Jaruwan Siritapetawee

A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex

A protease was isolated and purified from Artocarpus heterophyllus (jackfruit) latex and designated as a 48-kDa antimicrobial protease (AMP48) in a previous publication. In this work, the enzyme was characterized for more biochemical and medicinal properties. Enzyme activity of AMP48 was strongly inhibited by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, indicating that the enzyme was a plant serine protease. The N-terminal amino acid sequences (A-Q-E-G-G-K-D-D-D-G-G) of AMP48 had no sequence similarity matches with any sequence databases of BLAST search and other plant serine protease. The secondary structure of this enzyme was composed of high α-helix (51%) and low β-sheet (9%). AMP48 had fibrinogenolytic activity with maximal activity between 55 and 60°C at pH 8. The enzyme efficiently hydrolyzed α followed by partially hydrolyzed β and γ subunits of human fibrinogen. In addition, the fibrinolytic activity was observed through the degradation products by SDS-PAGE and emphasized its activity by monitoring the alteration of secondary structure of fibrin clot after enzyme digestion using ATR-FTIR spectroscopy. This study presented the potential role to use AMP48 as antithrombotic for treatment thromboembolic disorders such as strokes, pulmonary emboli and deep vein thrombosis.

Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion porin

In the present paper, we describe cloning and expression of two outer membrane proteins, BpsOmp38 (from Burkholderia pseudomallei) and BthOmp38 (from Burkholderia thailandensis) lacking signal peptide sequences, using the pET23d(+) expression vector and Escherichia coli host strain Origami(DE3). The 38 kDa proteins, expressed as insoluble inclusion bodies, were purified, solubilized in 8 M urea, and then subjected to refolding experiments. As seen on SDS/PAGE, the 38 kDa band completely migrated to approximately 110 kDa when the purified monomeric proteins were refolded in a buffer system containing 10% (w/v) Zwittergent 3-14, together with a subsequent heating to 95 degrees C for 5 min. CD spectroscopy revealed that the 110 kDa proteins contained a predominant beta-sheet structure, which corresponded completely to the structure of the Omp38 proteins isolated from B. pseudomallei and B. thailandensis. Immunoblot analysis using anti-BpsOmp38 polyclonal antibodies and peptide mass analysis by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS confirmed that the expressed proteins were BpsOmp38 and BthOmp38. The anti-BpsOmp38 antibodies considerably exhibited the inhibitory effects on the permeation of small sugars through the Omp38-reconstituted liposomes. A linear relation between relative permeability rates and M(r) of neutral sugars and charged antibiotics suggested strongly that the in vitro re-assembled Omp38 functioned fully as a diffusion porin.

Functional reconstitution, gene isolation and topology modelling of porins from Burkholderia pseudomallei and Burkholderia thailandensis.

The sequences for Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis have been deposited in the DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases under the accession numbers AY312416 and AY312417 respectively. The intracellular pathogen Burkholderia pseudomallei is the causative agent of tropical melioidosis, and Burkholderia thailandensis is a closely-related Gram-negative bacterium that does not cause serious disease. Like other bacteria, the major outer membrane (OM) porins of Burkholderia strains, Bps Omp38 and Bth Omp38 may have roles in antibiotic resistance and immunity. We purified both proteins and found them to be immunologically related, SDS-resistant, heat-sensitive trimers with M (r) of approx. 110000. In functional liposome-swelling assays, both proteins showed similar permeabilities for small sugar molecules, compatible with a pore diameter of between 1.2 and 1.6 nm. Secondary structure analysis by FTIR (Fourier-transform infrared) spectroscopy revealed almost identical spectra with predominantly beta-sheet structures, typical of bacterial porins. MALDI-TOF (matrix-assisted laser-desorption ionization-time of flight) MS and ESI/MS (electrospray ionization MS) analysis of each protein showed extensive sequence similarities to the OpcP1 porin from Burkholderia cepacia (later found to be 76.5% identical). Based on information from the incomplete B. pseudomallei genome-sequencing project, the genes encoding Omp38 were identified and amplified by PCR from B. pseudomallei and B. thailandensis genomic DNA. The nucleotide sequences are 99.7% identical, and the predicted processed proteins are 100% identical. Topology prediction and molecular modelling suggest that this newly-isolated and cloned porin is a 16-stranded beta-barrel and the external loops of the protein could be important determinants of the immune response to infection.

Biochemical characterization of a new glycosylated protease from Euphorbia cf. lactea latex

A dimeric protease designated as EuP-82 was purified from Euphorbia cf. lactea latex. Since its proteolytic activity was inhibited by a serine protease specific inhibitor (PMSF), EuP-82 was classified as a serine protease. N-glycan deglycosylation tests revealed that EuP-82 was a glycosylated protein. MALDI-TOF MS showed that EuP-82 was a homodimer, which was its active form. The optimal conditions for fibrinogenolytic activity were at pH 11 and 35 °C. EuP-82 enzyme had broad range of pH stability from 4 to 12. Moreover, the enzyme was still active in the presence of reducing agent (β-mercaptoethanol). EuP-82 was a proline-rich enzyme (about 20.69 mol%). Increased proline production can be found in higher plants in response to both biotic and abiotic stresses, high proline in the molecule of EuP-82 might stabilize its activity, structure and folding. Based on the N-terminal amino acid sequences and peptide mass fingerprint (PMF) of EuP-82, the enzyme was identified as a new serine protease. The digested products from EuP-82 cleavage of human fibrinogen were analyzed by SDS-PAGE and PMF. The results confirmed that EuP-82 could digest all subunits of human fibrinogen. EuP-82 cleaved fibrinogen with a Michaelis constant (Km) of 3.30 ± 0.26 μM; a maximal velocity (Vmax) of 400.9 ± 0.85 ng min(-1); and a catalytic efficiency (Vmax/Km) of 121.5 ± 9.25 ng μM(-1) min(-1). EuP-82 has potential for use in medicinal treatment, for example thrombosis, since the enzyme had fibrinogenolytic activity and high stability.

The 1.9 Å X-Ray Structure of Egg-white Lysozyme from Taiwanese Soft-Shelled Turtle (Trionyx Sinensis Wiegmann) Exhibits Structural Differences from the Standard Chicken-Type Lysozyme

Lysozyme from Taiwanese soft-shelled turtle (SSTLB) has been purified from turtle egg white and crystallized. The crystals diffract X-rays beyond 2 A resolution and belong to the orthorhombic P2(1)2(1)2(1) space group containing one molecule per asymmetric unit. The structure was elucidated using pheasant egg-white lysozyme as the molecular replacement search template. The overall structure of SSTLB is very similar to that of hen egg-white lysozyme (HEWL). Nevertheless, Pro104 in the substrate subsite A and other amino acids in the substrate subsites E and F differ from those of HEWL. These substitutions result in local conformational differences in the substrate binding sites of the two enzymes, effecting substrate binding and transglycosylation efficiency, translating into differing ranges of products.

Characterization of the binding of a glycosylated serine protease from Euphorbia cf. lactea latex to human fibrinogen

In this study, the binding of a glycosylated serine protease (EuP‐82) with human fibrinogen was investigated by isothermal titration calorimetry (ITC). ITC analysis indicated that the binding of EuP‐82 to fibrinogen in the conditions with or without the activator (Ca2+) was an exothermic reaction (dominant negative enthalpy), which tended to be driven by hydrogen bonding and van der Waals interactions. In contrast, the binding of fibrinogen−EuP‐82 in the condition with the inhibitor (Zn2+) was an unfavorable endothermic reaction. EuP‐82 could not inhibit the platelet activity in citrated whole blood via the ADP–receptor pathways (mainly, P2Y1 and P2Y12), but it could enhance the platelet aggregation. The ITC together with whole blood platelet aggregation suggested that EuP‐82 provided multiple fibrinogen‐binding sites that were not related to the arginine‐glycine‐aspartate (RGD) and the dodecapeptide sequences of fibrinogen. In addition, EuP‐82 had neither thrombin‐like activity nor anticoagulant activity. The SR‐FTIR spectra revealed that EuP‐82 was a glycoprotein. Deglycosylation of EuP‐82 did not affect its proteolytic activity. Moreover, EuP‐82 did not exhibit any toxicity to the living cells (NIH‐3T3). This study supports that EuP‐82 may be useful for wound‐healing material through stabilizing the clot via the platelet induction for the first process.

Silver Carp Bone Powder as Natural Calcium for Fish Sausage

ABSTRACT Although silver carp bones (SCB) are generated as waste, they are a natural source of calcium and have the potential to be a food ingredient. When the SCB had been soaked in hot sodium hydroxide (0.8%), autoclaved, and ground into SCB powder (SCBP), the total calcium content, analyzed by inductive coupled plasma-optical emission spectroscopy, was about 32%. X-ray absorption near-edge structure (XANES) spectroscopy specified hydroxyapatite as the major calcium component. The soluble calcium in SCBP was found to be 0.59% in de-ionized water. SCBP induced the cross-linking of proteins in minced fish after incubation at 40°C, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fortification of SCBP at 0.0, 0.5, and 1.0% (w/w) in fish emulsion sausage resulted in a decrease of moisture content, while the fat and protein contents increased slightly. Total ash content in the sausage increased significantly, and total calcium content increased 15-fold in fortified SCBP. The hardness and gumminess values of sausage were improved with the addition of SCBP, while the microstructure was not altered by SCBP.

Isolation and characterization of a galactose-specific lectin (EantH) with antimicrobial activity from Euphorbia antiquorum L. latex

A homodimeric 75 kDa lectin with hemagglutination activity (HA) was purified from the crude latex of Euphorbia antiquorum L. by two types of chromatography, on cation exchange (HiTrap SP FF) and hydrophobic HiTrap Phenyl FF (high sub) columns. The purified protein was designated EantH, and is classified as a galactose-specific thermostable lectin. The HA of EantH was stable at pH values of 5-9 and temperature 5-65 °C. The lectin had bacteriostatic action on the Gram-positive bacteria Staphylococcus aureus and S. epidermidis, with a minimum inhibitory concentration (MIC) of 2000 μg/ml and on a Gram-negative bacterium Samonella typhimurium, with a MIC of 1000 μg/ml. EantH inhibited the growth of Propionibacterium acnes and Streptococcus agalactiae with MIC of 125 μg/ml and 250 μg/ml, respectively. EantH killed P. acnes and S. agalactiae with a minimum microbicidal concentration (MMC) of 1000 μg/ml and 2000 μg/ml, respectively. Scanning electron microscopy indicated that binding of EantH to the carbohydrates in the cell walls of P. acnes and S. typhimurium drastically altered the bacterial cells, and led to inhibition of growth and/or cell death. The antimicrobial activity of EantH could be neutralized by d‑galactose, indicating that its bactericidal action involves binding to galactose in the cell wall.