N-terminal Amino Acid Sequence Research Articles

Purification and characterization of \u03b2-secretase inhibitory peptide from sea hare (Aplysia kurodai) by enzymatic hydrolysis

Amyloid plaque, also called senile plaque, the product of aggregation of β-amyloid peptides (Aβ), is observed in brains of the patients with Alzheimer’s disease (AD) and is one of the key factors in etiology of the disease. In this study, hydrolysates obtained from the sea hare (Aplysia kurodai) were investigated for β-secretase inhibitory peptide. The sea hare’s muscle protein was hydrolyzed using six enzymes in a batch reactor. Trypsin hydrolysate had highest β-secretase inhibitory activity compared to the other hydrolysates. β-secretase inhibitory peptide was separated using Sephadex G-25 column chromatography and high-performance liquid chromatography on a C18 column. β-secretase inhibitory peptide was identified as eight amino acid residues of Val-Ala-Ala-Leu-Met-Leu-Phe-Asn by N-terminal amino acid sequence analysis. IC50 value of purified β-secretase inhibitory peptide was 74.25 μM, and Lineweaver−Burk plots suggested that the peptide purified from sea hare muscle protein acts as a competitive inhibitor against β-secretase. Results of this study suggest that peptides derived from sea hare muscle may be beneficial as anti-dementia compounds in functional foods or as pharmaceuticals.

Isolation and characterization of a halotolerant and protease-resistant α-galactosidase from the gut metagenome of Hermetia illucens

Hermetia illucens is a voracious insect scavenger, decomposing food waste efficiently. To survey novel hydrolytic enzymes, we constructed a fosmid metagenome library using unculturable intestinal microorganisms from H. illucens in our previous study (Lee et al., 2014). Functional screening of the library on carboxymethyl cellulose plates identified a fosmid clone the product of which displayed hydrolytic activity. Sequence analysis of the fosmid revealed a novel α-galactosidase gene, Agas2. The Agas2 gene is composed of 2,007 base pairs encoding 668 amino acids with a deduced 25 amino acid N-terminal signal peptide sequence. The conceptual translation and domain analysis of Agas2 showed the highest sequence identity (84%) with the putative α-galactosidase of Dysgonomonas sp. HGC4, exhibiting well-conserved domain homology with glycosyl hydrolase family 97. Phylogenetic analysis indicated that Agas2 may be a currently uncharacterized α-galactosidase. The recombinant protein, rAgas2, was successfully expressed in E. coli. rAgas2 showed the highest activity at 40 °C and pH 7.0. It displayed great pH stability within a pH range of 5–11 for 15 h at 4 °C. rAgas2 was highly stable under stringent conditions, including polar organic solvents, non-ionic detergents, salt, and proteases. rAgas2 hydrolyzed α-d-galactose substrates, showing the maximum enzymatic activity toward p-nitrophenyl α-d-galactopyranoside (specific activity 128.37 U/mg). However, rAgas2 did not hydrolyze substrates linked with β-glucose moieties. Overall, Agas2 may be an attractive candidate for the degradation of α-galactose family oligosaccharides in high-salt, protease-rich and high-organic-solvent processes.

Properties and structure of a low-potential, penta-heme cytochrome c552 from a thermophilic purple sulfur photosynthetic bacterium Thermochromatium tepidum.

The thermophilic purple sulfur bacterium Thermochromatium tepidum possesses four main water-soluble redox proteins involved in the electron transfer behavior. Crystal structures have been reported for three of them: a high potential iron-sulfur protein, cytochrome c', and one of two low-potential cytochrome c552 (which is a flavocytochrome c) have been determined. In this study, we purified another low-potential cytochrome c552 (LPC), determined its N-terminal amino acid sequence and the whole gene sequence, characterized it with absorption and electron paramagnetic spectroscopy, and solved its high-resolution crystal structure. This novel cytochrome was found to contain five c-type hemes. The overall fold of LPC consists of two distinct domains, one is the five heme-containing domain and the other one is an Ig-like domain. This provides a representative example for the structures of multiheme cytochromes containing an odd number of hemes, although the structures of multiheme cytochromes with an even number of hemes are frequently seen in the PDB database. Comparison of the sequence and structure of LPC with other proteins in the databases revealed several characteristic features which may be important for its functioning. Based on the results obtained, we discuss the possible intracellular function of this LPC in Tch. tepidum.

Cloning cDNA sequence coding the KSA-1 lectin from red alga Kappaphycus striatum cultivated in Vietnam

The red alga Kappaphycus striatum is economically important food species and extensively cultivated in Vietnam. This cultivated alga may thus now contribute as a source of not only carrageenan, but also other bioactive compounds for biochemical and medicinal uses. KSA-1 lectin from this alga has been evaluated for biochemical properties, including high-mannose binding specificity, N-terminal amino acid sequence and molecular mass. In this study, we have cloned the cDNA clone encoding the lectin KSA-1. The full-length sequence of cDNA clone of KSA-1 lectin encoded a polypeptide of 268 amino acids including initiating methionine, with four tandemly repeated domains of about 67 amino acids, and sharing 43% sequence identity. The primary structure and the amino acid residues interact with mannopentaose core structure per a repeat domain of the KSA-1 lectin highly resemble and match those of anti-virus the lectin family in lower organinsms, including bacteria [BOA from Burkholderia oklahomensis EO147, MBHA from Myxococcus xanthus and PFA from Pseudomonas fluorescens Pf0-1], cyanobacteria [OAA from Oscillatoria agardhii] and red algae [ESA-2 from Eucheuma serra, EDA-2 from Eucheuma denticulatum, KAA-1 and KAA-2 from Kappaphycus alvarezii and KSA-2 from K. striatum]. Analysis of predicted secondary structure of KSA-1 showed twenty β-strands. Each repeated domain comprises the five β-strands and matches with number of β-strands of BOA lectin. Therefore, the red alga K. striatum could be a good source for application in biomedicine and biochemistry as materials for production of the functional lectin(s).

N-Terminal Cu-Binding Motifs (Xxx-Zzz-His, Xxx-His) and Their Derivatives: Chemistry, Biology and Medicinal Applications.

Peptides and proteins with N-terminal amino acid sequences NH2 -Xxx-His (XH) and NH2 -Xxx-Zzz-His (XZH) form well-established high-affinity CuII -complexes. Key examples are Asp-Ala-His (in serum albumin) and Gly-His-Lys, the wound healing factor. This opens a straightforward way to add a high-affinity CuII -binding site to almost any peptide or protein, by chemical or recombinant approaches. Thus, these motifs, NH2 -Xxx-Zzz-His in particular, have been used to equip peptides and proteins with a multitude of functions based on the redox activity of Cu, including nuclease, protease, glycosidase, or oxygen activation properties, useful in anticancer or antimicrobial drugs. More recent research suggests novel biological functions, mainly based on the redox inertness of CuII in XZH, like PET imaging (with 64 Cu), chelation therapies (for instance in Alzheimer's disease and other types of neurodegeneration), antioxidant units, Cu transporters and activation of biological functions by strong CuII binding. This Review gives an overview of the chemical properties of Cu-XH and -XZH motifs and discusses the pros and cons of the vastly different biological applications, and how they could be improved depending on the application.

Production of a novel laccase from Paraphoma Sp

By using a laccase-secretion indicator for screening laccase-producing microorganisms, a novel laccase-producing strain was isolated and identified as Paraphoma sp. strain GZS18, it produced increased laccase and mycelia at 34 °C. Further investigations showed that the production of laccase by Paraphoma sp. GZS18 was greatly enhanced by less toxic inducers copper sulphate and methyl orange. Copper sulphate and methyl orange were added into the cultivation medium at 12 and 60 h, respectively, and the maximum laccase production was obtained. Through Plackett–Burman design and response surface methodology, we obtained the optimum production conditions as follows: methyl orange, 39.90 μM; addition time of copper sulphate, 11.95 h; addition time of methyl orange, 51.40 h. Under the above conditions, the experimental value of laccase production was 12,250.76 U/L. The extracellular laccase from Paraphoma sp. GZS18 was purified to homogeneity, which showed a molecular mass of 75 kDa. N-terminal amino acid sequences was AXaVSVASREMT.

An extracellular yellow laccase from white rot fungus Trametes sp. F1635 and its mediator systems for dye decolorization

A novel extracellular laccase was purified from fermentation broth of the white rot fungus Trametes sp. F1635 by a three-step protocol including two consecutive ion-exchange chromatography steps on DEAE-Sepharose and SP-Sepharose, and a final gel-filtration on Superdex 75. The purified laccase (TsL) was a monomeric protein with the molecular mass of 64.8 kDa. It demonstrated high oxidation activity of 4.00 × 104 U/mg towards ABTS. Its N-terminal amino acid sequence was AIGPVADLTIINNAV which was unique and sharing high similarity of other fungal laccases. TsL was a yellow laccase based on absorption spectrum analysis. It demonstrated an acidic pH optimum of 2.6 and temperature optimum of 50 °C towards ABTS. The Km and Vmax values towards ABTS were estimated to 18.58 μM and 1.35 μmol/min, respectively. TsL manifested effective decolorization activity towards eriochrome black T (EBT), remazol brilliant blue R (RBBR), malachite green (MG), and eriochrome black T (EBT) (over 60%). Violuric acid (VA) and acetosyringone (AS) were the optimal mediators for the laccase in dye decolorization. Results suggest that TsL demonstrates great potential for dye decolorization and water treatment.

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Improved heterologous expression of the membrane-bound quinoprotein quinate dehydrogenase from Gluconobacter oxydans

Gluconobacter oxydans produces 3-dehydroquinate by oxidation of quinate through a reaction catalyzed by the quinate dehydrogenase (QDH), membrane-bound, pyrroloquinoline quinone (PQQ)-dependent dehydrogenase. We previously reported the nucleotide and deduced amino acid sequence of QDH and constructed a heterologous expression system of QDH in Pseudomonas sp. (A.S. Vangnai, W. Promden, W. De-Eknamkul, K. Matsushita, H. Toyama, Biochemistry (Moscow) 75:452–459, 2010). Through this study, we aim to update the sequences of QDH and improve the heterologous expression of QDH in Gluconobacter strains using a broad-host-range plasmid. Expression of QDH using a plasmid containing a long 5′-UTR was higher than that using a plasmid with a short 5′-UTR. In addition, the usage of the putative promoter region of the membrane-bound, alcohol dehydrogenase (ADH) of Gluconobacter resulted in higher expression levels compared to the usage of the lacZ promoter. Base substitution experiments allowed to identify the correct TTG initiation codon between two possibilities, and the result of these experiments were consistent with the N-terminal amino acid sequence of the expressed QDH. However, change of the TTG codon to ATG did not increase QDH expression. Therefore, the optimal plasmid for QDH expression included the structural gene with a long 5′-UTR and the ADH promoter. Cell membrane of the recombinant Gluconobacter strain presented approximately 10-times higher specific QDH activity than that observed in the wild-type strain.

Transcriptional profiling, molecular cloning, and functional analysis of C1 inhibitor, the main regulator of the complement system in black rockfish, Sebastes schlegelii

C1-inhibitor (C1inh) plays a crucial role in assuring homeostasis and is the central regulator of the complement activation involved in immunity and inflammation. A C1-inhibitor gene from Sebastes schlegelii was identified and designated as SsC1inh. The identified genomic DNA and cDNA sequences were 6837 bp and 2161 bp, respectively. The genomic DNA possessed 11 exons, interrupted by 10 introns. The amino acid sequence possessed two immunoglobulin-like domains and a serpin domain. Multiple sequence alignment revealed that the serpin domain of SsC1inh was highly conserved among analyzed species where the two immunoglobulin-like domains showed divergence. The distinctiveness of teleost C1inh from other homologs was indicated by the phylogenetic analysis, genomic DNA organization, and their extended N-terminal amino acid sequences. Under normal physiological conditions, SsC1inh mRNA was most expressed in the liver, followed by the gills. The involvement of SsC1inh in homeostasis was demonstrated by modulated transcription profiles in the liver and spleen upon pathogenic stress by different immune stimulants. The protease inhibitory potential of recombinant SsC1inh (rSsC1inh) and the potentiation effect of heparin on rSsC1inh was demonstrated against C1esterase and thrombin. For the first time, the anti-protease activity of the teleost C1inh against its natural substrates C1r and C1s was proved in this study. The protease assay conducted with recombinant black rockfish C1r and C1s proteins in the presence or absence of rSsC1inh showed that the activities of both proteases were significantly diminished by rSsC1inh. Taken together, results from the present study indicate that SsC1inh actively plays a significant role in maintaining homeostasis in the immune system of black rock fish.

Construction and Characterization of Cell-Penetrating Peptide-Fused Fibroblast Growth Factor and Vascular Endothelial Growth Factor for an Enhanced Percutaneous Delivery System.

The exogenous administration of growth factors has been examined for treating wounds and such factors serve as cosmetic agents for skin regeneration. However, the topical application of growth factors is hampered by their limited percutaneous absorption. In this study, we genetically modified basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A) using a cell-penetrating peptide to facilitate their permeation into skin and to avoid multiple steps in the chemical or physical modification of biomaterials. Low-molecular-weight protamine (LMWP)-fused bFGF and VEGF-A (LMWP-bFGF and LMWP-VEGF-A) were designed by serial polymerase chain reaction (PCR)-mediated addition of the LMWP codons onto the bFGF and VEGF-A genes and then produced in Escherichia coli. The purified LMWP-bFGF and LMWP-VEGF-A represented >90% of the total proteins, as determined by SDS-PAGE and densitometric quantification, and their actual molecular weights, determined by mass spectroscopy, were 19,026 and 15,715 Da, respectively, corresponding to the theoretical values. N-terminal amino acid sequencing analyses confirmed the N-terminal conjugation of LMWP to bFGF and VEGF-A. Immunoblotting analyses using antibodies against bFGF and VEGF-A, together with the proliferative effects of LMWP-bFGF and LMWP- VEGF-A on human keratinocytes and fibroblasts, demonstrated that the original biological activity of each growth factor was not altered by LMWP conjugation. In addition, the LMWP conjugation did not induce further cytotoxic effects on the skin cells, while the cell membrane-penetrating activities of LMWP-bFGF and LMWP-VEGF-A were significantly enhanced compared with the respective unconjugated growth factors. These results suggest that LMWP-bFGF and LMWP-VEGF-A can be used as effective topical therapeutic or cosmetic agents for skin regeneration and anti-aging treatments.

Preparation and In Vitro Evaluation of Elastic Nanoliposomes for Topical Delivery of Highly Skin-Permeable Growth Factors.

Percutaneous delivery of growth factors is often used to treat wounds, and for cosmetic purposes, as a way of accelerating healing and skin regeneration, respectively. However, the therapeutic effects of growth factors are diminished by their poor absorption when delivered percutaneously, in addition to their rapid degradation by proteinases. To overcome these obstacles, we constructed two skin-permeable compounds. Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A) were both genetically paired with low-molecular-weight protamine (LMWP), to yield the compounds LMWP-bFGF and LMWP-VEGF-A, respectively. The molecular weights and N-terminal amino acid sequences of LMWP-bFGF and LMWP-VEGF-A confirmed that the N-terminus-specific conjugation of LMWP with bFGF and VEGF-A had been successful. The biological abilities of the native factors to stimulate human fibroblast (CCD-986sk) and endothelial cell proliferation were preserved. Both compounds significantly promoted wound (scratch) recovery and enhanced procollagen type I C-peptide synthesis in CCD-986sk cells (to levels 184 and 133% those of the native compounds, respectively). The LMWP-conjugated growth factors were significantly more permeable than the native forms (by 7.29- and 29.22-fold, respectively). Finally, encapsulation of the compounds in positively charged elastic nanoliposomes (115 ± 1.54 nm in diameter with a zeta potential of 57.2 ± 3.05 mV) further improved both permeation and stability. Thus, nanoliposomes loaded with LMWP-conjugated growth factors are expected to enhance skin regeneration; the materials will find applications in wound-healing therapies and anti-wrinkle cosmetics.

The Dichotomy of the Poly(ADP-Ribose) Polymerase-Like Thermozyme from Sulfolobus solfataricus

The first evidence of an ADP-ribosylating activity in Archaea was obtained in Sulfolobus solfataricus(strain MT-4) where a poly(ADP-ribose) polymerase (PARP)-like thermoprotein, defined with the acronymous PARPSso, was found. Similarly to the eukaryotic counterparts PARPSso cleaves beta-nicotinamide adenine dinucleotide to synthesize oligomers of ADP-ribose; cross-reacts with polyclonal anti-PARP-1 catalytic site antibodies; binds DNA. The main differences rely on the molecular mass (46.5 kDa) and the thermophily of PARPSso which works at 80 °C. Despite the biochemical properties that allow correlating it to PARP enzymes, the N-terminal and partial amino acid sequences available suggest that PARPSso belongs to a different group of enzymes, the DING proteins, an item discussed in detail in this review.This finding makes PARPSso the first example of a DING protein in Archaea and extends the existence of DING proteins into all the biological kingdoms. PARPSsohas a cell peripheral localization, along with the edge of the cell membrane. The ADP-ribosylation reaction is reverted by a poly(ADP-ribose) glycohydrolase-like activity, able to use the eukaryotic poly(ADP-ribose) as a substrate too. Here we overview the research of (ADP-ribosyl)ation in Sulfolobus solfataricus in the past thirty years and discuss the features of PARPSso common with the canonical poly(ADP-ribose) polymerases, and the structure fitting with that of DING proteins.

Purification and characterization of fibrinolytic enzyme from a bacterium isolated from soil.

A novel extracellular enzyme with strong fibrinolytic activity, produced by Bacillus tequilensis, which was isolated from the soil of Zhuhai City (China) was purified and characterized. The enzyme was secreted by cultured B. tequilensis in solid state and purified at a high efficiency using the combination of salting out, ion exchange chromatography, and size exclusion chromatography. The enzyme was estimated to have a molecular weight of approximately 27kDa, pI of 8.9±0.1, to stable at pH 5.0-12.0 and up to 50°C; the optimum pH and temperature are 10.5 and 45°C (2373.59±54.81 U/mg), respectively. The fibrinolytic activity was enhanced by K+, Na+, Mg2+, Mn2+, Ca2+, and Ba2+ and inhibited by Cu2+, Zn2+, and Fe3+. Moreover, the activity was slightly enhanced by PMSF and EDTA at low concentrations and inhibited by β-mercaptoethanol. The N-terminal amino acid sequence is AQSVPYGISQI. The enzyme has a higher enzymatic activity than most other fibrinolytic enzymes. The high thermal stability indicated that it is easy to preserve and could be activated under high-temperature conditions.

Venom characterization of the Amazonian scorpion Tityus metuendus

The soluble venom from the scorpion Tityus metuendus was characterized by various methods. In vivo experiments with mice showed that it is lethal. Extended electrophysiological recordings using seven sub-types of human voltage gated sodium channels (hNav1.1 to 1.7) showed that it contains both α- and β-scorpion toxin types. Fingerprint analysis by mass spectrometry identified over 200 distinct molecular mass components. At least 60 sub-fractions were recovered from HPLC separation. Five purified peptides were sequenced by Edman degradation, and their complete primary structures were determined. Additionally, three other peptides have had their N-terminal amino acid sequences determined by Edman degradation and reported. Mass spectrometry analysis of tryptic digestion of the soluble venom permitted the identification of the amino acid sequence of 111 different peptides. Search for similarities of the sequences found indicated that they probably are: sodium and potassium channel toxins, metalloproteinases, hyaluronidases, endothelin and angiotensin-converting enzymes, bradykinin-potentiating peptide, hypothetical proteins, allergens, other enzymes, other proteins and peptides.

A novel epididymal quiescence factor inhibits sperm motility by modulating NOS activity and intracellular NO-cGMP pathway.

Mature and potentially motile spermatozoa stored in cauda epididymis in an inactive state for approximately 30 days; however, during ejaculation they regain motility. To understand the actual molecular mechanism of the sperm quiescence during caudal stay, a proteinaceous quiescence factor (QF) has been purified from caprine epididymal plasma to apparent homogeneity. In the present study complete purification, detailed characterization as well as mechanistic pathway of QF has been described. QF is purified to 215-fold with 45% activity recovery. It is a 59 kDa monomeric protein with isoelectric point 5.8 and optimally active at pH 7.5. Circular dichroism spectroscopy and atomic force microscopy study confirm its α-helical secondary structure and globular tertiary conformation. QF is a thermo-stable protein as higher temperature does not alter its helical structure. N-terminal amino acid sequencing and MALDI analysis of QF did not find 100% similarity with any available protein of the database, proved its novelty. QF at 2 μM dose inhibits sperm progressive forward motility within 10 min. This motility inhibitory activity of QF is mediated by reducing NOS enzyme activity and subsequently decreasing the intracellular NO and cGMP concentration. It does not modulate intracellular Ca++ and cAMP concentration. QF has no adverse effect on DNA integrity and morphology of spermatozoa. Motility inhibitory action of QF is reversible. Thus, the role of QF in maintaining energy saving quiescence state of mature cauda spermatozoa and its reactive nitrogen species reducing activity may lead to a new direction for storage of spermatozoa and idiopathic male infertility.

Enzymatic and molecular characterization of an acidic and thermostable chitinase 1 from Streptomyces thermodiastaticus HF 3-3.

Chitinase 1 (Chi1) is an acidic and thermostable hydrolytic enzyme capable of the breakdown of chitin, a resilient biopolymer that is the primary building block of fungi cell walls and marine exoskeletons. In this study, Chi1 was purified from the bacterium Streptomyces thermodiastaticus HF 3-3, and its properties were carefully characterized. The molecular mass of Chi1 was estimated to be approximately 46 kDa and, through sequencing, its N-terminal amino acid sequence was identified as ADSGKVKL. Although the optimal operating temperature and pH for Chi1 were determined to be 65°C and pH 5.5, respectively, the purified enzyme was stable over wide pH (1.5-9) and temperature ranges. Moreover, Chi1 retained 87% of its activity in the presence of 15% NaCl. While Chi1 activity was inhibited by Ag+ and Mn2+, other chemicals tested had no significant effect on its enzymatic activity. The Km and Vmax values of Chi1 for the substrate colloidal chitin were 1.23 ± 0.7 mg/mL and 6.33 ± 1.0 U/mg, respectively. Thin-layer chromatography analysis of the enzymatic reaction end products mainly detected diacetylchitobiose. We also cloned the Chi1 gene and purified the recombinant protein; the properties of the recombinant enzyme were nearly identical to those of the native enzyme. Therefore, Chi1 purified from S. thermodiastaticus HF 3-3 is unique, as it is highly stable under broad range of pH values, temperatures, and chemical exposures. Combined, these properties make this enzyme attractive for use in the industrial bioconversion of chitin.

Physicochemical Properties and Biological Activity of the Peptide–Protein Complex from Bovine Scleral Tissue

The peptide–protein complex from bovine sclera was studied. It is shown that it contained a protein with a molecular weight of 66387 Da with the partial N-terminal amino acid sequence DTHKSEIAHRFKDLG-, which is homologous to the mature molecule of bovine serum albumin, and polypeptides with molecular weights of 1300–5080 Da. With a model of the organotypic cultivation of posterior eye tissues of the newt Pleurodeles waltl in vitro, it was shown that the effect of this peptide–protein complex in low doses increased the viability of scleral fibroblasts.

Studies on localization and protein ligands of Galleria mellonella apolipophorin III during immune response against different pathogens

A lipid-binding protein apolipophorin III (apoLp-III), an exchangeable component of lipophorin particles, is involved in lipid transport and immune response in insects. In Galleria mellonella, apoLp-III binding to high-density lipophorins and formation of low-density lipophorin complexes upon immune challenge was reported. However, an unanswered question remains whether apoLp-III could form different complexes in a pathogen-dependent manner. Here we report on pathogen- and time-dependent alterations in the level of apoLp-III free and lipophorin-bound form that occur in the hemolymph and hemocytes shortly after immunization of G. mellonella larvae with different pathogens, i.e. Gram-negative bacterium Escherichia coli, Gram-positive bacterium Micrococcus luteus, yeast-like fungus Candida albicans, and filamentous fungus Fusarium oxysporum. These changes were accompanied by differently persistent re-localization of apoLp-III in the hemocytes. The apoLp-III-interacting proteins were recovered from immune hemolymph by affinity chromatography on a Sepharose bed with immobilized anti-apoLp-III antibodies. ApoLp-I, apoLp-II, hexamerin, and arylphorin were identified as main components that bound to apoLp-III; the N-terminal amino acid sequences of G. mellonella apoLp-I and apoLp-II were determined for the first time. In the recovered complexes, the pathogen-dependent differences in the content of individual apolipophorins were detected. Apolipophorins may thus be postulated as signaling molecules responding in an immunogen-dependent manner in early steps of G. mellonella immune response.

Characterization of porcine sapelovirus isolated from Japanese swine with PLC/PRF/5 cells.

Porcine sapelovirus (PSV) is a causative agent of neurological disorders, fertility disorders and dermal lesions of swine. In this study, we isolated two PSV strains, Jpsv477 and Jpsv1315, from swine faecal specimens using a PLC/PRF/5 cell culture system. The PSV infection of PLC/PRF/5 cells induced a cytopathic effect (CPE). Two types of virus particles with identical diameter (~35nm) but different densities (1.300 and 1.285g/cm3 ) were observed in the cell culture supernatants. Analysis of the entire genome sequence of Jpsv477 and Jpsv1315 revealed that both strains possess 7,558 nucleotides and the poly (A) tail and have a typical PSV genome organization consisting of a 5' terminal untranslated region (5'UTR), a large open reading frame (ORF), and a 3' terminal untranslated region (3'UTR). The ORF encodes a single polyprotein that is subsequently processed into a leader protein (L), four structural proteins (VP1, VP2, VP3 and VP4) and seven functional proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). The structural proteins VP1, VP2, VP3 and VP4 have molecular masses of ~35, ~26, ~25 and ~6kDa. The N-terminal amino acid sequence analysis of VP1, VP2, VP3 and VP4 confirmed that the cleavage sites between VP4 and VP2, VP2 and VP3, and VP3 and VP1 are K/A, Q/G and Q/G, respectively. We further confirmed that HepG2/C3A, Vero E6 and primary green monkey kidney cells (PGMKC) were also susceptible to PSV infection. The stability assay demonstrated that PSV was inactivated by heating at 60°C for 10min or 65°C for 5min. The virus also lost infectivity by incubation with 62.5ppm of NaClO for 30min.