Effects of Vibro harveyi and Staphyloccocus aureus infection on hemocyanin synthesis and innate immune responses in white shrimp Litopenaeus vannamei.

Hemocyanin, a multifunctional oxygen-carrying protein, has critical effects on immune defense in crustaceans. To explore the role of hemocyanin in anti-pathogen mechanism, effects of Vibrio harveyi (V. harvey) and Staphyloccocus aureus (S. aureus) on hemocyanin synthesis and innate immune responses were investigated in Litopenaeus vannamei (L. vannamei) during infection in vivo. Results showed that 105 and 106 cells mL-1V. harveyi and 106 cells mL-1S. aureus significantly affected plasma hemocyanin concentration, hepatopancreas hemocyanin mRNA and subunits expressions, plasma phenol oxidase (PO), hemocyanin-derived PO (Hd-PO), antibacterial, and bacteriolytic activities during the experiment under bacterial stress, while these parameters did not change remarkably in control group. The concentration of hemocyanin in plasma fluctuated, with a minimum at 12 h and a maximum at 24 h. Moreover, the expression of hemocyanin mRNA peaked at 12 h, while the level of hemocyanin p75 and p77 subunits reached maximum at 24 h. Besides, plasma PO and Hd-PO activities peaked at 24 h, and antimicrobial and bacteriolytic activities peaked at 12 h and 24 h, respectively. In addition, 105 cells mL-1S. aureus had no significant effect on the synthesis of hemocyanin and prophenoloxidase activating (pro-PO) system, but significantly increased antimicrobial activity at 12 h and bacteriolytic activity at 24 h. Therefore, these results suggest that the hemocyanin synthesis was initiated after invasion of pathogen, and the newly synthesized hemocyanin, acted as an immune molecule, can exerts PO activity to regulate the immune defense in L. vannamei in vivo.

The spider hemolymph clot proteome reveals high concentrations of hemocyanin and von Willebrand factor-like proteins.

Arthropods include chelicerates, crustaceans, and insects that all have open circulation systems and thus require different properties of their coagulation system than vertebrates. Although the clotting reaction in the chelicerate horseshoe crab (Family: Limulidae) has been described in details, the overall protein composition of the resulting clot has not been analyzed for any of the chelicerates. The largest class among the chelicerates is the arachnids, which includes spiders, ticks, mites, and scorpions. Here, we use a mass spectrometry-based approach to characterize the spider hemolymph clot proteome from the Brazilian whiteknee tarantula, Acanthoscurria geniculata. We focused on the insoluble part of the clot and demonstrated high concentrations of proteins homologous to the hemostasis-related and multimerization-prone von Willebrand factor. These proteins, which include hemolectins and vitellogenin homologous, were previously identified as essential components of the hemolymph clot in crustaceans and insects. Their presence in the spider hemolymph clot suggests that the origin of these proteins' function in coagulation predates the split between chelicerates and mandibulata. The clot proteome reveals that the major proteinaceous component is the oxygen-transporting and phenoloxidase-displaying abundant hemolymph protein hemocyanin, suggesting that this protein also plays a role in clot biology. Furthermore, quantification of the peptidome after coagulation revealed the simultaneous activation of both the innate immune system and the coagulation system. In general, many of the identified clot-proteins are related to the innate immune system, and our results support the previously suggested crosstalk between immunity and coagulation in arthropods.

Distribution and characterization of rhogocyte cell types in the mantle tissue of Haliotis laevigata.

Molluscan rhogocytes are known to be the only cells able to synthesize hemocyanin that is one of the largest respiratory proteins in nature. However, investigation of rhogocyte cells in vitro is limited due to difficulty in isolating and establishing marine cell culture. The aim of this study was to investigate the nature and distribution of rhogocyte cells of Haliotis laevigata in the mantle tissue with respect to the expression of the two known isoforms of hemocyanin. Rhogocyte cells were identified using immunofluorescence-fluorescence in situ hybridization (IF-FISH) that involved simultaneous staining of localized hemocyanin by a polyclonal antibody while the mRNA was hybridized with FISH probes. The distribution of rhogocyte cells was demonstrated using flow cytometry, followed by cell sorting with fluorescence-activated cell sorter (FACS) and confocal microscope imaging for further characterization. Our results suggested that the mantle tissue is dominated by two distinct populations of rhogocyte cells that synthesize hemocyanin type 1. Observation with confocal microscopy of both populations revealed hemocyanin localization in the periphery of the cell membrane. Cell population with higher antibody signal had irregular and elongated cell morphology with punctate mRNA probe signals. The second population with lower antibody signal had ovoid morphology and wide distribution of mRNA probe signals. We suggest that these populations represent two distinct phases of hemocyanin biosynthesis of a single isoform, which is closely related to Haliotis tuberculata type 1 hemocyanin (HtH1). The knowledge acquired in this study enhances the understanding of the biology of rhogocyte cells and biosynthesis of hemocyanin.

Concholepas hemocyanin biosynthesis takes place in the hepatopancreas, with hemocytes being involved in its metabolism.

Hemocyanins are copper-containing glycoproteins in some molluscs and arthropods, and their best-known function is O(2) transport. We studied the site of their biosynthesis in the gastropod Concholepas concholepas by using immunological and molecular genetic approaches. We performed immunohistochemical staining of various organs, including the mantle, branchia, and hepatopancreas, and detected C. concholepas hemocyanin (CCH) molecules in circulating and tissue-associated hemocytes by electron microscopy. To characterize the hemocytes, we purified them from hemolymph. We identified three types of granular cells. The most abundant type was a phagocyte-like cell with small cytoplasmic granules. The second type contained large electron-dense granules. The third type had vacuoles containing hemocyanin molecules suggesting that synthesis or catabolism occurred inside these cells. Our failure to detect cch-mRNA in hemocytes by reverse transcription with the polymerase chain reaction (RT-PCR) led us to propose that hemocytes instead played a role in CCH metabolism. This hypothesis was supported by colloidal gold staining showing hemocyanin molecules in electron-dense granules inside hemocytes. RT-PCR analysis, complemented by in situ hybridization analyses with single-stranded antisense RNAs as specific probes, demonstrated the presence of cch-mRNA in the hepatopancreas; this was consistent with the specific hybridization signal and confirmed the hepatopancreas as the site of CCH synthesis. Finally, we investigated the possibility that CCH catabolism in hemocytes was involved in the host immune response and in the generation of secondary metabolites such as antimicrobial peptides and phenoloxidase.

Role of a copper-specific metallothionein of the blue crab, Callinectes sapidus, in copper metabolism associated with degradation and synthesis of hemocyanin.

We have identified three MT encoding genes in the blue crab: MT-I, inducible by cadmium, zinc and copper; MT-II, inducible by cadmium and zinc; and MT-III, inducible by copper only [Syring et al., Comp. Biochem. Physiol. C, 125 (2000) 325-332]. To examine the role of the CuMT-I and CuMT-III isoforms in copper metabolism associated with the synthesis and degradation of the oxygen-binding copper protein, hemocyanin, we (1) cloned and sequenced hemocyanin cDNA, (2) examined interaction of the CuMTs with endoplasmic reticulum (ER) vesicles and (3) measured changes in levels of hemocyanin, MT-I, MT-III protein and mRNA that occur in crabs during different stages of the molt cycle. The cDNA-derived hemocyanin amino-acid sequence revealed the presence of a leader peptide indicating that hemocyanin is a secretory protein that is synthesized on the ER. Copper uptake studies show that ER vesicles take up both Cu1+ and Cu2+ in an ATP-independent process. The copper transporter has a Km of 10.8+/-2.4 microM copper and a Vmax of 6.1+/-0.5 nmol Cu/mg protein/10 min. ER vesicles contain hemocyanin, and bind CuMT-I and, preferentially, CuMT-III. However, binding does not result in copper transfer to the ER. There are statistically significant changes in hepatopancreas MT-III and hemocyanin mRNA, and in hemolymph hemocyanin concentrations during the molt cycle. MT-I mRNA remains constant. Changes in MT-III mRNA are positively correlated with changes in hemocyanin mRNA and hemocyanin protein, which points to coordinate control of MT-III and hemocyanin transcription. No CuMT-III protein is observed in hepatopancreas of intermolt crabs when levels of both MT-III and hemocyanin mRNA are high, suggesting rapid utilization of copper bound to MT-III when cells are actively synthesizing hemocyanin. CuMT-III is present in premolt and softshell crabs, and its emergence appears to coincide with a decrease in hemocyanin synthesis and increase in hemocyanin degradation. These results support the hypothesis that the copper-specific metallothionein is intricately involved in copper homeostasis associated with both the synthesis and degradation of hemocyanin.

Rhogocytes (pore cells) as the site of hemocyanin biosynthesis in the marine gastropod Haliotis tuberculata.

Rhogocytes (pore cells) are specific molluscan cell types that are scattered throughout the connective tissues of diverse body parts. We have identified rhogocytes in large numbers in tissue taken from mantle, foot and midgut gland of the abalone Haliotis tuberculata (Vetigastropoda). Within cisternae of the endoplasmic reticulum, particles are visible that resemble, in shape and size, hemocyanin molecules, the respiratory protein of many molluscs. Immunohistochemical experiments using hemocyanin-specific antibodies demonstrated that these cells contain hemocyanin. In situ hybridization with a cDNA probe specific for Haliotis hemocyanin showed that hemocyanin-specific mRNA is present in rhogocytes, which confirmed that they are the site of hemocyanin biosynthesis in this gastropod. A possible path of hemocyanin release into the hemolymph is discussed. Also in the vetigastropod Megathura crenulata, many rhogocytes could be detected. However, they lacked hemocyanin molecules which, together with published data, indicates a seasonal expression of hemocyanin in this animal.

Molecular cloning and evolution of lobster hemocyanin.

In the American lobster, Homarus americanus, oxygen is transported by a hemocyanin that is composed 2 x 6 subunits. N-terminal sequencing show the presence of three distinct subunit types (alpha, beta and gamma). We cloned the cDNA of one of these subunits that belong to the alpha-type. It encodes a hemocyanin subunit of 654 amino acids with a molecular mass of 84.8 kDa, which is synthesized in the hepatopancreas. Phylogenetic analyses of the crustacean hemocyanin sequences show two well-separated clades, which correspond to the alpha and gamma-type subunits. Sequences of beta-type subunits are still unknown. The gamma-sequences have evolved about 15% faster than the alpha-subunits, consistent with the proposed conservative function of the latter. Under the assumption of a molecular clock we calculated that alpha- and gamma-subunits split about 214 +/- 14 million years ago, suggesting their divergence only in the decapod Crustacea.

Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and htH.

Keyhole limpet hemocyanin (KLH) is a well-established immune stimulant and hapten carrier, and Haliotis tuberculata hemocyanin (HtH) is a related product. Biologically, KLH and HtH are blue copper proteins which serve as oxygen carriers in the blood of the keyhole limpet Megathura crenulata and the abalone H. tuberculata, respectively, two marine gastropods. Both hemocyanins occur as two distinct isoforms, termed KLH1 KLH2, HtH1, and HtH2. Each of these molecules is based on a very large polypeptide chain, the subunit (molecular mass ca 400 kDa), which is folded into a series of eight globular functional units (molecular mass ca 50 kDa each). Twenty copies of this subunit form a cylindrical quaternary structure (molecular mass ca 8 MDa). This article reviews the recent data on the biosynthesis, quaternary structure, subunit architecture, amino acid sequence, gene structure, and recombinant production of KLH and HtH.

Keyhole limpet hemocyanin: molecular structure of a potent marine immunoactivator. A review.

OBJECTIVES: In this short review we present a survey of the available biochemical and electron microscopic data on keyhole limpet hemocyanin (KLH). RESULTS: The biosynthesis of KLH and its biological role are discussed and the purification of the two isoforms of KLH (KLH1 and KLH2) presented in some detail. The determination of the molecular mass of KLH, its functional unit structure, carbohydrate content, immunological analysis and aspects of the molecular biology of KLH are all dealt with. Transmission electron microscopy (TEM) and crossed immunoelectrophoresis have played a significant part in the understanding of KLH structure. We present a summary of TEM studies on the native oligomers of KLH, the experimental manipulation of the different oligomeric states, immunological analysis and subunit reassociation. CONCLUSION: This fundamental structural information provides the scientific background upon which the understanding of the in vivo immunostimulatory function of KLH can be based.

Immunocytochemical reaction of a haemocyanin antibody in the midgut gland of Nautilus (Cephalopoda, Tetrabranchiata).

The branchial gland of the dibranchiate cephalopods is described as the site of haemocyanin synthesis. Because there is no equivalent to this organ in tetrabranchiate cephalopods the localization of haemocyanin synthesis remained unknown for a long time. In this study we could confirm the conclusions from preliminary investigations concerning the copper content of the midgut gland of Nautilus, which gave the first indications for a possible localization of haemocyanin synthesis in this organ. We developed a polyclonal antibody against Nautilus haemocyanin, tested its specificity, and used it on ultra-thin sections of the tissue of the midgut gland. It could be shown that there is a clear immunogold precipitation only on the triangular basal cells in the terminal alveoli. All the other types of cell in this organ were free of any immunoreactivity. It can be supposed that the triangular basal cells in the terminal alveoli of the midgut gland are the sites of haemocyanin synthesis in Nautilus.

Characterization of N-linked carbohydrate chains of the crayfish, Astacus leptodactylus hemocyanin.

The primary structure of the carbohydrate chains of hemocyanin from the crayfish Astacus leptodactylus were investigated. The carbohydrate content is 0.2% (w/w) as referred to total hemocyanin content, resp. 1.8% as referred only to the one subunit which is glycosylated. Mannose and N-acetylglucosamine are present in a molar ratio of 6:2. The carbohydrate chains are N-glycosidically linked as revealed by dot blot analysis using various lectins and enzymatic deglycosylation. Furthermore, they are part of only one hemocyanin subunit of A. leptodactylus. After enzymatic deglycosylation with PNGase F, the oligosaccharide pool was separated by FPLC on Mono Q and subsequent HPLC on Lichrosorb-NH2, the subfractions were characterized by 1H NMR spectroscopy. A total of six oligosaccharides, ranging from Man4GlcNAc2 to Man9GlcNAc2 is present, Man6GlcNAc2 representing the most abundant one with 57% of all oligosaccharides.

Hemocyanin synthesis in the blue crab Callinectes sapidus.

1. The synthesis of hemocyanin in the blue crab, Callinectes sapidus, was investigated by molecular techniques. 2. Polyadenylated mRNA was prepared from the hepatopancreas and translated in rabbit reticulocyte lysate. 3. Translation products were immunoprecipitated with immobilized antihemocyanin IgG. Analysis of the IgG-bound polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two polypeptides comigrating with authentic Callinectes hemocyanin. 4. A synthetic degenerate 17-mer oligonucleotide mixture derived from the amino acid sequence of the "copper B" binding site of crustacean hemocyanins was prepared. Northern blotting showed the probe to hybridize with one major 2.3 kb RNA species. 5. These results indicate that the hepatopancreas is the site of hemocyanin synthesis.

Protein production and the molting cycle in the crayfish Astacus leptodactylus (Nordmann, 1842). II. Hemocyanin and protein synthesis in the midgut gland.

The midgut gland from the crayfish Astacus leptodactylus synthesizes and secretes hemocyanin and proteins linearly for at least 24 hr in vitro. There are no sex-specific differences in the rate of protein and hemocyanin synthesis. During a standard incubation of 5 hr, up to 64% of the newly synthesized protein represents hemocyanin, which is also the predominant secretory protein. There are pronounced changes in the rate of hemocyanin synthesis during the molting cycle which coincide with corresponding changes in total protein synthesis. The titer of protein and hemocyanin synthesis during an intermolt phase exhibits a biphasic profile with the highest values in intermolt stage C4 and in premolt stage D1.

Immortalization of cloned mouse splenic macrophages with a retrovirus containing the v-raf/mil and v-myc oncogenes.

The recombinant retrovirus J2, which contains the v-raf/mil and v-myc oncogenes, was used to immortalize mouse splenic macrophages that had been cloned in soft agar. When added to freshly harvested colonies, J2 failed to yield cell lines but it immortalized up to 30% of the clones if they had been maintained for at least 4 months in medium containing colony-stimulating factor 1 (CSF-1). All of the cell lines grew in agar in a CSF-1-independent manner, and they produced tumors in nude and syngeneic mice. The cell lines were judged to be macrophage based on morphological criteria and because they secreted lysozyme, were phagocytic for antibody-coated particles, and expressed both the Mac-1 antigen and the CSF-1 receptor. The cell lines could be divided into three groups based on their expression of Ia and their ability to present an antigen to a T-cell hybridoma. The majority of the lines did not constitutively express Ia or present antigen, but a lymphokine did induce Ia in all of the lines, with most of them also acquiring antigen-presenting activity. However, a small proportion of lymphokine-treated lines continued to lack antigen-presenting activity despite their ability to express Ia. The third and smallest group of cell lines constitutively expressed both Ia and antigen-presenting activity. These results show that the J2 recombinant retrovirus is a useful means of immortalizing functionally distinct populations of cloned splenic macrophages.

Isolation of mRNA from membrane-bound polysomes synthesizing Sepia officinalis haemocyanin in Xenopus laevis oocytes.

Isolation and translation in Xenopus laevis oocytes of the mRNAs from the nuclear pellet and the cytoplasmic supernatant of the branchial glands from Sepia officinalis, homogenized in the presence of vanadyl-ribonucleoside complexes, revealed that the membrane-bound polysomes for haemocyanin sedimented together with the nuclei. Centrifugation on a 15-50% sucrose gradient of these polysomes, released by treatment with Triton X-100, yielded a major peak of heavy ones. The messenger fraction, isolated from this heavy fraction, directed the synthesis of the large 390 000 Mr haemocyanin polypeptide chain in oocytes. A large mRNA on heavy membrane-bound polysomes thus synthesizes the whole haemocyanin chain of S. officinalis.

Cell-free synthesis of hemocyanin from the scorpion Androctonus australis. Characterization of the translation products by monospecific antisera.

Translation of Androctonus australis poly(A)-RNA in vitro led to a number of polypeptides products (8-10) of 70-73 kDa analyzed by two-dimensional gel electrophoresis and identified by immunoprecipitation with an anti-(dissociated hemocyanin) antiserum. The translated hemocyanin polypeptides have the same physico-chemical characteristics as authentic hemocyanin subunits. Subunits Aa 2 and Aa 4 have been identified with monospecific antisera characterized (a) by their capability of reacting with their homologous subunit and (b) by their inability of binding to cross-reacting subunits. Each polypeptide chain is coded by a different messenger without significant post-translational events. Hemocyanin could be detected among the translation products of the poly(A)-RNA isolated from the cuticle under the carapace.

Translation of mRNA for Limulus polyphemus haemocyanin polypeptides in vitro: studies on subunit heterogeneity.

The haemocyanin of Limulus polyphemus is composed of a number (possibly 10-15) of polypeptides and is believed to be synthesised in cells called cyanoblasts. In vitro translation in the rabbit reticulocyte haemolysate system and in Xenopus oocytes, of mRNA isolated from cyanoblast-containing tissue, allowed the detection of several haemocyanin polypeptides amongst the products of translation. At least seven polypeptides with molecular weights in the range 68 000-71 000 were identified by an immunological method followed by electrophoretic characterisation on two-dimensional polyacrylamide gels. Comparison of the polypeptide patterns of authentic haemocyanin, reticulocyte lysate translation products and Xenopus oocyte translation products led to the conclusion that the polypeptides are unlikely to undergo significant post-translational modification or to possess cleavable signal sequences. It is proposed that release of haemocyanin into the haemolymph in vivo may involve bursting of the cyanoblasts.