Immune challenge induces N-terminal cleavage of the Drosophila serpin Necrotic
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)
Theoretical and practical aspects of proteinase inhibition kinetics
Meth. Enzymol.
(1995)- et al.
Physical characterization of serpin conformations
Methods
(2004) - et al.
Prophenoloxidase-activating proteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serine proteinase regulated by serpin-1J and serine proteinase homologs
Insect Biochem. Mol. Biol.
(2003) Serine proteinase inhibitors in arthropod immunity
Dev. Comp. Immunol.
(1999)- et al.
6-mer peptide selectively anneals to a pathogenic serpin conformation and blocks polymerization. Implications for the prevention of Z α1-antitrypsin-related cirrhosis
J. Biol. Chem.
(2002) - et al.
Characterization of the necrotic protein that regulates the Toll-mediated immune response in Drosophila
J. Biol. Chem.
(2003) - et al.
Serine proteases and their homologs in the Drosophila melanogaster genome: an initial analysis of sequence conservation and phylogenetic relationship
Gene
(2003) - et al.
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.
(2001) - et al.
Expression and purification of Manduca sexta prophenoloxidase-activating proteinase precursor (proPAP) from baculovirus-infected insect cells
Protein Exp. Purif.
(2001) - et al.
Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism
Proc. Natl. Acad. Sci. USA
(2002)
Inhibition of human pancreatic proteinases by mucus proteinase inhibitor, eglin c and aprotinin
Biochem. J.
In vivo significance of kinetic constants of macromolecular proteinase inhibitors
Adv. Exp. Med. Biol.
Targeted gene expression as a means of altering cell fates and generating dominant phenotypes
Development
Serpin structure, mechanism, and function
Chem. Rev.
Dual activation of the Drosophila toll pathway by two pattern recognition receptors
Science
Cited by (27)
Inhibition of immune pathway-initiating hemolymph protease-14 by Manduca sexta serpin-12, a conserved mechanism for the regulation of melanization and Toll activation in insects
2020, Insect Biochemistry and Molecular BiologyCitation Excerpt :Immune challenge causes an amino-terminal truncation of Necrotic at an unknown site. There is no major difference in association rate constant (kass) or stoichiometry of inhibition (molar ratio serpin behaving as inhibitor vs. substrate) between full-length Necrotic (Nec-fl) and NecΔN (Pelte et al., 2006), except that the later has a 13-fold increase in kass and 5-fold decrease in SI to porcine pancreatic elastase (PPE). Thus, removing the amino-terminal extension does not have a major effect on the biochemical activity of this serpin.
Drosophila melanogaster clip-domain serine proteases: Structure, function and regulation
2016, BiochimieCitation Excerpt :While Psh has been suggested to be the nec target protease, no direct interaction has been demonstrated so far. SPN43Ac has a long N-terminal extension that is cleaved in vivo upon microbial challenge [132]. The recombinant protein lacking this extremity has a very broad specificity and inhibited in vitro a wide range of proteases such as elastase, thrombin and chymotrypsin-like SPs, but failed to regulate tryspin or kallikrein [133].
The Drosophila serpins: Multiple functions in immunity and morphogenesis
2011, Methods in EnzymologyCitation Excerpt :Nec has a broad inhibitory spectrum for elastase, thrombin, and chymotrypsin-like proteases, in vitro (Robertson et al., 2003). The N-terminal extension of the Nec core serpin is cleaved on immune challenge, which increases the substrate specificity for porcine pancreatic elastase (Pelte et al., 2006). The length and sequence of this N-terminal is poorly conserved within the Drosophilidae, being about 50 amino acids in Drosophila sechellia and 125 amino acids in Drosophila grimshawi (Garrett et al., 2009).
Serpins flex their muscle: I. Putting the clamps on proteolysis in diverse biological systems
2010, Journal of Biological ChemistryCitation Excerpt :The serpin SPN43Ac neutralizes Persephone and negatively regulates the Toll pathway (15). Unusually, SPN43Ac has a glutamine-rich N-terminal extension that is cleaved upon microbial challenge (19). Flies with Spn43Ac mutations accumulate cleaved Spaetzle, exhibit black melanotic necrotic spots on the body, and undergo premature death.
- 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.