Martin Bilej

Identification and Cloning of a Glucan- and Lipopolysaccharide-binding Protein from Eisenia foetidaEarthworm Involved in the Activation of Prophenoloxidase Cascade

Coelomic fluid of Eisenia foetidaearthworms contains a 42-kDa protein named coelomic cytolytic factor 1 (CCF-1) that was described previously to be involved in cytolytic, opsonizing, and hemolytic properties of the coelomic fluid. Cloning and sequencing of CCF-1 reveal significant homology with the putative catalytic region of β-1,3- and β-1,3–1,4-glucanases. CCF-1 also displays homology with coagulation factor G from Limulus polyphemus and with Gram-negative bacteria-binding protein ofBombyx mori silkworm, two proteins involved in invertebrate defense mechanisms. We show that CCF-1 efficiently binds both β-1,3-glucan and lipopolysaccharide. Moreover, CCF-1 participates in the activation of prophenoloxidase cascade via recognition of yeast and Gram-negative bacteria cell wall components. These results suggest that the 42-kDa CCF-1 protein of E. foetida coelomic fluid likely plays a role in the protection of earthworms against microbes.

On the existence of cytokines in invertebrates.

Abstract. Based on the assumption that invertebrates, like vertebrates, possess factors regulating responses to infection or wounding, studies dealing with the evolution of immunity have focussed on the isolation and characterisation of putative cytokine-related molecules from invertebrates. Until recently, most of our knowledge of cytokine- and cytokine receptor-like molecules in invertebrates relies on functional assays and similarities at the physicochemical level. As such, a phylogenetic relationship between invertebrate cytokine-like molecules and vertebrate counterparts could not be convincingly demonstrated. Recent genomic sequence analyses of interleukin-1-receptor-related molecules, that is Toll-like receptors, and members of the transforming growth factor-β superfamily suggest that the innate immune system of invertebrates and vertebrates evolved independently. In addition, data from protochordates and annelids suggest that invertebrate cytokine-like molecules and vertebrate factors do not have the same evolutionary origin. We propose instead that the convergence of function of invertebrate cytokine-like molecules and vertebrate counterparts involved in innate immune defences may be based on similar lectin-like activities.

Identification of a cytolytic protein in the coelomic fluid of Eisenia foetida earthworms

Total coelomic fluid of earthworms Eisenia foetida (Oligochaeta, Annelida) is capable of lysing different mammalian tumor cell lines. This cytolytic activity is different from tumor necrosis factor (TNF)-mediated lysis and is not due to proteolysis. Total coelomic fluid was subjected to ion-exchange chromatography separation and a fraction with prominent cytolytic activity was used to elicit monoclonal antibodies that were screened for their capacity to neutralize the cytolytic effect of total coelomic fluid. One of the prepared neutralizing IgG antibodies was used for the immunoaffinity purification of a cytolytic factor from total coelomic fluid. SDS-PAGE and Western blot analyses revealed a protein band with an apparent molecular weight of 42 kDa. This cytolytic protein (termed CCF-1 or coelomic cytolytic factor 1) can be adsorbed on the surface of opsonized particles and may be involved in opsonizing and hemolytic effects of coelomic fluid.

Convergent evolution of cytokines

Functional analogues of vertebrate inflammatory cytokines have been described in a variety of invertebrates. The analogy is based mainly on the crossreactivity of antibodies elicited against vertebrate cytokines, the sensitivity of invertebrate immunocytes to the action of vertebrate cytokines, and the responsiveness of vertebrate immune cells to invertebrate factors. But without knowing the amino-acid or gene sequences of the putative invertebrate cytokine analogues, it has not been possible to demonstrate unequivocally a phylogenetic relationship between vertebrate cytokines and their invertebrate functional analogues. Here we show that, although a defence molecule from the earthworm Eisenia foetida (Oligochaeta, Annelida) and the mammalian tumour-necrosis factor TNF-α perform similar functions, they emerged independently during evolution.

Antimicrobial Defense of the earthworm

Discrimination of self and nonself is one of the features of all animal species but the ways of elimination of nonself are different. Defense strategies of invertebrates, which lack antibodies and lymphocytes, are based on innate defense mechanisms. The study of such, undoubtedly less complex, defense mechanisms in invertebrates may shed a new light on the more sophisticated immunity of vertebrates. The main aim of this review is to show on one experimental model—an oligochæte annelid—cellular and humoral defense pathways protecting against microbial infection.

Cellular expression of the cytolytic factor in earthworms Eisenia foetida

Coelomic fluid of earthworms contains a 42 kDa protein designated CCF-1 (coelomic cytolytic factor 1), which accounts for approximately 40% of cytolytic activity of the entire coelomic fluid. CCF-1 was documented to be present on cells of the mesenchymal lining of the coelomic cavity as well as on free coelomocytes. Both cellular and humoral levels of CCF-1 were significantly increased after parenteral injection of endotoxin. Moreover, CCF-1 seems to be involved in cell mediated cytotoxicity, because cytotoxic activity is blocked in the presence of anti-CCF-1 monoclonal antibody (mAb).

Identification and cloning of an invertebrate-type lysozyme from Eisenia andrei.

Lysozyme is a widely distributed antimicrobial protein having specificity for cleaving the beta-(1,4)-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (GlcNAc) of peptidoglycan of the bacterial cell walls and thus efficiently contributes to protection against infections caused mainly by Gram-positive bacteria. In the present study, we assembled a full-length cDNA of a novel invertebrate-type lysozyme from Eisenia andrei earthworm (EALys) by RT-PCR and RACE system. The primary structure of EALys shares high homology with other invertebrate lysozymes; however the highest, 72% identity, was shown for the destabilase I isolated from medicinal leech. Recombinant EALys expressed in Escherichia coli exhibited the lysozyme and isopeptidase activity. Moreover, real-time PCR revealed increased levels of lysozyme mRNA in coelomocytes of E. andrei after the challenge with both Gram-positive and Gram-negative bacteria.

Functional Convergence of Invertebrate and Vertebrate Cytokine-Like Molecules Based on a Similar Lectin-Like Activity

It is generally accepted that the action of cytokines results from their binding to specific receptors. However, many cytokines possess lectin-like activity that may be essential for the expression of their full biological activities. This review focuses on the physiological relevance of the lectin-like activity of cytokines during the innate immune response in mammals, using TNF as an illustrative example. Moreover, we will show that TNF displays functional analogies with a defense molecule from the earthworm Eisenia foetida termed CCF. These analogies are not reflected by primary sequence homology between CCF and TNF but are particularly based on a similar lectin-like activity/domain. Hence, from a phylogenetic point of view, the lectin-like activity/domain of CCF and TNF may represent an essential recognition mechanism that has been functionally conserved during the innate immune response of invertebrates and vertebrates as a result of convergent evolution.

Evidence for proteins involved in prophenoloxidase cascade Eisenia fetida earthworms

The prophenoloxidase cascade represents one of the most important defense mechanisms in many invertebrates. Following the recognition of microbial saccharides by pattern recognition molecules, proteinases cleave inactive prophenoloxidase to its active form, phenoloxidase. Phenoloxidase is a key enzyme responsible for the catalysis of the melanization reaction. Final product melanin is involved in wound healing and immune responses. Prophenoloxidase cascade has been widely described in arthropods; data in other invertebrate groups are less frequent. Here we show detectable phenoloxidase activity in 90-kDa fraction of the coelomic fluid of earthworms Eisenia fetida. Amino acid sequencing of peptides from the active fraction revealed a partial homology with invertebrate phenoloxidases and hemocyanins. Moreover, the level of phenoloxidase activity is lower and the activation slower as compared to other invertebrates.

Identification of a coelomic mitogenic factor in Eisenia foetida earthworm.

Coelomic fluid of earthworms Eisenia foetida (Oligochaeta, Annelida) exerts a mitogenic activity on murine splenocytes. Total coelomic fluid was subjected to size-exclusion chromatography and a semi-purified mitogenic fraction (fraction 5) was isolated and further characterized. Both coelomic fluid and the semi-purified fraction 5 block concanavalin A (ConA)-induced spleen cell proliferation but exert a synergistic effect on LPS-triggered spleen cell proliferation. Using a polyclonal antiserum neutralizing the mitogenic activity of the semi-purified fraction 5, a 60-kDa component was identified and named CMF (coelomic mitogenic factor). CMF was found to bind ConA which could account for its ability to inhibit ConA-induced spleen cell proliferation. CMF is present in the coelomic fluid as a trimer of a 20-kDa protein. N-terminal amino acid sequence of monomeric CMF reveals partial sequence homology with phospholipase A2 (PLA2). Moreover, CMF-enriched coelomic fluid fraction 5 exerts phospholipase activity comparable with that of bovine pancreatic PLA2. Our results suggest that coelomic fluid of E. foetida contains a ubiquitous PLA2-like enzyme which might be involved in immune reactions in earthworms such as anti-bacterial mechanisms.