Backbone and side chain chemical shift assignment of diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris, an organophosphorus-degrading enzyme.

NMR chemical shift assignments are reported for backbone (15N, 1H) and partial side chain (13Cα and ß, side chain 1H) atoms of diisopropyl fluorophosphatase (DFPase), a calcium-dependent phosphotriesterase capable of hydrolyzing phosphorus - fluorine bonds in a variety of toxic organophosphorus compounds. Analysis of residues lining the active site of DFPase highlight a number of residues whose chemical shifts can be used as a diagnostic of binding and detection of organophosphorus compounds.

Packaging of Diisopropyl Fluorophosphatase (DFPase) in Bacterial Outer Membrane Vesicles Protects Its Activity at Extreme Temperature.

Enzymatic decontamination of organophosphate compounds offers a biofriendly pathway to the neutralization of highly dangerous compounds. Environmental dissemination of enzymes, however, is an ongoing problem considering the costly process of production and chemical modification for stability that can diminish catalytic activity. As a result, there is interest in the potential for enzymatic encapsulation in situ or into nascent bacterial membrane vesicles to improve catalytic stability across various environmental challenges associated with storage and field deployment. In this study, we have engineered bacterial outer membrane vesicles (OMVs) to encapsulate the diisopropyl fluorophosphatase (DFPase), an enzyme originally isolated from squid Loligo vulgaris and capable of hydrolyzing diisopropyl fluorophosphate (DFP) and other organophosphates compounds. Here we employed a recombinant lipopeptide anchor to direct recruitment of DFPase into OMVs, which were isolated from culture media and tested for catalytic activity against both diisopropyl fluorophosphate and paraoxon. Our encapsulation strategy prevented the loss of catalytic activity despite lyophilization, extended storage time (2 days), and extreme temperatures up to 80 °C. These data underscore the appeal of DFPase as a biodecontaminant of organophosphates as well as the potential for OMV packaging in stabilized field deployment applications.

Mercury bioaccumulation in the long-fin squid Loligo forbesi near the Mid-Atlantic Ridge: Implications to human exposure.

Due to growing commercial interest as a fishing resource and its intermediate position in the marine trophic chains as both prey and predator, cephalopods can play an important role in mercury (Hg) transfer along the marine food webs, since they can bioaccumulate this metal in its tissues. Our study aims to analyze Hg accumulation in Loligo forbesi caught in the Azores Archipelago (Portugal) quantifying Hg in different tissues (mantle and stomach), as well in the squid stomach content, in order to evaluate the efficiency of Hg transfer from prey to predator. Hg data from the tissues was used to estimate the weekly tolerable Hg intake due to squid consumption. Overall data indicate that Hg measured in the stomach tissue (0.1 ± 0.01 µg g-1) was significantly higher than Hg levels found in the mantle (0.04 ± 0.001 µg g-1) and stomach contents (0.01 ± 0.001 µg g-1). BMF (bioaccumulation factor) was >1 for all the samples, indicating a biomagnification process from prey to predator. Hg concentration in the mantle tissue was correlated with mantle size; although females present higher Hg levels than males, the difference was found to be not related to gender but rather to the fact that females had larger bodies. Finally, considering the Hg concentration found in the mantle and the permitted Hg levels, it is advisable to consume up to 1050-1890g of squid per week, according to the regulatory agencies. Thus, our results indicate that, since these doses are respected, consumption of squids from the Azorean waters do not pose a risk to humans.

Halogenated natural products and anthropogenic persistent organic pollutants in chokka squid (Loligo reynaudii) from three sites along the South Atlantic and Indian Ocean coasts of South Africa.

Chokka squid (Loligo reynaudii) from three sites along the South African coast were analyzed for halogenated natural products (HNPs) and anthropogenic persistent organic pollutants (POPs). HNPs were generally more than one order of magnitude more abundant than POPs. The most prevalent pollutant, i.e. the HNP 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1), was detected in all chokka squid samples with mean concentrations of 105, 98 and 45 ng/g lipid mass, respectively, at the Indian Ocean (site A), between both oceans (site B) and the South Atlantic Ocean (site C). In addition, bromine containing polyhalogenated 1'-methyl-1,2'-bipyrroles (PMBPs), 2,4,6-tribromophenol (2,4,6-TBP, up to 28 ng/g lipid mass), polybrominated methoxy diphenyl ethers, MHC-1, TBMP and other HNPs were also detected. Polychlorinated biphenyls (PCBs) were the predominant class of anthropogenic POPs. PCB 153 was the most abundant PCB congener in chokka squid from the Indian Ocean, and PCB 138 in samples from the South Atlantic Ocean and between both oceans.

Multifunctional and biodegradable self-propelled protein motors.

A diversity of self-propelled chemical motors, based on Marangoni propulsive forces, has been developed in recent years. However, most motors are non-functional due to poor performance, a lack of control, and the use of toxic materials. To overcome these limitations, we have developed multifunctional and biodegradable self-propelled motors from squid-derived proteins and an anesthetic metabolite. The protein motors surpass previous reports in performance output and efficiency by several orders of magnitude, and they offer control of their propulsion modes, speed, mobility lifetime, and directionality by regulating the protein nanostructure via local and external stimuli, resulting in programmable and complex locomotion. We demonstrate diverse functionalities of these motors in environmental remediation, microrobot powering, and cargo delivery applications. These versatile and degradable protein motors enable design, control, and actuation strategies in microrobotics as modular propulsion sources for autonomous minimally invasive medical operations in biological environments with air-liquid interfaces.

Integrative omics analysis reveals differentially distributed proteins in dimorphic euspermatozoa of the squid, Loligo bleekeri.

In the coastal squid Loligo bleekeri, each male produces one of two types of fertilization-competent spermatozoa (eusperm) that exhibit morphological and behavioral differences. Large "consort" males produce short-tailed spermatozoa that display free-swimming behavior when ejaculated into seawater. Small "sneaker" males, on the other hand, produce long-tailed spermatozoa that exhibit a self-swarming trait after ejaculation. To understand the molecular basis for adaptive traits employed by alternative male mating tactics, we performed the transcriptome deep sequencing (RNA-seq) and proteome analyses to search for differences in testicular mRNAs and sperm proteins, respectively. From mature male testes we identified a total of 236,455 contigs (FPKM ≧1) where 3789 and 2789 were preferentially (≧10-fold) expressed in consort and sneaker testes, respectively. A proteomic analysis detected 4302 proteins in the mature sperm as post-translational products. A strongly biased (≧10-fold) distribution occurred in 55 consort proteins and 61 sneaker proteins. There was no clear mRNA-protein correlation, making a ballpark estimate impossible for not only overall protein abundance but also the degree of biased sperm type expressed in the spermatozoa. A family encoding dynein heavy chain gene, however, was found to be biased towards sneakers, whereas many enzymes involving energy metabolism were heavily biased towards consort spermatozoa. The difference in flagellar length matched exactly the different amount of tubulins. From these results we hypothesize that discrete differential traits in dimorphic eusperm arose from a series of innovative alterations in the intracellular components of spermatozoa.

Ocean warming enhances malformations, premature hatching, metabolic suppression and oxidative stress in the early life stages of a keystone squid.

BACKGROUND: The knowledge about the capacity of organisms' early life stages to adapt to elevated temperatures is very limited but crucial to understand how marine biota will respond to global warming. Here we provide a comprehensive and integrated view of biological responses to future warming during the early ontogeny of a keystone invertebrate, the squid Loligo vulgaris. METHODOLOGY/PRINCIPAL FINDINGS: Recently-spawned egg masses were collected and reared until hatching at present day and projected near future (+2°C) temperatures, to investigate the ability of early stages to undergo thermal acclimation, namely phenotypic altering of morphological, behavioural, biochemical and physiological features. Our findings showed that under the projected near-future warming, the abiotic conditions inside the eggs promoted metabolic suppression, which was followed by premature hatching. Concomitantly, the less developed newborns showed greater incidence of malformations. After hatching, the metabolic burst associated with the transition from an encapsulated embryo to a planktonic stage increased linearly with temperature. However, the greater exposure to environmental stress by the hatchlings seemed to be compensated by physiological mechanisms that reduce the negative effects on fitness. Heat shock proteins (HSP70/HSC70) and antioxidant enzymes activities constituted an integrated stress response to ocean warming in hatchlings (but not in embryos). CONCLUSIONS/SIGNIFICANCE: The stressful abiotic conditions inside eggs are expected to be aggravated under the projected near-future ocean warming, with deleterious effects on embryo survival and growth. Greater feeding challenges and the lower thermal tolerance limits of the hatchlings are strictly connected to high metabolic demands associated with the planktonic life strategy. Yet, we found some evidence that, in the future, the early stages might support higher energy demands by adjusting some cellular functional properties to increase their thermal tolerance windows.

Effects of ocean acidification on trace element accumulation in the early-life stages of squid Loligo vulgaris.

The anthropogenic release of carbon dioxide (CO(2)) into the atmosphere leads to an increase in the CO(2) partial pressure (pCO(2)) in the ocean, which may reach 950 µatm by the end of the 21st century. The resulting hypercapnia (high pCO(2)) and decreasing pH ("ocean acidification") are expected to have appreciable effects on water-breathing organisms, especially on their early-life stages. For organisms like squid that lay their eggs in coastal areas where the embryo and then paralarva are also exposed to metal contamination, there is a need for information on how ocean acidification may influence trace element bioaccumulation during their development. In this study, we investigated the effects of enhanced levels of pCO(2) (380, 850 and 1500 µatm corresponding to pH(T) of 8.1, 7.85 and 7.60) on the accumulation of dissolved (110m)Ag, (109)Cd, (57)Co, (203)Hg, (54)Mn and (65)Zn radiotracers in the whole egg strand and in the different compartments of the egg of Loligo vulgaris during the embryonic development and also in hatchlings during their first days of paralarval life. Retention properties of the eggshell for (110m)Ag, (203)Hg and (65)Zn were affected by the pCO(2) treatments. In the embryo, increasing seawater pCO(2) enhanced the uptake of both (110m)Ag and (65)Zn while (203)Hg showed a minimum concentration factor (CF) at the intermediate pCO(2). (65)Zn incorporation in statoliths also increased with increasing pCO(2). Conversely, uptake of (109)Cd and (54)Mn in the embryo decreased as a function of increasing pCO(2). Only the accumulation of (57)Co in embryos was not affected by increasing pCO(2). In paralarvae, the CF of (110m)Ag increased with increasing pCO(2), whereas the (57)Co CF was reduced at the highest pCO(2) and (203)Hg showed a maximal uptake rate at the intermediate pCO(2). (54)Mn and (65)Zn accumulation in paralarvae were not significantly modified by hypercapnic conditions. Our results suggest a combined effect of pH on the adsorption and protective properties of the eggshell and of hypercapnia on the metabolism of embryo and paralarvae, both causing changes to the accumulation of metals in the tissues of L. vulgaris.

The DFPase from Loligo vulgaris in sugar surfactant-based bicontinuous microemulsions: structure, dynamics, and enzyme activity.

The enzyme diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris is of great interest because of its ability to catalyze the hydrolysis of highly toxic organophosphates. In this work, the enzyme structure in solution (native state) was studied by use of different scattering methods. The results are compared with those from hydrodynamic model calculations based on the DFPase crystal structure. Bicontinuous microemulsions made of sugar surfactants are discussed as host systems for the DFPase. The microemulsion remains stable in the presence of the enzyme, which is shown by means of scattering experiments. Moreover, activity assays reveal that the DFPase still has high activity in this complex reaction medium. To complement the scattering experiments cryo-SEM was also employed to study the microemulsion structure.

Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid.

Many cephalopods exhibit remarkable dermal iridescence, a component of their complex, dynamic camouflage and communication. In the species Euprymna scolopes, the light-organ iridescence is static and is due to reflectin protein-based platelets assembled into lamellar thin-film reflectors called iridosomes, contained within iridescent cells called iridocytes. Squid in the family Loliginidae appear to be unique in which the dermis possesses a dynamic iridescent component with reflective, coloured structures that are assembled and disassembled under the control of the muscarinic cholinergic system and the associated neurotransmitter acetylcholine (ACh). Here we present the sequences and characterization of three new members of the reflectin family associated with the dynamically changeable iridescence in Loligo and not found in static Euprymna iridophores. In addition, we show that application of genistein, a protein tyrosine kinase inhibitor, suppresses ACh- and calcium-induced iridescence in Loligo. We further demonstrate that two of these novel reflectins are extensively phosphorylated in concert with the activation of iridescence by exogenous ACh. This phosphorylation and the correlated iridescence can be blocked with genistein. Our results suggest that tyrosine phosphorylation of reflectin proteins is involved in the regulation of dynamic iridescence in Loligo.

A novel 65 kDa RNA-binding protein in squid presynaptic terminals.

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing.

The role of protein assembly in dynamically tunable bio-optical tissues.

Cephalopods are nicknamed the "masters of disguise" for their highly evolved camouflage mechanisms, including the hallmark ability to rapidly change the color and reflectance of their skin. Previously, reflectin proteins were identified as the major biomaterial component of iridosomes [1], specialized light-reflecting architectures that contribute intense structural color to squid skin, eyes, and organs [2-5]. Supramolecular assembly of reflectin has been recognized as a key property in the protein's function [6]. Here, we report the first cloning and expression of a specific reflectin protein found in the responsive iridophore cells of the squid Loligo pealeii, which are unique in their ability to switch on/off and change color. We demonstrate that these iridophores can be chemically tuned to reflect the entire visible spectrum. By examining recombinant reflectin, we show that this dynamic optical function is facilitated by the hierarchical assembly of nanoscale protein particles that elicit large volume changes upon condensation. These findings provide insight into the design and synthesis of biomaterials for complex, responsive function in optical applications.

Molecular cloning and expression analysis of a GnRH-like dodecapeptide in the swordtip squid, Loligo edulis.

In vertebrates, gonadotropin-releasing hormone (GnRH), which is synthesized in the brain, is a key peptide involved in gonadal maturation regulated by the brain-pituitary-gonadal axis. GnRH isoforms and their primary structures have recently been determined in two species of non-chordates, the octopus (Octopus vulgaris) and sea hare (Aplysia californica), which are mollusks. Octopus and sea hare GnRHs are dodecapeptides that contain the structural core of chordate GnRH; however, chordate GnRHs, including tunicate GnRH, are decapeptides. in this study, we examined a GnRH-like peptide in the swordtip squid, Loligo edulis, to provide information on the structural evolution of GnRH in non-chordates. We isolated the full-length cDNA of a GnRH-like molecule from the central nervous system (CNS) of the squid. The open reading frame of this cDNA encodes a protein of 90 amino acids, which consists of a putative signal peptide, a GnRH dodecapeptide, a processing site, and a GnRH-associated peptide. This architecture is generally conserved in chordates. Compared to octopus GnRH, Squid GnRH is identical in the deduced amino acid sequence of the peptide, and 80.5% similar in base sequence. in a phylogenetic analysis, prepro-GnRHs of octopus, sea hare, and squid were segregated from all chordate prepro-GnRHs, in a group designated GnRHS. The squid prepro-GnRH mRNA was expressed mainly in the CNS. This study is the first report of GnRH cDNA cloning in squid and the third in non-chordates.

Receptor-coupling properties of the invertebrate visual guanine nucleotide binding protein iGqalpha.

Invertebrate visual iG(q)alpha is homologous to mammalian mG(q)alpha in two of the three domains important for G protein interaction with receptors; the C-terminus and the linker regions that connect the helical and ras-like domains of the alpha subunit. The third receptor-interacting domain, the N-terminus, contains a six amino acid extension MTLESI in mG(q)alpha that is not present in iG(q)alpha. In co-expression studies we assessed the promiscuity and efficacy of receptor coupling to phospholipase C (PLC) by iG(q)alpha, a non-palmitoylated mutant iG(q)alpha(C3,4A), mG(q)alpha and G(15)alpha. The invertebrate G proteins and mG(q)alpha only coupled to G(q)-coupled receptors (m1 muscarinic acetylcholine receptor (mChR1), alpha(1A)-adrenergic receptor (alpha1-AR)) and not to the G(i/s)-coupled receptors (CCR1 receptor, beta2-adrenergic receptor or dopamine D1 receptor) while G(15)alpha coupled to all receptors. iG(q)alpha and iG(q)alpha(C3,4A) both had double the efficacy for PLC activation compared to the mammalian G proteins when co-expressed with mChR1 and alpha1-AR. The increased efficacy of iG(q)alpha compared to mG(q)alpha was also seen downstream of PLC with carbachol stimulation of the mitogen-activated protein kinase, ERK1/2. Addition of the MTLESI extension onto the N-terminus of iG(q)alpha decreased its efficacy by 35% whereas deletion of this sequence from mG(q)alpha increased its efficacy by 60% in the PLC and ERK1/2 assays. iG(q)alpha, iG(q)alpha(C3,4A) and mG(q)alpha all displayed similar receptor-independent AlF(4)(-)activation of PLC and guanosine triphosphate hydrolysis (GTPase) activity. iG(q)alpha, and iG(q)alpha(C3,4A) both had increased receptor-activated guanosine 5'-[gamma-[(35)S]thio]triphosphate ([(35)S]GTPgammaS) binding when compared to mG(q)alpha when co-expressed with the mChR1. These results demonstrate that G(q) protein efficacy is at least partially determined by the presence of the amino-terminal MTLESI extension. Comparison of [(35)S]GTPgammaS binding rates helps explain the increased efficacy of the invertebrate G proteins.

Thermodynamic aspects of the heterogeneous deacetylation of beta-chitin: reaction mechanisms.

This paper aims at giving a better understanding of the reaction mechanisms involved in the heterogeneous deacetylation of beta-chitin in relation with the influence of soda concentration (30-55% (w/v)) and the type of sodium hydroxide hydrates formed in solution. The role of temperature (35-110 degrees C) and of the amount of sodium acetate generated in the reaction medium was also investigated. We demonstrated that the type of soda hydrate formed before deacetylation starts and its relative abundance drive the reaction efficiency. Thus, in the first part of this work, we evidenced that activation energies and the global reaction order associated to sodium hydroxide varied as a function of soda concentration. Therefore, we revealed that deacetylation efficiency was emphasized when the less hydrated soda was used, whereas anhydrous soda showed no or very low activity. We also pointed out that various parameters could be responsible for the progressive dehydration of the reaction medium, responsible for the transformation of the most reactive hydrates into less effective species. We underlined that this progressive dehydration could be caused by either one or all of the three following phenomena: alkaline hydrolysis of the polymer, the delivery of sodium acetate in the medium, and the evaporation of water when we process deacetylation at high temperatures and in open reactors. Beside kinetics reasons, we revealed that the transformation of soda hydrates as the deacetylation proceeded was also ascribable for the low reaction efficiency at long reaction times. Thanks to our investigations, we concluded that the amount of water present in the system chitin/soda/water/sodium acetate was the angle stone of complex equilibriums governing the reaction, and we propose soda mono- and dihydrates to be the most active reactants for the chitin deacetylation.

Protamines and other basic proteins from spermatozoa of molluscs.

The basic proteins obtained from spermatozoa of different species of the phylum Mollusca have been extracted and fractionated. The amino acid analysis and electrophoretic mobility of these proteins show a considerable variation in the types and relative amounts of the components present in different species. In some case-(Gibbula, Haliotis, Loligo, Octopus) the main components are similar to the protamines found in the salmonid fishes, although they appear to be larger in size (40-80 amino acids) and show significant differences in amino acid composition. In other cases (Mytilus, Chiton) a complex mixture of proteins is present, which including somatic-like histones and proteins intermediate in size and composition betweeln protamines and histones. Other molluscs (Ostrea, Spisula, Patella) also contain proteins intermediate in composition between protamines and histones, but their molecular weight appears to be larger than in histones. In Eledone a complex mixture of proteins containing cystine is obtained, with some components rich in arginine. In most species, somatic-like histones are also present. Their type and relative amount are different in each species. The significance of these results towards an understanding of the evolutionary history of these proteins is discussed. It is suggested that these proteins evolved from histone precursors.