A Spätzle-Processing Enzyme Required for Toll Signaling Activation in Drosophila Innate Immunity

Jang, In-Hwan; Chosa, Naoyuki; Kim, Sunghee; Nam, Hyuck‐Jin; Lemaître, Bruno; Ochiai, Masanori; Kambris, Zakaria; Brun, Sylvain; Hashimoto, Carl; Ashida, Masaaki; Brey, Paul T.; Lee, Won–Jae

doi:10.1016/j.devcel.2005.11.013

Cited by 263 publications

(225 citation statements)

References 57 publications

(74 reference statements)

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“…1B). HP6 may be an ortholog of Drosophila Persephone (35% identity), whereas HP8 has the highest identity to a group of proteinases whose substrate is the cytokine precursor prospätzle, including Drosophila Easter (42%) (42), SPE from Drosophila (39%) (18), and Tenebrio (45%) (11) and a predicted SPE named BAEEase from the silkworm, B. mori (70%) (18). The predicted proteolytic activation sites (2) are at 109 LDLH2ILGG in proHP6 and 111 NNDR2IVGG in proHP8 (15) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Functions of Manduca sexta Hemolymph Proteinases HP6 and HP8 in Two Innate Immune Pathways

Ishibashi

Ragan

et al. 2009

Journal of Biological Chemistry

154

178

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Serine proteinases in insect plasma have been implicated in two types of immune responses; that is, activation of prophenoloxidase (proPO) and activation of cytokine-like proteins. We have identified more than 20 serine proteinases in hemolymph of the tobacco hornworm, Manduca sexta, but functions are known for only a few of them. We report here functions of two additional M. sexta proteinases, hemolymph proteinases 6 and 8 (HP6 and HP8). HP6 and HP8 are each composed of an amino-terminal clip domain and a carboxyl-terminal proteinase domain. HP6 is an apparent ortholog of Drosophila Persephone, whereas HP8 is most similar to Drosophila and Tenebrio spätzle-activating enzymes, all of which activate the Toll pathway. proHP6 and proHP8 are expressed constitutively in fat body and hemocytes and secreted into plasma, where they are activated by proteolytic cleavage in response to infection. To investigate activation and biological activity of HP6 and HP8, we purified recombinant proHP8, proHP6, and mutants of proHP6 in which the catalytic serine was replaced with alanine, and/or the activation site was changed to permit activation by bovine factor Xa. HP6 was found to activate proPO-activating proteinase (proPAP1) in vitro and induce proPO activation in plasma. HP6 was also determined to activate proHP8. Active HP6 or HP8 injected into larvae induced expression of antimicrobial peptides and proteins, including attacin, cecropin, gloverin, moricin, and lysozyme. Our results suggest that proHP6 becomes activated in response to microbial infection and participates in two immune pathways; activation of PAP1, which leads to proPO activation and melanin synthesis, and activation of HP8, which stimulates a Toll-like pathway.Innate immune systems of mammals and arthropods include extracellular serine proteinase cascade pathways, which rapidly amplify responses to infection and stimulate killing of pathogens. These proteinase-driven processes include the complement system of vertebrates (1, 2) and pathways in arthropods involving proteinases containing amino-terminal clip domains (3). Clip domain proteinases function in blood coagulation (4, 5), activation of prophenoloxidase (proPO) that leads to melanin synthesis (6 -9), and stimulation of the Toll pathway to promote synthesis of antimicrobial peptides/proteins (AMPs) 2 secreted into the hemolymph (10, 11).The serine proteinase systems best characterized in arthropods are the horseshoe crab hemolymph coagulation pathway and the cascade leading to activation of the Toll pathway in dorsal-ventral development in Drosophila (12-14). Recent research also has led to better characterization of the proPO activation pathway in Manduca sexta (7,15,16) and the Tollsignaling pathway in the Drosophila immune response (17, 18) and to both the proPO and Toll pathways in the beetle Tenebrio molitor (11,19).In the proPO activation pathway, soluble pattern recognition proteins initially recognize pathogen-associated molecular patterns such as bacterial peptidoglycan or fungal ␤-1,3-glucan (...

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Section: Resultsmentioning

confidence: 99%

Functions of Manduca sexta Hemolymph Proteinases HP6 and HP8 in Two Innate Immune Pathways

Ishibashi

Ragan

et al. 2009

Journal of Biological Chemistry

154

178

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“…Perhaps, it is worth testing their roles in the proteolytic activation of M. sexta plasmatocyte-spreading peptide and spätzle precursors (Wang et al, 1999 and2007). Recently, Drosophila Sp4 (CG16705) was found to be a processing enzyme of spätzle, which activates the Toll-dependent antimicrobial response (Jang et al, 2006). HP21 is similar in amino acid sequence to D. melanogaster Snake (Fig.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor

Wang¹,

Jiang²

2007

Insect Biochemistry and Molecular Biology

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Upon wounding or infection, a serine proteinase cascade in insect hemolymph leads to prophenoloxidase (proPO) activation and melanization, a defense response against invading microbes. In the tobacco hornworm Manduca sexta, this response is initiated via hemolymph proteinase 14 (HP14), a mosaic protein that interacts with bacterial peptidoglycan or fungal β-1,3-glucan to autoactivate. In this paper, we report the expression, purification, and functional analysis of M. sexta HP21 precursor, an HP14 substrate similar to Drosophila Snake. The recombinant proHP21 is a 51.1 kDa glycoprotein with an amino-terminal clip domain, a linker region, and a carboxyl-terminal serine proteinase domain. HP14, generated by incubating proHP14 with β-1,3-glucan and β-1,3-glucan recognition protein-2, activated proHP21 by limited proteolysis between Leu 152 and Ile 153 . Active HP21 formed an SDS-stable complex with M. sexta serpin-4, a physiological regulator of the proPO activation system. We determined the P1 site of serpin-4 to be Arg 355 and, thus, confirmed our prediction that HP21 has trypsin-like specificity. After active HP21 was added to the plasma, there was a major increase in PO activity. HP21 cleaved proPO activating proteinase-2 precursor (proPAP-2) after Lys 153 and generated an amidase activity which activated proPO in the presence of serine proteinase homolog-1 and 2. In summary, we have discovered and reconstituted a branch of the proPO activation cascade in vitro: β-1,3-glucan recognition -proHP14 autoactivation -proHP21 cleavage -PAP-2 generation -proPO activation -melanin formation.

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“…However, functional cross-talk between Dm-PGRP-SA and Dm-GNBP1 during a Lys-type PGN recognition signal has not been determined yet. Recognition of Lys-type PG by the PG recognition complex has been suggested to activate the serine protease (SP) cascade, leading to the processing of Spätzle (Spz) and subsequent activation of the Toll signalling pathway (27,28).…”

Section: How Is Lys-type Pg Recognized In Drosophila?mentioning

confidence: 99%

“…At present, several SPs and SP homologues (SPHs) have been identified as regulators of the Toll pathway (28)(29)(30)(31). Among them, Dm-Spz-processing enzyme (SPE) was identified and reported to function as a processing enzyme to cleave Spz pro-protein to active Spz (28). Even though the biological functions of SPE have been characterized in Drosophila genetic studies, the identification of a direct upstream activator of SPE awaits further investigation.…”

Section: How Is Lys-type Pg Recognized In Drosophila?mentioning

confidence: 99%

Innate immune response in insects: recognition of bacterial peptidoglycan and amplification of its recognition signal

Kim¹,

Park²,

Ha³

et al. 2008

BMB Reports

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A Spätzle-Processing Enzyme Required for Toll Signaling Activation in Drosophila Innate Immunity

Cited by 263 publications

References 57 publications

Functions of Manduca sexta Hemolymph Proteinases HP6 and HP8 in Two Innate Immune Pathways

Functions of Manduca sexta Hemolymph Proteinases HP6 and HP8 in Two Innate Immune Pathways

Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor

Innate immune response in insects: recognition of bacterial peptidoglycan and amplification of its recognition signal

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