Structural dynamics of the heme pocket and intersubunit coupling in the dimeric hemoglobin from Scapharca inaequivalvis.

Cooperativity is essential for the proper functioning of numerous proteins by allosteric interactions. Hemoglobin from Scapharca inaequivalvis (HbI) is a homodimeric protein that can serve as a minimal unit for studying cooperativity. We investigated the structural changes in HbI after carbon monoxide dissociation using time-resolved resonance Raman spectroscopy and observed structural rearrangements in the Fe-proximal histidine bond, the position of the heme in the pocket, and the hydrogen bonds between heme and interfacial water upon ligand dissociation. Some of the spectral changes were different from those observed for human adult hemoglobin due to differences in subunit assembly and quaternary changes. The structural rearrangements were similar for the singly and doubly dissociated species but occurred at different rates. The rates of the observed rearrangements indicated that they occurred synchronously with subunit rotation and are influenced by intersubunit coupling, which underlies the positive cooperativity of HbI.

Comprehensive analysis on the regulation of differentially expressed of mRNA and ncRNA in different ovarian stages of ark shell Scapharca broughtonii.

BACKGROUND: Ovarian development is an important prerequisite and basis for animal reproduction. In many vertebrates, it is regulated by multiple genes and influenced by sex steroid hormones and environmental factors. However, relative information is limited in shellfish. To explore the biological functions and molecular mechanisms of mRNA and non-coding RNA that regulate ovarian development in Scapharca broughtonii, we performed whole transcriptome sequencing analysis on ovaries at three developmental stages. Furthermore, the biological processes involved in the differential expression of mRNA and ncRNA were analyzed. RESULTS: A total of 11,342 mRNAs, 6897 lncRNAs, 135 circRNAs, and 275 miRNAs were differentially expressed. By mapping the differentially expressed RNAs from the three developmental stages of Venn diagram, multiple groups of shared mRNAs and lncRNAs were found to be associated with ovarian development, with some mRNA and ncRNA functions associated with steroid hormone. In addition, we constructed and visualized the lncRNA/circRNA-miRNA-mRNA network based on ceRNA targeting relationships. CONCLUSIONS: These findings may facilitate our further understanding the mRNA and ncRNAs roles in the regulation of shellfish reproduction.

Effect of hypersalinic stress on hemocyte morphology and hemolymph cellular composition of the ark clam (Anadarakagoshimensis).

Bivalve mollusks as typical osmoconformers are unable to maintain a constant level of internal osmolarity in conditions of salinity stress. Adaptation to fluctuations of environmental salinity is achieved through cellular osmoregulatory responses, which are accompanied with a substantial shift in functional state of cells. In the present work we investigated the effect of hypersalinity stress on hemolymph cellular composition and morphology of the ark clam (Anadara kagoshimensis) hemocytes. Ark clams were subjected to a gradual increase of environmental salinity from 18‰ to 35‰ and 45‰ and maintained at those conditions for two days. Exposure to hypersalinity 35‰ induced changes in erythrocyte morphology and led to a decrease of their diameter. At salinity 45‰ a substantial increase of hemocyte average diameter was observed, whereas the shape of cells did not change (18‰). Hyperosmotic stress was not associated with changes in hemocyte viability as well as changes in hemolymph cellular composition. The results of the present work demonstrate high tolerance of A. kagoshimensis to short-time exposure to hypersalinic conditions.

Paired miRNA and RNA sequencing provides a first insight into molecular defense mechanisms of Scapharca broughtonii during ostreid herpesvirus-1 infection.

Ostreid herpesvirus 1 (OsHV-1) infection caused mortalities with relevant economic losses in bivalve aquaculture industry worldwide. Initially described as an oyster pathogen, OsHV-1 can infect other bivalve species, like the blood clam Scapharca broughtonii. However, at present, little is known about the molecular interactions during OsHV-1 infection in the blood clam. We produced paired miRNA and total RNA-seq data to investigate the blood clam transcriptional changes from 0 to 72 h after experimental infection with OsHV-1. High-throughput miRNA sequencing of 24 libraries revealed 580 conserved and 270 new blood clam miRNAs, whereas no genuine miRNA was identified for OsHV-1. Total 88-203 differently expressed miRNAs were identified per time point, mostly up-regulated and mainly targeting metabolic pathways. Most of the blood clam mRNAs, in contrast, were down-regulated up to 60 h post-injection, with the trend analysis revealing the activation of immune genes only when comparing the early and latest stage of infection. Taken together, paired short and long RNA data suggested a miRNA-mediated down-regulation of host metabolic and energetic processes as a possible antiviral strategy during early infection stages, whereas antiviral pathways appeared upregulated only at late infection.

Change in vibrational entropy with change in protein volume estimated with mode Grüneisen parameters.

For a small adjustment in average volume, due to a change in state of a protein or other macromolecule at constant temperature, the change in vibrational entropy is related to the mode Grüneisen parameters, which relate shifts in frequency to a small volume change. We report here values of mode Grüneisen parameters computed for two hydrated proteins, cytochrome c and myoglobin, which exhibit trends with mode frequency resembling those of glassy systems. We use the mode Grüneisen parameters to relate volumetric thermal expansion to previously computed values of the isothermal compressibility for several proteins. We also estimate changes in vibrational entropy resulting from the change in volume upon ligand bonding of myoglobin and the homodimeric hemoglobin from Scapharca inaequivalvis (HbI). We compare estimates of the change in entropy upon ligation obtained in terms of mode Grüneisen parameters with the results of normal mode analysis for myoglobin and earlier molecular dynamics simulations of HbI. The results illustrate how small changes in average volume can yield changes in entropy that contribute to ligand binding and allostery.

Transcriptomic analysis and expression of C-type lectins in response to Vibrio parahaemolyticus challenge in Scapharca subcrenata.

Little information is available on innate immune defense mechanisms of Scapharca subcrenata. C-type lectins (CTLs) are not only pattern recognition proteins that can bind pathogen-associated molecular patterns, but also crucial maternally-derived immune factors in mollusc egg. In this study, the comparative transcriptome analysis of Vibrio parahaemolyticus-infected and untreated hepatopancreas were performed to identify the key genes involved in maternal transfer of immunity. A total of 3514 and 9327 differentially expressed genes (DEGs) were identified at 6 and 48 h post challenge compared to control groups. Gene Ontology and Cluster of Orthologous Groups analysis showed that most DEGs were classified under regulation of signal transduction, regulation of the metabolic process of carbohydrates and secondary metabolites, while the processes of posttranscriptional modification and protein translation were inhibited manifestly. The DEGs were most enriched in pathways related to lysosome, phagosome and EMC-receptor interaction. Among the DEGs, 191 maternal immune-related genes that could provide developing embryos a better protection against pathogen infection were identified according to previous studies. Additionally, five CTLs (designated as SsCTL1-5) identified from the DEGs were cloned, and their expression patterns in different tissues and post immune stimulation were analyzed. These findings would be beneficial for understanding the innate immune defense mechanisms of S. subcrenata.

Water-mediated biomolecular dynamics and allostery.

Dynamic coupling with water contributes to regulating the functional dynamics of a biomolecule. We discuss protein-water dynamics, with emphasis on water that is partially confined, and the role of protein-confined water dynamics in allosteric regulation. These properties are illustrated with two systems, a homodimeric hemoglobin from Scapharca inaequivalvis (HbI) and an A2A adenosine receptor (A2AAR). For HbI, water-protein interactions, long known to contribute to the thermodynamics of cooperativity, are seen to influence the dynamics of the protein not only around the protein-water interface but also into the core of each globule, where dynamic and entropic changes upon ligand binding are coupled to protein-water contact dynamics. Similarly, hydration waters trapped deep inside the core region of A2AAR enable the formation of an allosteric network made of water-mediated inter-residue contacts. Extending from the ligand binding pocket to the G-protein binding site, this allosteric network plays key roles in regulating the activity of the receptor.

Purification and Characterization of a New CRISP-Related Protein from Scapharca broughtonii and Its Immunomodulatory Activity.

More and more attention has been paid to bioactive compounds isolated from marine organisms or microorganisms in recent years. At the present study, a new protein coded as HPCG2, was purified from Scapharca broughtonii by stepwise chromatography methods. The molecular weight of HPCG2 was determined to be 30.71 kDa by MALDI-TOF-MS. The complete amino acid sequence of HPCG2 was obtained by tandem mass spectrometry combined with transcriptome database analysis, and its secondary structure was analyzed using circular dichroism. HPCG2 comprised 251 amino acids and contained 28.4% α-helix, 26% ß-sheet, 18.6% ß-turn, and 29.9% random coil. HPCG2 was predicted to be a cysteine-rich secretory protein-related (CRISP-related) protein by domain prediction. Moreover, HPCG2 was proved to possess the immunomodulatory effect on the murine immune cells. MTT assay showed that HPCG2 promoted the proliferation of splenic lymphocytes and the cytotoxicity of NK cells against YAC-1 cells. Flow cytometry test revealed that HPCG2 enhanced the phagocytic function of macrophages and polarized them into M1 type in RAW264.7 cells. In particular, Western blot analysis indicated that the immunomodulatory mechanism of HPCG2 was associated with the regulation on TLR4/JNK/ERK and STAT3 signaling pathways in RAW 264.7 cells. These results suggested that HPCG2 might be developed as a potential immunomodulatory agent or new functional product from marine organisms.

Ark shell Scapharca broughtonii hemocyte response against Vibrio anguillarum challenge.

Scapharca broughtonii is one of the most important Arcidae aquaculture species in the Asia-Pacific region. We aimed to investigate the immune responses of hemocytes from ark shell S. broughtonii hemolymph against pathogens. Hemocyte ultrastructure and immunological activity in response to Vibrio anguillarum challenge were observed by scanning and transmission electron microscopy. Before ultrastructure observation, we used the API ZYM semi-quantitative kit to evaluate the levels of hydrolytic enzymes in the plasma and hemocytes following V. anguillarum infection. An enzyme-linked immunosorbent assay kit was used to investigate the variation in the lysozyme activity and hemocytes following bacterial infection. The results showed that hemocytes were the main defense cells against bacterial infection, whereas plasma played a role in the transport and support of hemocytes. It was presumed that an important function of lysozymes and hydrolytic enzymes in lysosomes was for bacterial digestion. Three major types of hemocytes were observed, namely, red blood cells (RBCs), white blood cells (WBCs), and thrombocytes (TCs). Scanning electron microscopy showed that the normal RBCs appeared pie-shaped with 10 µm diameter and 4 µm central thickness, whereas WBCs were spherical in shape with varying sizes, 4-8 µm diameter, and included small lymphocytes. TCs were long, spindle-shaped, and 12-20 µm in length. The cell membrane surface was smooth and even for all cells before pathogen challenge. Under transmission electron microscopy, RBCs displayed a limited ability to devour and digest bacteria adherent to the cell surface following infection. Many hemoglobin particles were observed in the RBC cytoplasm. WBCs were very active against bacterial invasion and showed a strong ability to digest and decompose infected and wrapped V. anguillarum through phagocytosis and lysosome fusion. Digestive vacuoles rapidly became transparent and were thought to contain increasing quantities of pathogen-induced lysozymes. WBCs that devoured pathogenic bacteria were prone to deformation as well as adhesion to each other. TCs were rich in endoplasmic reticulum (ER) content in their cytoplasm and were widely connected in a net-shaped structure. Mitochondria in TCs formed clusters upon invasion of V. anguillarum in the hemolymph. TCs disintegrated to release the ER into the plasma to form a mesh that facilitated clotting. The ability of circulating hemocytes to quickly modify their morphologies and stainability suggests that S. broughtonii is endowed with highly dynamic hemocyte populations capable of coping with environmental changes and rapidly growing pathogens.

Dual transcriptomic analysis of Ostreid herpesvirus 1 infected Scapharca broughtonii with an emphasis on viral anti-apoptosis activities and host oxidative bursts.

The ark shell, Scapharca (Anadara) broughtonii, is an economically important marine shellfish species in Northwestern Pacific. Mass mortalities of ark shell adults related to Ostreid herpesvirus-1 (OsHV-1) infection have occurred frequently since 2012. However, due to the lack of transcriptomic resource of ark shells, the molecular mechanisms underpinning the virus-host interaction remains largely undetermined. In the present study, we resolved the dual transcriptome changes of OsHV-1 infected ark shell with Illumina sequencing. A total of 44 M sequence reads were generated, of which 67,119 reads were mapped to the OsHV-1 genome. De novo assembly of host reads resulted in 276,997 unigenes. 74,529 (26.90%), 47,653 (17.20%) and 19, 611 (7.07%) unigenes were annotated into GO, KOG and KEGG database, respectively. According to RSEM expression values, we identified 2998 differentially expressed genes (DEGs) between control and challenged groups, which included 2065 up-regulated unigenes and 933 down-regulated unigenes. Further analysis of functional pathways indicated that OsHV-1 could inhibit host cell apoptosis mainly by the up-regulation of inhibitor of apoptosis protein (IAP), and thus facilitating its successful replication. While host hemoglobins could induce oxidative burst by suppressing its peroxidase activity, and thus defense against OsHV-1 infection. Although we reported a narrow expression of the OsHV-1 genome compared to Crassostrea gigas infection, we highlighted several common viral genes highly expressed in the two hosts, suggesting an important functional role. This study offers insights into the pathogenesis mechanisms of OsHV-1 infection in bivalve mollusks of the Arcidae family.

Molecular cloning, expression and biochemical characterization of hemoglobin gene from ark shell Scapharca broughtonii.

Hemoglobin, the main component of haemolymph, is widely distributed in animals. Although its important oxygen transport functions has been extensively reported, studies on the immune function of hemoglobin in mollusc are few. Research on immune of hemoglobin of ark shell Scapharca broughtonii attracted more and more attention due to its ownership of erythrocyte comparing with many other shellfish. In this study, the hemoglobin cDNA of S. broughtonii was cloned by EST and RACE methods (named as SbHb). Sequence analysis revealed that the cDNA was 946 bp in length, including an open reading frame (ORF) of 459 bp which encoded a polypeptide of 152 amino acid residues, and a 5'-untranslated region (UTR) of 313 bp, a 3'-UTR of 174 bp. Sequence and homology analysis showed that the SbHb shared similarity with that of other related species. The mRNA expression profiles of SbHb in tested tissues analyzed by quantitative real-time PCR (qRT-PCR) revealed that the mRNA of SbHb could be all detected in foot, gill, mantle, adductor muscle, haemocytes and hepatopancreas, and the highest level was found in the haemocytes, which is 163.2 times higher than that in adductor muscle. Vibrio anguillarum stimulation and hypoxia treatment both had significant impact on the expression of SbHb, which showed the same trends as increasing first to the highest at 16 h after treatment and then followed by declining. Recombinant protein of SbHb (rSbHb) was successfully obtained by prokaryotic expression, and further function analysis indicated obviously that the rSbHb had very strong phenoloxidase-like activity (PO-like activity) and it could remarkably inhibit growth of gram-negative bacteria V. anguillarum. All the data suggested that the SbHb plays a significant role in the process of antibacterial and anoxia tolerance reaction in S. broughtonii, providing the evidence that SbHb is a key immune factor.

Validation of housekeeping genes for quantitative mRNA expression analysis in OsHV-1 infected ark clam, Scapharca broughtonii.

Ostreid herpesvirus-1 (OsHV-1) presents interspecies transmission among bivalves. Recently, events of mass mortalities of ark clams (Scapharca broughtonii) infected with OsHV-1 have been recorded. To accurately assess the gene responding patterns of ark clams post OsHV-1 infection, constant stable housekeeping genes (HKGs) are needed as internal control to normalize raw mRNA expression data. In this study, ten candidate HKGs were selected, including 18S rRNA (18S), beta-actin (ACT), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), NADH dehydrogenase subunit (NADH), Elongation factor-1a (EF-1a), Elongation factor-1ß (EF-1ß), Elongation factor-1γ (EF-1γ), Ribosomal protein L7 (RL7), Ribosomal protein L15 (RL15) and Ribosomal protein S18 (S18). The expression levels of ten candidate HKGs were analyzed by real-time PCR under given experimental conditions, including various tissues, OsHV-1 challenge, temperature stress and OsHV-1 challenge at different temperature. Their expression stability values were further calculated using two different statistical models (geNorm and NormFinder). The results showed that different tissues presented distinct best pair genes combinations for gene expression analysis under OsHV-1 challenge. RL15 was comparatively more stable than other HKGs under various experimental conditions, while commonly used 18s and ACT seemed to be more greatly influenced by most given experimental conditions in ark clams. This study emphasized the necessity of prior validation of HKGs and would facilitate future gene expression analysis in ark clams or other shellfishes.

Interfacial water effect on cooperativity and signal communication in Scapharca dimeric hemoglobin.

Cooperativity is important in controlling the biological functions of allosteric proteins. Understanding the detailed mechanisms of cooperativity and allosteric regulation in such proteins is essential to understanding their function; however, the mechanism by which allosteric proteins undergo conformational transitions to aid the ligand escape process and its relevance to interfacial water molecules is not well understood. Here, we perform molecular dynamics simulations to examine these issues in Scapharca dimeric hemoglobin. The effects of interfacial water on dimeric motion, ligand escape probability, gate function, and cross-correlation are considered. The results reveal that interfacial water exhibits an unbalanced stress distribution in the interface region, leading to a bias helix bundle motion that not only can expedite the escape of the first ligand but also can increase the interval between the escape of both ligands. Correspondingly, the gate function follows the same time scale as the F-helix movement, and the gate opening is non-stochastic; moreover, the inconsistent motion between the gate parts resembles cooperative behavior. An explicit analysis of the intersubunit communication map provides at least 14 signal transduction pathways. Our results significantly aid in understanding the role of interfacial water in manipulating cooperativity and will lead to further applications involving molecular machines.

Experimental infection of adult Scapharca broughtonii with Ostreid herpesvirus SB strain.

We investigated the susceptibility of ark shell, Scapharca broughtonii, adults to Ostreid herpesvirus SB strain (OsHV-1-SB) through experimental infection by intramuscular injection assays. Results showed the onset of mortality occurred at 3days post injection, one day after the water turbidity became evident in rearing tanks. The mortality curves for the challenged group were similar to those observed at affected hatcheries. Histological lesions, herpesvirus-like particles and high OsHV-1-SB quantities were detected in challenged ark shells. This is the first study to successfully reproduce OsHV-1 disease in Arcoida species, and very few studies in adult bivalves (over 24months old).

Ocean acidification weakens the immune response of blood clam through hampering the NF-kappa ß and toll-like receptor pathways.

The impact of pCO2 driven ocean acidification on marine bivalve immunity remains poorly understood. To date, this impact has only been investigated in a few bivalve species and the underlying molecular mechanism remains unknown. In the present study, the effects of the realistic future ocean pCO2 levels (pH at 8.1, 7.8, and 7.4) on the total number of haemocyte cells (THC), phagocytosis status, blood cell types composition, and expression levels of twelve genes from the NF-kappa ß signaling and toll-like receptor pathways of a typical bottom burrowing bivalve, blood clam (Tegillarca granosa), were investigated. The results obtained showed that while both THC number and phagocytosis frequency were significantly reduced, the percentage of red and basophil granulocytes were significantly decreased and increased, respectively, upon exposure to elevated pCO2. In addition, exposure to pCO2 acidified seawater generally led to a significant down-regulation in the inducer and key response genes of NF-kappa ß signaling and toll-like receptor pathways. The results of the present study revealed that ocean acidification may hamper immune responses of the bivalve T. granosa which subsequently render individuals more susceptible to pathogens attacks such as those from virus and bacteria.

Complete genome sequence of Ostreid herpesvirus-1 associated with mortalities of Scapharca broughtonii broodstocks.

BACKGROUND: Ostreid herpesvirus-1 (OsHV-1) is the major bivalve pathogen associated with severe mortality events in a wide host range. In the early summer of 2012 and 2013, mass mortalities of blood clam (Scapharca broughtonii) broodstocks associated with a newly described variant of OsHV-1 (OsHV-1-SB) were reported. METHODS: In this study, the complete genome sequence of the newly described variant was determined through the primer walking approach, and compared with those of the other two OsHV-1 variants. RESULTS: OsHV-1-SB genome was found to contain 199, 354 bp nucleotides with 38.5% G/C content, which is highly similar to those of acute viral necrosis virus (AVNV) and OsHV-1 reference type. A total of 123 open reading frames (ORFs) putatively encoding functional proteins were identified; eight of which were duplicated in the major repeat elements of the genome. The genomic organization of OsHV-1-SB could be represented as TRL-UL-IRL-IRS-US-TRS, which is different from that of OsHV-1 reference type and AVNV due to the deletion of a unique region (X, 1.5Kb) between IRL and IRS. The DNA sequence of OsHV-1-SB is 95.2% and 97.3% identical to that of OsHV-1 reference type and AVNV respectively. On the basis of nucleotide sequences of 32 ORFs in OsHV-1-SB and the other nine OsHV-1 variants, results from phylogenetic analysis also demonstrated that OsHV-1-SB is most closely related to AVNV. CONCLUSIONS: The determination of the genome of OsHV-1 with distinguished epidemiological features will aid in our better understanding of OsHV-1 diversity, and facilitate further research on the origin, evolution, and epidemiology of the virus.

Roseovarius scapharcae sp. nov., isolated from ark shell Scapharca broughtonii.

A Gram-stain-negative, non-motile, aerobic and ovoid or rod-shaped bacterium, designated MA4-5T, was isolated from ark shell (Scapharca broughtonii) collected from the South Sea, South Korea. The novel strain grew optimally at 25 °C, at pH 7.0-8.0 and in the presence of 2.0 % (w/v) NaCl. Neighbour-joining and maximum-likelihood phylogenetic trees based on 16S rRNA gene sequences showed that strain MA4-5T forms a coherent cluster with the type strains of Roseovarius albus, Roseovarius aestuarii and Roseovarius nubinhibens, sharing 97.0-99.2 % sequence similarity. It exhibited 16S rRNA gene sequence similarity of 93.1-96.1 % to the type strains of other Roseovarius species. Strain MA4-5T contained Q-10 as the predominant ubiquinone and C18 : 1ω7c as the major fatty acid. The major polar lipids of strain MA4-5T were phosphatidylcholine, phosphatidylglycerol, one unidentified aminolipid and one unidentified lipid. The DNA G+C content of strain MA4-5T was 53.8 mol% and its mean DNA-DNA relatedness values with the type strains of R. albus, R. aestuarii and R. nubinhibens were 11-26 %. Differential phenotypic properties, together with phylogenetic and genetic distinctiveness, indicated that strain MA4-5T is separate from recognized species of the genus Roseovarius. On the basis of the data presented, strain MA4-5T is considered to represent a novel species of the genus Roseovarius, for which the name Roseovarius scapharcae sp. nov. is proposed. The type strain is MA4-5T ( = KCTC 42703T = NBRC 111226T).

A manganese superoxide dismutase (MnSOD) from ark shell, Scapharca broughtonii: Molecular characterization, expression and immune activity analysis.

Manganese superoxide dismutase (MnSOD) is one of the key members of the antioxidant defense enzyme family, however, data regarding to the immune function of MnSOD in mollusks still remain limited now. In this study, a full-length MnSOD cDNA was identified by rapid amplification of cDNA ends (RACE) method from cDNA library of ark shell Scapharca broughtonii (termed SbMnSOD). The cDNA contained an open reading frame (ORF) of 696 bp which encoded a polypeptide of 232 amino acids, a 5'-UTR with length of 32 bp and a 3'-UTR of 275 bp. Four putative amino acid residues (His-57, His-105, Asp-190 and His-194) responsible for manganese coordination were located in the most highly conserved regions of SbMnSOD and the signature sequence (DVWEHAYY) also existed in SbMnSOD. The deduced amino acid sequence of SbMnSOD shared high homology to MnSOD from other species. All those data revealed that the SbMnSOD was a novel member of the MnSOD family. The mRNA expression profiles of SbMnSOD in tissues of foot, gill, mantle, adductor muscle, hemocytes and hepatopancreas analyzed by quantitative real-time PCR (qRT-PCR) suggested the mRNA transcripts of SbMnSOD distributed in all the examined tissues. Importantly, Vibrio anguillarum challenge resulted in the increased expression of SbMnSOD mRNA with a regular change trend in all examined tissues, indicating SbMnSOD actively participated in the immune response process. What's more, further analysis on the antibacterial activity of the recombinant SbMnSOD showed that the fusion protein could remarkably inhibit growth of both Gram-positive and Gram-negative bacteria. The present results clearly suggested that SbMnSOD was an acute phase protein involved in the immune reaction in S. broughtonii.

Role of hemoglobin from blood clam Scapharca kagoshimensis beyond oxygen transport.

The evolutionary race between hosts and pathogens has led to a variety of adaptations. Little is known about the immunological role of hemoglobin (Hb) in antimicrobial immune responses. Results showed that a 31.2 kDa monodimer Hb (skHbI) and a 57.8 kDa heterotetramer Hb (skHbII) from the blood clam, Scapharca kagoshimensis, had phenoloxidase (PO)-like activities and antimicrobial activities. Both were found capable of oxidizing l-DOPA, catechol and hydroquinone. Their PO-like activities were visibly greatly inhibited by oxidase inhibitors, EDTA, and divalent metal ions, and greatly enhanced by isopropanol and Fe(2+), indicating that they have the properties of a metalloenzyme and a catecholase-type PO as well. They also showed obvious anti-bacterial activities against gram-positive bacteria but not against either gram-negative bacteria nor fungi. The anti-bacterial activities levels were a result of the generation of reactive oxygen species (ROS) of superoxide anions. These results indicate that skHbI and skHbII, not only function as iron-containing oxygen carriers, but also exert anti-bacterial activities and catecholase-type oxidizing activities. The fact that skHbII exerts high level of PO-like activity indicates different roles in the innate immunodefense system. These results may improve understanding of the multiple functions of invertebrate Hbs beyond serving as oxygen carriers and may provide insight into how the fundamental and universal mode of the innate immune system has persisted in respiratory proteins throughout the course of evolution.

Cooperative macromolecular device revealed by meta-analysis of static and time-resolved structures.

Here we present a meta-analysis of a large collection of static structures of a protein in the Protein Data Bank in order to extract the progression of structural events during protein function. We apply this strategy to the homodimeric hemoglobin HbI from Scapharca inaequivalvis. We derive a simple dynamic model describing how binding of the first ligand in one of the two chemically identical subunits facilitates a second binding event in the other partner subunit. The results of our ultrafast time-resolved crystallographic studies support this model. We demonstrate that HbI functions like a homodimeric mechanical device, such as pliers or scissors. Ligand-induced motion originating in one subunit is transmitted to the other via conserved pivot points, where the E and F' helices from two partner subunits are "bolted" together to form a stable dimer interface permitting slight relative rotation but preventing sliding.