Severe acute respiratory syndrome coronavirus 7a accessory protein is a viral structural protein

Severe acute respiratory syndrome coronavirus (SCoV) 7a protein is one of the viral accessory proteins. In expressing cells, 7a protein exhibits a variety of biological activities, including induction of apoptosis, activation of the mitogen-activated protein kinase signaling pathway, inhibition of host protein translation, and suppression of cell growth progression. Analysis of SCoV particles that were purified by either sucrose gradient equilibrium centrifugation or a virus capture assay, in which intact SCoV particles were specifically immunoprecipitated by anti-S protein monoclonal antibody, demonstrated that 7a protein was associated with purified SCoV particles. Coexpression of 7a protein with SCoV S, M, N, and E proteins resulted in production of virus-like particles (VLPs) carrying 7a protein, while 7a protein was not released from cells expressing 7a protein alone. Although interaction between 7a protein and another SCoV accessory protein, 3a, has been reported, 3a protein was dispensable for assembly of 7a protein into VLPs. S protein was not required for the 7a protein incorporation into VLPs, and yet 7a protein interacted with S protein in coexpressing cells. These data established that, in addition to 3a protein, 7a protein was a SCoV accessory protein identified as a SCoV structural protein.     

Formation of Protein Bodies in the Starchy Endosperm of Rice (Oryza sativa L.): A Re-investigation

The mechanism of protein body formation in the starchy endospermis described for two rice varieties, one of normal protein contentand the other high in protein. Three types of protein bodieswere found in both rices. While each protein body type was depositeddifferently, the mechanism of formation for the same type inthe two varieties was analogous. First secreted was the largespherical protein body. It was deposited within rough endoplasmicreticulum, had a dense centre and a concentric ring appearance,and was bounded by a single membrane. The second protein bodyto form was crystalline, was found in vacuoles, and was secretedvia the Golgi apparatus. The third type, the small sphericalprotein body, was secreted late in development, was depositedin vesicular rough endoplasmic reticulum, and lacked dense centresand concentric rings. The high protein rice had a thicker sub-aleuronelayer than the normal protein rice. Oryza sativa L., rice, protein bodies, starchy endosperm, ultrastructure     

Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions.     

Streptococcal protein G-gold complex: comparison with staphylococcal protein A-gold complex for spot blotting and immunolabeling

Protein G, a cell wall protein isolated from human group G streptococci strain G148, binds in a similar manner as protein A from Staphylococcus aureus to the Fc portion of IgG molecules. Indeed, protein G has been proposed as a superior Fc binding protein due to its broader species reactivity. Thus, we have prepared a complex of protein G with particles of colloidal gold and determined its applicability for spot-blot analysis and postembedding immunolabeling by comparing it with protein A-gold complex. By spot-blot analysis no difference in binding of protein G-gold or protein A-gold to IgG molecules from a whole spectrum of animal species was observed. Moreover, using rabbit, sheep, or goat anti-rat albumin antibodies to detect nitrocellulose-immobilized rat albumin or antigenic sites in paraffin and Lowicryl K4M thin sections from rat liver, no difference was found with protein G-gold or protein A-gold. Similarly, no difference in binding to protein G-gold or protein A-gold was observed with a battery of monoclonal antibodies. However, in contrast to expectations, protein A-gold reacted well with both sheep and goat IgG molecules; indeed, for the light and electron microscopic localization of albumin with sheep or goat antibodies it was as efficient as protein G-gold. These results demonstrate, therefore, that both protein G-gold and protein A-gold are useful second step reagents for immunolabeling and that protein G-gold was not a superior probe in the systems tested.     

Isolation and Characterization of a Novel Chitosan-Binding Protein from Non-Heading Chinese Cabbage Leaves

To know the mechanism of ammonia assimilation in non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis (L.) Makino) leaves regulated by chitosan (CTS), a CTS-binding protein was isolated from non-heading Chinese cabbage leaves using the chitosan affinity chromatography approach and this CTS-binding protein was partially characterized. The profile of the 53.1 kDa purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was compared with the native molecular weight of 106.5 kDa, which indicated that the purified protein was a dimer with identical subunits. After isoelectric focusing, a band was obtained at pH 8.25. The agglutination test and periodic acid-Schiff staining further revealed that the protein was a glycoprotein with lectin activity. Moreover, the purified protein contained 17.4% (w/w) neutral carbohydrate and 82.56% (w/w) protein. The comparison of this protein and the 67 kDa CTS-binding protein isolated previously from Rubus culture tissue exhibited some differences in characterization. According to results of peptide mass fingerprinting analysis, the protein purified in the present study does not show any similarity with any protein in the protein data bank. Thus, it was deduced that the protein purified in the present study is a novel CTS-binding protein.     

The cytoplasmic tail of the human respiratory syncytial virus F protein plays critical roles in cellular localization of the F protein and infectious progeny production

The importance of the F protein cytoplasmic tail (CT) for replication of human respiratory syncytial virus (HRSV) was examined by monitoring the behavior of viruses expressing F proteins with a modified COOH terminus. The F protein mutant viruses were recovered and amplified under conditions where F protein function was complemented by expression of a heterologous viral envelope protein. The effect of the F protein modifications was then examined in the context of a viral infection in standard cell types (Vero and HEp-2). The F protein modifications consisted of a deletion of the predicted CT or a replacement of the CT with the CT of the vesicular stomatitis virus (VSV) G protein. In addition, engineered HRSVs that lacked all homologous glycoprotein genes (SH, G, and F) and expressed instead either the authentic VSV G protein or a VSV G containing the HRSV F protein CT were examined. We found that deletion or replacement of the F protein CT seriously impaired the production of infectious progeny. Cells infected with viruses bearing CT modifications displayed increased F protein surface expression and increased syncytium formation. The distribution of F protein in the plasma membrane of infected cells was altered, resulting in an F protein that was evenly distributed rather than localized predominantly to virus-induced surface filaments. CT deletion or exchange also abrogated interaction of F protein with Triton-insoluble lipid rafts. Addition of the F protein CT to the VSV G protein, expressed as the only viral glycoprotein in an HRSV genome, had the opposite effects: the number of infectious progeny was higher, the surface distribution was changed from relatively even to localized, and the proportion of VSV G protein associated with lipid rafts was higher. Together, these results show that the HRSV F protein CT plays a critical role in F protein cellular localization and production of infectious virus and suggest that the function provided by the CT is independent of the F protein ectodomain and transmembrane domain and is mediated by F protein-lipid raft interaction.     

Determination of nonspecific lipid transfer protein in rat tissues and Morris hepatomas by enzyme immunoassay

Rat tissues contain a nonspecific transfer protein which in vitro mediates the transfer of diacylphospholipids as well as cholesterol between membranes. This protein appears identical to sterol carrier protein. A specific enzyme immunoassay for this protein was developed using antibodies raised in rabbits, against a homogeneous protein from rat liver. This assay was based on the very high affinity of the nonspecific lipid transfer protein for polyvinyl surfaces. A reproducible adsorption was achieved by presenting the protein to the surface in the presence of a large excess of bovine serum albumin. The adsorbed protein was detected with specific immunoglobulin (IgG) isolated by antigen-linked affinity chromatography and a goat anti-rabbit IgG-enzyme conjugate. Adsorption was proportional to the amount of protein present, giving rise to a linear standard curve. The enzyme immunoassay measured transfer protein levels in the range 0.2-2 ng. The highest concentrations of transfer protein were found in liver and intestinal mucosa. Levels in other tissues including brain, lung, kidney, spleen, heart, adrenals, ovary and testis were 5-10-fold lower than in liver. In the fast-growing Morris hepatoma 7777 the concentration of nonspecific lipid transfer protein was approximately one-tenth of that measured in the host liver, whereas a reduction of 65% was observed in the slow-growing Morris hepatomas 7787 and 9633. Subcellular distribution studies showed that approx. 70% of the transfer protein was present in the soluble supernatant fraction.     

Isolation and Characterization of a Novel Chitosan-Binding Protein from Non-Heading Chinese Cabbage Leaves

To know the mechanism of ammonia assimilation in non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis (L.) Makino) leaves regulated by chitosan (CTS), a CTS-binding protein was isolated from non-heading Chinese cabbage leaves using the chitosan affinity chromatography approach and this CTS-binding protein was partially characterized. The profile of the 53.1 kDa purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was compared with the native molecular weight of 106.5 kDa, which indicated that the purified protein was a dimer with identical subunits. After isoelectric focusing, a band was obtained at pH 8.25. The agglutination test and periodic acid-Schiff staining further revealed that the protein was a glycoprotein with lectin activity. Moreover, the purified protein contained 17.4 % (w/w) neutral carbohydrate and 82.56% (w/w) protein. The comparison of this protein and the 67 kDa CTS-binding protein isolated previously from Rubus culture tissue exhibited some differences in characterization. According to results of peptide mass fingerprinting analysis, the protein purified in the present study does not show any similarity with any protein in the protein data bank. Thus, it was deduced that the protein purified in the present study is a novel CTS-binding protein.     

Identification of a new protein involved in the regulation of the anticoagulant activity of activated protein C. Protein S-binding protein

The apparent molecular weight of functional protein S in citrated plasma was observed to be between 115,000 and 130,000 as measured by sedimentation equilibrium in the air-driven ultracentrifuge. The molecular weight of the functional protein decreased to approximately 62,000 when copper ions were added to the plasma. This suggested the presence of a protein S-binding protein in plasma, which was confirmed by gel filtration experiments. Frontal analysis of plasma indicated that functional protein S could exist in as many as three forms. Addition of copper ions to plasma reduced the number of forms to one. In order to isolate the binding protein, plasma was fractionated first on a column of immobilized iminodiacetic acid that had been equilibrated with copper ions. The proteins that eluted in a 0.6 M NaCl wash were passed over a column of protein S immobilized on agarose beads. A protein, eluted in the 0.6 M NaCl wash, was observed to bind to protein S in gel filtration experiments. When added to plasma depleted of both protein S and the binding protein, the binding protein was observed to enhance the anticoagulant activity of activated protein C only in the presence of protein S. Protein S-binding protein was also observed to enhance the rate of factor Va inactivation by activated protein C and protein S.     

Regulation of vitamin K-dependent protein S. Inactivation by thrombin

Thrombin treatment of the vitamin K-dependent protein S resulted in the loss of the activated protein C cofactor activity associated with protein S. The addition of phospholipid vesicles inhibited the inactivation. Thrombin treatment did not alter the molecular weight of the native protein. However, upon reduction, a peptide of approximately 3000 daltons was released from the treated protein. The interaction between calcium and protein S was reduced by thrombin treatment. When the calcium interaction was determined by the quenching of the intrinsic fluorescence of protein S, thrombin treatment appeared to inhibit the interaction between calcium and the protein. When the calcium interaction was observed by measuring the effect on the electrophoretic mobility of the protein, thrombin treatment reduced the interaction between calcium and protein S. However, the effect of thrombin treatment on the interaction between calcium and protein S was less than observed by the fluorescent method. This observation suggests that fluorescence quenching may be a result of a structural change induced by calcium binding. Thrombin treatment of protein S appears to uncouple the calcium binding from the structural change. In addition, the interaction between protein S and phospholipid vesicles was reduced by thrombin treatment. These results suggest that the thrombin conversion of protein S into a two-chain protein causes the loss of a calcium-induced change in protein structure, loss of the lipid-binding properties, and the loss of cofactor activity.     

Inhibition of human vitamin-K-dependent protein-S-cofactor activity by a monoclonal antibody specific for a Ca2+-dependent epitope

Protein S is an anticoagulant vitamin-K-dependent plasma protein functioning as a cofactor to activated protein C in the degradation of factors Va and VIIIa. A murine monoclonal antibody, HPS 7, specific for a calcium-stabilized epitope in human protein S, is described. The epitope was available in intact protein S, both in its free form and when protein S was bound to C4b-binding protein. It disappeared upon reduction of disulfide bridges and also after thrombin of chymotrypsin cleavage of protein S. Thrombin cleaves protein S close to the calcium-binding region containing gamma-carboxyglutamic acid (Gla). The cleaved protein still contains the Gla region, linked by a disulfide bridge, but it has a lower affinity for calcium and no protein C cofactor activity. The thrombin-mediated cleavage of protein S could be inhibited by HPS 7. The Ka for the interaction between protein S and the monoclonal was estimated to be approximately 0.7 X 10(8) M-1. Half-maximal binding between HPS 7 and protein S was observed at a calcium concentration of 0.50 mM, indicating that saturation of the Gla region with calcium was required for the interaction. The recently reported Gla-independent high-affinity calcium binding did not induce the epitope. The calcium-dependent binding of protein S to phospholipid vesicles as well as the protein C cofactor activity was inhibited by HPS 7. The data suggests that the epitope for HPS 7 is located in the Gla region of protein S or in the closely positioned thrombin-sensitive region.     

Newly Imported Rieske Iron-Sulfur Protein Associates with Both Cpn60 and Hsp70 in the Chloroplast Stroma

The precursor of the Rieske FeS protein, a thylakoid membrane protein, was imported by isolated pea chloroplasts, and the mature protein was shown to be integrated into the cytochrome bf complex of the thylakoid membranes. Insertion into the thylakoid membrane was sensitive to the ionophores nigericin and valinomycin, suggesting a requirement for a proton motive force. A considerable proportion of the imported Rieske protein was detected in the stromal fraction of the chloroplasts, and this increased when membrane insertion was blocked with ionophores. Electrophoresis of the stromal fraction under nondenaturing conditions resolved two distinct complexes containing the Rieske protein. One of these complexes was identified as an association of the Rieske protein with the chaperonin Cpn60 complex by its electrophoretic mobility, Mg-ATP-dependent dissociation, and immunoprecipitation with anti-Cpn60 antibodies. Coimmunoprecipitation of imported Rieske protein with anti-heat shock protein 70 (Hsp70) antibodies indicated that the Rieske protein was also associated, in an ATP-dissociable form, with a chloroplast Hsp70 homolog. Immunoprecipitation analysis of an import time course detected the highest amounts of the Cpn60-Rieske protein complex early in the time course, whereas highest amounts of the Hsp70-Rieske protein complex were formed much later. The disappearance of the Cpn60-Rieske protein complex correlated with increased amounts of the Rieske protein in the thylakoid fraction.     

The quaternary structure, antigenicity, and aggregational behavior of the secretory core protein of human hepatitis B virus are determined by its signal sequence.

Human hepatitis B virus encodes a secretory core protein, referred to as the HBe protein, whose secretion is mediated by the pre-C signal sequence. Here we examined whether this sequence is important only for translocation of the HBe precursor (the precore protein) or whether it also contributes to the structural and biophysical properties of the mature HBe protein. When a truncated hepatitis B virus precore protein, lacking the basic C-terminal domain which is cleaved from the wild-type protein during its conversion into HBe, was expressed in human hepatoma cells, only a small amount of HBe-like protein was produced. This protein was slightly smaller than the wild-type HBe protein, suggesting that C-terminal cleavage of the precore protein does not occur at the suggested site. When the authentic signal sequence of the precore protein (the pre-C sequence) was replaced by the unrelated signal sequence of an influenza virus hemagglutinin, not only the full-length but also the C-terminally truncated protein was expressed and secreted with high efficiency. Western blot (immunoblot) analyses with nonreducing gels and conformation-specific monoclonal antibodies revealed that the HBe protein secreted under control of the pre-C signal sequence was a monomer with HBe antigenicity, whereas the HBe-like protein secreted under control of the hemagglutinin signal sequence was a disulfide-bridge-linked dimer with both HBe and HBc antigenicity. Electron microscopic examination of gradient-purified particulate core gene products showed that HBe protein secreted under control of the hemagglutinin signal sequence forms core particles, whereas HBe protein secreted under control of the pre-C sequence does not. Thus, the pre-C sequence not only mediates the secretion but also determines the structural and aggregational properties of the HBe protein.     

Effect of Protein A on Adsorption of Bacteriophages to Staphylococcus aureus

Experiments were performed to determine if protein A influenced the association of bacteriophages with Staphylococcus aureus. Bacteriophage adsorption was compared in a S. aureus strain rich in protein A and mutants of this strain with very little protein A, in a strain with little protein A, and in mutants of this strain with increased protein A. In addition, the effect of growth in mannitol-salt broth and trypsin digestion (known to reduce protein A) on bacteriophage absorption was measured. There was an inverse relationship between protein A content of strains and the quantity of bacteriophage absorbed. However, no inhibition of staphylococcal phages was obtained with purified soluble protein A. Protein A as a surface component rendered the bacteria more resistant to adsorption of staphylococcal typing phages presumably by masking the phage receptor sites. When protein A-deficient mutants were incubated with bacteriophages, there was survival of staphylococci with increased protein A content probably due to a selective action.     

Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein.

The surface protein composition of members of a serogroup of Aeromonas hydrophila which exhibit high virulence for fish was examined. Treatment of whole cells of representative strain A. hydrophila TF7 with 0.2 M glycine buffer (pH 4.0) resulted in the release of sheets of a tetragonal surface protein array. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis showed that this sheet material was composed primarily of a protein of apparent molecular weight 52,000 (52K protein). A 52K protein was also the predominant protein in glycine extracts of other members of the high-virulence serogroup. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed the 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase 125I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size was required for A. hydrophila S-layer anchoring. The 52K S-layer protein exhibited hear-dependent SDS-solubilization behavior when associated with OM, but was fully solubilized at all temperatures after removal from the OM, indicating a strong interaction of the S layer with the underlying OM. The native S layer was permeable to 125I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behaviour characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein.     

Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. II. Immunological quantitation

The envelope of vesicular stomatitis virus was fused with the apical plasma membrane of Madin-Darby canine kidney cells by low pH treatment. The fate of the implanted G protein was then followed using a protein A- binding assay, which was designed to quantitate the amount of G protein in the apical and the basolateral membranes. The implanted G protein was rapidly internalized at 31 degrees C, whereas at 10 degrees C no uptake was observed. Already after 15 min at 31 degrees C, a fraction of the G protein could be detected at the basolateral membrane. After 60 min 25-48% of the G protein was basolateral as measured by the protein A-binding assay. At the same time, 25-33% of the implanted G protein was detected at the apical membrane. Internalization of G protein was not affected by 20 mM ammonium chloride or by 10 microM monensin. However, the endocytosed G protein accumulated in intracellular vacuoles and redistribution back to the plasma membrane was inhibited. We conclude that the implanted G protein was rapidly internalized from the apical surface of Madin-Darby canine kidney cells and a major fraction was routed to the basolateral domain.     

Expression and characterization of a tripartite fusion protein consisting of chimeric IgG-binding receptors and beta-galactosidase.

Using protein engineering, a tripartite fusion protein was constructed consisting of five IgG-binding regions of protein A from Staphylococcus aureus, two IgG-binding regions of protein G from Streptococcus strain G148 and beta-galactosidase from Escherichia coli. The resulting protein lacks the serum albumin binding regions of native protein G. The fusion protein, which is a tetramer of approximately 660 kDa, was designed as a tool for immunological assays taking advantage of its broad spectrum of antibody affinity. The gene was placed under control of two promoters, the PR promoter and the lac UV5 promoter and the expression from the two promoters was studied in a bioreactor. Induction of the PR promoter gave an intracellular product concentration corresponding to 20% of the cell dry weight. By utilizing the properties of beta-galactosidase, the protein was purified by extraction in an aqueous two-phase system. The fusion protein was not proteolytically degraded during the cultivation and purification steps. The biological activity of all three parts of the protein was demonstrated with a competitive ELISA.     

In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways

Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2(+)-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Km for C3 was 2.6 microM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis.     

Polyhedrin initiator codon altered to AUU yields unexpected fusion protein from a baculovirus vector

A recombinant baculovirus expression vector was constructed to express the core (capsid) protein of the hepatitis B virus. Along with the expected 21-kDa polypeptide, a second 24-kDa protein was observed. Immunoprecipitation and immunoblotting using a rabbit polyclonal anticore antiserum demonstrated that the two proteins were related. The core gene originally was cloned in-frame with the polyhedrin initiator codon that had been altered to AUU as a means of preventing fusion protein formation. A transient expression assay revealed expression of the 24-kDa protein was prevented if a frame-shift mutation was created upstream of the HBV core translation start site. These results suggest that the 24-kDa protein was the result of an unexpectedly high level of translation initiation at the AUU codon that gave rise to a polyhedrin-HBV core fusion protein. The 24-kDa core protein was shown to be a polyhedrin fusion protein by immunoblotting with an antipolyhedrin antiserum, and initiation at the AUU was demonstrated by amino terminal protein sequencing. Methods to prevent undesired fusion protein expression using this or similar vectors are discussed.     

A major outer membrane protein of Serratia marcescens which was easily solubilized and had a capacity to bind to calcium

Easily solubilized major outer membrane protein was found in Serratia marcescens. The protein was originally obtained as a membrane-associated, insoluble protein in the outer membrane when the cells were slightly disrupted. However, the amount of this protein in the outer membrane gradually decreased with the time of sonication. The decrease was not due to decomposition of the protein but to solubilization into a soluble fraction after a long period of disruption. The molecular weight of this protein was 47 kDa and it bound calcium. Another 40 kDa calcium binding protein was also found in the outer membrane of S. marcescens.