Positive charge of Arg-201 on hemagglutinin is required for the binding of H6N1 avian influenza virus to its target through a two-step process.

In our previous study, we produced a monoclonal antibody EB2 that recognized an epitope in the HA1 domain on the hemagglutinin (HA) of H6N1 influenza virus (A/chicken/Taiwan/2838 V/00). The residue Arg-201 (R201) on this epitope was protected by the glycan at Asn-167 (N167) from tryptic digestion; therefore, the infectivity of the virus was retained. R201 was extremely conserved in various subtypes of the influenza virus. To explore the role of R201 and the protecting glycan, we developed a bi-cistronic baculovirus expression system for the production of H6HA1 and H6HA0 (nearly full-length HA), which were glycosylated in insect cells. The expressed H6HA1 was mostly found in the trimeric form, and the H6HA0 protein was only found in the monomeric form. The trimeric H6HA1 was resistant to tryptic digestion; however, it could not bind to fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans. By contrast, the monomeric H6HA0 could bind to fetuin but was sensitive to tryptic digestion. We found that the positive charge on R201 was critical for binding HA to the negatively charged surface of host cells because the mutant R201A of H6HA0 lost its binding capacity substantially. Moreover, this binding capacity was dependent on the pH value and inhibited by free electrically charged amino acids. We propose a two-step model for binding the influenza virus with a host cell. The first step involved the specific recognition of the receptor binding site on HA to the sialylated glycan on the host cell. After the virus is engulfed by the acidic endosome, R201 could bind to the cell surface with stronger interactions and trigger the fusion process.

Viromimetic STING Agonist-Loaded Hollow Polymeric Nanoparticles for Safe and Effective Vaccination against Middle East Respiratory Syndrome Coronavirus.

The continued threat of emerging, highly lethal infectious pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV) calls for the development of novel vaccine technology that offers safe and effective prophylactic measures. Here, a novel nanoparticle vaccine is developed to deliver subunit viral antigens and STING agonists in a virus-like fashion. STING agonists are first encapsulated into capsid-like hollow polymeric nanoparticles, which show multiple favorable attributes, including a pH-responsive release profile, prominent local immune activation, and reduced systemic reactogenicity. Upon subsequent antigen conjugation, the nanoparticles carry morphological semblance to native virions and facilitate codelivery of antigens and STING agonists to draining lymph nodes and immune cells for immune potentiation. Nanoparticle vaccine effectiveness is supported by the elicitation of potent neutralization antibody and antigen-specific T cell responses in mice immunized with a MERS-CoV nanoparticle vaccine candidate. Using a MERS-CoV-permissive transgenic mouse model, it is shown that mice immunized with this nanoparticle-based MERS-CoV vaccine are protected against a lethal challenge of MERS-CoV without triggering undesirable eosinophilic immunopathology. Together, the biocompatible hollow nanoparticle described herein provides an excellent strategy for delivering both subunit vaccine candidates and novel adjuvants, enabling accelerated development of effective and safe vaccines against emerging viral pathogens.

A secretary bi-cistronic baculovirus expression system with improved production of the HA1 protein of H6 influenza virus in insect cells and Spodoptera litura larvae.

A bi-cistronic baculovirus expression vector was constructed to facilitate the expression, detection, and isolation of the hemagglutinin (HA) fragment HA1 of H6N1 avian influenza virus (AIV) in an insect and a culture of its cells. In this construct, the GP67sp signal peptide promoted the secretion of the recombinant protein into the culture medium, and improved protein expression and purification. Enhanced green fluorescent protein, co-expressed through an internal ribosome entry site, served as a visible reporter for protein expression detection. The hemolymph of Spodoptera litura larvae infected with the bi-cistronic baculovirus was collected for the purification of the recombinant HA1, which was found to be glycosylated, and monomeric and trimeric forms of the recombinant HA1 were identified. Proteins expressed in both the cell culture and larvae served as effective subunit vaccines for the production of antiserum against HA. The antiserum recognized the H6 subtype of AIV but not the H5 subtype.

Plastidial α-glucan phosphorylase 1 complexes with disproportionating enzyme 1 in Ipomoea batatas storage roots for elevating malto-oligosaccharide metabolism.

It has been proposed that malto-oligosaccharides (MOSs) are possibly recycled back into amylopectin biosynthesis via the sequential reactions catalyzed by plastidial α-glucan phosphorylase 1 (Pho1) and disproportionating enzyme 1 (Dpe1). In the present study, the reciprocal co-immunoprecipitation experiments using specific antibodies against Pho1 and Dpe1 demonstrated that these two enzymes can form a complex (the PD complex) in Ipomoea batatas storage roots. The immunohistochemistry analyses also revealed the co-localization of Pho1 and Dpe1 in the amyloplasts, and the protein levels of Pho1 and Dpe1 increased gradually throughout sweet potato storage root development. A high molecular weight PD complex was co-purified from sweet potato storage root lysates by size exclusion chromatography. Enzyme kinetic analyses showed that the PD complex can catalyze maltotriose and maltotetraose to generate glucose-1-phosphate in the presence of inorganic phosphate, and it also performs greater Dpe1 activity toward MOSs than does free form Dpe1. These data suggest that Pho1 and Dpe1 may form a metabolon complex, which provides elevated metabolic fluxes for MOS metabolism via a direct transfer of sugar intermediates, resulting in recycling of glucosyl units back into amylopectin biosynthesis more efficiently.

Multi-antigen avian influenza a (H7N9) virus-like particles: particulate characterizations and immunogenicity evaluation in murine and avian models.

BACKGROUND: Human infection with avian influenza A virus (H7N9) was first reported in China in March 2013. Since then, hundreds of cases have been confirmed showing severe symptoms with a high mortality rate. The virus was transmitted from avian species to humans and has spread to many neighboring areas, raising serious concerns over its pandemic potential. Towards containing the disease, the goal of this study is to prepare a virus-like particle (VLP) that consists of hemagglutinin (HA), neuraminidase (NA) and matrix protein 1 (M1) derived from the human isolate A/Taiwan/S02076/2013(H7N9) for potential vaccine development. RESULTS: Full length HA, NA, and M1 protein genes were cloned and expressed using a baculoviral expression system, and the VLPs were generated by co-infecting insect cells with three respective recombinant baculoviruses. Nanoparticle tracking analysis and transmission electron microscopy were applied to verify the VLPs' structure and antigenicity, and the multiplicity of infection of the recombinant baculoviruses was adjusted to achieve the highest hemagglutination activity. In animal experiments, BALB/c mice and specific-pathogen-free chickens receiving the VLP immunization showed elevated hemagglutination inhibition serum titer and antibodies against NA and M1 proteins. In addition, examination of cellular immunity showed the VLP-immunized mice and chickens exhibited an increased splenic antigen-specific cytokines production. CONCLUSIONS: The H7N9 VLPs possess desirable immunogenicity in vivo and may serve as a candidate for vaccine development against avian influenza A (H7N9) infection.

Glycosylation at hemagglutinin Asn-167 protects the H6N1 avian influenza virus from tryptic cleavage at Arg-201 and maintains the viral infectivity.

Cleavage of the hemagglutinin (HA) precursor (HA0) by trypsin, which produces the active HA1 and HA2 complex, is a critical step for activating the avian influenza virus (AIV). However, other tryptic cleavage sites on HA might also cause HA degradation and affect the virulence. Otherwise, HA is modified by glycosylation in the host cell. The conjugated glycans on HA may hinder the antigenic epitopes, and thus prevent the virus from being recognized and attacked by the antibodies. In this study, we observed that glycosylation at the Asn-167 (N167) site on the HA1 of the H6N1 AIV strain A/chicken/Taiwan/2838V/00 (2838V) protected Arg-201 (R201) from tryptic cleavage. The 2838V HA protein became sensitive to tryptic cleavage, whereas the glycans at N167 were removed by N-glycosidase F (PNGase-F). Furthermore, the infectivity of 2838V decreased when pretreated with PNGase-F and trypsin. Our observations suggest that the inaccessibility of the R201 residue of HA1 for tryptic cleavage, which is sterically hindered by glycosylation at N167, is a crucial factor for determining the infectivity of the AIV.

A monoclonal antibody recognizes a highly conserved neutralizing epitope on hemagglutinin of H6N1 avian influenza virus.

Neutralizing antibodies on the globular head of the hemagglutinin (HA) of avian influenza virus (AIV) are crucial for controlling this disease. However, most neutralizing antibodies lack cross reaction. This report describes the identification of a hemagglutinin epitope on the globular head near the receptor binding site of the H6N1 AIV. A monoclonal antibody named EB2 was prepared against the H6N1 AIV HA. Flow cytometry of AIV-infected chicken embryo fibroblast, DF-1 cells and specific-pathogen-free embryonated eggs were used to verify the neutralizing activity of this mAb. To narrow down the binding region, partially overlapping HA fragments and synthetic peptides were used to map the epitope by immune-blotting. The linear motif RYVRMGTESMN, located on the surface on the globular head of the HA protein, was identified as the epitope bound by EB2 mAb. Alignment of the EB2-defined epitope with other H6 AIVs showed that this epitope was conserved and specific to H6. We propose that this motif is a linear B-cell epitope of the HA protein and is near the receptor binding site. The identified epitope might be useful for clinical applications and as a tool for further study of the structure and function of the AIV HA protein.

Tentative identification of the second substrate binding site in Arabidopsis phytochelatin synthase.

Phytochelatin synthase (PCS) uses the substrates glutathione (GSH, γGlu-Cys-Gly) and a cadmium (Cd)-bound GSH (Cd∙GS2) to produce the shortest phytochelatin product (PC2, (γGlu-Cys)2-Gly) through a ping-pong mechanism. The binding of the 2 substrates to the active site, particularly the second substrate binding site, is not well-understood. In this study, we generated a structural model of the catalytic domain of Arabidopsis AtPCS1 (residues 12-218) by using the crystal structure of the γGlu-Cys acyl-enzyme complex of the PCS of the cyanobacterium Nostoc (NsPCS) as a template. The modeled AtPCS1 revealed a cavity in proximity to the first substrate binding site, consisting of 3 loops containing several conserved amino acids including Arg152, Lys185, and Tyr55. Substitutions of these amino acids (R152K, K185R, or double mutation) resulted in the abrogation of enzyme activity, indicating that the arrangement of these 2 positive charges is crucial for the binding of the second substrate. Recombinant AtPCS1s with mutations at Tyr55 showed lower catalytic activities because of reduced affinity (3-fold for Y55W) for the Cd∙GS2, further suggesting the role of the cation-π interaction in recognition of the second substrate. Our study results indicate the mechanism for second substrate recognition in PCS. The integrated catalytic mechanism of PCS is further discussed.

Preparation of monoclonal antibodies against poor immunogenic avian influenza virus proteins.

This study established a novel method of pre-screening peptides for monoclonal antibody (mAb) production. Whole virus particles were used as antigens to produce mAbs in the first stage. However, most mAbs obtained from this method were aimed toward hemagglutinin. For this reason, synthetic peptides were used as antigens for mAb production that aimed at the AIV proteins with low abundance or poor immunogenicity in the virus particle. The peptides that showed high immunogenicity were designed using bioinformatic tools for immunization. For high-throughput, a rabbit was used to screen the immunogenicities of the synthetic peptides. Those showed high immunity were used for mAb preparation in mice. Several new mAbs against PB2, PA, M1, M2, NS1 and NS2 proteins were successfully obtained in this study. Furthermore, the epitopes of M1 and NS1 mAbs were determined using competitive western blot assay and competitive ELISA. This study might simplify the mAb preparation and serves as the basis for developing mAb against poor immunogenic proteins.

Lemon protein disulfide isomerase: cDNA cloning and biochemical characterization.

BACKGROUND: Protein disulfide isomerases (PDIs), a family of structurally related enzymes, aid in protein folding by catalyzing disulfide bonds formation, breakage, or isomerization in newly synthesized proteins and thus. RESULTS: A ClPDI cDNA (1828 bp, GenBank accession HM641784) encoding a putative PDI from Citrus limonum was cloned by polymerase chain reaction (PCR). The DNA sequence encodes a protein of 500 amino acids with a calculated molecular mass of 60.5 kDa. The deduced amino acid sequence is conserved among the reported PDIs. A 3-D structural model of the ClPDI has been created based on the known crystal structure of Homo sapiens (PDB ID: 3F8U_A). The enzyme has two putative active sites comprising the redox-active disulfides between residues 60-63 and 405-408 (motif CGHC). To further characterize the ClPDI, the coding region was subcloned into an expression vector pET-20b (+), transformed into E. coli Rosetta (DE3)pLysS, and recombinant protein expressed. The recombinant ClPDI was purified by a nickel Sepharose column. PDI's activity was assayed based on the ability of the enzyme to isomerize scrambled RNase A (sRNase A) to active enzyme. The KM, kcat and kcat/KM values were 8.3 × 10-3 µM, 3.0 × 10-5 min-1, and 3.6 × 10-1 min-1 mM-1. The enzyme was most active at pH 8. CONCLUSIONS: The advantage of this enzyme over the PDI from all other sources is its low KM. The potential applications of this PDI in health and beauty may worth pursuing.

Plastidial starch phosphorylase in sweet potato roots is proteolytically modified by protein-protein interaction with the 20S proteasome.

Post-translational regulation plays an important role in cellular metabolism. Earlier studies showed that the activity of plastidial starch phosphorylase (Pho1) may be regulated by proteolytic modification. During the purification of Pho1 from sweet potato roots, we observed an unknown high molecular weight complex (HX) showing Pho1 activity. The two-dimensional gel electrophoresis, mass spectrometry, and reverse immunoprecipitation analyses showed that HX is composed of Pho1 and the 20S proteasome. Incubating sweet potato roots at 45°C triggers a stepwise degradation of Pho1; however, the degradation process can be partially inhibited by specific proteasome inhibitor MG132. The proteolytically modified Pho1 displays a lower binding affinity toward glucose 1-phosphate and a reduced starch-synthesizing activity. This study suggests that the 20S proteasome interacts with Pho1 and is involved in the regulation of the catalytic activity of Pho1 in sweet potato roots under heat stress conditions.

Increasing hybridoma viability and antibody repertoire after the cell fusion by the use of human plasma as an alternative supplement.

Prion diseases such as Bovine Spongiform Encephalopathy (BSE) and new variant Creutzfeldt-Jakob disease (nvCJD) have caused a major safety concern in cell cultures using fetal calf serum (FCS). In this study, we found that screened and tested human plasma (HP) obtained from blood centers may be an ideal alternate nutrient substitute to FCS for culturing hybridoma. In addition to the inherent safety, a ten-fold increase in the fusion efficiency has been observed if the HP was used as the nutrient supplement instead of FCS. Subsequently, a broader antibody repertoire may be recovered. The HP supplement was found to promote the growth of hybridoma cells but no impact on antibody secretion. Interestingly, this effect of enrichment was only observed for HP, but not plasma from other animals. Unidentified murine hybridoma cloning factors other than IL-6 may specifically reside in human blood.

Detection of H6 influenza antibody by blocking enzyme-linked immunosorbent assay.

Low pathogenic H6N1 avian influenza viruses (AIVs) have circulated in domestic chickens since 1972 in Taiwan. Detection of avian influenza (AI) antibody is a routine work for AI control in Taiwan. The currently available commercial enzyme-linked immunosorbent assays (ELISAs) are unable to differentiate antibody responses between different subtypes. To this end, a panel of monoclonal antibodies (mAbs) to A/chicken/Taiwan/2838V/00 (H6N1) was developed and implemented a blocking ELISA (bELISA) to detect the H6 antibody. These mAbs were confirmed specific to H6 AIVs. One monoclonal antibody was purified and labeled with horseradish peroxidase to set up a bELISA. The cut-off value was calculated to be 30% inhibition percentage from 138 H6-negative sera. The sensitivity and specificity of the bELISA were 100% and 97%, respectively. The bELISA detected seroconversion in H6-infected farms earlier than hemagglutination inhibition test. The results show that this bELISA could be used to detect H6 infection in the field.

Canine CD8 T cells showing NK cytotoxic activity express mRNAs for NK cell-associated surface molecules.

Natural killer (NK) cells have been considered to be a group of lymphocytes lacking clonally distributed receptors for antigens typical of T cells and B cells. In some mammalian species, including humans, a subpopulation of CD8(+) peripheral blood lymphocytes (PBLs) exhibits NK activity. This NK subpopulation has not been well characterized in mammals and its characterization is particularly poor in the dog. In this study, we demonstrated that a subset of canine CD8(+) cells derived from PBLs and lymphokine (IL-2)-activated killers (LAKs) of PBLs that was CD3(+), CD4(-), CD21(-), CD5(lo), alpha/betaTCR(+), and gamma/deltaTCR(-) contained substantially higher levels of mRNAs for NK cell-related receptors (NKp30, NKp44, NKG2D, 2B4, and CD16 for PBL, and NKG2D and CD56 for LAK) than the corresponding CD8(-) cells. This subset of CD8(+) lymphocytes derived from LAKs also displayed significantly higher NK cytotoxic activity than the corresponding CD8(-) cells. In contrast, CD8(+) cells derived from nonstimulated PBLs showed very low levels of NK cytotoxic activity. Our results indicate that, in IL-2-stimulated PBLs, canine CD8(+) cells are an important subset associated with NK cytotoxic activity.

Phytochelatin synthase is regulated by protein phosphorylation at a threonine residue near its catalytic site.

Heavy metals are toxic to most living organisms and cause health problems by contaminating agricultural products. In plants, phytochelatin synthase (PCS, EC 2.3.2.15) uses glutathione (GSH) as its substrate to catalyze the synthesis of heavy metal-binding peptides, known as phytochelatins (PC). PCS has been described as a constitutive enzyme that may be controlled by post-translational modifications. However, the detailed mechanism of its catalytic activity is not clear. In this study, in vitro experiments demonstrate that PCS activity increased following phosphorylation by casein kinase 2 (CK2) and decreased following treatment with alkaline phosphatase. Site-directed mutagenesis experiments at amino acids on AtPCS1 indicate that Thr 49 is the site for phosphorylation. This is further supported by fact that the mutant AtPCS1(T49A) cannot be phosphorylated, and its activity is significantly lower than that of the wild-type enzyme. In the modeled three-dimensional structure of AtPCS1, Arg 183 is within close proximity to Thr 49. The mutant AtPCS1(R183A) can be phosphorylated, but it shows much lower catalytic activity than the wild-type protein. This result suggested that Arg 183 may play an important role in the catalytic mechanism of AtPCS1. The possibility of the presence of a second substrate-binding site as a result of the interaction of these two amino acids is discussed. In addition, the activity of AtPCS1 was also found to be modulated by the C-terminal domain. The N-terminal catalytic domain of AtPCS1 was expressed (AtPCS1-N), and its catalytic activity was found to be even more sensitive to Cd or phosphorylation status than was the full-length enzyme.

A comprehensive evaluation of imidazole-zinc reverse stain for current proteomic researches.

In this paper, we comprehensively evaluated the capability of imidazole-zinc reverse stain (ZN) in comparative proteomics. Three commonly used protein gel staining methods, including silver (SN), SYPRO Ruby (SR), and CB stain were investigated alongside for comparison purpose. A transparency scanning procedure, which may deliver more even and contrasting gel images, was found best for documenting ZN stained gels. Our results showed that ZN was more sensitive than SN, SR, and CB. It may reveal as few as 1.8 ng of proteins in a gel. Moreover, ZN was found to provide a linear dynamic range of staining for revealing proteins up to 140 ng, and show an insignificant staining preference. To analyze a ZN stained 2-D gel image that generally comprises an apparent but even background, the Melanie 4 software was found more suitable than others. Furthermore, ZN demonstrated an equivalent or better MS compatibility than the other three staining methods. Intense and comprehensive MS profiles were frequently observed for ZN stained gel spots. Approximate two-third of ZN stained gel spots were successfully identified for protein identities. Taken together, our results suggest that the prompt, cost effective and versatile ZN is well suited for current proteomic researches.

Characterization of the high molecular weight Cd-binding complex in water hyacinth (Eichhornia crassipes) when exposed to Cd.

Water hyacinth ( Eichhornia crassipes) is a rapid-growing freshwater vascular plant that has been used to remove heavy metals in contaminated water. But the transportation and distribution of the absorbed heavy metal in the plant are not clear. In this study, water hyacinth was exposed to cadmium (Cd, 10 microM, pulse) and then transferred to a Cd-free solution (chase). The Cd content in the tissues was measured, and the Cd-binding complexes were isolated and identified. We found that (1) in two days, up to 80% of the Cd in the solution was absorbed by the plant, and the Cd could not be released back to the growth solution in the chase period; (2) approximately 1 mg of Cd was accumulated in the water hyacinth/g of dry weight in this condition; (3) invading Cd was bound rapidly as the low-molecular-weight (LMW) complex serving as the transient form for further sequestration; (4) the LMW complex in water hyacinth contained no phytochelatins and was different from the LMW complex in fission yeast; (5) the Cd absorbed in the plant was essentially stored in the high-molecular-weight (HMW) form after 1 week; (6) a small fraction of the absorbed Cd was found in the upper part of the plant (stem and leaves) in the form of complexes; (7) the HMW complex was composed of phytochelatins PC 3 and PC 4 primarily, with only a small amount of PC 2; (8) a rare PC-related peptide was found in the HMW complex that might be derived from PC 3. These observations contribute to the further understanding of water hyacinth in serving as an efficient and reliable accumulator for heavy metals.

Biochemical characterization of 1-Cys peroxiredoxin from Antrodia camphorata.

Antrodia camphorata is a unique medicinal mushroom found only in Taiwan. It has been used as a remedy for various diseases in folk medicine. Antrodia camphorata has been shown to exhibit antioxidative effects. Peroxiredoxins play important roles in antioxidation and cell signaling. A gene encoding an antioxidant enzyme, 1-cysteine peroxiredoxin (1-Cys Prx), was identified in an expressed sequence tag database of the A. camphorata and cloned by polymerase chain reaction. The 1-Cys Prx cDNA (837 bp, accession no. AY870325) contains an open reading frame encoding a protein of 223 amino acid residues with calculated molecular mass of 25,081 Da. The deduced protein shared 44-58% identity with 1-Cys Prx from Homo sapiens, Bos taurus, and Saccharomyces cerevisia. The sequence surrounding the conserved cysteine DFTPVCTTE is conserved. The coding sequence was subcloned into a vector, pET-20b (+), and transformed into Escherichia coli. The recombinant 1-Cys Prx was purified by Ni(2+)-nitrilotriacetic acid (Sepharose). The purified enzyme was characterized under various conditions. The enzyme is thermostable because its half-life of inactivation was 15.5 min at 60 degrees C. It was stable under alkaline pH range from 7.8 to 10.2. The enzyme showed decreased activity with increasing concentration of imidazole. The enzyme is sensitive to trypsin and chymotrypsin treatment.

Preparation of monoclonal antibody bank against whole water-soluble proteins from rapid-growing bamboo shoots.

An antibody bank against the whole proteins in a proteome is a useful tool for biological research. Using the standard cell fusion method, and a modified screening protocol, we produced an mAb bank against the total water-soluble proteins extracted from the rapid-growing green bamboo shoots. An improved two-stage strategy was employed to enrich those poor immunogenic or lower expressed proteins. Totally, we obtained a bank of 192 mAb which were identified as distinctive to each other by 2-DE and immunostaining.

Site-specific phosphorylation of L-form starch phosphorylase by the protein kinase activity from sweet potato roots.

A 78-amino acid insertion (L78) is found in the low-affinity type (L-form) of starch phosphorylase (L-SP, EC 2.4.1.1). This insertion blocks the starch-binding site on the L-SP molecule, and it decreases the binding affinity of L-SP toward starch. The computational analysis of the amino acid sequence on L78 predicts several phosphorylation sites at its Ser residues. Indeed, from the immunoblotting results using antibodies against phosphoamino acids, we observed that the purified L-SP from mature sweet potato (Ipomoea batatas) roots is phosphorylated. This observation led us to the detection of a protein kinase activity in the protein fraction of the crude extract from the sweet potato roots. The kinase was partially purified by liquid chromatography, and its native molecular mass was estimated as 338 kDa. An expressed peptide (L78P) containing the essential part of L78 was intensively phosphorylated by the kinase. However, H-SP (the high-affinity isomer of SP lacking the L78 insertion) and the proteolytic modified L-SP, which lost its L78 fragment, could not be phosphorylated. Furthermore, using L78P mutants by site-directed mutagenesis at Ser residues on L78, we demonstrate that only one Ser residue on L78 is phosphorylated by the kinase. These results imply that this kinase is specific to L-SP, or more precisely, to the L78 insertion. The in vitro phosphorylated L-SP shows higher sensitivity to proteolytic modification, but has no change in its kinetic parameters.