BIOLOGICAL OVERVIEW

In arthropods, the melanization reaction is associated with multiple host defense mechanisms leading to the sequestration and killing of invading microorganisms. Arthropod melanization is controlled by a cascade of serine proteases that ultimately activates the enzyme prophenoloxidase (PPO: in Drosophila PPO is called Black cells), which, in turn, catalyzes the synthesis of melanin. A Drosophila serine protease inhibitor protein, Serpin-27A regulates the melanization cascade through the specific inhibition of the terminal protease (prophenoloxidase-activating enzyme). Serpin-27A is required to restrict the phenoloxidase activity to the site of injury or infection, preventing the insect from excessive melanization (De Gregorio, 2002b). Evidence is presented for the coordination of hemolymph-borne melanization with activation of the Toll pathway in the Drosophila host defence (De Gregorio, 2002a; Ligoxygakis, 2002). The melanization reaction requires Toll pathway activation and depends on the removal of the Drosophila serine protease inhibitor Serpin27A. Flies deficient for this serpin exhibit spontaneous melanization in larvae and adults. Microbial challenge induces its removal from the hemolymph through Toll-dependent transcription of an acute phase immune reaction component (Ligoxygakis, 2002).

Insects have a sensitive mechanism for identifying pathogens and an array of strategies for defending themselves against microbial attack. To combat infection, the fruit fly relies on both constitutive and inducible defense mechanisms (Tzou, 2002). However, the first line of defense that prevents microbial invasion into the hemocoel is structural. It is comprised of the external cuticle, the gut peritrophic matrix, and the tracheal lining. When pathogens breach these barriers, they activate a wide range of inducible reactions. Perforation of the cuticle by injury or by microbial infection rapidly activates proteolytic cascades that lead to blood coagulation and melanization. Upon subsequent microbial or parasitic infection, a cellular immune response, mediated by different hemocyte types, is mounted and participates in pathogen/parasite clearance by phagocytosis or encapsulation. During systemic infection, a large set of inducible effector molecules, such as antimicrobial peptides (AMP), stress-responsive proteins, and other factors required for opsonization and iron sequestration, are secreted into the hemolymph (Tzou, 2002; De Gregorio, 2002b).

Apart from the regulation of AMP gene expression, very little is known about the other defense mechanisms that combat infection in Drosophila. The most dramatic and immediate immune response in Drosophila is the melanization reaction observed at the site of cuticular injury or on the surface of pathogens, parasites, and parasitoids invading the hemocoel. The blackening of the wounded area of the cuticle or the surface of invading organisms results from the de novo synthesis and deposition of melanin. In arthropods, melanization plays an important role in defense reactions, such as wound healing, encapsulation, sequestration of microbes, and the production of toxic intermediates, that are speculated to kill invading microorganisms (Ashida, 1998; Söderhäll, 1998). So far, no regulators of the melanization cascade have been identified at the molecular level in the fruit fly (De Gregorio, 2002b).

Although there is a paucity of information concerning the Drosophila melanization reaction, this response has been investigated in more detail in other arthropods (Söderhäll, 1998). It requires the activation of phenoloxidase (PO) (EC 1.14.18.1), an enzyme that catalyses the oxidation of mono- and diphenols to orthoquinones, which are then polymerized nonenzymatically to melanin. Insect phenoloxidase has been found to exist in the hemolymph plasma and the cuticle as an inactive form, called prophenoloxidase (PPO). Enzymatically inactive PPO is cleaved into active PO by a serine protease known as prophenoloxidase-activating enzyme (PPAE). PPAE also exists as an inactive zymogen that is activated through a stepwise process involving other serine proteases. PPAEs have been isolated from different insect species and other arthropods (Jiang, 1998; Lee, 1998; Satoh, 1999; Wang, 2001). Several studies indicate that the melanization cascade is triggered by injury or by recognition of microbial cell wall components, such as peptidoglycan, ß-1,3 glucan, and lipopolysaccharide (LPS), through pattern recognition proteins (Yoshida, 1996; Yu, 1999; Ma, 2000; Ochiai, 1999). Consequently, the PPO cascade is an efficient nonself-recognition system in invertebrates, and its immediate activation suggests that it may regulate aspects of the immune response other than PPO activation (De Gregorio, 2002b).

Although vertebrates do not possess an equivalent of a PPO system, the PPO cascade in insects is reminiscent of the blood clotting reactions in vertebrates. Both reactions involve serine protease cascades that must be tightly regulated to avoid a systemic activation, which is often fatal. One class of serine protease regulators ubiquitous among plants, animals, and viruses is serpins (Irving, 2000). Serpins compose a superfamily of proteins that fold into a conserved structure and employ a suicide substrate-like inhibitory mechanism (Ye, 2001). In the carboxyl terminal, they have a 30–40-residue-long 'reactive center loop' (RCL), which is exposed at the surface of protein that binds and covalently links to the active site of the target serine protease (Lawrence, 2000; Potempa, 1994). After cleavage of the RCL region, the P1 residue of the serpin forms a covalent acyl bond with the active site serine residue of the target protease (Cohen, 1977; Potempa, 1994). Like humans, fruit flies possess a high number of genes encoding serpins, which reflects the high number of serine protease genes found in this insect. However, the serpin gene family is poorly studied in Drosophila. To date, a mutation in only one serpin gene, spn43Ac, has been studied in relation to the necrotic phenotype (Levashina, 1999). The loss-of-function mutation in spn43Ac leads to constitutive activation of the Toll-mediated immune response, indicating that Spn43Ac inhibits a serine protease that functions upstream of the Toll ligand Spaetzle (De Gregorio, 2002b).

The identification of genes that regulate the melanization cascade in Drosophila has been complicated by the high number of serpin and serine protease candidate genes in the fly genome. Recently, a DNA microarray analysis helped in the selection of 5 serpin genes among the 30 encoded by the Drosophila genome that, like spn43Ac, are significantly upregulated in response to infection and might control one of the serine protease cascades associated to fly immune reactions (De Gregorio, 2001). Biochemical and genetic characterization of one of them, serpin-27A (spn27A), demonstrates that the protein encoded by this gene regulates the melanization cascade through the specific inhibition of PPO processing by the terminal serine protease PPAE (De Gregorio, 2002b).

Spn27A, like the antimicrobial peptides, is synthesized in the fat body tissue and secreted in the hemolymph. Biochemical assays show that recombinant Spn27A inhibits the activation of PPO in different insect species including Drosophila, pointing to a remarkable conservation of the melanization mechanisms. This finding is consistent with the sequence homology of PPO genes from the same species and allowed for an investigation of the role of Spn27A in heterologous systems for which the melanization cascade is better characterized than in Drosophila. The similarities between the PPO cleavage site and the reactive center loop of Spn27A prompted a test to see whether prophenoloxidase-activating enzyme PPAE was the target protease of Spn27A. Indeed recombinant Spn27A, but not a recombinant variant mutated in the reactive center loop, inhibits the PPAE purified from H. diomphalia. In agreement with the current models of serpin-serine protease interaction, the inhibition occurs through the formation of a specific covalent link between the two proteins (De Gregorio, 2002b).

These findings strongly suggest that, in Drosophila, the role of Spn27A is to modulate PPO processing through the inhibition of a still-unidentified PPAE. The role of Spn27A in melanization is supported by genetic analysis. spn27A¹ mutant flies frequently exhibit melanotic tumors and darkening of the cuticle, which may be the consequence of natural injury during the fly lifecycle. The most dramatic phenotype of spn27A¹ is, however, the excessive melanization reaction observed after injury or wasp infection. These observations indicate that the role of Spn27A is to limit the PPO cascade at the site of injury to prevent systemic melanization. The acute phase expression profile of spn27A in response to septic injury is probably critical for a tight spatial and temporal regulation of PPO activity (De Gregorio, 2002b).

The observation that some of the spn27A-deficient mutants do not present any form of constitutive melanization indicates the existence of multiple levels of regulation of the melanization reaction. An Spn27A-independant mechanism may limit the activation of PPO in the absence of stimulus. Although this has not been firmly demonstrated in Drosophila, this step is probably regulated by the availability of PPO in the hemolymph. At least in Drosophila, hemolymphatic PPO is localized in a hemocyte cell type called the crystal cell, which belongs to the oenocytoid lineage of hemocytes, known for their capacity to synthesize PPO. Crystal cells can be freely circulating in the hemolymph or sessile. Sessile crystal cells have been shown to be grouped in densely packed clusters along with plasmatocyte type hemocytes, in direct contact with the cuticular epithelial layer (Lanot, 2000). In response to different stimuli, like integumental injury or microbial invasion, crystal cells rupture and release their PPO content into the hemolymph plasma, especially around the area where stimulation occurs. The PPO may then be subjected to cleavage by PPAE in a process tightly regulated by Spn27A (De Gregorio, 2002b).

The melanization reaction is considered as an important facet of the insect host defense. Melanotic encapsulation can prevent completion of the malaria parasite lifecycle in Anopheles gambiae (Collins, 1986), while, in Drosophila, it plays an important role against infection by parasitoid wasps (Vass, 2000). However, little is known about the exact role of melanization during Drosophila immune response. The Black cells (Bc) mutation affects crystal cells and blocks the melanization reaction in the hemolymph but does not alter the inducibility of anti-microbial peptide genes. Interestingly, this mutation induces a higher susceptibility to microbial infection when combined with mutations affecting the Toll and Imd pathways. An experiment, showing that both Bc and spn27A¹ mutants are more susceptible to infection by the entomopathogenic fungus B. bassiana, indicates that a tight control of melanization is required for a proper antifungal response. The analysis of Bc and spn27A¹ mutants clearly shows that the terminal components of the melanization reaction (PPO, PPAE, and Spn27A) are not involved in the regulation of the antimicrobial peptide response. Hence, the PPO cascade and anti-microbial peptides provide two uncoupled defense systems that cooperate to fight microbial infection (De Gregorio, 2002b).

In mammals, a variety of proteolytic cascades, including blood coagulation, fibrinolysis, inflammation, and complement activation are regulated by serpins. In addition, serpins are also involved in various kinds of physiological processes, such as angiogenesis, apoptosis, neoplasia, and viral pathogenesis (Silverman, 2001). Several serpin genes have been inactivated by targeted mutation in the mouse. Some of these mutations have failed to reveal an overt phenotype, whereas others show various physiological and developmental alterations (Silverman, 2001). Spn27A and Spn43Ac (Necrotic) are two serpins characterized by genetic studies in Drosophila (Green, 2000; Levashina, 1999; De Gregorio, 2002b). Interestingly the phenotype induced by the deletion of these serpins shows similarities and differences. Both spn27A¹ and necrotic mutations result in a similar pleiotropic phenotype: female sterility, low viability at the pupal stage, and ectopic melanization in unchallenged animals. In addition, these two genes are both induced upon infection and are mainly regulated by the Toll pathway. Mutation in spn43Ac, however, induces a constitutive activation of the Toll pathway (Levashina, 1999), whereas mutation in spn27A causes an uncontrolled melanization reaction. Biochemical study clearly indicates a difference in target specificity between these two serpins: recombinant Spn43Ac is unable to inhibit the PPO cascade. It would be interesting to know whether the phenotype of each serpin mutant is mediated by more than one target protease. The observation that Bc is epistatic to (functions downstream of) spn27A indicates that the pleiotropic phenotype of spn27A¹ mutation can be explained by the multiple functions of melanization reaction in Drosophila. The role of Spn27A in early embryogenesis, which is not Bc dependent, remains to be investigated (De Gregorio, 2002b).

Since the Drosophila genome encodes five serine proteases for every serpin, it is very likely that serpins can inhibit multiple target proteases. Microarray analysis has shown that about 19 serine proteases are upregulated upon infection in Drosophila (De Gregorio, 2001). Among them, new targets of Spn27A and Spn43Ac activities might be identified (De Gregorio, 2002b).

The discovery of a regulator of the PPO pathway in Drosophila opens the way for the molecular characterization of the insect melanization cascade that plays a central role in host defense. The dramatic phenotype of spn27A¹ mutant flies underlines the critical role of serpin molecules in the tight regulation of proteolytic cascades activating host defense reactions. Furthermore, the high number of serine protease and serpin genes encoded in the fruit fly genome suggests that Drosophila is a remarkable model to understand the role of proteolytic cascades in the integrated host defense (De Gregorio, 2002b).

GENE STRUCTURE

cDNA clone length - 1567

Bases in 5' UTR - 169

Exons - 1

Bases in 3' UTR - 54

PROTEIN STRUCTURE

Amino Acids - 447

Structural Domains

Serpins belong to a family of proteins, which preferentially inhibit serine proteinases (Silverman, 2001). Each serpin interacts with its target enzyme via an exposed reactive center loop. This loop places the reactive center in an accessible position for interaction with its target, allowing the formation of a stable complex between enzyme and inhibitor. The amino acid sequence and conformation of the loop largely dominate the selectivity of inhibition. More specifically, inhibition relies primarily on the residues P1-P1' of the reactive center, which provide a cleavage site for the target protease. Recently, an immune inducible serpin whose P1-P1' reactive site sequence is reminiscent of the activation cleavage site in insect prophenoloxidases was cloned from the fall webworm Hyphantria cunea (Park, 2000). A Manduca sexta serpin with similar P1-P1' sequence was subsequently shown to inhibit PPAEs in vitro (Zhu, 2001). A gene encoding a homologous protein, designated Spn27A (CG11331), has been identified in the Drosophila genome by BLAST search. The three serpins have P1-P1' reactive sites that resemble the P1-P1' cleavage sites of their respective PPOs, suggesting that the PPAE of each species could be the target of these serpins. It is relevant to note here that the P4 (hydrophobic) and P2 (Asn) residues are also strikingly similar among these PPOs and the corresponding serpins (Ligoxygakis, 2002).

date revised: 20 January 2002

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