Study on Purification and Characterization of Polyphenol Oxidase from Acetes chinensis.

Acetes chinensis (belonging to the Decapoda Sergestidae genus) is widely distributed in East Asian waters and is extremely widespread and present in the shallow coastal areas of China. Polyphenol oxidase (PPO), which was extracted from Acetes chinensis, was purified in a four-step procedure involving phosphate-buffered saline treatment, ammonium sulphate precipitation, DEAE-Cellulose chromatography, and Phenyl-Sepharose HP chromatography, and then, its biochemical characterization was measured. The specific activity of the purified enzyme was increased to 643.4 U/mg, which is a 30.35 times increase in purification, and the recovery rate was 17.9%. L-dopa was used as the substrate, the enzymatic reactions catalyzed by PPO conformed to the Michaelis equation, the maximum reaction velocity was 769.23 U/mL, and the Michaelis constant Km was 0.846 mmol/L. The optimal pH of PPO from Acetes chinensis was 7.5, and the optimal temperature was 35 °C. The metal ions experiment showed that Mn2+ and K+ could enhance the activity of PPO; that Ba2+ and Ca2+ could inhibit the activity of PPO; and that Cu2+ had a double effect on PPO, increasing the PPO activity at low concentrations and inhibiting the PPO activity at high concentrations. The inhibitor experiment showed that the inhibitory effects of EDTA and kojic acid were weak and that ascorbic acid and sodium pyrophosphate had good inhibitory effects. The purification and characterization of Acetes chinensis serve as guidelines for the prediction of enzyme behavior, leading to effective prevention of enzymatic browning during processing.

Angiotensin I-Converting Enzyme (ACE-I) Inhibition and Antioxidant Peptide from a Squilla Species.

BACKGROUND: Oratosquilla woodmasoni is one of the marine squilla species, which is found in the entire Asia-Pacific region. This current study assesses the species as the main basis of both ACEi and antioxidant peptide. OBJECTIVE: To isolate the ACEi peptide derived from O. woodmasoni and examine its ACE inhibition along with antioxidant potential. MATERIALS AND METHODS: The squilla muscle protein was hydrolysed using alcalase and trypsin enzymes for 12 hours and tested for DH. The hydrolysates were examined for their ACEi activity and then the best hydrolysate was sequentially purified in various chromatographical methods. The purified peptide was studied for anti-oxidant and functional properties, followed by amino acid sequencing. The purified peptide was also evaluated for its toxicity by in vitro cell viability assay. RESULTS: The DH% was found to be 47.13 ± 0.72% and 89.43 ± 2.06% for alcalase and trypsin, respectively. The alcalase 5th-hour hydrolysate was detected with potent activity (65.97 ± 0.56%) using ACEi assay and was primarily fractionated using ultrafiltration; the maximum inhibitory activity was found with 77.04 ± 0.52% in 3-10 kDa fraction. Subsequently, the fraction was purified using IEC and GFC, in which the AC1-A2 fraction had higher antihypertensive activity (70.85 ± 0.78%). The non-toxic fraction showed hexapeptide HVGGCG with molecular weight 529 Da with great potential of antioxidant activity along with functional property. CONCLUSION: This peptide could be developed as a potential ACE-inhibitory and antioxidant agent.

Proteomic Analysis of the Hemolymph After Metschnikowia bicuspidata Infection in the Chinese Mitten Crab Eriocheir sinensis.

The "milky disease" of the Chinese mitten crab, Eriocheir sinensis, is a highly lethal fungal disease caused by Metschnikowia bicuspidata infection. To elucidate the immune responses of the hemolymph of E. sinensis to M. bicuspidata infection, a comparative analysis of the hemolymph of E. sinensis infected with M. bicuspidata and that treated with phosphate buffered saline was performed using label-free quantitative proteomics. A total of 429 proteins were identified. Using a 1.5-fold change in expression as a physiologically significant benchmark, 62 differentially expressed proteins were identified, of which 38 were significantly upregulated and 24 were significantly downregulated. The upregulated proteins mainly included cytoskeleton-related proteins (myosin regulatory light chain 2, myosin light chain alkali, tubulin α-2 chain, and tubulin ß-1 chain), serine protease and serine protease inhibitor (clip domain-containing serine protease, leukocyte elastase inhibitor, serine protein inhibitor 42Dd), catalase, transferrin, and heat shock protein 70. Upregulation of these proteins indicated that phenoloxidase system, phagocytosis and the ROS systems were induced by M. bicuspidata. The downregulated proteins were mainly organ and tissue regeneration proteins (PDGF/VEGF-related factor protein, integrin-linked protein kinase homing pat-4 gene) and hemagglutination-associated proteins (hemolymph clottable protein, hemocyte protein-glutamine gamma-glutamyltransferase). Downregulation of these proteins indicated that M. bicuspidata inhibited hemocyte regeneration and hemolymph agglutination. Fifteen differentially expressed proteins related to immunity were verified using a parallel reaction monitoring method. The expression trend of these proteins was similar to that of the proteome. To the best of our knowledge, this is the first report on the proteome of E. sinensis in response to M. bicuspidata infection. These results not only provide new and important information on the immune response of crustaceans to yeast infection but also provide a basis for further understanding the molecular mechanism of complex host pathogen interactions between crustaceans and fungi.

Overexpression and purification of a toxic peptide LaIT2 from Japanese scorpion, Liocheles australasiae.

In Japan, there are two species of scorpions, Madara scorpion (Isometrus maculatus) and Yaeyama scorpion (Liocheles australasiae), and both of them are living in Yaeyama island. It has been shown that Liocheles australasiae has venom including ß-toxin acting on K+-channels (ß-KTx) (Juichi et al., 2018) [1]. Interestingly, LaIT2, one of the toxins found in the venom of Liocheles australasiae, displays the virulence for insects but almost not for mammals. Until now, molecular mechanism of the functional specificity of LaIT2 is unknown. To clear this issue, we tried to establish the overexpression system of LaIT2 in Rosetta-gami B (DE3) pLysS, which have trxB/gor mutations to induce the disulfide bond formation. In this study, we have succeeded to overexpress the recombinant LaIT2 (rLaIT2) as a thioredoxin (Trx)-tagged protein, and established the purification protocol with Ni2+-NTA column chromatography, enterokinase digestion, and HPLC. We succeeded to obtain approximately 0.5 mg of rLaIT2 from the E. coli cells cultured in 1 L of M9 culture medium. Intramolecular disulfide bonding pattern of rLaIT2 was identified by endopeptidase fragmentation and mass spectrometry. rLaIT2 showed insecticidal activity and antimicrobial activity, and these are almost identical to those of natural LaIT2. 1H-15N HSQC spectrum of 15N-labeled rLaIT2 indicated that the rLaIT2 has a stable conformation.

Biochemical Study of Fibrinolytic Protease from Euphausia superba Possessing Multifunctional Serine Protease Activity.

BACKGROUND: Fibrinolytic protease from Euphausia superba (EFP) was isolated. OBJECTIVE: Biochemical distinctions, regulation of the catalytic function, and the key residues of EFP were investigated. METHODS: The serial inhibition kinetic evaluations coupled with measurements of fluorescence spectra in the presence of 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF) was conducted. The computational molecular dynamics (MD) simulations were also applied for a comparative study. RESULTS: The enzyme behaved as a monomeric protein with a molecular mass of about 28.6 kD with Km BApNA = 0.629 ± 0.02 mM and kcat/Km BApNA = 7.08 s-1/mM. The real-time interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to a biphase. Measurements of fluorescence spectra showed that serine residue modification by AEBSF directly caused conspicuous changes of the tertiary structures and exposed hydrophobic surfaces. Some osmolytes were applied to find protective roles. These results confirmed that the active region of EFP is more flexible than the overall enzyme molecule and serine, as the key residue, is associated with the regional unfolding of EFP in addition to its catalytic role. The MD simulations were supportive to the kinetics data. CONCLUSION: Our study indicated that EFP has an essential serine residue for its catalyst function and associated folding behaviors. Also, the functional role of osmolytes such as proline and glycine that may play a role in defense mechanisms from environmental adaptation in a krill's body was suggested.

Effects of ultrasound-assisted basic electrolyzed water (BEW) extraction on structural and functional properties of Antarctic krill (Euphausia superba) proteins.

A novel protein extraction method of ultrasound-assisted basic electrolyzed water (BEW) was proposed, and its effects on the structural and functional properties of Antarctic krill proteins were investigated. Results showed that BEW reduced 30.9% (w/w) NaOH consumption for the extraction of krill proteins, and its negative redox potential (-800 ~ -900 mV) protected the active groups (carbonyl, free sulfhydryl, etc.) of the proteins from oxidation compared to deionized water (DW). Moreover, the ultrasound-assisted BEW increased the extraction yield (9.4%), improved the solubility (8.5%), reduced the particle size (57 nm), favored the transition of α-helix and ß-turn to ß-sheet, promoted the surface hydrophobicity and disulfide bonds formation of krill proteins when compared to BEW without ultrasound. These changes contributed to the enhanced foam capacity, foam stability and emulsifying capacity of the krill proteins. Notably, all the physicochemical, structural and functional properties of the krill proteins were comparable to those extracted by the traditional ultrasound-assisted DW. This study suggests that the ultrasound-assisted BEW can be a potential candidate to extract proteins, especially offering an alternative way to produce marine proteins with high nutritional quality.

Liquid-liquid phase separation promotes animal desiccation tolerance.

Proteinaceous liquid-liquid phase separation (LLPS) occurs when a polypeptide coalesces into a dense phase to form a liquid droplet (i.e., condensate) in aqueous solution. In vivo, functional protein-based condensates are often referred to as membraneless organelles (MLOs), which have roles in cellular processes ranging from stress responses to regulation of gene expression. Late embryogenesis abundant (LEA) proteins containing seed maturation protein domains (SMP; PF04927) have been linked to storage tolerance of orthodox seeds. The mechanism by which anhydrobiotic longevity is improved is unknown. Interestingly, the brine shrimp Artemia franciscana is the only animal known to express such a protein (AfrLEA6) in its anhydrobiotic embryos. Ectopic expression of AfrLEA6 (AWM11684) in insect cells improves their desiccation tolerance and a fraction of the protein is sequestered into MLOs, while aqueous AfrLEA6 raises the viscosity of the cytoplasm. LLPS of AfrLEA6 is driven by the SMP domain, while the size of formed MLOs is regulated by a domain predicted to engage in protein binding. AfrLEA6 condensates formed in vitro selectively incorporate target proteins based on their surface charge, while cytoplasmic MLOs formed in AfrLEA6-transfected insect cells behave like stress granules. We suggest that AfrLEA6 promotes desiccation tolerance by engaging in two distinct molecular mechanisms: by raising cytoplasmic viscosity at even modest levels of water loss to promote cell integrity during drying and by forming condensates that may act as protective compartments for desiccation-sensitive proteins. Identifying and understanding the molecular mechanisms that govern anhydrobiosis will lead to significant advancements in preserving biological samples.

Biological activities of a recombinant fortilin from Fenneropenaeus merguiensis.

Human Fortilin, an antiapoptotic protein, has also been implicated in several diseases; however, several potential uses of fortilin have also been proposed. Bearing the implications of fortilin in mind, fortilin analog, which has no complication with diseases, is required. Since a recombinant full-length fortilin from Fenneropenaeus merguiensis (rFm-Fortilin (FL)) reported only 44% (3e-27) homologous to human fortilin, therefore the biological activities of the Fm-Fortilin (FL) and its fragments (F2, F12, and F23) were investigated for potential use against HEMA toxicity from filling cement to pulp cell. The rFm-Fortilin FL, F2, 12, and F23 were expressed and assayed for proliferation activity. The rFm-Fortilin (FL) showed proliferation activity on human dental pulp cells (HDPCs) and protected the cells from 2-hydroxy-ethyl methacrylate (HEMA) at 1-20 ng/ml. In contrast, none of the rFm-Fortilin fragments promoted HDPC growth that may be due to a lack of three conserved amino acid residues together for binding with the surface of Rab GTPase for proliferative activity. In addition, rFm-Fortilin (FL) activated mineralization and trend to suppressed production of proinflammatory cytokines, including histamine (at 10 ng/ml) and TNF-α (at 100 ng/ml). Besides, the rFm-Fortilin (FL) did not mutate the Chinese hamster ovary (CHO) cell. Therefore, the rFm-Fortilin (FL) has the potential use as a supplementary medical material to promote cell proliferation in patients suffering severe tooth decay and other conditions.

Currently Available Recombinant Alternatives to Horseshoe Crab Blood Lysates: Are They Comparable for the Detection of Environmental Bacterial Endotoxins? A Review.

Endotoxin testing by recombinant factor C (rFC) is increasing with the addition of new suppliers of reagents. By use of a recombinantly produced factor C , based on the sequence of a coagulation enzyme present in horseshoe crab amebocyte lysates, the rFC tests are designed as substitutes for the traditional Limulus amebocyte lysate (LAL)/Tachypleus amebocyte lysate tests based on horseshoe crab blood. Comparative testing of samples with both the LAL and recombinant reagents has shown a high degree of correlation, suggesting that use of rFC is comparable to the more traditional LAL tests and may be technologically superior. Recombinant factor C does not recognize the factor G pathway, the alternate coagulation pathway that the lysate reagents detect. This feature allows rFC to detect endotoxin more selectively. As a recombinantly produced material, it avoids the use of the horseshoe crabs required for lysate production, thereby protecting this species, which is at risk in some parts of the world. Recombinant factor C is expected to further benefit from a more sustainable supply chain based upon a robust biotechnological production process. We summarize here the results of many studies that evaluated the use of recombinant technology for the detection of environmental endotoxin. Additionally, we include a review of the current compendia and regulatory status of the recombinant technologies for use in the quality control of pharmaceutical manufacturing. Our analysis confirms that the recombinant technologies are comparable in protecting patient safety.

Antibiofilm and immunological properties of lectin purified from shrimp Penaeus semisulcatus.

Penaeid prawns are considered as most demanding fishery resources. The current study aims to purify and characterize lectin from the haemolymph of Penaeus semisulcatus. The semisulcatus-lectin was purified by affinity chromatography using mannose coupled Sepharose CL-4B column and purified lectin exhibited a single band of 66 kDa in SDS-PAGE. The purity and crystalline structure of purified lectin was confirmed by HPLC and X-ray diffraction analysis. Semisulcatus-lectin exhibited yeast agglutination activity against Saccharomyces cerevisiae and agglutinated human erythrocytes. Semisulcatus-lectin was evaluated for phenol oxidase activation and phagocytic activities. It was observed that semisulcatus-lectin had antibacterial activity against Gram-negative Vibrio parahaemolyticus and Aeromonas hydrophila, suggesting a potential therapeutic strategy in aquaculture industry for disease management.

Lectin-Like Activity of Hemocyanin in Freshwater Prawn, Macrobrachium rosenbergii.

Lectins are proteins that bind to the carbohydrate moieties on surface of bacteria, erythrocytes and other cells of invertebrates causing agglutination and mediate in recognition of foreign substances. In the present study, we isolated and characterized a lectin molecule present in the hemolymph of Macrobrachium rosenbergii, an important cultured freshwater prawn. Lectin in serum samples of adult prawns was assessed through hemagglutination (HA) test using rabbit RBC that showed a titre ranging from 16 to 64. This serum hemagglutinin was confirmed as a C-type lectin based on its dependency on calcium ions towards binding to rabbit RBCs. The hemagglutinin was also found to be stable at the pH range of 5.0-10.0 and temperature range of 10-40 °C. Of various sugars and glycoproteins tested in hemagglutination inhibition assay, the serum lectin was found specific only to N-acetylneuraminic acid and fetuin with respective minimum inhibitory concentrations at 50 mM and 0.31 mg/ml. Further, the lectin was purified by affinity chromatography on rabbit erythrocyte stroma, which showed hemagglutination with rabbit RBC. In electrophoretic analyses, the purified lectin showed one band with molecular weight of ~ 427 kDa in native gradient PAGE, and its two constituent polypeptide chains of ~ 81 and ~ 73 kDa in SDS-PAGE. These polypeptides were analysed in MALDI-TOF/TOF mass spectrometry and identified as hemocyanins. It was hence, concluded that hemocyanin in M. rosenbergii possesses lectin-like activity.

Tyrophagus putrescentiae group 4 allergen allergenicity and epitope prediction.

INTRODUCTION AND OBJECTIVES: Allergen-specific immunotherapy (ASIT) is the only allergic disease-modifying therapy available for children and adults, and recombinant allergens are an interesting approach to improve allergy diagnosis and ASIT. Tyrophagus putrescentiae is a common storage mite that produces potent allergens. The aim of this study was to express and characterize recombinant group 4 allergen protein of T. putrescentiae (Tyr p 4), and to further investigate allergenicity and potential epitopes of Tyr p 4. MATERIALS AND METHODS: The cDNA encoding Tyr p 4 was generated by RT-PCR and subcloned into pET-28a(+) plasmid. The plasmid was then transformed into E. coli cells for expression. After purification by nickel affinity chromatography and identification by SDS-PAGE, recombinant Tyr p 4 protein was used for a skin prick test and an ELISA to determine the allergic response. RESULTS: Study participants' allergic response rate to Tyr p 4 protein was 13.3% (16/120). Eight B-cell epitopes and three T-cell epitopes of Tyr p 4 were predicted. CONCLUSIONS: Similar to group 4 allergens of other species of mite, allergenicity of Tyr p 4 is weak. The expression, characterization and epitope prediction of recombinant Tyr p 4 protein provide a foundation for further study of this allergen in the diagnosis and ASIT of storage mite allergy.

A shrimp glycosylase protein, PmENGase, interacts with WSSV envelope protein VP41B and is involved in WSSV pathogenesis.

Viral glycoproteins are expressed by many viruses, and during infection they usually play very important roles, such as receptor attachment or membrane fusion. The mature virion of the white spot syndrome virus (WSSV) is unusual in that it contains no glycosylated proteins, and there are currently no reports of any glycosylation mechanisms in the pathogenesis of this virus. In this study, we cloned a glycosylase, mannosyl-glycoprotein endo-ß-N-acetylglucosaminidase (ENGase, EC 3.2.1.96), from Penaeus monodon and found that it was significantly up-regulated in WSSV-infected shrimp. A yeast two-hybrid assay showed that PmENGase interacted with both structural and non-structural proteins, and GST-pull down and co-immunoprecipitation (Co-IP) assays confirmed its interaction with the envelope protein VP41B. In the WSSV challenge tests, the cumulative mortality and viral copy number were significantly decreased in the PmEngase-silenced shrimp, from which we conclude that shrimp glycosylase interacts with WSSV in a way that benefits the virus. Lastly, we speculate that the deglycosylation activity of PmENGase might account for the absence of glycosylated proteins in the WSSV virion.

Cloning and characterization of DOPA decarboxylase in Litopenaeus vannamei and its roles in catecholamine biosynthesis, immunocompetence, and antibacterial defense by dsRNA-mediated gene silencing.

Catecholamines (CAs) play critical roles in regulating physiological and immunological homeostasis in invertebrates and vertebrates under stressful environments. DOPA decarboxylase (DDC), an enzyme responsible for the decarboxylation step of dopamine synthesis, participates in neurotransmitter metabolism and innate immunity. In shrimp, two genes encoding CA-related enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase, were further identified and characterized as neuroendocrine-immune regulators. In this study, full-length complementary DNA of DDC cloned from the thoracic ganglia of shrimp, Litopenaeus vannamei, (LvDDC) was predicted to encode a 452-amino acid protein with a pyridoxal-dependent decarboxylase-conserved domain, and this deduced protein of LvDDC was phylogenetically closely related to insect DDC. LvDDC messenger RNA expression was analyzed by a semiquantitative RT-PCR and a real-time quantitative RT-PCR and found to be abundant in the hepatopancreas and nervous system but at low levels in haemocytes, heart, stomach, and gills. To determine the role of LvDDC, double-stranded (ds)RNA was used for in vivo assessments. LvDDC-depleted shrimp revealed significant increases in the total haemocyte count, hyaline cells, granular cells, phenoloxidase activity, and respiratory bursts of haemocytes per unit of haemolymph, and phagocytic activity and clearance efficiency toward Vibrio alginolyticus. Further, decreased LvDDC mRNA expression was accompanied by decreases in dopamine, glucose, and lactate levels in haemolymph. In shrimp that received LvDDC-dsRNA for 3 days and were then challenged with V. alginolyticus, the survival rate of LvDDC-depleted shrimp was significantly higher than that of shrimp that received diethyl pyrocarbonate-water or non-targeted dsRNA. In conclusion, the cloned LvDDC was responsible for controlling dopamine synthesis, which then regulated physiological and immune responses in L. vannamei.

Molecular cloning and functional analysis of three STAT isoforms in red swamp crayfish Procambarus clarkii.

The Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway is associated with the innate immune system and plays crucial roles in the mediation of immune response to viral infections. In this study, three STAT isoform cDNAs were cloned from the red swamp crayfish Procambarus clarkii, and they were designated as PcSTATa, PcSTATb, and PcSTATc. PcSTATa and PcSTATb were generated through the alternative splicing of the last exon, and PcSTATc was produced by intron retention. PcSTATa, PcSTATb, and PcSTATc contained 2382, 2337, and 2274 bp open reading frames encoding proteins with 793, 778, and 757 amino acid residues, respectively. Domain prediction analysis revealed that three isoforms of PcSTATs contain a STAT interaction domain, a STAT all-alpha domain, a STAT DNA binding domain, and a Src-homology 2 domain. The mRNA transcripts of three PcSTAT isoforms were detected in all examined tissues of male and female crayfish. The expression levels of the three PcSTAT isoforms in the hemocytes, gills, and intestines significantly changed after the white spot syndrome virus (WSSV) challenge. PcSTAT silencing by dsRNA interference could positively regulate the expression levels of three anti-lipopolysaccharide factors (PcALF1, PcALF2, and PcALF6) and two crustins (PcCrus1 and PcCrus2) and negatively regulate the expression levels of three ALFs (PcALF3, PcALF4, and PcALF5) and two crustins (PcCrus3 and PcCrus4). These results suggest that all three PcSTAT isoforms are involved in the host defense against WSSV infection.

Identification and Molecular Characterization of a Pellino Protein in Kuruma Prawn (Marsupenaeus Japonicus) in Response to White Spot Syndrome Virus and Vibrio Parahaemolyticus Infection.

Kuruma prawn, Marsupenaeus japonicus, has the third largest annual yield among shrimp species with vital economic significance in China. White spot syndrome virus (WSSV) is a great threat to the global shrimp farming industry and results in high mortality. Pellino, a highly conserved E3 ubiquitin ligase, has been found to be an important modulator of the Toll-like receptor (TLR) signaling pathways that participate in the innate immune response and ubiquitination. In the present study, the Pellino gene from Marsupenaeus japonicus was identified. A qRT-PCR assay showed the presence of MjPellino in all the tested tissues and revealed that the transcript level of this gene was significantly upregulated in both the gills and hemocytes after challenge with WSSV and Vibrio parahaemolyticus. The function of MjPellino was further verified at the protein level. The results of the three-dimensional modeling and protein-protein docking analyses and a GST pull-down assay revealed that the MjPellino protein was able to bind to the WSSV envelope protein VP26. In addition, the knockdown of MjPellino in vivo significantly decreased the expression of MjAMPs. These results suggest that MjPellino might play an important role in the immune response of kuruma prawn.

Valorization of snow crab (Chionoecetes opilio) cooking effluents for food applications.

BACKGROUND: Seafood processing generates significant amounts of solid and liquid waste in the environment. Such waste represents a potential source of high-value biomolecules for food, pharmaceutic and cosmetic applications. There are very few studies on the valorization of wastewaters compared to solid by-products. However, cooking waters are characterized by a high organic polluting load, which could contain valuable molecules such as proteins, pigments and flavor compounds. Snow crab (Chionoecetes opilio) processing is included among the most important processes in Canadian fisheries, although its cooking effluent composition is not well characterized. RESULTS: The present study concentrated and valorized the biomass in snow crab cooking wastewaters for the development of products for food applications. A membrane process was designed and optimized to concentrate the effluents. The chemical composition of the concentrates was analyzed, including characterizing the flavor profile compounds. The extracts were mainly composed of proteins (592 g kg-1 ) and minerals (386 g kg-1 ) and contained desirable flavor compounds. Their functional properties (solubility, water-holding capacity, oil-holding capacity) and antioxidant activities were also assessed, and their safety was verified. CONCLUSION: The cooking effluents generated by snow crab processing facilities, usually considered as waste, can be concentrated and turned into a natural aroma for the food industry. © 2019 Society of Chemical Industry.

The immunosuppressive functions of two novel tick serpins, HlSerpin-a and HlSerpin-b, from Haemaphysalis longicornis.

Serpins are evolutionarily conserved serine protease inhibitors that are widely distributed in animals, plants and microbes. In this study, we reported the cloning and functional characterizations of two novel serpin genes, HlSerpin-a and HlSerpin-b, from the hard tick Haemaphysalis longicornis of China. Recombinant HlSerpin-a and HlSerpin-b displayed protease inhibitory activities against multiple mammalian proteases. Similar to other tick serpins, HlSerpin-a and HlSerpin-b suppressed the expression of inflammatory cytokines such as TNF-α, interleukin (IL)-6 and IL-1ß from lipopolysaccharide-stimulated mouse bone-marrow-derived macrophages (BMDMs) or mouse bone-marrow-derived dendritic cells (BMDCs). The minimum active region (reaction centre loop) of HlSerpin-a, named SA-RCL, showed similar biological activities as HlSerpin-a in the protease inhibition and immune suppression assays. The immunosuppressive activities of full-length HlSerpin-a and SA-RCL are impaired in Cathepsin G or Cathepsin B knockout mouse macrophages, suggesting that the immunomodulation functions of SA and SA-RCL are dependent on their protease inhibitory activity. Finally, we showed that both full-length HlSerpins and SA-RCL can relieve the joint swelling and inflammatory response in collagen-induced mouse arthritis models. These results suggested that HlSerpin-a and HlSerpin-b are two functional arthropod serpins, and the minimal reactive peptide SA-RCL is a potential candidate for drug development against inflammatory diseases.

Purification, characterization and antibacterial activities of red color-related protein found in the shell of kuruma shrimp, Marsupenaeus japonicus.

The well-known red color change plays a significant role in consumer acceptability of crustacean species. In this study, we described the purification of the red color-related protein named MjRCP75 from the shell of Marsupenaeus japonicus. It was a homogeneous monomer with molecular mass of 75 kDa and rich in α-helix conformation. The α-helix content decreased within the increasing of heating temperature and was transformed dominantly to ß types. Identification and structural analysis revealed that MjRCP75 belonged to hemocyanin family. The released pigment from heated MjRCP75 showed a λmax at 483 nm in acetone. MjRCP75 showed clearly antibacterial activity against Escherichia coli, Staphylococcus aureus, and Vibrio parahaemolyticus. These findings identify MjRCP75 as the red color-related protein in M. japonicus shell and reveal its involvement in antibacterial activities.

Crystal structure of jumping spider rhodopsin-1 as a light sensitive GPCR.

Light-sensitive G protein-coupled receptors (GPCRs)-rhodopsins-absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release retinal upon isomerization as opposed to the retinal in bistable rhodopsins that "reisomerize" upon absorption of a second photon. Understanding the mechanistic differences between these light-sensitive GPCRs has been hindered by the scarcity of recombinant models of the latter. Here, we reveal the high-resolution crystal structure of a recombinant bistable rhodopsin, jumping spider rhodopsin-1, bound to the inverse agonist 9-cis retinal. We observe a water-mediated network around the ligand hinting toward the basis of their bistable nature. In contrast to bovine rhodopsin (monostable), the transmembrane bundle of jumping spider rhodopsin-1 as well that of the bistable squid rhodopsin adopts a more "activation-ready" conformation often observed in other nonphotosensitive class A GPCRs. These similarities suggest the role of jumping spider rhodopsin-1 as a potential model system in the study of the structure-function relationship of both photosensitive and nonphotosensitive class A GPCRs.