A lectin from Crenomytilus grayanus as a probe for the detection of widespread cancerous and metastatic cells.

A novel Gal-binding lectin from mussels (Crenomytilus grayanus, CGL) with 6 binding sites in the dimeric structure has been previously shown to have antifungal, anticancer, and antibacterial activities. In this study, a glycan array was used to confirm that CGL recognizes a range of non-reducing end α- or ß-linked Gal glycans on normal cells but not sialic acid-capped glycans. This finding suggests that CGL has potential in the tumor detection due to the hyper-sialylation present in cell surface glycans from cancer cells. To evaluate the feasibility of this possibility, we labeled CGL with biotin and then mixed it with streptavidin-horseradish peroxidase (HRP) to create a CGL-biotin-SP complex as a probe for use in enzyme-linked lectin assays. CGL-biotin-SP successfully distinguished not only HeLa cells and de-sialylated HeLa cells that mimic normal cell surface glycans but also lung and breast cancer cells with different metastatic abilities. This work provides the insights into a new Gal-binding lectin by establishing its specificity and also demonstrates practical applications in cancer diagnosis greater than other reported lectins.

Modeling of the mussel catch muscle contractile apparatus in actomyosin suspension.

In this paper, we tried to create a contractile model from proteins of the catch muscle of the Gray mussel, similar to the well-described suspension contractile model of vertebrate skeletal muscles. This model makes it possible to characterize the processes in the reconstructed contractile apparatus with the help of monitoring the two characteristics of muscle suspensions - the optical density and the particle size. Contractile model of the catch muscle we constructed was the simplest model consisting of two proteins, actin and myosin. During this work we compared the optical manifestations of the contraction and relaxation states of constructed model with earlier data on the actomyosin suspension of skeletal muscles. It appeared that the approach used in the study of skeletal muscle actomyosin relaxing - the use of an increased amount of ATP - cannot be applied to the contractile model of the molluscan catch muscle. Nevertheless we managed to reach relaxed state of this model with modifying calcium concentration. As a result, we laid the foundation for further reconstruction of the third state of the catch muscle - the catch tone.

A GM1b/asialo-GM1 oligosaccharide-binding R-type lectin from purplish bifurcate mussels Mytilisepta virgata and its effect on MAP kinases.

A 15-kDa lectin, termed SeviL, was isolated from Mytilisepta virgata (purplish bifurcate mussel). SeviL forms a noncovalent dimer that binds strongly to ganglio-series GM1b oligosaccharide (Neu5Acɑ2-3Galß1-3GalNAcß1-4Galß1-4Glc) and its precursor, asialo-GM1 (Galß1-3GalNAcß1-4Galß1-4Glc). SeviL also interacts weakly with the glycan moiety of SSEA-4 hexaose (Neu5Acα2-3Galß1-3GalNAcß1-3Galα1-4Galß1-4Glc). A partial protein sequence of the lectin was determined by mass spectrometry, and the complete sequence was identified from transcriptomic analysis. SeviL, consisting of 129 amino acids, was classified as an R(icin B)-type lectin, based on the presence of the QxW motif characteristic of this fold. SeviL mRNA is highly expressed in gills and, in particular, mantle rim tissues. Orthologue sequences were identified in other species of the family Mytilidae, including Mytilus galloprovincialis, from which lectin MytiLec-1 was isolated and characterized in our previous studies. Thus, mytilid species contain lectins belonging to at least two distinct families (R-type lectins and mytilectins) that have a common ß-trefoil fold structure but differing glycan-binding specificities. SeviL displayed notable cytotoxic (apoptotic) effects against various cultured cell lines (human breast, ovarian, and colonic cancer; dog kidney) that possess asialo-GM1 oligosaccharide at the cell surface. This cytotoxic effect was inhibited by the presence of anti-asialo-GM1 oligosaccharide antibodies. With HeLa ovarian cancer cells, SeviL showed dose- and time-dependent activation of kinase MKK3/6, p38 MAPK, and caspase-3/9. The transduction pathways activated by SeviL via the glycosphingolipid oligosaccharide were triggered apoptosis. DATABASE: Nucleotide sequence data have been deposited in the GenBank database under accession numbers MK434191, MK434192, MK434193, MK434194, MK434195, MK434196, MK434197, MK434198, MK434199, MK434200, and MK434201.

Protein composition of thick filaments from molluscan catch muscle and the role of twitchin in the catch-state formation.

In the work, we performed densitometry of thick filaments of the Gray's mussel catch muscle; densitometry included determination of electrophoretic dye binding constants of proteins. The results of densitometry showed that the content of twitchin in thick filaments is significantly (10 times) lower than the content of myosin. We performed an in vitro simulation of the contractile apparatus of the catch muscle and showed that with such content, links formed by twitchin cannot stop "relaxation". So, we doubt that the role of twitchin in the formation of the catch state is to form load-bearing links between thin and thick filaments that keep the muscle in the contracted state. At the same time, densitometry has shown that the content of the unique catch-muscle protein - myorod - significantly exceeds the content of twitchin and reaches the level of myosin. Like twitchin, myorod is capable of forming regulated cross-links between thick and thin filaments. Such a high content of this protein may indicate that it is myorod, and not twitchin, that is responsible for the formation of catch load-bearing cross-links.

Protein expression profiles in Bathymodiolus azoricus exposed to cadmium.

Proteomic changes in the "gill-bacteria complex" of the hydrothermal vent mussel B. azoricus exposed to cadmium in pressurized chambers ((Incubateurs Pressurises pour l'Observation en Culture d'Animaux Marins Profonds - IPOCAMP) were analyzed and compared with the non-exposed control group. 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) showed that less than 1.5% of the proteome of mussels and symbiotic bacteria were affected by a short-term (24 h) Cd exposure. Twelve proteins of the more abundant differentially expressed proteins of which six were up-regulated and six were down-regulated were excised, digested and identified by mass spectrometry. The identified proteins included structural proteins (actin/actin like proteins), metabolic proteins (calreticulin/calnexin, peptidyl-prolyl cis-trans isomerase, aminotransferase class-III, electron transfer flavoprotein, proteasome, alpha-subunit and carbonic anhydrase) and stress response proteins (chaperone protein htpG, selenium-binding protein and glutathione transferases). All differently expressed proteins are tightly connected to Cd exposure and are affected by oxidative stress. It was also demonstrated that B. azoricus was well adapted to Cd contamination therefore B. azoricus from hydrothermal vent areas may be considered a good bioindicator.

Activity Dependence of a Novel Lectin Family on Structure and Carbohydrate-Binding Properties.

A GalNAc/Gal-specific lectins named CGL and MTL were isolated and characterized from the edible mussels Crenomytilus grayanus and Mytilus trossulus. Amino acid sequence analysis of these lectins showed that they, together with another lectin MytiLec-1, formed a novel lectin family, adopting ß-trefoil fold. In this mini review we discuss the structure, oligomerization, and carbohydrate-binding properties of a novel lectin family. We describe also the antibacterial, antifungal, and antiproliferative activities of these lectins and report about dependence of activities on molecular properties. Summarizing, CGL, MTL, and MytiLec-1 could be involved in the immunity in mollusks and may become a basis for the elaboration of new diagnostic tools or treatments for a variety of cancers.

Mutagenesis Studies and Structure-function Relationships for GalNAc/Gal-Specific Lectin from the Sea Mussel Crenomytilus grayanus.

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) with anticancer activity represents а novel lectin family with ß-trefoil fold. Earlier, the crystal structures of CGL complexes with globotriose, galactose and galactosamine, and mutagenesis studies have revealed that the lectin contained three carbohydrate-binding sites. The ability of CGL to recognize globotriose (Gb3) on the surface of breast cancer cells and bind mucin-type glycoproteins, which are often associated with oncogenic transformation, makes this compound to be perspective as a biosensor for cancer diagnostics. In this study, we describe results on in silico analysis of binding mechanisms of CGL to ligands (galactose, globotriose and mucin) and evaluate the individual contribution of the amino acid residues from carbohydrate-binding sites to CGL activity by site-directed mutagenesis. The alanine substitutions of His37, His129, Glu75, Asp127, His85, Asn27 and Asn119 affect the CGL mucin-binding activity, indicating their importance in the manifestation of lectin activity. It has been found that CGL affinity to ligands depends on their structure, which is determined by the number of hydrogen bonds in the CGL-ligand complexes. The obtained results should be helpful for understanding molecular machinery of CGL functioning and designing a synthetic analog of CGL with enhanced carbohydrate-binding properties.

Biochemical responses of the golden mussel Limnoperna fortunei under dietary glyphosate exposure.

The aim of this study was to analyze the biochemical alterations in the golden mussel Limnoperna fortunei under dietary glyphosate exposure. Mussels were fed during 4 weeks with the green algae Scenedesmus vacuolatus previously exposed to a commercial formulation of glyphosate (6 mg L-1 active principle) with the addition of alkyl aryl polyglycol ether surfactant. After 1, 7, 14, 21 and 28 days of dietary exposure, glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), acetylcholinesterase (AChE), carboxylesterases (CES) and alkaline phosphatase (ALP) activities, glutathione (GSH) content and damage to lipids and proteins levels were analyzed. A significant increase (72%) in the GST activity and a significant decrease (26%) in the CES activity in the mussels fed on glyphosate exposed algae for 28 days were observed. The ALP activity was significantly increased at 21 and 28 days of dietary exposure (48% and 72%, respectively). GSH content and CAT, SOD and AchE activities did not show any differences between the exposed and non exposed bivalves. No oxidative damage to lipids and proteins, measured as TBARS and carbonyl content respectively, was observed in response to glyphosate dietary exposure. The decrease in the CES activity and the increases in GST and ALP activities observed in L. fortunei indicate that dietary exposure to glyphosate provokes metabolic alterations, related with detoxification mechanisms.

A GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus modulates immune response in macrophages and in mice.

A GalNAc/Gal-specific lectin (CGL) from the edible mussel Crenomytilus grayanus has been demonstrated to exhibit antibacterial properties. However, the mechanism of immune modulation by CGL in mammalian cells remains unclear. Here, we demonstrated that CGL can activate immune responses in macrophages and in mice. In the in vitro cell models, CGL induced tumour necrosis factor-α and interleukin-6 secretion in mouse RAW264.7 macrophages, mouse bone marrow-derived macrophages, human THP-1 macrophages, human peripheral blood mononuclear cells and human blood monocyte-derived macrophages. The CGL-mediated cytokine production was regulated by reactive oxygen species, mitogen-activated protein kinases, protein kinase C-α/δ and NF-κB. Interestingly, in lipopolysaccharide-activated macrophages, CGL induced endotoxin tolerance (characterized by the downregulation of nitric oxide, inducible nitric oxide synthase, interleukin-6 and cyclooxygenase II) via the downregulation of IRAK2 expression, JNK1/2 phosphorylation and NF-κB activation. CGL also slightly increased the bactericidal activity of macrophages and induced cytokine production in mouse models. Overall, our data indicate that CGL has the potential to be used as an immune modulator in mammals.

Vibrio diabolicus challenge in Bathymodiolus azoricus populations from Menez Gwen and Lucky Strike hydrothermal vent sites.

Menez Gwen (MG) and Lucky Strike (LS) deep-sea hydrothermal vents are located at 850 m and 1730 m depths respectively and support chemosynthesis-based ecosystems partially differing in heavy metal concentration, temperature range, and faunistic composition. The successfully adapted deep-sea vent mussel Bathymodiolus azoricus is found at both vent locations. In such inhospitable environments survival strategies rely on the establishment of bacteria-vent animal symbiosis In spite of the toxic nature of deep-sea vents, the problem of microbial threat and the need for immunity exist in B. azoricus. This study aims at investigating the immune system of B. azoricus from MG and LS populations by comparing immune gene expressions profiles using the deep-sea vent-related Vibrio diabolicus. Expression of nineteen immune genes was analyzed from gill, digestive gland and mantle tissues upon 3 h, 12 h and 24 h V. diabolicus challenges. Based on quantitative-Polymerase Chain Reaction (qPCR) significant gene expression differences were found among MG and LS populations and challenge times MG mussels revealed that gill and digestive gland gene expression levels were remarkably higher than those from LS mussels. Expression of Carcinolectin, Serpin-2, SRCR, IRGs, RTK, TLR2, NF-κB, HSP70 and Ferritin genes was greater in MG than LS mussels. In contrast, mantle tissue from LS mussels revealed the highest peak of expression at 24 h for most genes analyzed. The activation of immune signaling pathways demonstrated that gene expression profiles are distinct between the two mussel populations. These differences may possibly ensue from intrinsic immune transcriptional activities upon which host responses are modulated in presence of V. diabolicus. mRNA transcript variations were assessed during 24 h acclimatization taking into account the partial depuration to which mussels were subjected to. Additionally, gene expression differences may reflect still accountable effects from the presence of vent remaining microfluidic environments within the tissues analyzed.

Troponin-like regulation in muscle thin filaments of the mussel Crenomytilus grayanus (Bivalvia: Mytiloida).

Muscles of bivalve molluscs have double calcium regulation--myosin-linked and actin-linked. While the mechanism of myosin-linked regulation is sufficiently studied, there is still no consensus on the mechanism of actin-linked regulation. Earlier we showed a high degree of Ca2+-sensitivity of thin filaments from the adductor muscle of the mussel Crenomytilus grayanus (Mytiloida). In order to elucidate the nature of this regulation, we isolated the fraction of minor proteins from the mussel thin filaments, which confers Ca2+-sensitivity to reconstituted actomyosin-tropomyosin. Proteins of this fraction, ABP-19, ABP-20, and ABP-28, were chromatographically purified and identified. According to the results of mass spectrometry and Western blot analysis, as well as by their functional properties, these mussel actin-binding proteins appeared to correspond to the troponin components from the skeletal muscles of vertebrates (TnC, TnI and TnT). The reconstituted mussel troponin complex confers to actomyosin-tropomyosin more than 80% Ca2+-sensitivity. The in vivo molar ratio of actin/tropomyosin/troponin was calculated to be 7:1:0.5, i.e., the content of troponin in mussel thin filaments is two times lower than in thin filaments of skeletal muscles of vertebrates. These data demonstrate that troponin-like regulation found in the catch muscle of the mussel C. grayanus is present at least in two suborders of bivalves: Pectinoida and Mytiloida.

Antioxidant defense responses in Mytella guyanensis (Lamarck, 1819) exposed to an experimental diesel oil spill in Paranaguá Bay (Paraná, Brazil).

We evaluated the effects of diesel oil on the bivalve Mytella guyanensis using biomarkers of oxidative stress (glutathione S-transferase, glutathione peroxidase, and reduced glutathione) after an experimental in situ spill in a mangrove area in southern Brazil. A linear model was developed for the Multiple Before-After Control-Impact (MBACI) experimental design to assess the significance of biological responses. Control and impacted sites were sampled seven and two days before as well as two and seven days after the spill. With the exception of a late response of reduced glutathione (GSH) levels on day seven, none of the biomarkers were significantly altered by the impact. This result was attributed to the high environmental variability of the experimental sites combined with a low sensitivity of Mytella guyanensis to diesel oil at short time-scales. The high resistance of M. guyanensis suggests that its antioxidant response is triggered only after a medium- to long-term exposure to contaminants.

Effect of seasonality on oxidative stress responses and metal accumulation in soft tissues of Aulacomya atra, a mussel from the South Atlantic Patagonian coast.

This study investigated the effects of pollution and its interaction with temperature on the oxidative status of the ribbed mussel Aulacomya atra in the southern Atlantic Patagonian coast. Animals were collected from four sites with different degree and type of human activity impact, during the summer and winter of 2011. Seawater chromium, copper, manganese, nickel and zinc concentrations were measured, as well as metal accumulation, lipid peroxidation, protein oxidation, reduced glutathione levels, and enzymatic activities of superoxide dismutase and glutathione-S-transferase in gills and digestive glands. Metal bioaccumulation and oxidative stress responses in both tissues were generally higher in mussels from harbor areas. Water temperature had a remarkable effect on gill SOD activity and protein oxidation during winter in mussels from all locations. Methodologically, we conclude that measuring both metal bioaccumulation and oxidative stress responses allowed for a more accurate assessment of the biological effects of metal present in seawater.

40-kDa actin-binding protein of thin filaments of the mussel Crenomytilus grayanus inhibits the strong bond formation between actin and myosin head during the ATPase cycle.

Mobility and spatial orientation of a novel 40-kDa actin-binding protein from the smooth muscle of the mussel Crenomytilus grayanus was studied by polarized fluorometry. The influence of this protein on orientation and mobility of the myosin heads was investigated during modeling the different stages of the ATPase cycle. The 40-kDa actin-binding protein affected the strong actin-myosin binding. We suggest that the 40-kDa actin-binding protein is involved in regulation of the actin-myosin interaction in the smooth muscle of the mussel.

Monitoring the biochemical and cellular responses of marine bivalves during thermal stress by using biomarkers.

The present work aimed to study the cellular, biochemical and molecular biomarkers in the digestive glands and hemocytes of Modiolus barbatus and whether there is a hierarchy in their response to thermal stress. We determined a) the neutral red retention assay (NRR) in heamotocytes and b) the lysosomal membrane stability (LMS), the levels of second messenger cAMP, the activity of acetylcholinesterase (AChE) in the digestive glands of Modiolus barbatus after acclimation to 18 °C, 24 °C, 28 °C or 30 °C for 30 days. Moreover, in order to estimate the threshold of temperature inducing expression of stress proteins we determined the levels of Hsp70 and Hsp90 in the digestive glands. Hsps are expressed at lower temperature than those causing reduction in the LMS and NNR times. The reduction in the LMS and NNR times at high temperatures of acclimation might be related to inability of Modiolus barbatus to gain energy from the ingested food.

2-D difference gel electrophoresis approach to assess protein expression profiles in Bathymodiolus azoricus from Mid-Atlantic Ridge hydrothermal vents.

Hydrothermal vent mussels Bathymodiolus azoricus are naturally exposed to toxic chemical species originated directly from vent chimneys. The amount of toxic elements varies significantly among vent sites along the Mid-Atlantic Ridge and B. azoricus must be able to adapt to changes in hydrothermal fluid composition, temperature and pressure. The aim of this work was to study changes in the proteome in the "gill-bacteria complex" of mussels B. azoricus from three hydrothermal vent sites with distinct environmental characteristics using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE). Results showed that 31 proteins had different expression profiles among vent sites and both cluster and principal component analysis confirm a clear separation of mussels between sites. This suggests the existence of specific parameters grouping individuals from the same hydrothermal site. Protein spots of the more abundant differentially expressed proteins were excised, digested with trypsin and identified by mass spectrometry. All identified proteins (actin, ubiquinone, S-adenosylhomocysteine hydrolase, cysteine peptidases, chaperonin and catalase) have been related previously with oxidative stress conditions and are known to be affected by ROS inducing stressors, including metals. Results point out to specific adaptations at the proteome level of B. azoricus depending on the level of toxicants present in their environment.

Molluscan catch muscle myorod and its N-terminal peptide bind to F-actin and myosin in a phosphorylation-dependent manner.

Myorod is expressed exclusively in molluscan catch muscle and localizes on the surface of thick filaments together with twitchin and myosin. This protein is an alternatively spliced product of the myosin heavy-chain gene containing the C-terminal rod part of myosin and a unique N-terminal domain. We have recently reported that this unique domain is a target for phosphorylation by gizzard smooth muscle myosin light chain kinase (MLCK) and molluscan twitchin, which contains a MLCK-like domain. To elucidate the role of myorod phosphorylation in catch muscle, a peptide corresponding to the specific N-terminal region of the protein was synthesized in phosphorylated and unphosphorylated form. We report, for the first time, that unphosphorylated full-length myorod and its unphosphorylated N-terminal synthetic peptide are able to interact with rabbit F-actin and thin filaments from molluscan catch muscle. The binding between thin filaments and the peptide was Ca²+-dependent. In addition, we found that phosphorylated N-terminal peptide of myorod has higher affinity for myosin compared to the unphosphorylated peptide. Together, these observations suggest the direct involvement of the N-terminal domain of myorod in the regulation of molluscan catch muscle.

[Thin filaments of molluscan catch muscles contain a calponin-like protein].

A novel 40 kDa protein was detected in native thin filaments from catch muscles of the mussel Crenomytilus grayanus. The MALDY-TOF analysis of the protein showed a 40% homology with the calponin-like protein from the muscle of Mytilus galloprovincialis (45 kDa), which has a 36% homology with smooth muscle calponin from chicken gizzard (34 kDa). The amount of the calponin-like protein in thin filaments depends on isolation conditions and varies from the complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the contact of the protein in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22 degrees C and optimal values of both pH and ionic strength of the extraction solution, total calponin-like protein coprecipitates with thin filaments. At 2 degrees C it remains in the supernatant. The 40 kDa calponin-like protein from the mussel Crenomytilus grayanus has similar properties with smooth muscle calponin (34 kDa). It is thermostable and inhibits the actin-activated Mg -ATPase activity of actomyosin. In addition, the 40 kDa calponin-like protein isolated without using thermal treatment contains endogenous kinases. It was found that the calponin-like protein can be phosphorylated by endogenous kinases in the Ca -independent manner. These results indicate that the calponin-like protein from the catch muscle of the mussel Crenomytilus grayanus is a new member of the calponin family. The role of proteins from this family both in muscle and ponmuscle cells is still obscure. We suggest that the calponin-like protein is involved in the Ca -independent regulation of smooth muscle contraction.

Sub-lethal effects of cadmium on the antioxidant defence system of the hydrothermal vent mussel Bathymodiolus azoricus.

The mussel Bathymodiolus azoricus is one of the most abundant species in the Mid-Atlantic Ridge hydrothermal vents and is continually exposed to the high-temperature venting fluids containing high metal concentrations and enriched in sulphides and methane, which constitute a potential toxic environment for marine species. The aim of this study was to assess the effects of a sub-lethal Cd concentration on the antioxidant defence system of this mussel. B. azoricus were collected at Menez Gwen vent site (37 degrees 51'N, 32 degrees 31'W) and exposed to Cd (50 microg l(-1)) during 24 days, followed by a depuration period of six days. A battery of stress related biomarkers including antioxidant enzymes (superoxide dismutase-SOD, catalase-CAT; glutathione peroxidases-GPx), metallothioneins (MT), lipid peroxidation (LPO) and total oxyradical scavenging capacity (TOSC) were measured in the gills and mantle of B. azoricus. Cd was accumulated linearly during the exposure period in both tissues and no significant elimination occurred after the 6 days of depuration. Antioxidant enzymes activities were significantly higher in the gills. Cyt-SOD, T-GPx and Se-GPx were induced during the experiment but this was also observed in control organisms. Mit-SOD and CAT activities remained relatively unchanged. MT levels increased linearly in the gills of exposed mussels in the first 18 days of exposure. No significant differences were observed between LPO levels of control and exposed mussels. TOSC levels remained unchanged in control and exposed mussels. This suggests that although Cd is being accumulated in the tissues of exposed mussels, MT defence system is enough to detoxify the effect of Cd accumulated in the tissues. Furthermore, other factors besides the presence of Cd are influencing the antioxidant defence system in B. azoricus.

A new property of twitchin to restrict the "rolling" of mussel tropomyosin and decrease its affinity for actin during the actomyosin ATPase cycle.

A new evidence on the regulatory function of twitchin, a titin-like protein of molluscan muscles, at muscle contraction has been obtained at studying the movements of IAF-labeled mussel tropomyosin in skeletal ghost fibers during the ATP hydrolysis cycle simulated using nucleotides and non-hydrolysable ATP analogs. For the first time, myosin-induced multistep changes in mobility and in the position of mussel tropomyosin strands on the surface of the thin filament during the ATP hydrolysis cycle have been demonstrated directly. Unphosphorylated twitchin shifts the tropomyosin towards the position typical for muscle relaxation, decreases the tropomyosin affinity to actin and inhibits its movements during the ATPase cycle. Phosphorylation of twitchin by the catalytic subunit of protein kinase A reverses this effect. These data imply that twitchin is a thin filament regulator that controls actin-myosin interaction by "freezing" tropomyosin in the blocked position, resulting in the inhibition of the transformation of weak-binding states into strong-binding ones during ATPase cycle.