Protease cascades in immune responses

Extracellular serine protease cascades have evolved in animals for signalling that stimulates rapid responses to infection or wounding. Mammalian blood coagulation, fibrinolysis, and complement activation are prominent examples of such pathways. Haemolymph coagulation in horseshoe crabs in response to LPS or P-1,3-glucan is a well-characterized serine protease cascade in arthropods (Iwanaga, 2007). Melanization and activation of the Toll pathway are immune responses of insects, which implement a cascade of serine proteases to generate an effector response. The horseshoe crab and insect cascades have in common the involvement of clip-domain serine proteases, which have a C-terminal protease domain from the S1 (chymo-trypsin) family and an N-terminal clip domain thought to function in localization or regulation of the proteases (Jiang and Kanost, 2000). Clipdomain proteases are synthesized and secreted into haemolymph as inactive zymogens, which are activated by a specific proteolytic cleavage carried out by another protease. These pathways also often involve serine protease homologues (SPHs), which contain a domain with sequence similarity to serine proteases, but with the active-site serine changed to an inactive residue, most often glycine. SPHs may also contain N-terminal clip domains and are fairly abundant in insect genomes (Ross et al, 2003; Waterhouse et al, 2007). Although their functions are not yet well understood, SPHs appear to interact with clip-domain proteases and their substrates to modulate or regulate immune cascade pathways. SPHs in M. sexta and Holotrichia diomphalia bind phenoloxidase (PO) and form high-molecular-weight complexes (Yu et al., 2003; Wang and Jiang, 2004a; Gupta et al, 2005; Piao et al, 2005). We have identified more than 25 clip-domain proteases and four SPHs expressed in M. sexta fat body or haemocytes (Jiang et al, 2005). Described below are the results of efforts to understand the functions of these haemolymph proteases (HPs) in innate immune responses.

3.4.1 Proteases and stimulation of antimicrobial peptide synthesis

The Toll pathway in D. melanogaster triggers synthesis of drosomycin and other antimicrobial peptides in response to fungi and many Gram-positive bacteria (Lemaitre and Hoffmann, 2007). The response is initiated by recognition proteins that bind pathogens or microbial patterns, as discussed briefly above. Such binding triggers an extracellular serine protease cascade involving multiple serine proteases, terminating in cleavage and activation of the cytokine Spätzle by a clip-domain protease called Spätzle-processing enzyme (SPE) (Jang et al., 2006). Upstream of SPE in this pathway are clipdomain proteases called Persephone (Ligoxygakis et al, 2002), Spirit, and Grass (Kambris et al, 2006; El Chamy et al, 2008) (Figure 3.1). At this point it is not known what protease activates SPE in D. mela-nogaster, although a SPE-activating enzyme (SAE) has been identified in the beetle T. mollitor (Kim et al, 2008). Activated Spätzle binds to the Toll membrane receptor in the fat body and haemocytes, and triggers an intracellular signal transduction cascade that activates Rel-family transcription factors.

The Toll pathway was initially discovered for its role in dorsal-ventral patterning during embryogenesis. However, the embryonic pathway leading to Spätzle processing involves different serine proteases (Dissing et al., 2001; LeMosy et al., 2001; Rose et al., 2003). The last two proteases in this pathway, Snake and Easter, each contain a clip domain. The initiation of this embryonic serine protease cascade is poorly understood, but seems to be triggered by a product of Pipe, a heparin sulphate 2-O-sulfotrans-ferase that is specifically expressed on the ventral side of the follicular epithelium (Sen et al, 1998).

D. melanogaster Spätzle can be activated in response to fungi in at least two ways. GNBP3 («65% similar in its N-terminal domain to that of M. sexta ßGRPs) binds to ß-1,3-glucan and induces expression of drosomycin in response to Candida albicans. However, certain entomopathogenic fungi, which secrete serine proteases to penetrate the cuticle, activate the Toll pathway by directly cleaving the protease Persephone (Gottar et al.,

2006). A complete pathway for Spätzle activation in response to Lys-peptidoglycan containing Grampositive bacteria has been worked out in T. molitor. It requires the recognition proteins PGRP-SA and GNBP1, modular serine protease (which has 3600 sequence identity with M. sexta HP14), SAE, and SPE (Kim et al., 2008).

A Toll pathway also appears to function in stimulating antimicrobial peptide synthesis in lepidop-teran insects, including M. sexta. A Toll receptor is constitutively expressed in several tissues of M. sexta and is present on the surface of haemo-cytes. Its mRNA is upregulated in response to yeast and bacteria (Ao et al., 2008). B. mori Spätzle-1 has been shown to upregulate antimicrobial gene expression in both B. mori and M. sexta (Wang et al.,

2007). We recently isolated a cDNA for a M. sexta homologue of Spätzle (C. An, H. Jiang, and M.R. Kanost, unpublished results) and are investigating its activation and biological function. M. sexta HP8, a clip-domain protease most similar to Drosophila Easter and SPE (the known Spätzle activators), has been identified as a protease that cleaves and activates M. sexta Spätzle. HP6 is a clip-domain protease that appears to be the M. sexta orthologue of Drosophila Persephone. Biochemical analysis using purified recombinant proteins indicates that HP6 is


Embryonic development

Pipe product i

Immune response Gram+ bacteria or fungi

Manduca Immune response

Bacteria or fungi

Gastrulation defective (Gd)



Snake Snake* 1

Easter Easter* SPE* proSPE \ /

HP6* proHP6

HP8* proHP8




Figure 3.1 A model of Toll-activation pathways in D. melanogaster and M. sexta. D. melanogaster Spätzle activation occurs during embryonic development and in haemolymph during immune challenge. During embryonic development, gastrulation defective (Gd) becomes active (*) in the ventral region of the perivitelline space in response to Pipe expression via an unknown mechanism. Activation of Snake by Gd leads to activation of Easter, which cleaves Spätzle to form an active Toll ligand. In immune-related Toll activation, pattern-recognition proteins recognize Gram-positive bacteria or fungi and activate, through an unknown mechanism, serine protease cascades that involve serine proteases Grass and Spirit, and the terminal protease, Spätzle-processing enzyme (SPE). The serine protease Persephone is also involved upstream of SPE and may be activated directly by fungal or bacterial proteases. How these proteases are activated or what their targets are remains unknown, with the exception of SPE cleavage of Spätzle. In M. sexta we identified the two terminal proteases in Spätzle activation. Haemolymph proteinase 6 (HP6) activates proHP8 and HP8 cleaves Spätzle, creating the active Toll ligand. HP6 also is involved in a cascade leading to PO activation and the melanization response.

an activator of HP8, which in turn activates Spätzle (Figure 3.1). ProHP6 and proHP8 in plasma both become activated after exposure to bacteria, and injection of either HP6 or HP8 into M. sexta larvae results in induced expression of several antimicrobial peptides (C. An, H. Jiang, and M.R. Kanost, unpublished results).

3.4.2 ProPO activation

M. sexta pathways for proPO activation are now relatively well understood. PO catalyses oxidative reactions involved in melanin synthesis, which has been implicated in microbial killing through the generation of toxic compounds (Cerenius et al., 2008). PO activation and melanization in response to microbial exposure can occur faster than antimicrobial peptide synthesis. Oxidation reactions catalysed by PO lead to the formation of reactive quinone intermediates, cytotoxic molecules like 5,6-dihydroxyindole (DHI), and reactive oxygen or nitrogen intermediates that may contribute to the killing of invading pathogens (Nappi and Christensen, 2005; Zhao et al, 2007). Melanization can also occur in haemocyte nodules or on the surface of encapsulated objects or parasites (Cerenius and Söderhäll, 2004).

PO hydroxylates tyrosine to form an o-diphenol, 3,4-dihydroxyphenylalanine (DOPA), using its monophenol monooxygenase (tyrosinase) activity. PO also oxidizes o-diphenols such as DOPA to their corresponding quinones (Kanost and Gorman, 2008). The orthoquinones resulting from this process polymerize to form melanin. Specifically, dopaquinone can non-enzymically cyclize to form dopachrome. Dopachrome-converting enzyme catalyses the decarboxylation of dopachrome to DHI, which can be converted by PO to i ndole-5,6-quinone, which then polymerizes to form DHI eumelanin (Nappi and Christensen, 2005).

Tyrosine hydroxylase is an intracellular enzyme with monophenoloxidase activity that also can convert tyrosine to DOPA. Tyrosine hydroxylase expression is upregulated in M. sexta upon immune challenge and may have a significant role in melanin synthesis in immune responses (Gorman et al., 2007a). Another intracellular, immune-induced enzyme important in the early stages of melanin synthesis is Dopa decarboxylase (Kim et al., 2000a; Zhu et al., 2003a), which converts DOPA to dopa-mine. Dopamine is a better PO substrate than DOPA and is present at higher concentration in M. sexta haemolymph, and thus probably contributes significantly as a precursor for quinone and melanin synthesis.

Two proPO genes are expressed constitutively in M. sexta oenocytoids, each producing an inactive, approximately 80 kDa zymogen (Hall et al, 1995; Jiang et al; 1997, Gorman et al, 2007a). The two M. sexta proPOs are approximately 50% identical in sequence and form heterodimers. M. sexta proPOs lack signal peptides, which is common among arthropod proPOs. They are apparently released into plasma by lysis of oenocytoids, a process whose regulation needs investigation. PPO is activated through specific proteolytic cleavage. In M. sexta, three proPO-activating proteases (PAPs) have been discovered: PAP1, PAP2, and PAP3 (Jiang et al, 1998, 2003a, 2003b). Each of the three PAPs is synthesized as a proPAP zymogen, and they must be activated by specific proteolytic cleavage. PAP1 contains a single N-terminal clip domain and is activated by cleavage after Arg-127. PAP2 and PAP3 each contain two N-terminal clip domains and are activated by cleavage after Lys-153 and Lys-146, respectively. The solution structure of the dual clip domains from PAP-2 has been solved (Huang et al., 2007). Each clip domain adopts a fold in which a three-stranded antiparallel ß-sheet is flanked by two a-helices. This structure should be helpful in designing experiments to examine functions of the clip domains in formation of protein complexes in proPO activation. The M. sexta PAPs require a cofac-tor composed of SPHs assembled in a high Mr form (Yu et al, 2003; Gupta et al, 2005). The SPHs must also be activated by limited proteolysis at a specific

Arg or Lys residue. ProPO activation includes a complex network of proteins that are sequentially activated by recognition of damaged tissue or invading microbes (Figure 3.2) (Jiang, 2008).

Recently, serine protease cascades that lead to proPO activation in M. sexta have been elucidated. At the top of one identified pathway is HP14, a complex protein that contains five low-density lipoprotein receptor class A domains, a Sushi domain, a cysteine-rich region, and a C-terminal protease domain (Ji et al., 2004). HP14 can autoactivate in the presence of peptidoglycan or P-1,3-glucan and PGRP2 (Wang and Jiang, 2006). HP14 then activates proHP21, and HP21 cleaves and activates proPAP2 or proPAP3 (Gorman et al., 2007b; Wang and Jiang, 2007), which then activates proPO in the presence


bacteria g


bacteria g proHP14 HP14*

proHP14 HP14*

proHP21 HP21*

HP6* proHP6

HP6* proHP6


SPH1* J proSPH1

proPAP2/3 PAP2/3* PAP1* proPAP1


SPH1* J proSPH1

Figure 3.2 Protease cascades involved in M. sexta proPO activation. An initiator protease, proHP14, is activated subsequent to interactions between haemolymph plasma pattern-recognition proteins and microbial patterns. HP14 activates proHP21 and HP21 then activates proPAP2 and proPAP3. In another branch, HP6 activates proPAP1. HP6 is also involved in M. sexta Toll activation (see Figure 3.1). The activator(s) of proHP6 are not yet known but proHP6 activation is stimulated by PAP1 in a positive-feedback loop. SPHs are not catalytically active proteases but still must be cleaved to function in PO activation. PAP1 can cleave SPH2; other SPH activators are unknown. Any one of the active PAPs can interact with cleaved SPHs to form a functional PO activation complex, which cleaves proPO to active PO. PO oxidizes catechols in the haemolymph that, after further reactions, form melanin. HP, haemolymph proteinase; PAP, prophenoloxidase-activating proteinase; PO, phenoloxidase; SPH, serine proteinase homologue.

of cleaved SPHs (Figure 3.2). ProPAP1 is not activated by HP21 (Wang and Jiang, 2007). However, HP6 can cleave and activate proPAP1 (C. An, H. Jiang, and M.R. Kanost, unpublished results), indicating at least two protease cascades which can lead to proPO activation, as well as potential cross-talk between pathways for melanization and Toll activation, both involving HP6. As an additional positive regulatory mechanism, active PAP1 can cleave SPH2 and also stimulates activation of proHP6, suggesting that a self-reinforcing, positive feedback mechanism helps to promote rapid proPO activation (Wang and Jiang, 2008). In T. molitor, active SPE can cleave proPO and SPH1, which together are sufficient for melanization (Kan et al, 2008).

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