Cloning and biochemical characterization of astacin-like squid metalloprotease

We have cloned four cDNAs encoding astacin-like squid metalloproteases (ALSMs)-I and -II from the Japanese common squid and ALSMs-I and -III from the spear squid. Analysis of the deduced amino acid sequences revealed that ALSMs possess a signal peptide and a pro-sequence followed by an astacin-like catalytic domain and an MAM (meprin, A5 protein, receptor protein-tyrosine phosphatase mu) domain. Phylogenetic analysis revealed that ALSM corresponds to a new cluster of astacins. To analyze the function of the MAM domain, wild-type ALSM and an MAM-truncated mutant were expressed in a baculovirus expression system. The expressed protein encoding full-length ALSM hydrolyzed myosin heavy chain as effectively as native ALSM, whereas the MAM-truncated mutant possessed no protease activity, suggesting that the MAM domain contributes to substrate recognition. ALSM has been isolated from squid liver and mantle muscle. However, analysis with a specific antibody generated against ALSM indicated the presence of ALSM in a wide variety of tissues. ALSM was located in the extracellular matrix of mantle muscle cells. Thus, ALSM is a secreted protease, as are other members of the astacin family. The extracellular localization raises the possibility of substrates other than myosin. The physiological role of ALSM remains unknown, at this time.     

Expression and tissue distribution of astacin-like squid metalloprotease (ALSM)

Astacin metalloprotease family members function in a wide variety of biologic events, including cell differentiation and morphogenesis during embryonic development and adult tissue differentiation. We previously isolated and characterized an astacin-like squid metalloprotease (ALSM). To elucidate the embryonic expression of ALSM, we performed immunohistochemical analysis with specific antibodies and examined the expression profiles of ALSM isoforms by in situ hybridization analysis. Tissue distribution and expression were also examined in adult spear squid. mRNA expression of ALSM isoforms I and III was first detected in newly hatched squid and was restricted to the liver. No mRNA signals were detected in other tissues even in adult squids. At the protein level, both isoforms were prominent in the liver of embryos and later in digestive organs of adult squid. Both isoforms were also detected in muscle tissues, including mantle and tentacle muscle. Staining for ALSM III was also identified in the iris and in tissues near the eye in squid embryos. However, no reactive bands were detected by immunoblotting of adult squid eyes. Thus, ALSM is initially expressed at the late stage of embryogenesis in spear squid, and expression is restricted to the liver. Thereafter, ALSM isoforms function in various tissues in an isoform-dependent manner.     

Solubility properties of a 65-kDa peptide prepared by restricted digestion of myosin with astacin-like squid metalloprotease

Substructure of the myosin rod and its correlation to filament formation is largely based on studies of proteolytic digests and expressed proteins. However, tryptic digestion of myosin always produces polymorphous peptides. Consequently, it is difficult to determine the relation between myosin substructure and filament formation. Similarly, filament formation with recombinant myosin protein is also difficult to interpret because it is never clear whether the recombinant protein folds like the native protein. We recently reported a novel metal protease isolated from squid liver, astacin-like squid metalloprotease (ALSM), which can specifically hydrolyze in vitro myosin heavy chain. In the present study, we examined the solubility properties of the 65-kDa peptide and light meromyosin (LMM) prepared by ALSM isoform II and trypsin digestion, respectively. The 65-kDa peptide is much less soluble than LMM under physiological conditions, even though the length of 65-kDa peptide is shorter than that of LMM. These results suggest that a novel substructure of myosin drives filament assembly.     

The secret life of the giant Australian cuttlefish Sepia apama (Cephalopoda): Behaviour and energetics in nature revealed through radio acoustic positioning and telemetry (RAPT)

Sepia apama were tagged with acoustic transmitters and monitored on their native House Reef, Boston Bay, South Australia, with a radio acoustic positioning telemetry (RAPT) system. Cuttlefish were tagged with position-only and intra-mantle jet pressure transmitters. New data analyses were developed to handle problem data that arise with an uneven reef environment. Maximum range for the cuttlefish varied from 90 m to 550 m. Cuttlefish home range was between 5300 m² and 23,700 m². S. apama were found to be diurnal as average distance travelled was higher in the day than at night, and cuttlefish were active for 32 days, but only 18 nights. After the cuttlefish settled into reef crevices, activity spectrum and positioning analysis showed foraging behaviour at only 3.7% per day and 2.1% per night. Cuttlefish were found to spend more than 95% of the day resting, which suggests that their bioenergetics are more akin to those of octopus than of squid. The cuttlefish combination of predator avoidance, efficient foraging and quiescent lifestyle allows energy to be channelled into growth and fulfillment of the live-fast-die-young cephalopod philosophy.     

Control of the spatial distribution of sodium channels in giant fiber lobe neurons of the squid

Na+ channels are present at high density in squid giant axon but are absent from its somata in the giant fiber lobe (GFL) of the stellate ganglion. GFL cells dispersed in vitro maintain growing axons and develop a Na+ channel distribution similar to that in vivo. Tunicamycin, a glycosylation inhibitor, selectively disrupts the spatially appropriate, high level expression of Na+ channels in axonal membrane but has no effect on expression in cell bodies, which show low level, inappropriate expression in vitro. This effect does not appear to involve alteration in Na+ channel turnover or axon viability. K+ channel distribution is unaffected. Thus, glycosylation appears to be involved in controlling Na+ channel localization in squid neurons.     

XRD studies of beta-chitin from squid pen with calcium solvent

The crystalline structure of β-chitin from squid pen was investigated by X-ray diffraction (XRD). The purified β-chitin was prepared from bigfin reefsquid pen. β-Chitin was treated with saturated calcium chloride dihydrate/alchohol (CaCl₂·2H₂O/MeOH) solvent system at different conditions for XRD studies. The change of crystallinity of β-chitin from squid pen was studied by using the fiber photographs on imaging plates. The results showed that the diffraction peak (0 1 0) was shifted. It means that the lattice plane (0 1 0) interplanarilly spreaded to 3.4 Å, when the squid pen was washed with water after treatment of Ca solvent. Furthermore, when the squid pen was dried after treatment of Ca solvent and washing with water, interplanar spacing of (0 1 0) inversely shrank to 1.1 Å. These results suggested that Ca solvent especially influences the plane (0 1 0) of β-chitin structure.     

Collagen from diamondback squid (Thysanoteuthis rhombus) outer skin

Collagens (acid-solubilized and pepsin-solubilized collagens) were prepared from diamondback squid outer skin and partially characterized. The yields of acid-solubilized and pepsin-solubilized collagens were about 1.3 and 35.6%, respectively, on a dry weight basis. Pepsin-solubilized collagen was heterotrimer with a chain composition of ala2a3. The patterns of peptide fragments were different from that of porcine skin collagen. Denaturation temperature was 27.5 degrees C, about 10 degrees C lower than that of porcine collagen. The amino acid composition of pepsin-solubilized collagen from diamondback squid outer skin was similar to that from cuttlefish outer skin. This squid is big among squid species, and its skin is thick. It is clear that diamondback squid outer skin has a potential as an alternative source of collagen to bovine skin and bone. At present, collagen using aquatic materials such as skin (cod and a deep-sea fish) and scale (sea bream and anchovy) is the development stage in the related industries. Unless the problem of BSE infection in land animals is resolved aquatic materials as an alternative source of collagen will attract much attention in the cosmetic and medical fields.     

Expression and Localization of sPLA2-III in the Rat CNS

Phospholipases A₂ (PLA₂) are enzymes that cleave the sn-2 bond of membrane phospholipids to yield free fatty acids and lysophospholipids. Secretory PLA₂-III (sPLA₂-III) has been suggested to be important for neuronal differentiation, growth and survival, and is highly expressed in the spinal cord. The aim of this study is to elucidate its expression and distribution in different regions of the adult rat CNS. Quantitative RT-PCR analyses showed high levels of sPLA₂-III mRNA expression in the brainstem and spinal cord and low expression in the olfactory bulb. Western blot analyses showed high level of expression in the brainstem, spinal cord and cerebral neocortex. A dense band corresponding to the catalytically active, mature/cleaved form, and a faint band corresponding to the full length sPLA₂-III were detected in post-mitochondrial supernatants, from different parts of the CNS. Subcellular fractionation of spinal cord homogenates showed that sPLA₂-III protein is present in the ‘light membrane/cytosol’ fraction, but not the nucleus, synaptosomal membrane or synaptic vesicle-enriched fractions. sPLA₂-III was immunolocalized to neurons in the cerebral neocortex, Purkinje neurons in the cerebellar cortex, periaqueductal gray, red nucleus, spinal trigeminal nucleus and dorsal horn of the spinal cord. Electron microscopy of the spinal cord and cerebral neocortex showed that sPLA₂-III was localized in dendrites or dendritic spines, that formed asymmetrical synapses with unlabeled, putatively glutamatergic, axon terminals. The localization of mature/cleaved form of sPLA₂-III in postsynaptic structures suggest a physiological role of the enzyme in neurotransmission or synaptic plasticity.     

Both d- and l-specific Lactate Dehydrogenases Co-exist in Individual Cephalopods

Both d-lactate-specific and l-lactate-specific lactate dehydrogenases coexist in individual cephalopods, contrary to the commonly held view that invertebrate species may contain one or other, but not both. We describe the tissue distribution of these lactate dehydrogenases and of octopine dehydrogenase, the major pyruvate reductase activity in cephalopods, in three species: common squid (Loligo vulgaris), cuttlefish (Sepia officinalis) and lesser octopus (Eledone cirrhosa). The l-specific lactate dehydrogenase of squid is shown to be a dimer of 36,000 dalton subunits.     

Halide peroxidase in tissues that interact with bacteria in the host squid Euprymna scolopes

An enzyme with similarities to myeloperoxidase, the antimicrobial halide peroxidase in mammalian neutrophils, occurs abundantly in the light organ tissue of Euprymna scolopes, a squid that maintains a beneficial association with the luminous bacterium Vibrio fischeri. Using three independent assays typically applied to the analysis of halide peroxidase enzymes, we directly compared the activity of the squid enzyme with that of human myeloperoxidase. One of these methods, the diethanolamine assay, confirmed that the squid peroxidase requires halide ions for its activity. The identification of a halide peroxidase in a cooperative bacterial association suggested that this type of enzyme can function not only to control pathogens, but also to modulate the interactions of host animals with their beneficial partners. To determine whether the squid peroxidase functions under both circumstances, we examined its distribution in a variety of host tissues, including those that typically interact with bacteria and those that do not. Tissues interacting with bacteria included those that have specific cooperative associations with bacteria (i.e., the light organ and accessory nidamental gland) and those that have transient nonspecific interactions with bacteria (i.e., the gills, which clear the cephalopod circulatory system of invading microorganisms). These bacteria-associated tissues were compared with the eye, digestive gland, white body, and ink-producing tissues, which do not typically interact directly with bacteria. Peroxidase enzyme assays, immunocytochemical localization, and DNA-RNA hybridizations showed that the halide-dependent peroxidase is consistently expressed in high concentration in tissues that interact bacteria. Elevated levels of the peroxidase were also found in the ink-producing tissues, which are known to have enzymatic pathways associated with antimicrobial activity. Taken together, these data suggest that the host uses a common biochemical response to the variety of types of associations that it forms with microorganisms.     

Isolation and ultrastructural characterization of squid synaptic vesicles

Synaptic vesicles contain a variety of proteins and lipids that mediate fusion with the pre-synaptic membrane. Although the structures of many synaptic vesicle proteins are known, an overall picture of how they are organized at the vesicle surface is lacking. In this paper, we describe a better method for the isolation of squid synaptic vesicles and characterize the results. For highly pure and intact synaptic vesicles from squid optic lobe, glycerol density gradient centrifugation was the key step. Different electron microscopic methods show that vesicle membrane surfaces are largely covered with structures corresponding to surface proteins. Each vesicle contains several stalked globular structures that extend from the vesicle surface and are consistent with the V-ATPase. BLAST search of a library of squid expressed sequence tags identifies 10 V-ATPase subunits, which are expressed in the squid stellate ganglia. Negative-stain tomography demonstrates directly that vesicles flatten during the drying step of negative staining, and furthermore shows details of individual vesicles and other proteins at the vesicle surface.     

Growth and tissue composition as a function of feeding history in juvenile cephalopods

We present the results of a series of experiments that examined the effect of feeding history on the growth and tissue composition of juveniles of two tropical cephalopods; the squid Sepioteuthis lessoniana and the cuttlefish Sepia elliptica. Juveniles were reared in individual containers for between 35 and 42 days at different ration levels, three ration levels for the squid and two levels for the cuttlefish. Although differences in ration were sufficient to cause different growth rates, both in body length and mass, the effects on tissue composition were less definitive. Sepioteuthis juveniles on the highest rations had higher concentrations of water, but no difference in lipid, carbohydrate or protein when compared with their lower ration siblings. In the case of juvenile cuttlefish no difference in tissue composition was detected between the two ration levels. RNA:protein ratios were also determined for the juveniles to provide an estimate of instantaneous growth. A significant correlation was found between body size and RNA:protein ratio in the squid; those juveniles that ate more had higher RNA:protein ratios than lower ration individuals. Significantly, the juvenile cuttlefish showed no relationship between growth rate and RNA:protein ratios, which means that we are unable to use this measure to estimate the growth rates of wild individuals. In conclusion, ration level did affect growth rates and food availability is an important factor in modifying growth rates of wild individuals. However, we could not find, at the individual level, an index or measure that could be used to explain the variability of observed differences in growth rates as a function of nutritional history.     

Human group III secreted phospholipase A2 promotes neuronal outgrowth and survival

Human sPLA2-III [group III secreted PLA2 (phospholipase A2)] is an atypical sPLA2 isoenzyme that consists of a central group III sPLA2 domain flanked by unique N- and C-terminal domains. In the present study, we found that sPLA2-III is expressed in neuronal cells, such as peripheral neuronal fibres, spinal DRG (dorsal root ganglia) neurons and cerebellar Purkinje cells. Adenoviral expression of sPLA2-III in PC12 cells (pheochromocytoma cells) or DRG explants facilitated neurite outgrowth, whereas expression of a catalytically inactive sPLA2-III mutant or use of sPLA2-III-directed siRNA (small interfering RNA) reduced NGF (nerve growth factor)-induced neuritogenesis. sPLA2-III also suppressed neuronal death induced by NGF deprivation. Lipid MS revealed that sPLA2-III overexpression increased the cellular level of lysophosphatidylcholine, a PLA2 reaction product with neuritogenic and neurotropic activities, whereas siRNA knockdown reduced the level of lysophosphatidylcholine. These observations suggest the potential contribution of sPLA2-III to neuronal differentiation and its function under certain conditions.     

Characterization of a Vibrio fischeri aminopeptidase and evidence for its influence on an early stage of squid colonization

Vibrio fischeri cells are the sole colonists of a specialized light organ in the mantle cavity of the sepiolid squid Euprymna scolopes. The process begins when the bacteria aggregate in mucus secretions outside the light organ. The cells eventually leave the aggregate, enter the light organ, and encounter a rich supply of peptides. The need to dissociate from mucus and presumably utilize peptides led us to hypothesize that protease activity is integral to the colonization process. Protease activity associated with whole cells of Vibrio fischeri strain ES114 was identified as the product of a putative cell membrane-associated aminopeptidase (PepN). To characterize this activity, the aminopeptidase was cloned, overexpressed, and purified. Initial steady-state kinetic studies revealed that the aminopeptidase has broad activity, with a preference for basic and hydrophobic side chains and k(cat) and K(m) values that are lower and smaller, respectively, than those of Escherichia coli PepN. A V. fischeri mutant unable to produce PepN is significantly delayed in its ability to colonize squid within the first 12 h, but eventually it establishes a wild-type colonization level. Likewise, in competition with the wild type for colonization, the mutant is outcompeted at 12 h postinoculation but then competes evenly by 24 h. Also, the PepN-deficient strain fails to achieve wild-type levels of cells in aggregates, suggesting an explanation for the initial colonization delay. This study provides a foundation for more studies on PepN expression, localization, and role in the early stages of squid colonization.     

Retinal phospholipase C from squid is a regulator of Gq alpha GTPase activity

The phospholipase C (PLC) pathway is the major signaling mechanism of photoactivation in invertebrate photoreceptors. Here we report the cloning of a cDNA encoding a 140-kDa retinal PLC that is uniquely expressed in squid photoreceptors. This cDNA encodes a protein with multiple distinct modular domains: PH, X and Y catalytic, and C2 domains, as well as G- and P-box motifs and two GTP/ATP binding motifs. The PLC was stimulated by activated squid Gq alpha but not by squid Gq beta gamma or mammalian beta gamma subunits. The PLC was inhibited by monophosphate, diphosphate and triphosphate nucleotides but not cyclic nucleosides. We also tested the ability of PLC-140 to regulate the GTPase activity of Gq alpha in the rhabdomeric membranes. Depletion of PLC-140 from the rhabdomeric membranes decreased the GTP hydrolysis but not GTP gamma S binding to the membranes. Reconstitution of purified PLC-140 with membranes accelerated Gq alpha GTPase activity by fivefold at a concentration of 2.5 microM. Our data suggest that PLC-140 plays an important role in both the activation and inactivation pathways of invertebrate visual transduction.     

Dye-coupling in the early squid embryo

Summary Functional cell-to-cell communicaton pathways were studied in pre-organogenetic squid embryos using the fluorescent dyes Lucifer Yellow-CH (LY) and Fluorescein-isothiocyanate Dextran (FITC-D). LY (M.Wt.450) micro-injected into ectoderm or mes-endoderm cells spread extensively over the embryo, flowing laterally along each germ layer, crossing germ layer boundaries and also the cell-to-yolk syncytium boundary. In contrast, when FITC-D (M.Wt.9000 or 17,500) was micro-injected into ectoderm or mes-endoderm cells, spread was limited to a maximum of 7 cells within the respective germ layer.FITC-D did not cross germ-layers or flow into the yolk syncytium. Our results suggest there are two classes of cell-to-cell communication pathways in the germ-layer stage squid embryo; a ubiquitously distributed junction that is permeable to small molecules and a larger junction that allows the transport of both small molecules and macromolecules upto 17.500 M.Wt. The former pathway, which may correspond to the gap junction, is operative throughout the mitotic cycle, while the latter, possibly a cytoplasmic bridge, is functional while cells are in interphase, but does not allow the transport of large molecules during mitotic activity.     

Kinesin-3 is an organelle motor in the squid giant axon

Conventional kinesin (Kinesin-1), the founding member of the kinesin family, was discovered in the squid giant axon, where it is thought to move organelles on microtubules. In this study, we identify a second squid kinesin by searching an expressed sequence tag database derived from the ganglia that give rise to the axon. The full-length open reading frame encodes a 1753 amino acid sequence that classifies this protein as a Kinesin-3. Immunoblots demonstrate that this kinesin, unlike Kinesin-1, is highly enriched in chaotropically stripped axoplasmic organelles, and immunogold electron microscopy (EM) demonstrates that Kinesin-3 is tightly bound to the surfaces of these organelles. Video microscopy shows that movements of purified organelles on microtubules are blocked, but organelles remain attached, in the presence Kinesin-3 antibody. Immunogold EM of axoplasmic spreads with antibody to Kinesin-3 decorates discrete sites on many, but not all, free organelles and localizes Kinesin-3 to organelle/microtubule interfaces. In contrast, label for Kinesin-1 decorates microtubules but not organelles. The presence of Kinesin-3 on purified organelles, the ability of an antibody to block their movements along microtubules, the tight association of Kinesin-3 with motile organelles and its distribution at the interface between native organelles and microtubules suggest that Kinesin-3 is a dominant motor in the axon for unidirectional movement of organelles along microtubules.     

Limitations on locomotor performance in squid

An empirical equation relating O2 consumption (power input) to pressure production during jet-propelled swimming in the squid (Illex illecebrosus) is compared with hydrodynamic estimates of the pressure-flow power output also calculated from pressure data. Resulting estimates of efficiency and stress indicate that the circularly arranged obliquely striated muscles in squid mantle produce maximum tensions about half those of vertebrate cross-striated muscle, that "anaerobic" fibers contribute to aerobic swimming, and that peak pressure production requires an instantaneous power output higher than is thought possible for muscle. Radial muscles probably contribute additional energy via elastic storage in circular collagen fibers. Although higher rates of aerobic power consumption are only found in terrestrial animals at much higher temperatures, the constraint on squid performance is circulation, not ventilation. Anaerobic power consumption is also among the highest ever measured, but the division of labor between "aerobic" and "anaerobic" fibers suggests a system designed to optimize the limited capacity of the circulation.     

Heritability and fitness-related consequences of squid personality traits

Dumpling squid, Euprymna tasmanica, show consistent individual differences in behaviour that can be classified according to indices reflecting shy-bold, activity and reactivity responses. Using crosses of wild-caught single males to multiple females with known behavioural phenotypes, this study estimated patterns of additive genetic and residual variance in these behavioural traits from offspring of squid in two contexts, a threat (antipredator) and feeding (foraging) test. Genetic contributions to behavioural expression were dependent on test context. Behaviours in antipredator contexts had significant heritabilities (h(2) = 0.2-0.8) while behaviours from foraging contexts had lesser additive genetic and greater residual components (h(2) = 0.05-0.08). Personality trait variation in females was not related to her fecundity. Female boldness in foraging situations, which co-varied with body size, explained small but significant variation ( approximately 21%) in brood hatching success, while successful fertilization was determined by positive assortion of mate pairs according to their shy-bold phenotype. These results are discussed in terms of the ecological and evolutionary significance of animal "personality" traits in wild populations of animals.     

Hydrogen sulfide as a precursor for the synthesis of isethionate in the squid giant axon.

Cysteine is taken up by the squid giant axon to about 200% of equivalent distribution, whereas sulfide is taken up (probably as hydrogen sulfide) to about 40% of equivalence. Thereafter, the squid axon synthesizes its major anion, isethionate, in about equal amounts from the sulfide, or from the sulfur of cysteine, but not at all from the carbons of cysteine. Squid nerve also contains rhodanese, an enzyme which transfers the outer (sulfane) sulfur of thiosulfate to cyanide to produce thiocyanate. It is speculated that, instead of “detoxifying cyanide,” as the reaction involving rhodanese is commonly described, the physiological role of this enzyme is the formation of a carbon-sulfur bond, leading finally, in the squid, to the formation of isethionate. This is the first evidence concerning the pathway for the synthesis of isethionate in squid nerve where this compound is normally present at a concentration of 150 mm.