Identification of a novel metal binding protein, segon, in plasma of the eastern oyster, Crassostrea virginica

The second most abundant protein of eastern oyster plasma was purified, characterized and named segon. The 39 kDa protein as determined by SDS-PAGE under reducing conditions made up about 17% of plasma proteins and was found in extrapallial fluid. RACE reactions with primers designed from an EST sequence identified by BLAST search in GenBank using the N-terminal amino acid sequence obtained by Edman degradation of the purified protein, predicted a 997 bp complete cDNA that encoded 277 amino acids including a 16-residue signal peptide at the N-terminus. The deduced mature protein, composed of 261 amino acids, had a calculated molecular mass of 30,483.9 Da which was lower than the molecular mass of the purified protein measured by MALDI. The difference was likely due to post-translational modifications as the protein was predicted to have multiple sites for glycosylation and phosphorylation. The protein mRNA was detected in hemocytes by in situ hybridization and quantified in oyster tissues by RT-qPCR. Immunohistochemistry revealed that the protein was most abundant in tissues rich in blood sinuses like the gills and dorsally along the base of the mantle. ICP metal analysis of purified protein indicated highest association with zinc, calcium and iron and much greater metal content than in purified dominin, the most abundant protein of eastern oysters. Results of N-terminal and internal peptide sequencing of SDS-PAGE separated plasma proteins from Pacific, Suminoe and European flat oysters indicated that the second most abundant plasma protein is conserved. Several possible functions of segon in metal transport and detoxification, host defense, antioxidation and shell mineralization are proposed as they relate to its capacity to bind metals.     

Allograft Inflammatory Factor 1 Functions as a Pro-Inflammatory Cytokine in the Oyster,Crassostrea ariakensis

The oyster Crassostrea ariakensis is an economically important bivalve species in China, unfortunately it has suffered severe mortalities in recent years caused by rickettsia-like organism (RLO) infection. Prevention and control of this disease is a priority for the development of oyster aquaculture. Allograft inflammatory factor-1 (AIF-1) was identified as a modulator of the immune response during macrophage activation and a key gene in host immune defense reaction and inflammatory response. Therefore we investigated the functions of C. ariakensis AIF-1 (Ca-AIF1) and its antibody (anti-CaAIF1) in oyster RLO/LPS-induced disease and inflammation. Ca-AIF1 encodes a 149 amino acid protein containing two typical Ca²⁺ binding EF-hand motifs and shares a 48–95% amino acid sequence identity with other animal AIF-1s. Tissue-specific expression analysis indicates that Ca-AIF1 is highly expressed in hemocytes. Significant and continuous up-regulation of Ca-AIF1 is detected when hemocytes are stimulated with RLO/LPS (RLO or LPS). Treatment with recombinant Ca-AIF1 protein significantly up-regulates the expression levels of LITAF, MyD88 and TGFβ. When anti-CaAIF1 antibody is added to RLO/LPS-challenged hemocyte monolayers, a significant reduction of RLO/LPS-induced LITAF is observed at 1.5–12 h after treatment, suggesting that interference with Ca-AIF1 can suppress the inflammatory response. Furthermore, flow cytometric analysis indicated that anti-CaAIF1 administration reduces RLO/LPS-induced apoptosis and necrosis rates of hemocytes. Collectively these findings suggest that Ca-AIF1 functions as a pro-inflammatory cytokine in the oyster immune response and is a potential target for controlling RLO infection and LPS-induced inflammation.     

Characterization of the major plasma protein of the eastern oyster,Crassostrea virginica,and a proposed role in host defense

The major plasma protein of the eastern oyster, Crassostrea virginica, was purified, characterized and named dominin. SDS-PAGE analyses revealed that dominin consistently made up more than 40% of eastern oyster plasma and extrapallial fluid proteins. Three different forms of dominin were observed under non-reducing conditions. PCR and RACE primers designed from partial amino acid sequences obtained by tandem mass spectrometry of purified dominin identified 720 bp of complete cDNA encoding 192 amino acid residues. Based on the deduced amino acid sequence of mature dominin, its molecular mass was calculated to be 19,389 Da and was lower than the molecular mass of purified dominin measured by MALDI. This difference is likely due to post-translational modifications of dominin as the purified protein was found to be glycolysated, phosphorylated and likely sulfated. The amino acid sequence showed high similarity to the major plasma protein of the Pacific oyster (Crassostrea gigas), cavortin, and of the green-lipped mussel (Perna canaliculus), pernin, and to a recently described protein labeled as an extracellular superoxide dismutase from the Sydney rock oyster Saccostrea glomerata. While dominin was found to possess a Cu/Zn superoxide dismutase (SOD) domain, the domain was not completely conserved which explained why purified dominin lacked SOD activity. Dominin mRNA was detected in hemocytes by in situ hybridization and its expression measured by quantitative real time RT-PCR was significantly higher in winter than summer. Although the function(s) of dominin and homologous proteins is uncertain, the reported ability of cavortin to sequester iron and possibly limit the availability of this essential metal to pathogens suggests a potential role in host defense for this group of dominant plasma proteins. Other possible functions of dominin in antioxidation, wound repair, metal transport and shell mineralization are discussed leading us to conclude that dominin is likely a multifunctional protein.     

A single WAP domain-containing protein from Litopenaeus vannamei hemocytes

A cDNA clone coding for a single WAP domain (SWD) protein was isolated from a hemocyte cDNA library of Litopenaeus vannamei. The full-length cDNA sequence is 0.4kb long and encodes a 93-amino acid protein. Using this sequence as a probe a similar clone coding for a 92-amino acids protein was found in a cDNA library from Penaeus monodon hemocytes. The mRNA size was confirmed by Northern blot as well as that gene is expressed in hemocytes, but not in hepatopancreas. mRNA levels of the shrimp SWD protein were modified after injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response. Although amino acid sequence seems to be similar to those of other WAP domain-containing proteins, shrimp SWD protein does not have any other functional domain, similar to a mouse single WAP motif (SWAM) protein reported in mouse; however, the phylogenetic analysis shows that shrimp SWD is more related to other WAP proteins than to mouse SWAM.     

Molecular characterization and expression analysis of lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) from pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis>

The lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) plays an important function in the innate immune response of invertebrates as a pattern recognition receptor (PRR). Herein, we described the isolation and characterization of pearl oyster Pinctada fucata LGBP (designated as poLGBP). The poLGBP cDNA was 2,075 bp long and consisted of a 5′-untranslated region (UTR) of 18 bp, a 3′-UTR of 299 bp with one cytokine RNA instability motifs (ATTTA), and an open reading frame (ORF) of 1,758 bp encoding a polypeptide of 585 amino acids with an estimated molecular mass of 65.1 kDa and a theoretical isoelectric point of 5.80. Homology analysis of the deduced amino acid sequence of the poLGBP with other known LGBP sequences by MatGAT software revealed that the poLGBP shared 26.3–56.7% identity and 40.5–70.9% similarity to the other known LGBP sequences. SMART and alignment analysis revealed that the poLGBP possessed a potential polysaccharide-binding motif, a glucanase motif, a LPS-binding site, a β-1,3-linkage of polysaccharide, a glycine-rich region, a threonine-rich region and two N-glycosylation sites. In healthy pearl oyster, the poLGBP mRNA was specifically expressed in digestive gland, and not detected in gill, adductor muscle, gonad, intestine, mantle and hemocytes. However, after bacteria stimulation, the expression of the poLGBP mRNA was significantly up-regulated in digestive gland and also weakly detected in haemocytes, gonad and intestine. After LPS stimulation, the poLGBP mRNA expression was significantly up-regulated at 8 and 12 h in digestive gland, and the expression level was 10.7-fold higher than the PBS group at 12 h. After bacteria stimulation, the expression level of the poLGBP mRNA was also significantly up-regulated in digestive gland and was 12.9-fold higher than the PBS group at 8 h. However, during the experiment, the poLGBP mRNA expression was not detected in gill after LPS or bacteria stimulation. The tissue-specific expression and the expression up-regulation after LPS or bacteria stimulation in digestive gland suggested that the poLGBP was an inducible acute-phase protein and might play an important function in digestion as digestive enzyme and pattern recognition receptor.     

Primary structure of molluscan metallothioneins deduced from PCR-amplified cDNA and mass spectrometry of purified proteins

The primary structure of metallothioneins (MT) of a mollusc, the oyster Crassostrea virginica, was determined by molecular cloning and mass spectrometry of purified proteins. The cloning strategy included PCR amplification of the responsible cDNAs from total cDNA using completely degenerate oligonucleotides (derived from the N-terminal amino acid sequence) and oligo(dT)20 as primers. Primer extension off mRNA was used as an independent determination of the nucleotide sequence represented by the degenerate PCR primers. The deduced amino acid sequence was consistent with characteristics of class I MT. Twenty-one cysteine residues, were arranged in nine Cys-X-Cys motifs, five as Cys-Lys-Cys. A single Cys-X-X-Cys motif was also observed. Two MTs that differ only in the presence or absence of an N-acetyl group exist in this organism. Masses of tryptic peptides of purified MTs corresponded with those of peptides predicted from tryptic cleavages of the deduced amino acid sequence. Allowing for known N-terminal modifications, 96% of the deduced sequence was confirmed by mass spectrometry. Comparison (FASTA algorithm) of the primary structure of the oyster MTs with those of other species indicated a higher similarity with vertebrate MTs than with those of other invertebrates.     

红螯螯虾酚氧化酶原及其特性研究

采用同源克隆策略和RACE技术, 从红螯螯虾Cherax quadricarinatus血细胞中克隆得到酚氧化酶原基因的全长cDNA序列, 共2951 bp, 开放读码框为1995 bp, 编码665个氨基酸. 预测的分子量和等电点分别为75.7 kD和6.23. 酚氧化酶原含有两个推测的tyrosinase copper-binding motifs (带有六个组氨酸残基)和一个thiol-ester-like motif, 这些特征和其他甲壳动物的酚氧化酶原特征相同. 红螯螯虾酚氧化酶原氨基酸序列与通讯螯虾Pacifastacus leniusculus、欧洲龙虾Homarus gammarus、美洲龙虾Homarus americanus 和克氏原螯虾Procambarus clarkii 酚氧化酶原的相似率分别为68%、63%、63%和59%. 酚氧化酶原基因双酶切后连接入pET-28a原核表达载体, 转化到大肠杆菌BL21后重组表达酚氧化酶原蛋白. 在重组蛋白纯化后, 免疫新西兰大耳兔制备得到的酚氧化酶原多克隆抗体, 其效价大于1:12800. 红螯螯虾血淋巴、肝和鳃组织中的酚氧化酶原mRNA表达和酚氧化酶活性较高, 而神经、心、肠和肌肉中较低. 中华绒螯蟹螺原体和嗜水气单胞菌免疫红螯螯虾后, 血淋巴细胞、肝和鳃组织中的酚氧化酶原和酚氧化酶活性在免疫后的不同时间均出现了显著性的增加, 此结果表明酚氧化酶原和酚氧化酶在红螯螯虾对抗细菌感染的过程中起到重要的免疫作用. 此结果为进一步深入研究酚氧化酶原基因和酚氧化酶的功能及其调控机理奠定基础.       

A human protein specific for the immunoglobulin octamer DNA motif contains a functional homeobox domain

The homeobox domain is shared by Drosophila homeotic proteins, yeast mating type proteins, and some functionally uncharacterized mammalian proteins. A lymphoid-restricted human protein that binds to the immunoglobulin octamer regulatory motif was shown to contain an amino acid sequence that has 33% amino acid identity with the consensus sequence of the previously cloned homebox domains. This homeobox gene was localized to chromosome 19, thus mapping separately from other human homebox genes. A mutant protein containing amino acid substitutions within a putative helix-turn-helix motif in the homeobox domain did not bind DNA detectably. This human homeobox protein was shown to bind the same DNA sequence as the homeobox domains of the yeast mating type proteins and Drosophila homeotic protein, suggesting that homeobox proteins may have closely related DNA binding characteristics.     

The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u-PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported. The protein has a high content of cysteine residues. The NH2-terminal sequence is not closely related to any known sequence. The identification of the purified and sequenced protein with the human u-PA receptor is based on the following findings: 1) the ability of the purified protein to bind u-PA and its amino-terminal fragment; 2) the identical electrophoretic mobilities observed for cross-linked conjugates, formed between either the purified protein or the u-PA receptor on intact U937 cells and the above ligands; 3) the identity of the apparent molecular weight of the purified protein to that predicted for the u-PA receptor in the same cross-linking studies; 4) the identical extent of glycosylation of the purified protein and of the u-PA receptor in crude membrane fractions, as detected after cross-linking; 5) the ability of antibodies raised against the purified protein to inhibit cellular binding of the amino-terminal fragment of u-PA.     

Primary structure of anti-lipopolysaccharide factor from American horseshoe crab, Limulus polyphemus

The complete amino acid sequence of anti-lipopolysaccharide (LPS) factor purified from the hemocytes lysate of the American horseshoe crab, Limulus polyphemus, was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, clostripain, and Staphylococcus aureus V8 protease. Upon sequencing the peptides by the automated Edman method, the following primary structure was obtained: (Sequence: in text). During the sequence analysis, two species of the protein, which differed from each other at one locus, were found and characterized. L. polyphemus anti-LPS factor was a basic protein consisting of a single polypeptide chain of 101 residues and a calculated molecular weight of 11,786 or 11,800. The hydrophobic NH2-terminal sequence and the clustering of positive charges found in the disulfide loop yielded a typical amphipathic character of this protein. Moreover, L. polyphemus anti-LPS factor showed 83% sequence identity with the Tachypleus tridentatus protein, and the sequence similar to that observed in the EF-hand structure was found to contain in the COOH terminal portions of these proteins, although its function is unknown.     

A novel putative lipoprotein receptor (CasLpR) in the hemocytes of the blue crab, Callinectes sapidus: cloning and up-regulated expression after the injection of LPS and LTA

The full-length cDNA encoding a putative lipoprotein receptor (CasLpR) was isolated from the hemocytes of Callinectes sapidus using 5′ and 3′ RACEs. The open reading frame for CasLpR contains a precursor of putative CasLpR consisting of 1710 amino acid residues including 22 amino acid residues of the signal peptide (22 amino acids). Mature CasLpR (1688 amino acids with 5.6% of phosphorylation sites) has multiple, putative functional domains: five low-density lipoprotein receptor domains in the N-terminus, and a G-protein-coupled receptor proteolysis site domain and a 7 transmembrane receptor (secretin family) domain in the C-terminus. To date, there are no proteins with a similar domain structure in the GenBank. The expression pattern of CasLpR was exclusive in hemocytes among all tested tissues obtained from a juvenile female at intermolt stage: brain, eyestalk ganglia, pericardial organs, and thoracic ganglia complex (nervous system); hepatopancreas (digestive system); heart, artery and hemocytes (circulatory system); gill and antennal gland (excretory system), hypodermis; and Y-organ (endocrine organ). There was no CasLpR expression in the ovary of an adult female. A putative function of CasLpR was examined after challenges of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) in vivo using qRT-PCR assays. Animals at 24 h after injection of LPS or LTA up-regulated the expression of CasLpR in hemocytes by ∼3.5 and 1.4 folds, respectively, compared to the controls that received saline injection. LPS challenge also caused the greatest increment (∼55 folds) of heat shock protein 90 (Hsp90) expression in these samples. These data indicate that putative CasLpR and CasHsp90 may be involved in the defense system or the stress response of C. sapidus.     

Characterization of a cytotoxic pilin subunit of Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogenic bacterium, known to produce protein toxins that kill the larval host. We have described a cytotoxic pilin subunit of X. nematophila, which is expressed on the cell surface and also secreted in the extracellular medium associated with outer membrane vesicles. A 17kDa pilin subunit was isolated and purified from X. nematophila cell surface. The protein showed cytotoxicity to larval hemocytes of Helicoverpa armigera in an in vitro assay, causing agglutination of the cells, and releasing cytoplasmic enzyme lactate dehydrogenase in the medium. The pilin protein was able to bind to the surface of larval hemocytes. The binding and cytotoxicity of the purified 17kDa protein to hemocytes was inhibited by antiserum raised against the pilin protein. The study demonstrates for the first time a cytotoxic structural subunit of pilin from an entomopathogenic bacterium X. nematophila that is excreted in the extracellular medium with outer membrane vesicles.     

Purification and Amino Terminal Sequencing of Human Melanoma Nerve Growth Factor Receptor

Abstract: The nerve growth factor (NGF) receptor, solubilized with Triton X-100 detergent, has been purified from human melanoma cell line A875. Purification to near-homogeneity was achieved by chromatography on wheat germ agglutinin-agarose, followed by immunoaffinity chromatography on Sepharose columns coupled with anti-NGF receptor monoclonal antibody (MAb). The purified receptor, a 75, 000-dalton protein, retains the capacity to bind NGF as well as anti-receptor MAbs. Final purification was achieved by sodium dodecyl sulfate-polyacryiamide gel electrophoresis. The sequence of amino acid residues at the amino terminus has been determined. Possible sequence homology between the NGF receptor and several other proteins is discussed. Using the purified receptor as immunogen, new MAbs to the NGF receptor have been produced. The NGF receptor was visualized by immunoperoxidase staining in tissue sections of dorsal root ganglia from monkeys.     

Interaction of the sliding clamp beta-subunit and Hda, a DnaA-related protein

In Escherichia coli, interactions between the replication initiation protein DnaA, the beta subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro. A new beta-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified beta-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind beta. A 10-amino-acid peptide containing the E. coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay. These results establish that the interaction of Hda with beta is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.     

Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum binds specifically to sulfated glycoconjugates and to HepG2 hepatoma cells suggesting a role for this molecule in sporozoite invasion of hepatocytes.

Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum contains an amino acid motif based around the sequence WSPCSVTCG which is also found in region II of the circumsporozoite (CS) proteins of different species of Plasmodium. This amino acid motif confers on the CS protein the ability to bind specifically to sulfated glycoconjugates and to hepatocytes. This suggests that the interaction of CS protein with sulfated glycoconjugates on the surface of the hepatocytes may represent the first molecular event of sporozoite invasion of liver cells. Experimental evidence indicates that TRAP is localized both on the micronemes and on the surface of P. falciparum sporozoites implying that TRAP with its putative sulfated glycoconjugate binding motif may also be involved in recognition and/or entry of hepatocytes by the sporozoite. We show here that different TRAP constructs expressed in Escherichia coli bind to sulfogalactosyl-cerebrosides (sulfatides) and to the surface of HepG2 cells. These interactions are dependent on the presence of the conserved amino acid motif WSPCSVTCG within the sequences of the constructs and are completely inhibited by several sulfated glycoconjugates as well as by suramin, a polysulfonated drug with anti-protozoan activity. Moreover, sporozoite invasion of HepG2 cells is inhibited by antisera raised against these different TRAP constructs and by the presence of low concentrations of suramin. We concluded that TRAP may be one of the parasite encoded molecules in the host-parasite interaction that results in sporozoite invasion of hepatocytes.     

A novel,anchorless streptococcal surface protein that binds to human immunoglobulins

We have characterized a novel surface protein from urea extract of whole cells of group A Streptococcus pyogenes (GAS). A major protein band (35kD) was found to hybridize with human IgG by Western blotting. A search of the N-terminal amino acid sequence of this protein by using the GAS genome sequence database revealed an open reading frame that encoded a 38-kDa protein with a signal peptide sequence. We have named this protein streptococcal immunoglobulin-binding protein 35 (Sib35). It was found to be an anchorless protein with no LPXTG motif, distinct from the M protein superfamily exhibiting immunoglobulin-binding activity, and partially secreted in the culture supernatant. Recombinant Sib35 was also shown to bind human IgA and IgM. The sib35 gene was found in all GAS strains examined, but not in oral, group B, C, or G streptococcal strains. These results suggest that Sib35 is a unique immunoglobulin-binding protein in GAS.     

Complementary DNA sequence of rabbit CAP18--a unique lipopolysaccharide binding protein

CAP18 is a novel 18 kDa cationic protein [pI approximately 10] originally purified from rabbit granulocytes using as an assay the agglutination of lipopolysaccharide (LPS) coated erythrocytes. cDNA clones encoding CAP18 were isolated from a rabbit bone marrow cDNA library using a PCR generated oligonucleotide probe derived from the N-terminal amino acid sequence. The deduced amino acid sequence reveals a putative signal sequence of 29 amino acids and a mature protein of 142 amino acid residues. The predicted size of the encoded protein is 16.6 kDa with a pI of 10. There are no N-linked glycosylation sites. The CAP18 sequence bears no homology with other known LPS-binding proteins including human bacterial permeability increasing protein (BPI)(1) and rabbit LPS binding protein (LBP)(2).