Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 286 (40), 35087-95

93-kDa Twin-Domain Serine Protease Inhibitor (Serpin) Has a Regulatory Function on the Beetle Toll Proteolytic Signaling Cascade

Affiliations

93-kDa Twin-Domain Serine Protease Inhibitor (Serpin) Has a Regulatory Function on the Beetle Toll Proteolytic Signaling Cascade

Rui Jiang et al. J Biol Chem.

Abstract

Serpins are protease inhibitors that play essential roles in the down-regulation of extracellular proteolytic cascades. The core serpin domain is highly conserved, and typical serpins are encoded with a molecular size of 35-50 kDa. Here, we describe a novel 93-kDa protein that contains two complete, tandemly arrayed serpin domains. This twin serpin, SPN93, was isolated from the larval hemolymph of the large beetle Tenebrio molitor. The N-terminal serpin domain of SPN93 forms a covalent complex with the Spätzle-processing enzyme, a terminal serine protease of the Toll signaling cascade, whereas the C-terminal serpin domain of SPN93 forms complexes with a modular serine protease and the Spätzle-processing enzyme-activating enzyme, which are two different enzymes of the cascade. Consequently, SPN93 inhibited β-1,3-glucan-mediated Toll proteolytic cascade activation in an in vitro system. Site-specific proteolysis of SPN93 at the N-terminal serpin domain was observed after activation of the Toll proteolytic cascade in vivo, and down-regulation of SPN93 by RNAi sensitized β-1,3-glucan-mediated larval death. Therefore, SPN93 is the first serpin that contains twin tandemly arrayed and functionally active serpin domains that have a regulatory role in the larval Toll proteolytic signaling cascade.

Figures

FIGURE 1.
FIGURE 1.
Purification of SPN93. A, chromatographic steps to purify native SPN93 from the larval hemolymph. B, chromatogram of hydroxylapatite column chromatography. Each fraction (5 μl, Fractions 3–11) was incubated with aSPE. The aSPE-mediated amidase activity measured using a synthetic α-thrombin substrate conjugated with fluorescence dye was inhibited by addition of fractions 3–5 (gray bars). C indicates control, i.e. aSPE amidase was assayed without column fraction. A280 (line with filled circles) and sodium phosphate (NaPi) gradient (dot line) are shown. C, SDS-PAGE pattern stained with CBB for the chromatography fractions shown in B. Fractions 3–5 were pooled. D, identification of covalently bound serpin-protease complex between aSPE and native SPN93. Both aSPE and SPN93 were incubated and separated by SDS-PAGE followed by CBB staining (left panel) or by Western blotting using an anti-SPE antibody (right panel). Arrows indicate the generated 76-kDa serpin-protease complexes.
FIGURE 2.
FIGURE 2.
Complex formation between SPN93 and aSPE, aMSP, or aSAE. A, twin-domain serpin organization of SPN93. RCL indicates the reactive center loop. Red arrows indicate the putative cleavage sites. Cloned regions for recombinant proteins are indicated. B, SDS-PAGE patterns of purified rSPN93 proteins. Column 1, whole 93-kDa serpin (rSPN93-NC); column 2, N-terminal domain of SPN93 (rSPN93-N); column 3, C-terminal domain (rSPN93-C); column 4, rSPN93-N-K371Q mutant. C, inhibition of SPE-mediated amidase by rSPN93s. Samples of rSPN93 as indicated (2 μg) were incubated with 50 ng of aSPE for 15 min at 30 °C, and then the amidase activity of aSPE was determined. D, complex formation between rSPN93-NC and aSPE. Both aSPE (100 pmol) and rSPN93-NC (50 pmol) were incubated for 4 h at room temperature and separated by SDS-PAGE. The new bands that were generated are indicated as (a) and (b). E, N-terminal sequence of band (a) showed a mixture sequence of rSPN93-NC and aSPE proteins. The blue color-highlighted GHM is the cloning artifact of rSPN93-NC. F, N-terminal sequence of band (b) was identical to the amino acid sequence of Phe-372 to Asn-380 of SPN93. G, molecular diagram of the 76-kDa aSPE-SPN93 complex formation and release of the C-terminal 53-kDa fragment. N-ter and C-ter indicate N-terminal and C-terminal serpin domain, respectively.
FIGURE 3.
FIGURE 3.
Complex formation between rSPN93-C and aMSP or aSAE. A–C, combinations including aMSP, aSAE, or aSPE (each 500 ng) and rSPN93-NC, rSPN93-N, or rSPN93-C (1 μg), as indicated, incubated for 1 h at 30 °C and separated on SDS-PAGE under reducing conditions. The specific serine protease-serpin complexes indicated by red arrows were visualized by Western blotting using anti-SPN93-NC antibody (α-SPPN93-NC), anti-SPN93-N-antibody (α-SPN93-N), or anti-SPN93-C-antibody (α-SPN93-C) as indicated. D, identification of C-terminal fragments of rSPN93-C released after serpin/protease complex formation. Either aMSP, aSPE (50 pmol) and/or rSPN93-C (100 pmol), as indicated, was incubated for 4 h at room temperature and then analyzed by Tricine SDS-PAGE and stained with CBB. Bands 1 and 2 were generated as well as the specific serine protease-serpin complexes indicated by red arrows. E, N-terminal sequence of band 1 or 2 corresponding to Met-778 to Ala-782 of SPN93.
FIGURE 4.
FIGURE 4.
SPN93-N blocked β-1,3-glucan-mediated pro-Spätzle processing in an in vitro system. When the six Toll cascade-regulating factors, GNBP3, pro-MSP, pro-SAE, pro-SPE, pro-Spätzle, and Ca2+, were co-incubated with β-1,3-glucan (100 ng) for 5 min, processed Spätzle (12 kDa) was detected by Western blotting (WB) with an affinity-purified anti-Spätzle antibody (α-Spätzle, column 2). When serpins were added to the reaction mixture, pro-Spätzle processing was inhibited by rSPN93-NC (column 3) and rSPN93-N (column 4), but not by rSPN93-C (column 5). The 30-kDa pro-Spätzle and the 12-kDa processed Spätzle are indicated with arrows. Data shown are representative of at least three independent experiments.
FIGURE 5.
FIGURE 5.
β-1,3-Glucan-induced proteolysis of SPN93 in vivo. A, localization of SPN93. Proteins (40 μg) from plasma, fat bodies, and hemocytes were separated using SDS-PAGE and visualized by Western blotting (WB) against SPN93 (left panel) or by CBB staining (right panel). B–D, proteolysis processing of SPN93 and other Toll cascade-regulating serpins after β-1,3-glucan injection. Larval hemolymph of T. molitor was collected after injection of β-1,3-glucan (600 ng) at the indicated times, and 40 μg of proteins from collected hemolymph was separated by SDS-PAGE under reducing conditions and visualized by Western blotting using each SPN antibody (B, C) or by CBB staining (D). C lane (lane 6 in B) contains in vitro complex formation products between rSPN93-NC (100 pmol) and aSPE (50 pmol).
FIGURE 6.
FIGURE 6.
Activated SPE preferred to make complex SPN93 rather than SPN48 in vitro. Combinations including rSPN93-NC, rSPN48 (each 4.5 pmol), and active form of SPE (aSPE, 1.5 pmol) as indicated, were incubated for 0, 2, 5, or 15 min at 30 °C in 20 mm Tris-HCl, pH 7.8, and separated using SDS-PAGE under reducing conditions. The specific serpin-protease complexes indicated by red arrows were visualized by Western blotting (WB) using anti-SPN93 antibody (left panel) or anti-SPE48 antibody (right panel).
FIGURE 7.
FIGURE 7.
Functional analysis of SPN93 in vivo. A, dsRNA-mediated silencing of SPN93 in T. molitor larva. Four micrograms of SPN93 dsRNA or control dsRNA was injected into a fifth instar T. molitor larva. After 3 days of dsRNA injection, silencing of target mRNA was confirmed by RT-PCR. B, effect of β-1,3-glucan-mediated melanin synthesis on RNAi silenced larvae. After confirming a silencing of the SPN93 mRNA, the pretreated larvae by SPN93 dsRNA (circles) or control RNA (squares), along with nontreated larvae (triangles) were injected with 600 ng of β-1,3-glucan per larva (closed symbols) or with PBS (open symbols). The melanization of larvae was monitored for 3 days (20 larvae/group).
FIGURE 8.
FIGURE 8.
SPN93 targets serine proteases of the Toll proteolytic cascade in beetle larvae. The β-1,3-glucan bound by GNBP3 recruits zymogen form of MSP and activates the protease cascade composed of MSP, SAE, and SPE (17). Activated SPE converts pro-Spätzle into processed Spätzle leading to the production of AMPs via the Toll receptor-mediated signaling pathway and also initiates the melanin synthesis by activation of pro-phenoloxidase (pro-PO) and pro-serine protease homolog 1 (pro-SPH1) via a PO-SPH1 melanization complex. The N-terminal serpin domain of twin-domain serpin SPN93 inactivates aSPE, whereas the C-terminal domain inactivates aMSP and aSAE.

Similar articles

See all similar articles

Cited by 8 articles

See all "Cited by" articles

Publication types

Associated data

LinkOut - more resources

Feedback