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).
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, 2002).
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).
Ashida M. and Brey P.T. (1998) Recent advances in research on the insect prophenoloxidase cascade. In: Brey P.T. and Hultmark D. (Eds.) Molecular Mechanisms of Immune Response in Insects. London: Chapman and Hall
Cohen, A. B., Gruenke, L. D., Craig, J. C. and Geczy, D. (1977). Specific lysine labeling by 18-OH during alkaline cleavage of the alpha-1-antitrypsin-trypsin complex. Proc. Natl. Acad. Sci. 74: 4311-4314. 303770
Collins, F., et al. (1986). Genetic selection of a plasmodium-refractory strain of the malaria vector Anopheles gambiae. Science, 234: 607-610. 3532325
De Gregorio, E., Spellman, P. T., Rubin, G. M. and Lemaitre, B. (2001). Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays. Proc. Natl. Acad. Sci. 98: 12590-12595. 11606746
De Gregorio, E., Spellman, P. T., Tzou, P., Rubin, G. M. and Lemaitre, B. (2002a). The Toll and Imd pathways are the majors regulators of the immune response in Drosophila. EMBO J. 21: 2568-2579. 12032070
De Gregorio, E., et al. (2002b). An immune-responsive serpin regulates the melanization cascade in Drosophila. Dev. Cell 3: 581-592. 12408809
Green C., et al. (2000). The necrotic gene in Drosophila corresponds to one of a cluster of three serpin transcripts mapping at 43A1.2. Genetics 156: 1117-1127. 11063688
Hashimoto, C., et al. (2003). Spatial regulation of developmental signaling by a serpin. Dev. Cell 5: 945-950. 14667416
Irving, J. A., Pike, R. N., Lesk, A. M. and Whisstock, J. C. (2000). Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res. 10: 1845-1864. 11116082
Irving, P., et al. (2001). A genome-wide analysis of immune responses in Drosophila. Proc. Natl Acad. Sci. 98: 15119-15124. 11742098
Jiang H., Wang Y. and Kanost M.R. (1998) Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: a bacteria-inducible protein similar to Drosophila easter. Proc. Natl. Acad. Sci. 95: 12220-12225. 9770467
Kwon, T. H., Kim, M. S., Choi, H. W., Joo, C. H., Cho, M. Y. and Lee, B. L. (2000). A masquerade-like serine protease homologue is necessary for PO activation in the coleopteran insect Holotrichia diomphalia larvae. Eur. J. Biochem. 267: 6188-6196. 11012672
Lanot, R., Zachary, D., Holder, F. and Meister, M. (2000). Post-embryonic hematopoiesis in Drosophila. Dev. Biol. 230: 243-257. 11161576
Lawrence D. A., et al. (2000). Partitioning of serpin-proteinase reactions between stable inhibition and substrate cleavage is regulated by the rate of serpin reactive center loop insertion into beta-sheet A. J. Biol. Chem. 275: 5839-5844. 10681574
Lee, S. Y., et al. (1998). In vitro activation of pro-phenol-oxidase by two kinds of pro-phenol-oxidase-activating factors isolated from hemolymph of coleopteran, Holotrichia diomphalia larvae. Eur. J. Biochem. 254: 50-57. 9652393
Levashina, E. A., et al. (1999). Constitutive activation of toll-mediated antifungal defense in serpin-deficient Drosophila. Science, 285: 1917-1919. 10489372
Ligoxygakis, P., et al. (2002). A serpin mutant links Toll activation to melanization in the host defence of Drosophila. EMBO J. 21: 6330-6337. 12456640
Ma, C. and Kanost, M. R. (2000). A beta1,3-glucan recognition protein from an insect, Manduca sexta, agglutinates microorganisms and activates the phenoloxidase cascade. J. Biol. Chem. 275: 7505-7514. 10713054
Ochiai, M. and Ashida, M. (1999). A pattern recognition protein for peptidoglycan. Cloning the cDNA and the gene of the silkworm, Bombyx mori. J. Biol. Chem. 274: 11854-11858. 10207004
Park, D. S., Shin, S. W., Hong, S. D. and Park, H. Y. (2000). Immunological detection of serpin in the fall webworm, Hyphandria cunea and its inhibitory activity on the prophenoloxidase system. Mol. Cells 10: 186-192. 10850660
Potempa, J., Korzus, E. and Travis, J. (1994). The serpin superfamily of proteinase inhibitors: structure, function, and regulation. J. Biol. Chem. 269: 15957-15960. 8206889
Rämet, M., Lanot, R., Zachary, D. and Manfruelli, P. (2001). JNK signaling pahway is required for efficient wound healing in Drosophila. Dev. Biol. 241: 145-156. 11784101
Satoh, D., Horii, A., Ochiai, M. and Ashida, M. (1999). Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori. Purification, characterization, and cDNA cloning. J. Biol. Chem. 274: 7441-7453. 10066809
Silverman, G. A., et al. (2001). The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J. Biol. Chem. 276: 33293-33296. 11435447
Söderhäll, K. and Cerenius, L. (1998). Role of prophenoloxidase-activating system in invertebrate immunity. Curr. Opin. Immunol. 10: 23-28. 9523106
Tzou, P., De Gregorio, E. and Lemaitre, B. (2002). How Drosophila combats microbial infection: a model to study innate immunity and host-pathogen interactions. Curr. Opin. Microbiol. 5: 102-110.
Vass, E. and Nappi, A. J. (2000). Developmental and immunological aspects of Drosophila-parasitoid relationships. J. Parasitol. 86: 1259-1270. 11191902
Wang, R., Lee, S. Y., Cerenius, L. and Soderhall, K. (2001). Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus. Eur. J. Biochem. 268: 895-902. 11179955
Ye, S. and Goldsmith, E. J. (2001) Serpins and other covalent protease inhibitors. Curr. Opin. Struct. Biol. 11: 740-745. 11751056
Yoshida, H., Kinoshita, K. and Ashida, M. (1996). Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori. J. Biol. Chem. 271: 13854-13860. 8662762
Yu, X. Q., Gan, H. and Kanost, M. R. (1999). Immulectin, an inducible C-type lectin from an insect, Manduca sexta, stimulates activation of plasma prophenol oxidase. Insect Biochem. Mol. Biol. 29: 585-597. 10436935
Zhu, Y. (2001). Identification of immune-related genes from the tobacco hornworm, Manduca sexta, and characterization of two immune-inducible proteins, serpin-3 and leureptin. PhD thesis, Kansas State University, Manhattan, KS.
date revised: 20 May 2004
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