InteractiveFly: GeneBrief

serpin-27A : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References


Gene name - serpin-27A

Synonyms - Spn27A

Cytological map position - 36F5--6

Function - protease inhibitor

Keywords - immune response, melanization

Symbol - spn27A

FlyBase ID: FBgn0028990

Genetic map position -

Classification - serpin

Cellular location - secreted



NCBI links: Precomputed BLAST | Entrez Gene
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).


REGULATION

Characterization of functional activity

Interestingly, close examination of the deduced amino acids revealed that Spn27A contains a C-terminal region that is homologous to the region around the conserved cleavage site of insect prophenoloxidases (PPO). Serine proteases have been shown to cleave the propeptide of PPO between Arg and Phe within the NRFG motif to generate active PO for melanin synthesis. Furthermore, the cleavage sites of PPO from most insects and Spn27A are very similar (NRFG versus NKFG, respectively). It is hypothesized that both these proteins serve as substrates for a common serine protease. To investigate the possible involvement of Spn27A in regulating PPO activation, a PPO activation assay was performed using Drosophila pupal homogenate in the presence of recombinant Spn27A (rSpn27A). Time-course analysis shows that PO activity in the pupal homogenate increases over time, reaching a plateau after 30 min. The addition of rSpn27A to the pupal homogenates completely blocks PO activity, regardless of incubation time. Given that insect PPO activation cascades are very similar, it is postulated that the Drosophila rSpn27A could perhaps affect the activation of PPO in various insect systems. The hemolymph plasma from larvae of two lepidopterans (Bombyx mori and Galleria mellonella) were used and Spn27A was shown to be able to significantly block PPO activation in both species (De Gregorio, 2002b).

Even though there is a striking amino acid sequence similarity between Spn27A and PPOs in and around the cleavage site, the cleavage site itself is not identical. Specifically, Spn27A has a Lys at the cleavage site, whereas insect PPOs have an Arg at this position. Generally, trypsin-like serine proteases are known to cleave their substrates after a basic amino acid, such as Arg or Lys. The putative P1 site of rSpn27A was mutated from Lys to Ala (rSpn27AK406A). Following this point mutation, rSpn27AK406A completely lost its PO activation inhibitory effect. In a second set of experiments, to mimic the PPO cleavage site, the lysine of the putative P1 site was mutated to arginine (rSpn27AK406R). Interestingly, rSpn27AK406R inhibits PPO activation with a lower efficiency than rSpn27A wild-type in Drosophila pupal homogenate (De Gregorio, 2002b).

Serpins bind their target proteases through an RCL domain that mimics their substrate. Therefore the sequence similarity between the Spn27A RCL and the insect PPO cleavage site suggests that the target of Spn27A might be prophenoloxidase-activating enzyme (PPAE). Highly purified prophenoloxidase-activating enzyme from the coleopteran insect Holotrichia diomphalia (HdPPAE) can convert PPO to PO (Lee, 1998). Because the Drosophila PPAE has not yet been identified, highly purified PPAE from H. diomphalia was employed. To investigate whether HdPPAE is the target protease of Spn27A, a protease activity assay was conducted in the presence of rSpn27A and its mutants using chromogenic substrates. When HdPPAE and rSpn27A were incubated in a 1:1 molar ratio, the HdPPAE enzymatic activity was greatly inhibited. A similar result was obtained using the rSpn27AK406R mutant. However, the rSpn27AK406A mutant was unable to inhibit HdPPAE enzymatic activity (De Gregorio, 2002b).

To further demonstrate that Spn27A is a natural inhibitor of PPAE, the final step of the PPO activation system (HdPPO plus active HdPPAE with cofactors) was reconstituted in the presence of rSpn27A. The result showed that rSpn27A and the rSpn27AK406R mutant could almost completely inhibit de novo-generated PO activity, while the rSpn27AK406A mutant had no effect (De Gregorio, 2002b).

It is generally accepted that de novo-generated PO activity is due to limited proteolysis of PPO by the activating enzyme (PPAE). To demonstrate that the cleavage of PPO by HdPPAE is blocked by rSpn27A the effect of rSpn27A variants on the conversion of PPO to PO in the reconstituted system was checked. According to the PO activity assay it was found that rSpn27A and the rSpn27AK406R completely inhibit the cleavage of PPO, while rSpn27AK406A mutant has no effect. In summary, the biochemical analysis strongly suggests that Spn27A functions in Drosophila as a negative regulator of PPO activation through the inhibition of PPO-activating enzyme (PPAE) (De Gregorio, 2002b).

Expression of Spn27A is responsive to bacterial challenge

Genetic data suggest a role of Spn27A in blood-borne melanization. An antibody was developed against the C-terminal part of the protein. With this antibody, a marked protein band of ~50 kDa was detected in the hemolymph of wild-type adults. This molecular weight corresponds to that of the amino acid sequence deduced of the Spn27A gene. This 50 kDa band is absent from the hemolymph of the Spn27Aex32 mutant flies. The band corresponding to Spn27A disappears from the hemolymph 3 h after bacterial challenge and reappears around 17 h post challenge. This disappearance is interpreted as resulting from the interaction of the serpin with its cognate protease, followed by removal or degradation of the complex. Moreover, this disappearance is infection dependent since injury with a sterile needle did not have a similar effect (Ligoxygakis, 2002b).

To determine whether the time window in which Spn27A is absent from the hemolymph correlates with increasing PO activity after infection, PO activity was examined in the hemolymph of immune-challenged wild-type flies. A rapid increase in PO activity was observed that reached its maximum at 3 h post challenge. From then on, PO activity decreased significantly following the time of serpin re-emergence in the hemolymph (17 h post challenge) and reaching basal levels 24 h following immune challenge. In parallel to each PO measurement, the hemolymph of the same infected flies was examined for the presence of the serpin. In every case, the increase in PO activity correlated with the absence of Spn27A. Conversely, reduction in PO activity coincided with Spn27A reappearance (Ligoxygakis, 2002b).

In non-infected flies deficient for Spn27A, the same elevated PO levels were observed as in wild-type flies subjected to bacterial challenge. This was even observed in some of the Spn27A mutant flies that did not show a spontaneous melanization phenotype. Finally, overexpression of Spn27A through a UAS-Spn27A construct, using the ubiquitous driver daGA4, or in the fat body of transgenic flies using yolkGAL4 inhibited the increase in PO activity normally following bacterial challenge (Ligoxygakis, 2002b).

Regulation of the melanization reaction and its activation through the Toll pathway

Depletion of Serpin-27A (Spn27A) from the hemolymph following immune challenge is best explained by assuming that it binds to a cognate protease and that the resulting complex is either removed from circulation or degraded. Two intracellular signaling pathways control the expression of challenge-induced genes in the fat body, the predominant immuno-responsive tissue of Drosophila. DNA microarray data indicate that the infection-induced expression of several putative prophenoloxidase-activating enzyme (PPAE) genes in Drosophila is under the control of the Toll pathway (De Gregorio, 2002a). Moreover, Spn27A transcription is also regulated in an immune-dependent manner (De Gregorio, 2001). It was of interest to examine whether the melanization observed in the Spn27Aex32 mutants could be correlated to the well-defined Toll-mediated host response in Drosophila. In Dif or spaetzle (spz) mutants, in which the Toll pathway is blocked, the serpin is not removed from the circulating hemolymph. Interestingly, the serpin is removed in kenny (key) mutants that block the Imd pathway, which is not involved in the control of PPAE gene expression (De Gregorio, 2002a). A testable prediction that can be derived from these results is that in a Toll pathway mutant background PO enzymatic activity following bacterial challenge should be at very low levels. It as indeed observed that Dif- and spz-infected flies have a PO activity comparable to the basal level of non-challenged wild-type flies in contrast to key-infected flies, which show normal PO levels. The data furthermore imply that the protease (or the factor that triggers its activation), which removes Spn27A, may not be present as a zymogen activated by infection, but may need de novo protein synthesis dependent on Toll signal transduction. This was confirmed by infecting wild-type flies with bacteria in the presence of an inhibitor of translation (cycloheximide). Protein synthesis following bacterial infection was examined by mass spectrometry; none of the induced antimicrobial peptides were synthesized in the experimental conditions indicating that protein synthesis was efficiently blocked. Importantly, it was noted that in these flies during the same infection procedures the serpin was not removed from circulation. Taken together, these results on the requirement of Toll signaling pathway and protein synthesis for serpin removal show that the protease(s) removing Spn27A from circulation is synthesized de novo in response to Toll signaling. Alternatively, a component that activates it is dependent on Toll signaling-driven de novo protein synthesis (Ligoxygakis, 2002b).

The question emerging from these data is whether the above-mentioned protease or activating component is directly involved in activating melanization or whether it is required solely for Spn27A removal. In other words, does Toll signaling activate melanization directly? To address this issue, an spn27A; spz double mutant was generated. In larvae and flies homozygous for both mutations, constitutive melanization could still be observed. This means that the components of the PO cascade are already in the hemolymph, and Toll pathway activation tips the balance from an inhibitory state to protease triggering by removal of Spn27A (Ligoxygakis, 2002b).

It is concluded that Toll pathway mutants do not exhibit PO induction following bacterial challenge. In wild-type flies, PO activity reaches its maximum 3 h post-infection and decreases significantly at 17 h following immune challenge. Most of the larvae and a significant number of adults deficient for the serpin Spn27A show spontaneous melanization and increased PO activity. The results further indicate that this circulating serpin disappears from the hemolymph 3 h following bacterial challenge and re-emerges at 17 h post-infection. This time window correlates with PO activation kinetics. Moreover, overexpression of Spn27A in fat body cells of adults prevents PO activation by immune challenge. Serpin depletion (as PO activation) relies on the Toll pathway, since in mutants of this pathway Spn27A is not removed from the hemolymph. Finally, it has been shown that serpin depletion requires de novo protein synthesis, since infection in the presence of an inhibitor of translation (cycloheximide) does not result in its removal. Among the serpin-deficient flies, only those with melanotic tumors showed a constitutive expression of the antimicrobial peptide genes Drosomycin and diptericin. There is as yet no clear explanation for their expression, which is dependent on Toll and Imd respectively. Expression could be an indirect effect of the presence of the melanotic capsules in the body cavity. It is relevant to note here that other mutations, which are not specifically linked to the immune response but result in melanotic tumors, activate antimicrobial peptide gene expression in the absence of an overt microbial infection. An alternative model could be that Spn27A blocks both the Imd and Toll pathways and these pathways would be constitutively activated in flies deficient for the serpin. Although equally interesting, this possibility does not seem to be the case. If Spn27A negatively controlled these pathways, then all the serpin-deficient flies, and not only those with melanotic tumors, would exhibit expression of the peptides. Conversely, overexpression of the serpin would inhibit antimicrobial peptide induction following microbial challenge. Finally, bacterial infection of flies overexpressing the serpin (through a UAS-Spn27A transgene) results in expression of peptides at wild-type levels (Ligoxygakis, 2002b).

A surprising implication of these results is that at least one component of the melanization cascade is controlled by the Toll pathway and has to be synthesized de novo after infection. Two pieces of evidence support this hypothesis: (1) in a loss of function allele of Dif (Dif1), which encodes a protein able to translocate to the nucleus but unable to bind DNA, depletion of serpin27A and concomitant PO activation does not take place after bacterial challenge, and (2) in the presence of protein synthesis inhibitors, Spn27A is not removed. Since the sequence found in the hinge region of serpin27A suggests that it is an active serine protease inhibitor, it is proposed that Spn27A is removed by its target protease, forming a covalent complex. Such complexes have a half-life of a few minutes and are rapidly cleared from the hemolymph, which could explain in a simple manner the observed depletion (Ligoxygakis, 2002b).

A model is presented to explain these data. In non-infection conditions, Spn27A inhibits PPAE and blocks melanization. Circulating pathogen recognition receptors sense the bacterial infection and signal to the Toll pathway. Intracellular transduction of the signal is mediated by the Rel transcription factor DIF, which initiates an acute phase transcription. This leads to de novo production of a further amount of PPAE, which induces PO cleavage and targets Spn27A for removal. Alternatively, Toll activation could lead to the production of serine protease homologs that are co-factors of PPAE, as demonstrated in a beetle (Kwon, 2000), or a modifying enzyme that triggers the cascade (like the Drosophila pipe gene). Reappearance of the serpin in the hemolymph should inhibit any further melanization-associated proteolytic action. The question whether this reappearance is a consequence of transcription was not addressed. Nevertheless, DNA microarray data show that the Spn27A transcription level is strongly elevated after septic injury (De Gregorio, 2001). Finally, the fact that in the spn27A; spz double mutant spontaneous melanization is not suppressed evokes the possibility that the Toll pathway is activating a protease dedicated to the removal of Spn27A without taking part in the actual melanization cascade. Given that Spn27A has been shown to inhibit biochemically a PPAE of another insect (De Gregorio, 2002b) and that in turn Drosophila PPAEs are rapidly upregulated following infection (De Gregorio, 2001, 2002a; Irving, 2001), the simpler scenario is favored. Analysis of an XP element insertion in a putative PPAE gene (N. Pelte and J.-M. Reichhart, unpublished data reported in Ligoxygakis, 2002) will probably help to clarify the matter (Ligoxygakis, 2002b).

Two proteases defining a melanization cascade in the immune system of Drosophila

The melanization reaction is used as an immune mechanism in arthropods to encapsulate and kill microbial pathogens. In Drosophila, the serpin Spn27A regulates melanization apparently by inhibiting the protease that activates phenoloxidase, the key enzyme in melanin synthesis. This study describes the genetic characterization of two immune inducible serine proteases, MP1 and MP2, which act in a melanization cascade regulated by Spn27A. MP1 is required to activate melanization in response to both bacterial and fungal infection, whereas MP2 is mainly involved during fungal infection. Pathogenic bacteria and fungi may therefore trigger two different melanization cascades that use MP1 as a common downstream protease to activate phenoloxidase. The melanization reaction activated by MP1 and MP2 plays an important role in augmenting the effectiveness of other immune reactions, thereby promoting resistance of Drosophila to microbial infection (Tang, 2006).

Melanization is a conserved host defense reaction in insects and other arthropods, such as in the mosquito, where it is a critical determinant of resistance to the malarial parasite. A recent key finding revealed that Spn27A, a serpin-type protease inhibitor, is a negative regulator of melanization in Drosophila. This study used suppression of the spn27A melanization phenotype as a genetic strategy to identify two Drosophila proteases, MP1 and MP2, having essential roles in activating melanization in response to microbial infection (Tang, 2006).

The data are consistent with MP2 acting genetically upstream of MP1 and thus with MP2 activating MP1 in a protease cascade leading to melanization. Interestingly, the application of evolutionary markers suggests that MP1 and MP2 belong to the primordial class of serine proteases that tend to function most downstream in a protease cascade, as in the case of Easter and its direct activator involved in activating Toll during development. However, it has not been demonstrated that MP2 directly activates MP1, since MP2 failed to cleave the zymogen form of MP1 when co-expressed in transfected Drosophila S2 cells, thereby suggesting that another protease acts in between MP2 and MP1. Since the protease down-stream of MP2 that acts either in parallel to or upstream of Spn27A, MP1 is a candidate to be PPAE, the terminal protease in the melanization cascade that cleaves prophenoloxidase (PPO) as well as the putative direct target of Spn27A. Cleavage of PPO by MP1 was not detected when assayed by co-expression in transfected S2 cells. However, biochemical studies in the beetle and tobacco hornworm have identified a non-enzymatic cofactor required for PPO cleavage by PPAE to generate PO activity in vitro (Kanost, 2004; Kown, 2000), which may indicate that such a cofactor is required to demonstrate PPO cleavage by MP1 (Tang, 2006).

The data suggest that another melanization cascade exists in Drosophila besides the one involving MP1 and MP2. In activating melanization and PO activity, MP1 is essential during both bacterial and fungal infection, whereas MP2 is essential during fungal infection and partially required during bacterial infection. Thus, another protease may function analogously to MP2 in activating MP1 during bacterial infection. The convergence of two different melanization cascades on MP1 is consistent with the idea that MP1 is the shared terminal protease of both cascades that activates phenoloxidase (Tang, 2006).

It is presumed that MP1 and MP2 activate melanization in the hemolymph. Since both MP1 and MP2 have an N-terminal signal sequence for secretion, they may be secreted by the fat body and/or blood cells into the hemolymph. This possibility is consistent with the detection of MP1 and MP2 in extracted hemolymph when epitope-tagged versions of the full-length proteases are overexpressed with act-Gal4 and with constitutive melanization induced by overexpression of preactivated MP1 using the c564-Gal4 driver, which is expressed in the fat body and hemocytes (Tang, 2006).

MP1 and MP2 define a protease cascade distinct from the one that may activate the Toll pathway; they activate melanization independently of this pathway. However, MP2 (but not MP1) is important for the induction of Drosomycin expression and for viability of Drosophila following natural fungal infection. One explanation is that MP2 activates two distinct pathways, one leading to melanization and the other leading to the induction of Drosomycin expression, and that this dual role is important for resistance to natural fungal infection. Preliminary experiments indicate that overexpression of preactivated MP2 does not induce Drosomycin expression in non-infected adult flies, unlike in the case of the Persephone protease (Ligoxygakis, 2002a), thereby suggesting that MP2 does not induce Drosomycin expression by directly activating the Toll pathway. Nonetheless, there may exist cross-talk between melanization and the Toll pathway mediated by MP2 involving an as yet undefined mechanism (Tang, 2006).

Surprisingly, the melanization reaction does not appear to be critical for survival of Drosophila after bacterial or fungal infection. However, it was observed that the inability to activate melanization is detrimental when flies are also defective in the Toll or the Imd pathway controlling antimicrobial peptide synthesis. Melanization is an immediate immune response that temporally precedes the induction of antimicrobial peptide synthesis, which requires gene transcription. Consequently, melanization may play a crucial role in weakening a microbial infection, thereby enhancing the effectiveness of subsequent immune reactions. Having a single switch, such as MP2, to activate a temporal sequence of immune reactions would therefore seem to be an advantageous mechanism for ensuring a potent defense against a microbial pathogen (Tang, 2006).

In conclusion, two serine proteases were identified, among the large set of serine proteases encoded in the Drosophila genome, as being essential components of a melanization cascade activated by microbial infection. A major goal in future studies will be to delineate the entire cascade from the pattern recognition receptor that triggers the cascade to the putative cofactor required for phenoloxidase activation (Tang, 2006).


DEVELOPMENTAL BIOLOGY

Oligonucleotide microarray analysis performed on Drosophila adult males indicates that spn27A is one of the five serpin-encoding genes induced by septic injury with a mixture of gram-positive and gram-negative bacteria and, like spn43Ac, is also induced after natural infection by the entomopathogenic fungus Beauveria bassiana (De Gregorio, 2001). The expression profile in response to septic injury shows that spn27A is an acute response gene with the highest expression level at 3 hr postinfection. Northern analysis of the spn27A expression profile after septic injury and B. bassiana infection confirms the microarray data. The comparison of the expression profiles in wild-type and mutants deficient for the Toll (spaetzle, spz), Imd (relish, rel), or both (rel,spz) pathways reveals that the expression of spn27A is mainly controlled by the Toll pathway (De Gregorio, 2002a; De Gregorio, 2002b).

To examine the temporal expression profile of spn27A during Drosophila development, RT-PCR analysis and Western blot analysis were performed. spn27A mRNA is expressed throughout all developmental stages and also in cultured Drosophila Schneider cells (S2). However, it was most weakly expressed in both adult males and females. The calculated molecular weight of Spn27A is 48.12 kDa. However, Spn27A antiserum detected two major bands around 65 kDa in samples corresponding to the various developmental stages. The Western blot analysis of spn27A-deficient flies with the same antiserum indicates that only the lower band corresponds to Spn27A protein. By mass number analysis with MALDI spectrometry, the molecular mass of purified recombinant Spn27A was determined to be 66.59 kDa. Such a large difference between the observed molecular weight and the calculated mass number is most likely due to posttranslational modifications, such as glycosylation, which is often observed in the serpin family (Potempa, 1994). Spn27A was also detected in the culture medium of Drosophila Schneider cells, suggesting that Spn27A is a secreted protein. N-terminal amino acid sequencing with purified recombinant Spn27A showed the cleavage site of the signal peptide to be located between Gly25 and Asn26 (De Gregorio, 2002b).


EFFECTS OF MUTATION

A genetic analysis was carried out to investigate the role of Spn27A in vivo. For this, an XP element inserted 200 bp upstream of the translational start site of the gene was mobilized. An imprecise excision was obtained which removed along with the XP, a DNA fragment of 1.4 kb in total, comprising 1.2 kb of the serpin open reading frame. This mutant (Spn27Aex32) is a protein null. To eliminate possible contributions from other mutations in the genetic background of the excision allele, this mutation was combined with a chromosomal deletion, Df(2L)6374, which uncovers the region to which Spn27A maps. Larvae and adults homozygous or hemizygous for the Spn27Aex32 allele showed constitutive melanization (40% for larvae; 35% for adults). Melanization was particularly conspicuous around internal organs such as gut and fat body, but was never associated with barrier epithelia of the body wall. Statistics of repeated Cyo-GFP/Spn27Aex32 self crosses or Cyo-GFP/Spn27Aex32 with Cyo-GFP/Df(2L)6374 crosses revealed a high rate of lethality for Spn27Aex32 homozygous or hemizygous progeny. Most of the homozygous or hemizygous larvae (40%) that developed spontaneous melanization died in mid-pupal stages. To demonstrate that the above observations were due to the absence of the corresponding serpin, a wild-type copy of Spn27A was re-introduced into the mutant background using the UAS/GAL4 system. The UAS-Spn27A rescue construct was driven by daGAL4. Addition of the UAS-Spn27A transgene in an Spn27Aex32 genetic background suppressed lethality, since the homozygous progeny showed the expected Mendelian ratios. Moreover, in the presence of the UAS-Spn27A transgene, spontaneous melanization in these mutants was suppressed. Survival experiments were conducted with Spn27Aex32 homozygous flies infected with various classes of microorganisms. Survival of these mutants following bacterial challenge with Gram-negative or Gram-positive bacteria or fungal infection was comparable to wild-type flies. Nevertheless, the Spn27Aex32 mutants were particularly sensitive in terms of their melanization reaction to injury-coupled infection since this treatment resulted in the appearance of extended melanization around the wound, in stark contrast to wild-type flies. A clean injury with a sterile needle did not produce such an effect. Finally, expression of the antimicrobial peptide genes drosomycin and diptericin was analyzed. In the absence of any immune challenge, a constitutive expression of both peptide genes was observed in serpin-deficient flies. However, this expression was restricted to those individuals that developed spontaneous melanization (Ligoxygakis, 2002b).

In order to investigate the role of Spn27A in vivo, an spn27A-deficient mutant was constructed. The spn27A gene is nested within an intron of the cup gene. The cup gene is implicated in oogenesis. There are several reported alleles of cup that are all female sterile. A P element [EP(2)2349] located 900 bp upstream of the spn27A ORF and 110 bp from the last cup exon did not display any defect in oogenesis, suggesting that it does not interfere with cup expression. To generate a Drosophila strain deficient in spn27A, the P element EP(2)2349 was mobilized and lines were screened for the presence of a deletion uncovering the spn27A start codon. Out of 30 lines tested, one (spn27A1) was found bearing a deletion (E25), of 1175 bp downstream of the EP(2)2349 insertion site, which included the first 275 bp of the spn27A ORF. In agreement with the molecular characterization, it was found that spn27A1 larvae and adults express neither spn27A mRNA nor protein. In addition, spn27A1 mutants are female sterile, with the embryos dying at the very early developmental stage, further suggesting that the cup gene might be affected by the E25 deletion. However, spn27A1/cup females are not sterile; therefore, it was concluded that the sterility of spn27A1 female flies is a consequence of the lack of maternal spn27A expression during early embryogenesis. The spn27A1 homozygous larvae are viable, all reaching pupal stage. However, only 30% of spn27A1 adults emerged from the pupal stage, suggesting an implication of Spn27A during metamorphosis. spn27A1 adults often had a defect in wing expansion but are still viable. Only a modest rate of mortality was observed when the mutants were kept at 29°C. Consistent with an inhibitory function of Spn27A in the melanization cascade, constitutive melanization was observed in the cuticle and wings of most of the spn27A1 adults. Also, spn27A1 larvae sometimes had melanotic tumors (De Gregorio, 2002b).

Injury to wild-type larvae with a needle induced a melanization, at the wound site, whose extension is usually proportional to the injury size. Once the wound has efficiently healed, larvae progress to the pupal stage and sometimes to the adult stage. Interestingly, in spn27A1 mutant larvae, integumental injury with a standard needle leads to an uncontrolled hemocoelic melanization reaction visible within 2 hr of pricking. Fifty percent to 70% of spn27A1 larvae died in the first 5 hr after injury, while less than 10% of wild-type larvae succumbed. In most cases, the melanization reaction diffused throughout the larval body cavity of spn27A mutants, and dead larvae turned completely black. It is not clear whether the spn27A mutant larvae die because of the toxic effect of excessive melanization or from a defect in wound healing. In agreement with the second hypothesis, a recent study indicates that flies mutated in the melanization cascade exhibit poor ability to recover from an important injury, pointing to a link between melanization and clotting (Rämet, 2001). Interestingly, spn27A1 larvae survived after injury with a thin tungsten needle but exhibit a more intense melanization reaction at the wound site than the wild-type (De Gregorio, 2002b).

In adults, melanization at the wound site is more intense in spn27A1 flies than in the wild-type. Interestingly, the injection of rSpn27A proteins in the thorax of spn27A1 flies blocks the melanization at the injury site. This confirms the biochemical analysis showing that the function of Spn27A is to limit PPO activation. In the adults, the uncontrolled melanization reaction has only a weak effect on the survival rate of spn27A1 flies (De Gregorio, 2002b).

The invasion of Drosophila larvae by the parasitoid wasp, Leptopilina boulardi, is known to induce a melanization reaction associated with the encapsulation of the wasp's egg). Generally, in wild-type parasitized larvae, only one melanization spot is observed. L. Boulardi-parasitized spn27A1 larvae induce a strong systemic hemocoelic melanization reaction, but it is not specifically associated to the encapsulation process. This genetic analysis suggests that the role of Spn27A is to restrict the melanization reaction to the site of injury or encapsulation. These data taken together with the biochemical analysis further suggest that Spn27A inhibits Drosophila PPAE, thus limiting PPO activation in response to injury and parasitoid invasion (De Gregorio, 2002b).

A mutation in the Drosophila uncharacterized gene, Black cells (Bc), affects crystal cells and blocks the melanization reaction in the hemolymph. To confirm that the zygotic phenotype of spn27A is due to a misregulation of the melanization cascade, Bc,spn27A1 homozygous double mutant was generated. Interestingly all the phenotypes induced by the spn27A mutation except for female sterility are suppressed in a Bc mutant background. Bc,spn27A1 double mutants, like Bc, show a better viability at pupal stage compared with spn27A1 single mutants. Interestingly, mutation in spn27A does not enhance the weak melanization reaction observed in Bc homozygous larvae after injury. Bc adult flies, like spn27A1 mutants, are more susceptible than wild-type to infection by B. Bassiana, confirming a role for the melanization reaction to resist this fungus. Bc,spn27A1 double mutants showed the same survival curve as Bc flies. The absence of additive effect in the double mutant suggests that Spn27A contributes to fungal resistance through the control of the melanization cascade (De Gregorio, 2002b).

An extracellular serine protease cascade generates the ligand that activates the Toll signaling pathway to establish dorsoventral polarity in the Drosophila embryo. This cascade is regulated by a serpin-type serine protease inhibitor, which plays an essential role in confining Toll signaling to the ventral side of the embryo. This role is strikingly analogous to the function of the mammalian serpin antithrombin in localizing the blood-clotting cascade, suggesting that serpin inhibition of protease activity may be a general mechanism for achieving spatial control in diverse biological processes (Hashimoto, 2003).

In order to explicitly test the hypothesis that a serpin is involved in spatially regulating the Easter protease, by analogy to the role of antithrombin in blood clotting, the Drosophila genome was searched for candidate serpins. A serpin that inhibits the extracellular Easter protease should have, in addition to the C-terminal reactive center loop sequence characteristically found in known inhibitory serpins, a basic residue at the predicted reactive site to match the amino acid specificity of Easter and an N-terminal signal sequence for secretion. Eight serpins were identified that fulfilled all three criteria, out of about 25 encoded in the genome. The predicted reactive sites of two serpins, Spn1 and Spn27A, additionally showed provocative sequence similarity to the cleavage site of Spätzle, the Easter substrate (Hashimoto, 2003).

To determine whether any of the eight candidate serpins could inhibit Easter, their inhibitory activity was assessed in a cultured cell assay involving coexpression of the Easter catalytic domain and Spätzle. Both Spn1 and Spn27A efficiently blocked Easter cleavage of Spätzle, while the other six candidates had no appreciable effect. Based on these results, Spn1 and Spn27A emerged as the best candidates for a natural inhibitor of Easter (Hashimoto, 2003).

To investigate the role of Spn1 and Spn27A in regulating Easter in vivo, the genetic consequences of removing maternal serpin activity were examined. For Spn27A, an apparently protein null mutation has been generated to assess the zygotic role of Spn27A in regulating the melanization reaction during the immune response. Therefore, this mutation was used to remove maternal spn27A function and the resulting phenotype was characterized by scoring embryos for the expression of dorsoventral zygotic genes. In the wild-type embryo at the blastoderm stage, the zen gene is expressed in a dorsal domain, the rho gene in two ventrolateral stripes, and the twi gene in a ventral domain. By contrast, embryos lacking maternal spn27A function show a striking expansion of twi expression across the entire dorsoventral axis, with a corresponding loss of rho and zen transcription. In addition, the mutant embryos failed to differentiate a cuticle at the end of embryogenesis, consistent with the interpretation that all cells had adopted the ventral-most mesodermal fate, as dictated by uniform twi expression. Finally, the ventralized phenotype was completely rescued by injection of embryos with in vitro synthesized spn27A RNA. This result demonstrates that the mutant phenotype was caused by the loss of spn27A function, and is consistent with a requirement for spn27A in germline rather than somatic tissue. The genetic characterization and rescue experiments together demonstrate that the serpin Spn27A is essential for establishing embryonic dorsoventral polarity (Hashimoto, 2003).

The strongly ventralized phenotype produced by the loss of spn27A function requires wild-type easter activity, consistent with the interpretation that the Spn27A protein acts to regulate Toll signaling rather than another pathway important for establishing embryonic dorsoventral polarity. Females lacking spn27A and either easter or spätzle function produce dorsalized embryos lacking all ventral and lateral structures, indistinguishable from the phenotype produced by the easter or spätzle mutation alone (Hashimoto, 2003).

Although it has not yet been possible to examine the role of Spn1 in embryonic dorsoventral patterning, the nearly complete ventralization caused by loss of Spn27A suggests that Spn1 is not functionally redundant with Spn27A. Its ability to inhibit Easter activity in vitro may therefore indicate that the natural target of Spn1 is a protease sharing substrate specificity with Easter (Hashimoto, 2003).

The ventralized phenotype observed with the loss of maternal spn27A function implies that regulation of Easter following zymogen activation is required for maintaining embryonic polarity. If activated Easter were capable of diffusion, Spn27A may primarily act to maintain the initial asymmetry of zymogen activation, by inhibiting Easter before its diffusion to the dorsal side of the embryo. Alternatively, if Spn27A were itself localized to the dorsal side, it could be providing an opposing gradient of a signaling antagonist. In fact, the following experiments support the first model. First, embryos lacking Spn27A can be completely rescued by injection of cultured cell medium containing Spn27A protein into the perivitelline space surrounding the embryo, irrespective of whether injection occurred on the dorsal or the ventral side. This result demonstrates that Spn27A acts in the same extracellular compartment where Easter functions, and suggests that there is no requirement for Spn27A to be prelocalized to a specific region along the dorsoventral axis. Second, Spn27A was detected in perivitelline fluid extracted from embryos, thus providing evidence that Spn27A is a soluble and diffusible protein. Finally, by immunostaining, Spn27A was detected across the entire dorsoventral axis of the embryo. Thus, Spn27A appears to be a circulating component of the perivitelline space surrounding the embryo (Hashimoto, 2003).

In conclusion, these experiments demonstrate that the serpin Spn27A is essential for spatially regulating the signal that defines embryonic dorsoventral polarity in Drosophila. This role for Spn27A reveals another link between development and immunity. The Toll signaling pathway was discovered for its role in Drosophila embryonic development, but is now also appreciated as a key defense mechanism against pathogens in the innate immune systems of both insects and mammals, for example, activating the production of antifungal peptides in Drosophila. Spn27A was first described to have a zygotic role in regulating activation of the melanization reaction during the immune response, and now has been discovered to have a maternal role in regulating activation of the Toll signaling pathway during embryonic patterning. In the melanization reaction, Spn27A presumably regulates the protease that activates phenol oxidase, a key enzyme in melanin biosynthesis. This protease may be distinct from Easter, since easter mutant flies do not show any gross defect in their ability to mount a melanization reaction at the site of tissue injury. Interestingly, it appears that in development and in immunity the same ligand, processed Spätzle, activates the Toll signaling pathway, yet distinct serine protease cascades and serpins regulate the processing of Spätzle. The data suggest that the role of Spn27A in establishing embryonic dorsoventral polarity is to control the spatial distribution of Toll signaling. Although its target, the Easter protease, is apparently only activated on the ventral side of the embryo, this initial asymmetry is not sufficient to establish axial polarity. As a circulating component of the extracellular fluid surrounding the embryo, Spn27A acts either by controlling the level of active Easter on the ventral side or by preventing diffusion of active Easter toward the dorsal side, thereby ensuring that the Toll ligand is ventrally restricted. In the absence of Spn27A, excess Toll ligand not bound to receptor or active Easter itself spreads toward the dorsal side, ultimately resulting in nearly uniform activation of Toll signaling that ventralizes the embryo. Conversely, increased Spn27A inhibits activated Easter before it cleaves enough Spätzle precursor, leading to insufficient Toll signaling that dorsalizes the embryo. These studies reveal that an active mechanism for preventing Toll activation on the dorsal side of the embryo is required to establish embryonic dorsoventral polarity and depends on a critical level of Spn27A. More generally, the role of Spn27A in localizing a serine protease cascade that generates a developmental signal is very analogous to the role of the mammalian plasma serpin antithrombin in confining the blood-clotting cascade to the site of vascular damage. This striking parallel demonstrates how serine protease cascades and serpins are used to exert spatial control in two distinct biological processes that both rely on posttranslational mechanisms (Hashimoto, 2003).


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date revised: 12 January 2018

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