Isolation and purification of a cell adhesion factor from crayfish blood cells.

Isolated granular haemocytes (blood cells) from the crayfish Pacifastacus leniusculus attached and spread in vitro on coverslips coated with a lysate of crayfish haemocytes. No cell adhesion activity was detected in crayfish plasma. The cell adhesion activity was only present in haemocyte lysates in which the prophenoloxidase (proPO) activating system (Söderhäll and Smith, 1986a, b) had been activated; either by lipopolysaccharide (LPS), the beta-1,3-glucan laminarin, or by preparing the lysate in 5 mM Ca2+. Both lysates of granular or of semigranular haemocytes could mediate adhesion. After A23187-induced exocytosis of the granular cells, cell adhesion activity could be generated in the secreted material if it was incubated with laminarin. The factor responsible for cell adhesion was isolated from an active haemocyte lysate and purified by ammonium sulfate precipitation, cation exchange chromatography and Con A-Sepharose; it had a molecular mass of approximately 76 kD on an SDS-polyacrylamide gel. An antibody to this 76-kD band inhibited cell adhesion. Ca2+ was necessary in the medium for the cells to adhere to the adhesion factor. With cyanide or azide, the cells attached but failed to spread. It is suggested that in vivo the cell adhesion factor is stored in the secretory granules of the semigranular and the granular cells in a putative inactive pro-form, which can be released during exocytosis and, in the presence of beta-1,3-glucans or LPS, be activated outside the cells to mediate cell attachment and spreading, processes of essential importance in arthropod host defense.

Role of the prophenoloxidase-activating system in invertebrate immunity.

The melanization reaction, which is a common response to parasite entry in invertebrate animals, especially arthropods, is due to the activity of an oxidoreductase, phenoloxidase. This enzyme is part of a complex system of proteinases, pattern recognition proteins and proteinase inhibitors constituting the so-called prophenoloxidase-activating system. It is proposed to be a non-self recognition system because conversion of prophenoloxidase to active enzyme can be brought about by minuscule amounts of molecules such as lipopolysaccharide, peptidoglycan and beta-1, 3-glucans from micro-organisms. Several components of this system recently have been isolated and their structure determined.

The prophenoloxidase activating system and associated proteins in invertebrates.

In this review, we present arguments indicating that prophenoloxidase (proPO) activating system acts as a pattern recognition and defence system in invertebrate blood. Phenoloxidase (PO) activity has been found in the blood of many invertebrates. At least in arthropods, echinoderms and urochordates, the inactive pro-form, proPO has been found to be elicited by the microbial cell-wall components beta-1, 3-glucans, lipopolysaccharide and/or peptidoglycan. This activation seems to involve elicitor-binding proteins and serine protease(s). ProPO, the proPO-activating enzyme (ppA) and plasma elicitor-binding proteins, have been purified from some arthropods, and proPO and the beta-1, 3-glucan binding protein (beta GBP) have been cloned and sequenced from crayfish. Arthropod proPO has a molecular mass of 70-90 kDa and PO has a molecular mass of 60-70 kDa. The beta GBP also stimulates phagocytosis of fungal cells and, after reacting with beta-1, 3-glucan, blood-cell degranulation (and release of the proPO system). In addition, a cell-adhesion protein (of 70-100 kDa), apparently associated with the proPO system, has been purified from arthropods. This mediates blood-cell adhesion, degranulation, phagocytosis and encapsulation. The cell-adhesion protein and beta GBP bind to a common blood-cell membrane receptor. It would be interesting to see the sequences of more proPO system components and investigate whether the scheme for cellular communication and defence, involving the cell-adhesion protein, elicitor-binding proteins and the membrane receptor described in arthropods, applies to invertebrates in general.

Amino acid sequence around the thiolester of alpha 2-macroglobulin from plasma of the crayfish, Pacifastacus leniusculus.

Alpha 2-Macroglobulin (alpha 2 M) was isolated from plasma of the freshwater crayfish, Pacifastacus leniusculus, using ultracentrifugation, ion-exchange chromatography and gel filtration techniques. The Pacifastacus alpha 2 M molecule (P alpha 2 M) was radio-actively labeled in the thiol ester structure with iodo [14C]acetic acid in the presence of methylamine. After reduction and carboxymethylation of the protein, it was digested with trypsin. A 14C-labeled tryptic peptide was sequenced and contained an amino acid sequence very similar to other known thiol ester sequences from human alpha 2 M and related proteins. The N-terminal sequence of P alpha 2 M was related to that recently determined for lobster alpha 2 M [(1987) J. Biol. Chem. 262, 14606-14611].

Drosophila ferritin mRNA: alternative RNA splicing regulates the presence of the iron-responsive element.

Several mRNAs encoding the same ferritin subunit of Drosophila melanogaster were identified. Alternative RNA splicing and utilisation of different polyadenylation sites were found to generate the transcripts. The alternative RNA splicing results in ferritin transcripts with four unique 5' untranslated regions. Only one of them contains an iron-responsive element. The iron-responsive element was found to bind in vitro specifically to human recombinant iron regulatory protein 1. Furthermore, the ferritin subunit mRNAs are differentially expressed during development. Our data provides the first molecular evidence that the presence of iron-responsive element in a ferritin mRNA is regulated by alternative RNA splicing.

Carbon and nitrogen metabolism in ectomycorrhizal fungi and ectomycorrhizas.

The literature concerning the metabolism of carbon and nitrogen compounds in ectomycorrhizal associations of trees is reviewed. The absorption and translocation of mineral ions by the mycelia require an energy source and a reductant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid and carbohydrate syntheses during the growth of the mycelia. Competition for photosynthates occurs between the fungal cells and the various vegetative sinks in the host tree. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolites between the various sites of utilization are only poorly understood. Both ascomycetous and basidiomycetous ectomycorrhizal fungi synthesize and some, if not all, accumulate mannitol, trehalose and triglycerides. The fungal strains employ the Embden--Meyerhof pathway of glucose catabolism and the key enzymes of the pentose phosphate pathway (6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, transaldolase and transketolase). Anaplerotic CO2 fixation, via pyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, provides high pools of amino acids. This process could be important in the recapture and assimilation of respired CO2 in the rhizosphere. The ectomycorrhizas are thought to contain the Embden--Meyerhof pathway, the pentose phosphate pathway and the tricarboxylic acid cycle, which provide the carbon skeletons for the assimilation of ammonia into amino acids. The main route of assimilation of ammonia appears to be through the glutamine synthetase-glutamate synthase cycle in the ectomycorrhizas. Glutamate dehydrogenase plays a minor role in this process. Glutamate dehydrogenase and glutamine synthetase are present in free-living ectomycorrhizal fungi and they participate in the assimilation of ammonia and the synthesis of amino acids through the glutamate dehydrogenase/glutamine synthetase sequence. In both in vitro cultures of fungi and ectomycorrhizas, the assimilated nitrogen accumulates in glutamine. Glutamine, but also ammonia, are thought to be exported from the fungal tissues to the host cells. Studies on the metabolism of ectomycorrhizas and ectomycorrhizal fungi have focused on the metabolic pathways and compounds which accumulate in the symbiotic tissues. Studies on regulation of the overall process, and the control of enzyme activity in particular, are still fragmentary.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and characterization of a hemagglutinin with affinity for lipopolysaccharides from plasma of the crayfish Pacifastacus leniusculus.

A hemagglutinin with a high specific activity against trypsinized rabbit erythrocytes was identified in plasma of the freshwater crayfish Pacifastacus leniusculus. The activity of this crayfish hemagglutinin could be inhibited by sialoglycoproteins such as porcine stomach mucin, bovine submaxillary mucin, fetuin, and ovalbumin. However, the involvement of sialic acid in its binding specificity could not be unambiguously proven. Furthermore, the hemagglutinating activity in the crayfish plasma could be specifically inhibited by lipopolysaccharide from E. coli K-235, which might indicate a recognition role for this hemagglutinin. This hemagglutinin, which accounts for less than 0.01% of the total plasma protein, was purified to near homogeneity using affinity chromatography on a Fetuin-Sepharose 4B column. The molecular mass of the unreduced protein as revealed by sodium dodecyl sulphate electrophoresis in polyacrylamide gel was found to be 420,000 Da. Upon reduction with dithiothreitol the hemagglutinin dissociated to several subunits with masses ranging from 65,000 to 80,000 Da. Affinoblotting with peroxidase labelled lectins indicated that the hemagglutinin was likely to be a glycoprotein.

Intracellular signaling in arthropod blood cells: involvement of protein kinase C and protein tyrosine phosphorylation in the response to the 76-kDa protein or the beta-1,3-glucan-binding protein in crayfish.

Degranulation (regulated exocytosis) of crayfish granular blood cells and release of the prophenoloxidase activating system can be triggered by two endogenous ligands, a 76-kDa cell adhesion protein or a beta-1,3-glucan-binding protein when reacted with beta-1,3-glucan (beta GBP-L). These ligands bind a recently described membrane receptor. Degranulation triggered by these ligands was inhibited by the protein kinase C (pkC) inhibitor staurosporine or by the tyrosine kinase inhibitor herbimycin A. Incubation with the 76-kDa protein increased tyrosine phosphorylation of a granular cell protein of approximately 80 kDa. In addition, the pkC activating phorbol esters PMA or beta PDD degranulated the cells in a dose-dependent manner, whereas the control isomeric phorbol ester alpha PDD that does not activate pkC did not have any effect on the cells. Thus, we propose that binding of the 76-kDa protein or beta GBP-L to the receptor triggers cellular responses via a pathway that includes pkC activation and protein tyrosine phosphorylation.

Separation of the haemocyte populations of Carcinus maenas and other marine decapods, and prophenoloxidase distribution.

Three morphologically distinct populations of haemocytes; the granular, semigranular and hyaline cells; were isolated from the haemolymph of Carcinus maenas and other decapod crustaceans by density gradient centrifugation. Cell lysis and coagulation during separation were prevented, without significant loss of cell viability, by the use of citrate/EDTA buffer at low pH as an anticoagulant. Biochemical analyses of the haemocyte fractions revealed that prophenoloxidase is present in the granular and semigranular cells, but not the hyaline cells, and, thus, is a useful marker for cell purity in the hyaline haemocyte populations. A method for rapidly detecting prophenoloxidase contamination of the hyaline cells using L-dopa and trypsin in drop assay is described.

Molecular cloning and characterization of prophenoloxidase in the black tiger shrimp, Penaeus monodon.

A cDNA encoding shrimp, Penaeus monodon, prophenoloxidase (proPO) was obtained by screening a hemocyte library by plaque hybridization using a proPO cDNA fragment from freshwater crayfish, Pacifastaceus leniusculus, as a probe. The 3,002 bp cDNA contains an open reading frame of 2,121 bp and a 881 bp 3'-untranslated region. The molecular mass of the deduced amino acid sequence (688 amino acids) is 78,700 Da with an estimated pI of 5.8. Two putative copper binding sites are present and they have a highly conserved sequence around these sites. No signal peptide was detected in the shrimp proPO, as has been previously shown to be the case for all arthropod proPOs cloned so far. The cleavage site of zymogen activation is likely to be between Arg 44 and Val 45. A tentative complement-like motif (GCGWPQHM) is also present. Shrimp proPO mRNA is synthesized in the hemocytes and not in the hepatopancreas. Comparison of amino acid sequences showed that shrimp proPO is more closely related to another crustacean proPO, namely crayfish, than to the insect proPOs.

Hemocyte lysate enhancement of fungal spore encapsulation by crayfish hemocytes.

Crayfish hemocytes exhibited a stronger encapsulation reaction to fungal blastospores of Beauveria bassiana coated with hemocyte lysate, than to blastospores treated with plasma or buffer, indicating an opsonic function of hemocyte lysate proteins. Five proteins of the prophenoloxidase activating system in the hemocytes were attached to foreign surfaces (including the blastospores) after activation and it is suggested that these attaching proteins (one being phenoloxidase) are responsible for the opsonic function of the hemocyte lysate on crayfish blood cells.

A comparison of phenoloxidase activity in the blood of marine invertebrates.

A range of marine invertebrates were screened for prophenoloxidase (a marker protein of the prophenoloxidase activating system) in the coelomic fluid or hemolymph. The crustaceans and the ascidian, Ciona intestinalis, displayed strongest activity, with low levels found in the starfish, Asterias rubens, the sea urchin, D. antillarum, and the brachiopod, Liothyrella uva. The enzyme appeared to be absent from the isopod, Glyptonotus antarcticus. Further analyses of the blood cells of selected species revealed that prophenoloxidase tends to reside in the granular-type cells or their equivalent, and in the crustaceans and C. intestinalis is activated by lipopolysaccharides. In arthropods, prophenoloxidase is known to represent the terminal component of a complex cascade of enzymes that functions in non-self-recognition and host defence. The present study establishes that the enzyme is present in the blood cells of most, but not all, crustaceans and occurs in certain other invertebrate species, notably the urochordate, C. intestinalis.

Opsonic activity of cell adhesion proteins and beta-1,3-glucan binding proteins from two crustaceans.

A beta-1,3-glucan binding protein (beta GBP) from the shore crab Carcinus maenas was purified from plasma by precipitation of the protein at low ionic strength. The protein had a molecular mass of 110 kDa, and was shown to affinity precipitate with laminarin, a soluble beta-1,3-glucan, and to cross-react with an antiserum directed toward beta GBP from the crayfish Pacifastacus leniusculus. Also, a protein from the haemocytes of C. maenas with a molecular mass of 80 kDa was found to mediate cell attachment and cause degranulation of crab cells, similar to the 76 kDa protein present in the haemocytes of P. leniusculus. Antibodies against the crayfish 76-kDa protein reacted with the crab 80-kDa protein present in the granular cells. No 80-kDa protein could be found in the hyaline cells. Using a method with FITC-conjugated yeast particles in a phagocytosis assay, both the beta GBP and the 80-kDa protein from C. maenas were shown to have opsonic activity as had beta GBP and 76-kDa protein from P. leniusculus, resulting in higher levels of phagocytosis by the crab hyaline cells. Treatment of the yeast particles with beta GBP previously reacted with laminarin (beta GBP-L) only resulted in a minor increase of phagocytosis. Moreover, if the phagocytic cells were preincubated with beta GBP-L or with the 80-kDa protein, the enhancement of the phagocytic activity by beta GBP or the 80-kDa protein were abolished, indicating that a saturable number of one kind of cell surface receptor seem to be involved in phagocytosis.

A plasma protein isolated from brown shrimp (Penaeus californiensis) which enhances the activation of prophenoloxidase system by beta-1,3-glucan.

A beta-glucan-binding protein (BGBP) has been identified in brown shrimp plasma by using a polyclonal antiserum against a BGBP from the freshwater crayfish. The protein was purified by immunoaffinity chromatography, and its molecular and biological properties described. Brown shrimp BGBP is a monomeric protein with a molecular mass of 100 kDa, similar to those described for other crustacean BGBPs. This protein is capable of enhancing the prophenoloxidase (proPO) system activation induced by laminarin. Both amino acid composition and N-terminal sequence are markedly similar to those of crayfish BGBP.

Peroxinectin, a cell adhesive protein associated with the proPO system from the black tiger shrimp, Penaeus monodon.

Upon activation of the prophenoloxidase activating system in the shrimp, Penaeus monodon, a cell adhesion activity in the haemolymph is generated. A cell adhesion assay showed that a high number of granular cells (60%) adhered to coverslips coated with a shrimp haemocyte lysate supernatant, whereas a very low number of cells adhered to coverslips coated with bovine serum albumin. Inhibition of adhesion by an antiserum against crayfish peroxinectin, a cell adhesion protein, revealed that the cell adhesion activity detected in shrimp haemocyte lysate supernatant might result from a peroxinectin-like molecule in shrimp. A cDNA clone encoding shrimp peroxinectin was isolated, which had an open reading frame of 2337 nucleotides, with a polyadenylation sequence and a poly A tail. It encodes a protein of 778 amino acids including a 20 amino acid signal peptide. The mature protein (758 amino acids) has a predicted molecular mass of 84.8kDa and an estimated pI of 9.0. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and KGD (Lys-Gly-Asp), were found in shrimp peroxinectin. Sequence comparison shows that the shrimp protein is similar to crayfish peroxinectin (69%) and to various peroxidases and putative peroxidases from invertebrates and vertebrates. The shrimp peroxinectin cDNA also shows similarity (51%) to both Drosophila peroxinectin-related protein (AAF78217) and peroxidasin (S46224), an extracellular matrix protein combining an active peroxidase domain as well as immunoglobulin domains, leucine rich repeats and procollagen-like motif. However, the sequence similarity to both Drosophila molecules are mostly within the peroxidase domain. Northern blot analysis, using a non-peroxidase region in peroxinectin as a probe, revealed that peroxinectin is constitutively expressed in shrimp haemocyte and was reduced significantly in shrimp injected with a beta-1,3-glucan, laminarin, to mimic an infection with a fungus.

An atypical iron-responsive element (IRE) within crayfish ferritin mRNA and an iron regulatory protein 1 (IRP1)-like protein from crayfish hepatopancreas.

A putative crayfish iron-responsive element (IRE) is present in the 5'-untranslated region of the crayfish ferritin mRNA. The putative crayfish IRE is in a cap-proximal position and shares most of the structural features of the consensus IRE, but the RNA stem-loop structure contains a bulge of a guanine instead of a cytosine at the expected position, so far thought to be a hallmark of IREs. By using an electromobility shift assay this IRE was shown to specifically bind purified recombinant human iron regulatory protein 1 (IRP1) as well as a factor(s) present in a homogenate of crayfish hepatopancreas, likely to be a crayfish IRP1 homologue. With mutations in the crayfish IRE, the affinity of IRP to IRE was drastically decreased. A cDNA encoding an IRP1-like protein was cloned from the hepatopancreas of crayfish. This protein has sequence similarities to IRP, and contains all the active-site residues of aconitase, two putative RNA-binding regions and a putative contact site between RNA and IRP. These results show that a crayfish IRE, lacking the bulged C, can bind IRP1 in vitro and that an IRP1-like protein present in crayfish hepatopancreas may have both aconitase and RNA-binding activities.

Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR.

A diagnostic procedure, based on a polymerase chain reaction method (PCR) was developed to detect infection of crayfish with the Oomycete Aphanomyces astaci. A set of oligonucleotide primers was designed to specifically amplify A. astaci DNA in the ITS region surrounding the 5.8S rDNA gene. The PCR amplifies a 115bp amplicon. The specificity of the primers was demonstrated by testing on 27 A. astaci strains and against 20 non-A. astaci Oomycetes and 5 fungal species. Most of the non-A. astaci Oomycete or fungal species included in the study are either known parasites of freshwater crayfish cuticle or can be found in their natural environment. Specificity was also tested against crayfish tissue and some known parasites and bacteria infecting crayfish. A protocol for the extraction of A. astaci DNA from infected crayfish tissue was developed. The optimised method allows the detection of two genome equivalents of purified A. astaci genomic DNA. The method was tested on noble crayfish (Astacus astacus), artificially infected with A. astaci. Detection of A. astaci was possible at the very first time of sampling, which was 2 days after the beginning of spore exposure.

Isolation of cDNA encoding a novel serpin of crayfish hemocytes.

We have cloned a serpin-type proteinase inhibitor from a crayfish hemocyte cDNA library. The deduced amino acid sequence consists of 429 amino acids with a putative signal peptide of 21 amino acids. The mature protein has a calculated molecular mass of 45,029 daltons. Identities ranging up to 38% were observed between the crayfish serpin and other members of the serpin family. Phylogenetic analysis shows that the crayfish serpin has a closer relationship to insect serpins than to other animal serpins. Phe369-Ser370 were proposed to be the P1-P1' residues of the inhibitor reactive site. This protein was found to be expressed in hemocytes but not in the hepatopancreas of the crayfish Pacifastacus leniusculus.

Effect of fixed epimastigote forms of Trypanosoma cruzi on the hemocytes and the prophenoloxidase-activating system of the crayfish Pacifastacus leniusculus.

The effect of the parasite Trypanosoma cruzi on the hemocytes and the prophenoloxidase (proPO)-activating system of the crayfish Pacifastacus leniusculus was studied. Incubation of the crayfish hemocyte lysate with fixed epimastigote forms of the parasites (4 x 10(5) cells/ml) induced a marked activation of the crayfish proPO system, measured as phenoloxidase activity. The activation of proPO by the parasite was much stronger (7-fold) than that induced by beta-1, 3-glucans (1 mg/ml) which are known to be efficient elicitors of the proPO system. The fixed parasites promoted the spreading and degranulation of different populations of the crayfish hemocytes isolated by Percoll gradients, and were often observed to be attached to the crayfish hemocytes in rosette-like fashion. The attachment of the epimastigote forms of T. cruzi to the crayfish blood cell surface was not dependent on the adhesive 76-kDa protein released by the crayfish hemocytes, since the exocytotic inhibitor SITS and monospecific antibodies to the 76-kDa protein did not prevent parasite adhesion. The crayfish hemocytes apparently are able to phagocytose the fixed epimastigote forms of T. cruzi in vitro.

Experimental infection of white spot syndrome virus in freshwater crayfish Pacifastacus leniusculus.

The signal freshwater crayfish Pacifastacus leniusculus was found to be susceptible to infection with white spot syndrome virus (WSSV). Histopathological observations of various tissues of virus-injected crayfish showed similar symptoms to those from WSSV-infected penaeid shrimp, but no appearance of white spots on the cuticle or reddish body colour were observed, although these are the prominent gross signs of white spot disease in shrimp. A gene probe for detecting WSSV was developed in order to detect the virus in affected cells and tissues using in situ hybridisation. Strong signals were observed in cells of virus-injected crayfish, but not in control-injected crayfish. The number of granular haemocytes in virus-injected crayfish was significantly higher than in sham-injected and non-injected crayfish from Days 5 to 8 (p < or = 0.05) and Days 3 to 8 (p < 0.01) post-injection, respectively. The proportion of granular haemocytes in virus-injected crayfish was also significantly higher than in sham-injected controls from Days 3 to 8 (p < 0.01). These results indicate that WSSV has a significant effect on the proportion of different haemocyte types in the freshwater crayfish.