Role of the prophenoloxidase-activating system in invertebrate immunity

23

Role of the prophenoloxidase-activating system in invertebrate immunity Kenneth S6derh ill and Lage Cerenius 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 I]-l,3-glucans from micro-organisms. Several components of this system recently have been isolated and their structure determined.

is slightly delayed and will usually occur within a few hours after entry of a bacterium or fungus. Evidently, some sort of recognition of the foreign particle has to take place in order to transfer the message to the cells that will synthesize the appropriate immune factors, such as antimicrobial peptides. Recognition of foreign material is believed to occur through recognition molecules, in the blood (haemolymph) of invertebrates. These induce activation of the prophenoloxidase (proPO)-activating system (proPO-AS), and may also induce activation of other defence processes.

Figure 1 Addresses

Division of Physiological Mycology, University of Uppsala, Villav~igen 6, 752 36 Uppsala, Sweden e-mail:[email protected]

Q[~-l,S-glucansl I Peptidoglycans I ILipopolysaccharidel

Current Opinion in Immunology 1998, 10:23-28

http:l/biomednet.comlelecref10952791501000023 © Current Biology Ltd ISSN 0952-7915 Abbreviations BGBP ]~-1,3-glucan-binding protein GNBP Gram-negative bacteria binding protein

LPS PLD proPO proPO-AE proPO-AS

lipopolysaccharide pacifastinlight chain domain prophenoloxidase proPO-activating enzyme proPO-activating system

Introduction

~

Iiimmune defence| o' 1

e.g. peroxinectin))

N~ognitionmolecules ~

g ~ Serineproteinases1

Prophenoloxidase ,

~> Phenoloxidase O

Invertebrate animals are widely distributed and can be found in almost any kind of habitat. Their dispersal and survival depend to a large extent on successful defences against various kinds of micro-organisms and parasites, especially as many of these animals live in environments where micro-organisms are thriving. It is therefore clear that invertebrates must have very efficient means of recognizing and combating potentially harmful micro-organisms.

A scheme for prophenoloxidase-activationin arthropods.

Invertebrate animals lack true antibodies and, hence, also an adaptive immune response. Although they have proteins with immunoglobulin domains [1], they have to rely solely on innate immune mechanisms. One such innate defence is the production of antimicrobial peptides as a response to parasite entry. Antimicrobial peptides and their synthesis have been studied in great detail in some insect species and during the last few years excellent studies on how the genes for these antimicrobial peptides are regulated in Drosophila melanogaster have been published [2°]. T h e production of these peptides

The proPO-AS is an efficient non-self recognition system [3] in invertebrates that can recognize and respond to picograms per litre of lipopolysaccharides or peptidoglycans from bacteria and I]-l,3-glucans from fungi. T h e recognition of foreign material in invertebrates thus appears to be much more efficient than in vertebrates [4",5]. As a result of activation of the proPO-AS the parasite is blackened in the host haemolymph by the deposition of melanin due to the action of phenoloxidase,

CurrentOpinionin Immunology

24

Innateimmunity

an oxidoreductase. This reaction is called the melanization reaction and is easily observed around parasites in the haemolymph or the exoskeleton (cuticle). T h e proPO-AS consists of several different proteins among which are proteinases, proteinase inhibitors and recognition molecules that recognise structural features of the bacterial and fungal components. Upon activation of the system, the associated proteins gain biological activity and participate in the cellular defence reactions of the host animal.

Prophenoloxidase and phenoloxidase T h e enzyme involved in melanin formation is phenoloxidase (monophenyl L-dopa:oxygen oxidoreductase; EC 1.14.18.1). Phenoloxidase activity has been detected in the haemolymph or coelom of many invertebrate groups, both protostomes and deuterostomes. This enzyme catalyses the oxidation of phenols to quinones, which then will polymerize nonenzymatically to melanin. Both mono- and diphenols can be oxidized by phenoloxidase, and the intermediary compounds formed as well as melanin itself are toxic to micro-organisms [5]. Recently, it was demonstrated that a derivative of dihydroxyphenylalanine (N-13alanyl-3,4-dihydroxyphenylalanine (5-S-GAD) present in the blood of the insect Sarcophaga peregrina exhibited antibacterial activity [6"]. Its antibacterial activity is the result of H 2 0 2 production. 5-S-GAD could be produced by a tyrosinase and thus it is very likely that this or similar molecules are generated by the action of phenoloxidase in the haemolymph of invertebrates. Invertebrate phenoloxidase has, wherever carefully studied, been found to exist in the blood in an inactive form, proPO, which is activated in a stepwise process involving serine proteinases, which have previously been activated by microbial cell wall constituents. ProPOs have been isolated and characterized from several invertebrate animals; in all cases where the inactive proenzyme has been purified, the monomer has a mass of about 70-80kDa and, after proteolytic activation, the active enzyme, phenoloxidase, has a mass of 60-70kDa [7-12]. In native form the enzyme can exist as an oligomer. In the cockroach Blaberus discoidalis, endogenous lectins can trigger activation of the proPO-AS [13] and phospholipids have been shown to induce activation of phenoloxidase in a crude preparation from the horseshoe crab Limulus polyphemus [14]. In the latter case, crude proPO preparations were used and as a consequence the mechanism of activation is not yet understood. T h e primary structures of several arthropod proPOs have been determined and all contain two functional copper-binding sites and lack a signal peptide [15-18]. T h e first report of a proPO sequence was from crayfish [15]. It has two putative copper-binding sites and the purified molecule contains two copper atoms, showing that both sites are functional [15]. T h e overall similarity between the four cloned proPOs (from the freshwater crayfish Pacifastacus leniusculus, and from the insects

Manduca sexta, D. melanogaster and Bombyx mori) is about 40%, whereas the sequence similarity around the copper-binding sites is 60-70%. In some insects, two or more forms of proPO are present. T h e importance of this polymorphism is not known, but it may be that the different proPOs have different functions; for example, one form may be deposited in the cuticle and the other in the blood cells. ProPO and arthropod haemocyanins, the hexamerins and arylphorin receptors in insects [19",20"], and the cockroach Cr-PI allergen [21] appear to be homologous proteins. T h e tyrosinases make up another family of proteins, which includes molluscan haemocyanin [19",20",22"]. T h e first non-arthropod phenoloxidase to be be cloned, from the ascidian Halocynthia roretzi, more closely resembles vertebrate tyrosinases than arthropod proPOs [23]. In contrast to arthropod proPOs, the ascidian enzyme has a signal peptide and a transmembrane domain like vertebrate tyrosinases and is involved in producing melanin in the pigment cells. It therefore appears as if tyrosinases and proPOs diverged early in evolution from a common copper-binding protein. For a more detailed discussion of the tyrosinases see Gelder et aL [22"1. Crustacean proPO is synthesised in the blood cells [15], whereas in crayfish haemocyanin is known to be synthesised in the hepatopancreas. In insects the proPO also seems to be synthesised in the blood cells [18]. Recently, proPO has been shown to be transported and deposited in the cuticle of the silkworm, B. mori [24], where it may be involved in the sclerotization of the cuticle and in defence against invading parasites. It will be of interest to determine whether both silkworm proPOs or only one of the two forms is transported and deposited in the cuticle. ProPO is activated in arthropods by proteolytic cleavage by a native proteinase to yield an active prophenoloxidase, and the cleavage site has been identified in some proPOs [15,18,25]. ProPO-activating enzymes (proPO-AE), that is, serine proteinases, have been isolated from crayfish [26], D. melanogaster [25] and B. mori [27] and their molecular masses are about the same i 3 0 k D a . A thioester-like motif is present in proPO, although it is not yet known whether it is functional, and it may be that proteinases of the proPO-AS have complement-like motifs or domains like those in Factor C of the horseshoe crab clotting system and in cc2-macroglobulins in several protostome species. So far, no proPO-AE have been cloned. Interestingly, a molecule resembling the complement component C3 has been identified in a deuterostome, the sea urchin Strongylocentrotus purpuratus [28"], but the function of this protein is not yet known.

ProPO-associated proteins Several recognition proteins that bind to microbial molecules have been characterised from arthropods and some of these molecules have been shown to be involved

The prophenoloxidase-activating system in invertebrate immunity S6derh~ll and Cerenius

in activation of the proPO-AS and in cellular immune reactions, although they may also be involved in eliciting other defensive activities. Proteins that bind to LPS have been characterised in a number of different arthropods (Table 1). These LPSbinding proteins differ greatly in their chemical properties and protein structure. In the horseshoe crab Tachypleus tHdentatus for example, they are highly diverse and some are antimicrobial peptides (for example, tachypleusin, polyphemusin) while others are serine proteinases (for example factor C) [29"]. T h e primary structures have been determined for a number of LPS-binding proteins, but to date no common structure for any LPS-binding protein has been found. Haemolin, a protein present in the blood of some insects and a member of the immunoglobulin superfamily [1] can bind to LPS and upon binding prevents aggregation of haemocytes and functions as an opsonising factor [1,30]. A hemolin homologue has also been found in a mollusc [31]. Marmaras and co-workers suggest that phenoloxidase is involved in crosslinking LPS to an LPS receptor on the haemocyte surface, but this receptor has not yet been characterised [32]. Since LPS can adhere with different affinities to many proteins and surfaces, some information about the affinity between LPS and its protein ligand [33] is of value for interpreting the in vivo function of the LPS-binding proteins. A protein that binds

25

Gram-negative bacteria (GNBP) has been found in the silkworm B. mori. It has a mass of 50kDa and similarity to bacterial 13-1,3-glucanases. If comparisons are made with the active-site region of glucanases, the similarity is as high as 63%, which suggests that this protein may perhaps also be active in binding 13-1,3-glucans from fungal cells [34"]. Such proteins have been isolated from a few arthropods (Table 1) and have been cloned from the horseshoe crab and the freshwater crayfish. The 13-1,3-glucan-binding protein (BGBP) from horseshoe crab (Factor G) consists of two non-covalently linked subunits; the larger subunit contains the glucanase domain, as in GNBP, and the smaller subunit contains the serine proteinase domain [35]. After binding to glucans, factor G converts the proclotting enzyme to an active enzyme which then cleaves coagulogen to coagulin, resulting in the formation of a clot. In the freshwater crayfish P. leniusculus, the glucan-binding protein is a monomer of 100 kDa and, although it has some limited glucanase-like motifs, the sequence is completely different from that of Factor G and silkworm GNBP [36]. Although these proteins have some glucanase motifs none of them can function as a glucanase enzyme. A plausible hypothesis is that the BGBPs developed from a primitive glucanase and then evolved into proteins without glucanase activity, but which instead only bind the glucan and operate as elicitors of defence responses. An interesting finding was that the crayfish BGBP seems to be identical to the shrimp protein LP 1, which is involved in lipid transport to the

Table 1 Invertebrate pattern recognition molecules. Molecule

Size

Properties

References

100 kDa monomer

Binds to haemocytes; opsonin; no known enzymatic activities Mediates ~-l,3-glucan-induced clotting through limited proteolysis

[36]

Its cloning from a sea urchin indicates the presence of a mannanbinding protein in these animals

[40 °]

Opsonin

[49]

Opsonin

[49]

Prevents haemocyte aggregation; stimulates phagocytosis Binds to Gram-negative bacteria Mediates LPS removal from blood Mediates LPS-induced clotting through limited proteolysis

[1]

~-l,3-glucan binding Crustacean BGBP Horseshoe crab factor G

Dimer of 37 and 72 kDa

[35]

Mannan-binding MASP-related proteinase

LPS-binding

Periplaneta LBP Periplaneta lectin hemolin Silk worm GNBP lipophorin horseshoe crab factor C

450 kDa (28 kDa subunits) 190 kDa (32, 30 kDa subunits) 48 kDa 50 kDa - 6 0 0 - ? 0 0 kDa 123 kDa

[34"] [50] [29"]

The table only includes proteins for which the primary structure and some data regarding their function in immune reactions are available. LBP, lipopolysaccharide-binding protein; MASP, mannan-binding protein-associated serine protease.

26

Innateimmunity

ovary, showing that reproduction and immunity might be linked in crustaceans. Upon treatment with 13-glucans, BGBP binds to the haemocyte surface, possibly through an R G D motif (Arg-Gly-Asp) present in the molecule which indicates binding to integrin-like proteins. Both an 13-integrin [37] and another BGBP receptor have been characterised in crayfish [5]. One important function of BGBP is to act as an opsonizing protein in crayfish. A proteinase zymogen in B. mori can be activated by a glucan and in this respect resembles Factor G of the horseshoe crab [38]. So far only one invertebrate peptidoglycan-binding protein has been isolated. This was isolated from B. mori and has a low molecular weight, but its structure is yet unknown [39"]. Mannan-binding proteins may also be present in invertebrate animals, at least in the deuterostomes, as a mannan-binding protein-associated serine protease was recently cloned from the ascidian H. roretzi [40"]. A wealth of papers has been published regarding the biological function of proPO components and the system has been implicated in several defence reactions in arthropods and in many other invertebrate groups. But only in a few cases have components of the proPO-AS been shown to act directly as immune factors. In M. sexta it has been proposed that an interleukin-l-like molecule is associated with proPO and phenoloxidase and that tlais complex is a carrier for interleukin-1 in the blood [41]. A cell-adhesion protein present in the haemocytes of crayfish seems to be directly related to the proPO system as it gains its biological activity upon activation of the proPO-AS, although the exact mechanism of activation is unknown. This ceil-adhesion molecule, with a mass of 76kDa, is a peroxidase and accordingly has been named peroxinectin [42], but the peroxidase activity is not necessary for the cell adhesion function of the protein. A 170kDa extracellular matrix protein of D. melanogaster also has a functional peroxidase domain [43]. Peroxinectin also acts as an opsonin in crayfish. T h e finding that a peroxidase is a cell-adhesion protein was of interest since this enzyme is present in most living organisms and in higher animals is important as a producer of antibacterial factors. Recently, Johansson et al. [44"'] clearly demonstrated that myeloperoxidase also functioned as a cell-adhesion molecule, a finding which may have implications for the understanding of the role of peroxidases in immunity.

Regulation of the proPO-AS T h e proPO-AS has to be controlled and regulated to avoid the deleterious effects of active components of the system, and in particular active phenoloxidase which can produce highly toxic intermediates. Most proPOs are present as zymogens and are located in vesicles within the

blood cells. Their release from the blood cells has been shown in crayfish to involve the engagement of specific receptors followed by a regulated exocytosis of the proPO components from the cells [5]. In insects, low molecular weight proteinase inhibitors efficiently inhibit the activation of the proPO-AS. In the locust Locusta migratot~a, three such inhibitors have been characterised, and two (LICM I and LCIM II) have been cloned [45]. These low molecular weight inhibitors are derived from a two-domain precursor protein encoded by a single messenger RNA, indicating that the polypeptide is processed after translation into single-domain inhibitors. In the freshwater crayfish, a large molecular weight proteinase inhibitor, pacifastin, with a mass of 155kDa, was recently characterised in detail [46"]. Pacifastin is an inhibitor of the proPO-AS and inhibits crayfish proPO-AE in a 1:1 molar ratio. It is encoded in two mRNAs, one coding for a light chain consisting of nine proteinase inhibitor domain (pacifastin light chain domain, P L D ) and one coding for a heavy chain with three transferrin domains, two of which can bind iron. T h e P L D s are homologous to the LICM I and II of the locust, but also have sequence similarity to other proteins with cysteine-rich stretches, such as yon Willebrand factor, the Jagged protein and a cloned sequence of unknown function from Caenorhabditis elegans [46"]. It is also likely that pacifastin may have antimicrobial activity as do other transferrins. T h e pacifastin and the locust inhibitors are suggested to form a new class of proteinase inhibitors. In an insect, M. sexta, twelve serpins have been shown to be produced by alternative splicing from a single gene; they have a variable region in the carboxy terminal 39-46 residue. One of these serpins was found to be an efficient inhibitor of the proPO-AS [47"]. Another important way of controlling the activity of active phenoloxidase is through direct inhibition of the enzyme itself. In the house fly Musca domestica, a dopa-containing peptide with a mass of 4.2kDa was found to be a competitive inhibitor of the fly phenoloxidase [48].

Conclusions Several components of the so-called proPO-AS have been structurally determined during the past few years and new and important functions in defence have been attributed to some of them. But much still needs to be learnt about both the biochemical mechanism of activation of constituent factors of the system and their function in immune defence. It is tempting to speculate that there may be a link between the proPO-AS and the induction of antimicrobial syntheses, since a serine proteinase associated with the proPO-AS is likely to induce activation of the Toll-signalling pathway in Drosophila, leading to synthesis of antimicrobial peptides.

The prophenoloxidase-activating system in invertebrate immunity SSderh~ill and Cerenius

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •-

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2. An

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27

containing protein homologous to arthropod hemocyanin. Proc Nat/Acad Sci USA 1995, 92:7774-7778. 19. •

Burmester T, Scheller K: Common origin of arthropod tyrosinase, arthropod hemocyanin, insect hexamerin, and dipteran arylphorin receptor. J Mol Evol 1996, 42:713-728. The phylogeny of tyrosinases, prophenoloxidases, hemocyanin, hexamerins and related proteins are discussed in this paper and in [20"]. 20. •

Durstewitz G, Terwilliger NB: cDNA cloning of a developmentally regulated hemocyanin subunit in the crustacean Cancer magister and phylogenetic analysis of the hemocyanin gone family. Mo/ Biol Evo/1997, 14:266-276. The phylogeny of tyrosinases, prophenoloxidases, hemocyanin, hexamerins and related proteins are discussed in this paper and in [19"]. 21.

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6. •

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12.

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13.

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Aspan A, Huang TS, Cerenius L, SSderh~ll K: cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation. Proc Nat/Acad Sci USA 1995, 92:939-943.

16.

Fujimoto K, Okino N, Kawabata S-i, Iwanaga S, Ohnishi E: Nucleotide sequence of the eDNA encoding the proenzyme of phenol oxidase A 1 of Drosophila melanogaster. Proc Nat/Acad Sci USA 1995, 92:7769-7773.

1 7.

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Courtney Smith L: Sea urchin coelomocytes specifically express a C3 complement component and a complement receptor or regulatory protein. Dev Comp/mmunol 1997, 21:143. Only an abstract but the complete sequence was presented at the ISDCI meeting in July 1997 in Virginia. The work is important for our understanding of the phylogeny of the complement system. 29. •

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35.

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protein of Drosophila development EMBO J 1994, 13:34383447. 44. •.

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39. •

YoshidaH, Kinoshita K, Ashida M: Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori. J Biol Chem 1996, 271:13854-13860. The description of the first isolation of a peptidoglycan-binding protein from an invertebrate.

LiangZ, Sottrup-Jensen L, Aspen A, Hall M, S6derh~ll K: Pecifastin, a novel 155-kDa heterodimeric proteinase inhibitor containing a unique transferrin chain. Proc Natl Acad Sci USA 1997, 94:6882-6687. Pacifastin is a large proteinase inhibitor in crayfish blood which efficiently inhibits the prophenoloxidase activation through direct inhibition of the activating enzyme. This protein has a unique composition, in that it consists of a light chain with nine proteinase inhibitor domains covalently linked to a heavy chain with three transferrin domains.

40. •

47. •

Ji X, Azumi K, Sasaki M, Nonaka M: Ancient origin of the complement lectin pathway revealed by molecular cloning of mannen binding protein-associated serine protease from a urochordate, the Japanese ascidian, Halocynthia roretzi. Proc Nat/Acad Sci USA 1997, 94: 6340-6345. A report of the presence of a MASP-related proteinase in a deuterostome, which may indicate that a mannan-binding protein may also be present in this group of animals. 41.

42.

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