Immune challenge induces N-terminal cleavage of the Drosophila serpin Necrotic

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Abstract

The Drosophila Necrotic protein is a serine proteinase inhibitor, which regulates the Toll-mediated innate immune response. Necrotic specifically inhibits an extracellular serine proteinase cascade leading to activation of the Toll ligand, Spätzle. Necrotic carries a polyglutamine extension amino-terminal to the core serpin structure. We show here that cleavage of this N-terminal extension occurs following immune challenge. This modification is blocked in PGRP-SAsemmelweiss mutants after Gram-positive bacterial challenge and in persephone mutants after fungal or Gram-positive bacterial challenge, indicating that activation of either of the Toll pathway upstream branches induces N-terminal cleavage of the serpin. The absolute requirement of persephone gene product for this cleavage indicates that Gram-positive bacteria activate a redundant set of proteinases upstream of Toll. Both full-length Necrotic and the core serpin are active inhibitors of a range of serine proteinases: the highest affinity being for cathepsin G and elastases. We found a 13-fold increase in the specificity of the core serpin over that of full-length Necrotic for one of the tested proteinases (porcine pancreatic elastase). This finding indicates that cleavage of the Necrotic amino-terminal extension might modulate Toll activation following the initial immune response.

Introduction

Serine proteinase inhibitors (serpins) regulate a wide range of processes such as blood coagulation, complement activation, and inflammation in mammals (Gettins, 2002; Silverman et al., 2001) and similar defense responses in invertebrates (Kanost, 1999). One of the best characterized of these invertebrate responses is the activation of the Drosophila Toll pathway, which is triggered by fungal or Gram-positive bacterial infections. A member of the family of peptidoglycan recognition proteins (PGRPs), PGRP-SA, and the serine proteinase Persephone (Psh) have been shown genetically to delineate two separate signalling branches, upstream of Toll, and responsible, respectively, for the activation of the Toll pathway after Gram-positive bacterial challenge and natural fungal infection (Ligoxygakis et al., 2002a; Michel et al., 2001). Activation of the Toll receptor downstream of both these branches induces a signalling cascade leading to translocation of an NF-κB-like protein Dorsal-related immune factor (Dif) to the nucleus and synthesis of antimicrobial peptides (Hoffmann and Reichhart, 2002). The Toll receptor is activated by its ligand, a cysteine-knot growth factor called Spätzle (Spz), which is cleaved from its propeptide following infection (Levashina et al., 1999).

The Necrotic (Nec) serpin (previously called Spn43Ac) regulates Toll activation by inhibiting a proteinase involved in the cleavage of Spz (Levashina et al., 1999). Serpins are characterized by a highly conserved tertiary structure and a dynamic mechanism of inhibition. Native serpins have a folded core structure with an exposed reactive center loop (RCL), which is presented as an ideal substrate for the target proteinase. Cleavage of the RCL at the P1–P′1 position allows it to insert within a 5-strand β-sheet structure in the serpin core. During this process, the serpin relaxes and the proteinase is translocated by 70 Å from one pole of the serpin to the other. The proteinase molecule is distorted and trapped in a covalently linked serpin–proteinase complex, which is targeted for destruction (Gettins, 2002).

Nec has an alanine-rich hinge region and its active site is characterized by leucine and serine in the P1–P′1 positions. Nec is a highly unusual serpin in that the core structure carries an 80–100 amino acid N-terminal extension of unknown function. This extension has no obvious structure, but contains stretches of glutamines and prolines, including a 9-residue polyglutamine repeat. Following infection with a mixture of Gram-positive and Gram-negative bacteria, the Nec protein is cleaved (Levashina et al., 1999). We show here that, unexpectedly, this cleavage corresponds to the removal of the N-terminal polyglutamine-containing extension and is specifically linked to the induction of the Toll pathway following either Gram-positive bacterial or fungal infections. The cleavage of the N-terminal extension of Nec by both type of infections requires a wild-type psh gene, suggesting that Gram-positive bacteria activate a redundant proteinase upstream of Toll. We have been unable to express the full-length Nec protein (Nec-fl) in Escherichia coli cells, although the N-terminally truncated serpin (Nec-ΔN) is readily expressed and is a potent inhibitor of elastase- and chymotrypsin-like proteinases (Robertson et al., 2003). In this paper, we express Nec-fl in a baculovirus/insect cell system and show that it has similar stability and folding to the Nec-ΔN protein expressed in E. coli. The reaction kinetics and stoichiometry of inhibition (SI) ratios, for both forms of the serpin are compared with a range of serine proteinases. The observed change in specificity between Nec-fl and Nec-ΔN towards its target proteinase(s) could be a way to rapidly restore the initial conditions in the hemolymph after infection.

Section snippets

Fly stocks and genetics

Flies were cultured at 25 °C. Oregon-R was used as the wild-type strain. To identify the faster-migrating Nec species (Levashina et al., 1999) we constructed a Nectag transgene. Myc and hemaglutinin (Ha) tags were introduced into the Nec-coding sequence by PCR. This construct carries the Myc tag between the signal peptide and the N-terminus of the secreted peptide (SP), while the Ha tag is at the C-terminus of the protein (Fig. 1A). The construct was cloned into the pUAST plasmid (Brand and

Immune-induced cleavage of Nec released an N-terminal fragment

The double-tagged construct, P[UAS-nectag] (Fig. 1A, Materials and Methods), suppressed the nec phenotype of mutant flies when expressed under control of the ubiquitous da promoter (in nec; P[da-Gal4]/P[UAS-nectag] flies, data not shown). This result shows that the Nectag protein remains biologically active. As previously reported, the anti-GST-Nec antibody recognized a single band (of about 60 kDa) in hemolymph from unchallenged flies and double bands (of about 60 and 52 kDa) after

Discussion

The mammalian and invertebrate innate immune system share highly conserved signalling pathways. In both cases, a central receptor, Toll or one of the mammalian homologous Toll-like receptors (TLRs), signals downstream to a Rel family transcription factor that in turn activates hundreds of effector genes (Hoffmann, 2003). However, while mammalian TLRs bind to and directly recognize microbial molecules, Drosophila Toll receptor is activated by its endogenous ligand Spz (Weber et al., 2003). In

Acknowledgments

This work was supported by the French Centre National de la Recherche Scientifique and the National Institutes of Health. We thank Drs. M. Kanost and V. Leclerc for critical reading of the manuscript, Dr. Steve Hartson at Oklahoma State University Recombinant DNA/Protein Resource Facility for assistance in MALDI-mass spectrometry and A. Meunier for technical help. TRD is an MRC Career Development Fellow.

References (30)

  • D. Belorgey et al.

    Inhibition of human pancreatic proteinases by mucus proteinase inhibitor, eglin c and aprotinin

    Biochem. J.

    (1996)
  • J.G. Bieth

    In vivo significance of kinetic constants of macromolecular proteinase inhibitors

    Adv. Exp. Med. Biol.

    (1984)
  • A.H. Brand et al.

    Targeted gene expression as a means of altering cell fates and generating dominant phenotypes

    Development

    (1993)
  • P.G. Gettins

    Serpin structure, mechanism, and function

    Chem. Rev.

    (2002)
  • V. Gobert et al.

    Dual activation of the Drosophila toll pathway by two pattern recognition receptors

    Science

    (2003)
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    1

    Present address: Research Group Signaling and Functional Genomics, German Cancer Research Center (DKFZ/B110), Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany.

    2

    Authors who made equal contribution.

    3

    Present address: The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

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