examines, by means of a population balance technique, the kinetics of platelet aggregation in a shear field. The particle collision efficiency, epsilon, and the particle void volume fraction, phi, are estimated from particle number density data. The collision efficiency represents the fraction of particle collisions that result in the binding together of the involved particles. We term epsilon and phi population balance properties because they refer to physical characteristics of platelets and aggregates that are pertinent to their aggregation behavior. Experiments focused on the dependence of epsilon on platelet concentration, shearing rate, and time in a controlled shear field. The collision efficiency is lower in dilute platelet suspensions. This finding supports an ADP-mediated mechanism for shear aggregation. The collision efficiency passes through a maximum with respect to shearing rate, suggesting a competition between the opposing effects of increasing platelet activation and increasing collision violence. The collision efficiency is highest during the first ten seconds in the shear field and declines significantly thereafter. Even at its maximum, however, epsilon for shear aggregation is small: only about one in every thousand particle collisions results in binding.     

聚乙二醇伴随式离子交换层析分离重组乙肝病毒表面抗原

对由中国仓鼠卵巢细胞(CHO)表达的多聚亚基蛋白HBsAg在离子交换层析过程中容易因亚基解离而导致蛋白解聚和丧失生物活性的难题,实验中选择聚乙二醇（PEG）作为保护剂伴随式（Polyethylene Glycol-Accompanied）离子交换层析分离纯化HBsAg。实验表明,在流动相中加入1% PEG10000(W/V)作为纯化伴侣, HBsAg的回收率由55% 左右提高到80%以上,纯化倍数基本保持在12左右。对纯化产物进行SDS_PAGE分析表明,1% PEG10000的纯化伴侣伴随式离子交换层析能全部保留HBsAg的糖基化蛋白单体（27kD和30kD）,高效液相色谱联用多角度激光散射(High Performance Size Exclusion Chromatography_Multiangle Laser Light Scattering, HPSEC-MALLS )进一步分析阐明了PEG能促使HBsAg颗粒尺寸分布更均一,结构更接近天然乙肝表面抗原。     

Parameters of red blood cell aggregation as correlates of the inflammatory state

To identify clinically relevant parameters of red blood cell (RBC) aggregation, we examined correlations of aggregation parameters with C-reactive protein and fibrinogen in unstable angina (UA), acute myocardial infarction (AMI), and bacterial infection (BI). Aggregation parameters were derived from the distribution of RBC population into aggregate sizes (cells per aggregate) and changing of the distribution by flow-derived shear stress. Increased aggregation was observed in the following order: UA, AMI, and BI. The best correlation was obtained by integration of large aggregate fraction as a function of shear stress. To differentiate plasmatic from cellular factors in RBC aggregation, we determined the aggregation in the presence and absence of plasma and formulated a "plasma factor" (PF) ranging from 0 to 1. In AMI the enhanced aggregation was entirely due to PF (PF = 1), whereas in UA and BI it was due to both plasmatic and cellular factors (0 < or = PF < or = 1). It is proposed that clinically relevant parameters of RBC aggregation should express both RBC aggregate size distribution and aggregate resistance to disaggregation and distinguish between plasmatic and cellular factors.     

Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA).

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin-treated,32P-prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton-insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS-PAGE and autoradiography. Incorporation of actin into the Triton-insoluble fraction was used to quantify the formation of F-actin. Thrombin-stimulated platelet cytoskeletal composition was different from PMA-stimulated cytoskeletal composition. Thrombin-stimulated platelets contained not only the three major proteins: actin (43 kDa), myosin (200 kDa) and an actin-binding protein (250 kDa), but three additional proteins of Mr56 kDa, 80 kDa and 85 kDa in the cytoskeleton, which were induced in by thrombin dose-response relationship. In contrast, PMA-stimulated platelets only induced actin assembly, and the 56 kDa, 80 kDa and 85 kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56 kDa, 80 kDa and 85 kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin-activated platelets aggregation could involve additional cytoskeletal components (56 kDa, 80 kDa, 85 kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.     

The self-assembly of collagen molecules

The aggregation of native acid-soluble collagen (N-ASC) and of pronase-treated acid soluble collagen (P-ASC) was examined in solution under conditions which varied from those of minimum collagen-collagen interaction to those leading to incipient fiber formation. Molecular weights and weight distributions were determined in the analytical ultracentrifuge using the Yphantis high speed sedimentation equilibrium and Aarchiblad approach-to-equilibrim techniques. The aggregation was pH and ionic strength dependent in each case. Under conditions of minimum aggregation (low pH, low ionic strength), N-ASC showed the presence of permant aggregates. At higher pH and ionic strength, a higher fraction of aggregate was formed but these were of the same charcter and molecular weight as the permanent aggregates. The aggregates were of a single molecular size, with a weight of 1.5 × 10⁶ daltons, compared with a monomer collagen weight of 3.1 × 10⁵ daltons. The P-ASC formed aggregates also but to a much lower extent and the maximum aggregate size corresponded to dimers in molecular weight. These data show the major importance of molecular end-regions in collagen aggregation to form native type fibers and, by virtue of the discrete size of the N-ASC aggregates, support the microfibrillar hypothesis for the assembly of collagen fibrills.     

Sodium dodecylsulfate polyacrylamide gel electrophoresis of recombinant hepatitis B surface antigen particles

To investigate the factors leading to broadening of the recombinant hepatitis B surface antigen (HBsAg) peak in size-exclusion chromatography, the HBsAg particles eluting in different regions of the peak were subjected here to electrophoretic analysis. In nonreduced samples, the 24-kD band corresponding to the S monomer was detected when excessively large amounts of HBsAg were loaded onto the gel. Hence, some monomers are not disulfide-crosslinked in assembled particles. On the other hand, the results of alkylation experiments indicated the presence of free sulfhydryl group(s) in a little portion of freshly-purified HBsAg which was retarded on the size-exclusion chromatographic column and had significant antigenicity. This fraction of HBsAg was shown to be oligomeric and capable of spontaneous assembly into higher-order structures during aging.     

Role of the aggregation factor in the regulation of phosphoinositide metabolism in sponges. Possible consequences on calcium efflux and on mitogenesis

The aggregation factor (AF) of the marine sponge Geodia cydonium recognizes the aggregation receptor (AR) which is inserted in the plasma membrane, under formation of species-specific aggregates. The specific cell-binding fragment of the AF was used to investigate for the first time the phosphoinositide metabolism in a lower avertebrate system. We found that after binding of the cell-binding fragment to the aggregation receptor a strong and rapid stimulation of the phosphate incorporation into phosphatidylinositol occurs followed by an increased turnover of phosphoinositides in the Geodia cells. The consequences of an increased degradation of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol-1,4,5-trisphosphate and diacylglycerol are 2-fold. First, after the addition of the extracellular stimulus the cytosolic Ca2+ concentration rises, resulting in a rapid increased Ca2+ efflux rate. The functional consequence of the increase of the extracellular Ca2+ level is an initiation of the aggregate formation that is mediated by the collagen assembly factor (= primary aggregation factor). Second, some experimental evidences are presented, showing that the other second messenger formed, diacylglycerol, causes a translocation of protein kinase C within the cell. Incubation of Geodia cells with the cell-binding fragment of the AF, or with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, resulted within 5 min after treatment in a 70% decrease in protein kinase C activity in the cytosolic fraction and in a 700% increase in enzyme activity in the membrane fraction. It is proposed that by membrane association protein kinase C becomes activated. As a result of this event a series of cellular proteins are phosphorylated, a process which ultimately leads to an unusually strong induction of DNA polymerase alpha activity. From these data we conclude that inositol trisphosphate and protein kinase C also play a fundamental role in cellular signal transduction in lower eukaryotes.     

Membrane structure of the hepatitis B virus surface antigen particle

Expression of S protein, an envelope protein of hepatitis B virus, in the absence of other viral proteins, leads to the secretion of hepatitis B virus surface antigen (HBsAg) particles that are formed by budding from the endoplasmic reticulum membranes. The HBsAg particles produced by mouse fibroblast cells show a unique lipid composition, with 1,2-diacyl glycerophosphocholine being the dominant component. The lipid organization of the HBsAg particles was studied by measuring electron spin resonance (ESR) using various spin-labeled fatty acids, and the results were compared with a parallel study on HVJ (Sendai virus) and vesicles reconstituted with total lipids of the HBsAg particles (HBs-lipid vesicles). HVJ and the HBs-lipid vesicles showed typical ESR spectra of lipids arranged in a lipid bilayer structure. In contrast, the ESR spectra obtained with the HBsAg particles showed that the movement of lipids in the particle is severely restricted and a typical immobilized signal characteristic of tight lipid-protein interactions was also evident. Phosphatidylcholine (PC) in the HBsAg particles was not exchangeable by a PC-specific exchange protein purified from bovine liver, while phospholipase A(2) from Naja naja vemon was able to hydrolyze all the PC in the particles. These analyses suggest that the lipids in the HBsAg particles are not organized in a typical lipid bilayer structure, but are located at the surface of the particles and are in a highly immobilized state. Based on these observations we propose a unique lipid assembly and membrane structure model for HBsAg particles.     

Mutational analysis of hepatitis B surface antigen particle assembly and secretion.

Cells infected with hepatitis B virus produce both virions and 20-nm subviral (surface antigen or HBsAg) particles; the latter are composed of viral envelope proteins and host-derived lipid. Although hepatitis B virus encodes three envelope proteins (L, M, and S), all of the information required to produce an HBsAg particle resides within the S protein. This polypeptide spans the bilayer at least twice and contains three hydrophobic regions, two of which are known to harbor topogenic signal sequences that direct this transmembrane orientation. We have examined the effects of mutations in these and other regions of the S protein on particle assembly and export. Lesions in the N terminal signal sequence (signal I) can still insert into the endoplasmic reticulum bilayer but do not participate in any of the subsequent steps in assembly. Deletion of the major internal signal (signal II) completely destabilizes the chain. Deletion of the C-terminal hydrophobic domain results in a stable, glycosylated, but nonsecreted chain. However, when coexpressed with wild-type S protein this mutant polypeptide can be incorporated into particles and secreted, indicating that the chain is still competent for some of the distal steps in particle assembly. The correct transmembrane disposition of the N terminus of the molecule is important for particle formation: addition of a heterologous (globin) domain to this region impairs secretion, but the defect can be corrected by provision of an N-terminal signal sequence that restores the proper topology of this region. The resulting chimeric chain is assembled into subviral particles that are secreted with normal efficiency.     

Enhanced intracellular accumulation of recombinant HBsAg in CHO cells by dimethyl sulfoxide

The intracellular hepatitis B surface antigen (HBsAg) content per cell was significantly increased by 7.2-fold in the culture of recombinant CHO cells with 1.5% dimethyl sulfoxide (DMSO), while the HBsAg production and specific productivity were only improved by 70% and 3.2-fold, respectively. The significant accumulation of HBsAg within rCHO cells by DMSO stimulation was testified with flow cytometry measurements. Electron microscopy was applied to show that the dilated areas scattered over whole cytoplasm within rCHO cells in response to DMSO, and further revealed that intracellular HBsAg was localized to these areas with immunogold labeling. The failure of intracellular HBsAg virus-like particle assembly was revealed to be closely associated with the HBsAg accumulation within DMSO-stimulated rCHO cells on the basis of sucrose gradient analysis of cell extract. This work provided the details to further understand the HBsAg accumulation within rCHO cells in response to DMSO.