An evolutionarily conserved serine protease network mediates melanization and Toll activation in Drosophila

Melanization and Toll pathway activation are essential innate immune mechanisms in insects, which result in the generation of reactive compounds and antimicrobial peptides, respectively, to kill pathogens. These two processes are mediated by phenoloxidase (PO) and Spätzle (Spz) through an extracellular network of serine proteases. While some proteases have been identified in Drosophila melanogaster in genetic studies, the exact order of proteolytic activation events remains controversial. Here, we reconstituted the serine protease framework in Drosophila by biochemical methods. This system comprises 10 proteases, i.e., ModSP, cSP48, Grass, Psh, Hayan-PA, Hayan-PB, Sp7, MP1, SPE and Ser7, which form cascade pathways that recognize microbial molecular patterns and virulence factors, and generate PO1, PO2, and Spz from their precursors. Furthermore, the serpin Necrotic negatively regulates the immune response progression by inhibiting ModSP and Grass. The biochemical approach, when combined with genetic analysis, is crucial for addressing problems that long stand in this important research field.


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Figs. S1 to S17 Fig. S1.Transcript profiles and orthologous relationships of putative members of the Drosophila immune serine protease system.Candidates of the immune protease cascades in Drosophila were selected among the serine protease genes that show a close phylogenetic relationship with the known cascade members in Manduca and Tenebrio.The phylogenetic and RNA-seq data were retrieved from previous studies (8,46).The mRNA levels, as represented by log2(FPKM+1) values, are shown in the gradient heat map from blue (0) to maroon (10).The values are labeled as 0 for 0-0.49, 1 for 0.50-1.49, 2 for 1.50-2.49,3 for 2.50-3.49, 4 for 3.50-4.49,5 for 4.50-5.49,6 for 5.50-6.49,7 for 6.50-7.49,8 for 7.50-8.49,and 9 for 8.50-9.49.

Fig. S2. Autoactivation assay of ModSP zymogen. (A)
The 10% SDS/PAGE followed by immunoblot analyses of the purified proModSP* and proModSP.ProModSP* (200 ng) or proModSP (200 ng) was treated by SDS sample buffer, separated along with protein size markers, and detected by antibody to M. sexta HP14 (left) or anti-6xHis antibody (right).The ModSP zymogen, N-terminal fragment of ModSP, and catalytic domain of ModSP are marked with dots, arrowhead, and asterisk, respectively.The C-terminal catalytic domain (~31 kDa) was hardly detected by anti-HP14 antibody because of its low immunogenicity.(B) Purified proModSP (300 ng) was incubated with insoluble S. aureus peptidoglycan (500 ng) or soluble E. coli peptidoglycan (500 ng), PGRP-SA (500 ng), GNBP1 (500 ng), and buffer A (to 12 μL) for 2 h at 37°C.Alternatively, the zymogen was incubated with curdlan (5 μg), GNBP3 (500 ng), and buffer A (to 12 μL) for 2 h at 37°C.The reaction mixture and controls were subjected to 10% SDS-PAGE under reducing condition, followed by immunoblot analyses using antibody to M. sexta HP14.Sizes and positions of the Mr markers are indicated.The ModSP zymogen and the N-terminal fragment of ModSP produced after activation cleavage are marked with dot and arrowhead, respectively.Purified procSP48 (1 μg) was incubated with E. coli peptidoglycan (1 μg), PGRP-SA (500 ng), GNBP1 (500 ng), proModSP* (500 ng), and buffer A (to 25 μL) for 2 h at 37°C.The reaction mixture and controls were separated by 10% SDS-PAGE under non-reducing condition and detected by immunoblotting using anti-FLAG antibody.Sizes and positions of the Mr markers are indicated.The cSP48 precursor and active cSP48 are marked with dot and square, respectively.(B) ModSP does not cleave procSP42.Purified procSP42 (1 μg) was incubated with E. coli peptidoglycan (1 μg), PGRP-SA (500 ng), GNBP1 (500 ng), proModSP* (500 ng), and buffer A (to 25 μL) for 2 h at 37°C.The reaction mixture and controls were separated by 10% SDS-PAGE under reducing condition (left) or non-reducing condition (right) and detected by immunoblotting using anti-FLAG antibody.Sizes and positions of the Mr markers are indicated.The cSP42 precursor and its dimer are marked with dots.(C) ProGrass activation.Purified proGrass (1 μg) was incubated with E. coli peptidoglycan (1 μg), PGRP-SA (500 ng), GNBP1 (500 ng), proModSP* (500 ng), procSP48 (100 ng), and buffer A (to 25 μL) for 2 h at 37°C.The reaction mixture and controls were subjected to 12% SDS-PAGE under non-reducing condition and detected by immunoblotting using anti-FLAG antibody.The small amount of procSP48 added in the reaction was hardly detected by the antibody, even though it has a FLAG tag at the C-terminus.Sizes and positions of the Mr markers are indicated.The Grass precursor and active Grass are marked with dot and square, respectively.(A-C) Purified proPsh (400 ng) (A) or proHayan-PA (400 ng) (B), or proHayan-PB (400 ng) (C) was incubated with active cSP48 (100 ng), proGrass (500 ng), and buffer A (to 25 μL) for 1 h at 37°C.Active cSP48 was produced in a mixture of E. coli peptidoglycan (1 μg), PGRP-SA (500 ng), GNBP1 (500 ng), proModSP* (500 ng), procSP48 (100 ng), and buffer A (to 20 μL) for 1 h at 37°C.(D, E) Purified proPsh (500 ng) or proHayan-PA (500 ng) or proHayan-PB (500 ng) was incubated with 1 μL of serially diluted subtilisin purified from B. subtilis (D) or various amounts of purified recombinant M. anisopliae Pr1A (E), and buffer A (to 25 μL) for 30 min at 29°C.The reaction mixture and controls were resolved by 10% SDS-PAGE under non-reducing condition and detected by immunoblotting using anti-Myc antibody.Sizes and positions of the Mr markers are indicated.The precursors of Psh, Hayan-PA, and Hayan-PB are marked with dots, while the active forms of Psh and Hayan-PA are marked with squares.The active form of Hayan-PB migrated to a position greater than 37 kDa under non-reducing condition, and it's hard to distinguish that from the surrounding bands (marked with vertical lines).

Fig. S16. Peptides coverage map of ModSP and Grass.
In the duplicate experiment (fig.S13), the reaction mixture and zymogen controls were resolved by 10% SDS-PAGE under reducing condition.Following in-gel chymotrypsin digestion of the Nec-protease bands and subsequent LC-MS/MS analysis, the peptides coverage map of ModSP (A) and Grass (B) in the reaction containing proModSP*, procSP48, proGrass, and Nec, were visualized.

Fig. S17. Inhibitory effect of Nec on the proteolytic activity of ModSP (A) and Grass (B). (A)
Suppression of procSP48 activation by ModSP.Purified proModSP* (500 ng), procSP48 (500 ng), Nec (800 ng), and buffer A (to 25 μL) were incubated for 2 h at 37°C.The reaction mixture and controls were subjected to 10% SDS-PAGE and immunoblot analyses using anti-FLAG antibody.(B) Suppression of proPsh and proHayan activation by Grass.Active Grass was produced in a mixture of proModSP* (500 ng), procSP48 (100 ng), proGrass (800 ng), and buffer A (to 20 μL) for 1 h at 37°C.Then Nec (800 ng, 2 μL) and proPsh (500 ng, 2 μL) or proHayan-PA (500 ng, 1 μL) were added to the mixture and incubated for 1 h at 37°C.The reaction mixture and controls were subjected to reducing SDS-PAGE and immunoblot analyses using anti-Myc antibody.The precursor and catalytic domain of cSP48, Psh and Hayan-PA are marked with dots and arrowheads, respectively.Sizes and positions of the Mr markers are indicated.

Fig
Fig. S3.cSP48 but not cSP42 functions between ModSP and Grass.(A) ProcSP48 activation.Purified procSP48 (1 μg) was incubated with E. coli peptidoglycan (1 μg), PGRP-SA (500 ng), GNBP1 (500 ng), proModSP* (500 ng), and buffer A (to 25 μL) for 2 h at 37°C.The reaction mixture and controls were separated by 10% SDS-PAGE under non-reducing condition and detected by immunoblotting using anti-FLAG antibody.Sizes and positions of the Mr markers are indicated.The cSP48 precursor and active cSP48 are marked with dot and square, respectively.(B) ModSP does not cleave procSP42.Purified procSP42 (1 μg) was incubated with E. coli peptidoglycan (1 μg), PGRP-SA (500 ng), GNBP1 (500 ng), proModSP* (500 ng), and buffer A (to 25 μL) for 2 h at 37°C.The reaction mixture and controls were separated by 10% SDS-PAGE under reducing condition (left) or non-reducing condition (right) and detected by immunoblotting using anti-FLAG antibody.Sizes and positions of the Mr markers are indicated.The cSP42 precursor and its dimer are marked with dots.(C) ProGrass activation.Purified proGrass (1 μg) was incubated with E. coli peptidoglycan (1 μg), PGRP-SA (500 ng), GNBP1 (500 ng), proModSP* (500 ng), procSP48 (100 ng), and buffer A (to 25 μL) for 2 h at 37°C.The reaction mixture and controls were subjected to 12% SDS-PAGE under non-reducing condition and detected by immunoblotting using anti-FLAG antibody.The small amount of procSP48 added in the reaction was hardly detected by the antibody, even though it has a FLAG tag at the C-terminus.Sizes and positions of the Mr markers are indicated.The Grass precursor and active Grass are marked with dot and square, respectively.

Fig. S4 .
Fig. S4.Psh and two isoforms of Hayan integrate signals from Grass and microbial proteases.(A-C)Purified proPsh (400 ng) (A) or proHayan-PA (400 ng) (B), or proHayan-PB (400 ng) (C) was incubated with active cSP48 (100 ng), proGrass (500 ng), and buffer A (to 25 μL) for 1 h at 37°C.Active cSP48 was produced in a mixture of E. coli peptidoglycan (1 μg), PGRP-SA (500 ng), GNBP1 (500 ng), proModSP* (500 ng), procSP48 (100 ng), and buffer A (to 20 μL) for 1 h at 37°C.(D, E) Purified proPsh (500 ng) or proHayan-PA (500 ng) or proHayan-PB (500 ng) was incubated with 1 μL of serially diluted subtilisin purified from B. subtilis (D) or various amounts of purified recombinant M. anisopliae Pr1A (E), and buffer A (to 25 μL) for 30 min at 29°C.The reaction mixture and controls were resolved by 10% SDS-PAGE under non-reducing condition and detected by immunoblotting using anti-Myc antibody.Sizes and positions of the Mr markers are indicated.The precursors of Psh, Hayan-PA, and Hayan-PB are marked with dots, while the active forms of Psh and Hayan-PA are marked with squares.The active form of Hayan-PB migrated to a position greater than 37 kDa under non-reducing condition, and it's hard to distinguish that from the surrounding bands (marked with vertical lines).

Fig. S9 .
Fig. S9.Identification and quantification of the target peptide after proteolytic maturation.Purified proMP1 (2 μg) was incubated with 40 ng of Pr1A, 2 μg of proPsh, and buffer A in a 12 μL reaction volume for 30 min at 37°C.The reaction mixture and zymogen controls were resolved by 10% SDS-PAGE under reducing condition.Following in-gel Glu-C endopeptidase digestion and subsequent LC-MS/MS analysis, the second MS spectra (A) and intensity (B) of the target peptide were visualized.

Fig. S11 .
Fig. S11.No significant loss of Toll signaling in double-mutant flies for Sp7 and SPE.Wildtype w 1118 flies and Sp7 SK6 , SPE SK6 , and Sp7 SK6 , SPE SK6 mutant flies were immune challenged by septic injury with B. subtilis (OD600 = 10).Flies were collected 16 h after challenge and Drs gene expression was monitored by qRT-PCR in total RNA extracts.RpL32 mRNA was used as reference gene.Data represent mean ± SD of three independent experiments, each with two technical replicates.Results were normalized to the value in w 1118 control flies.Statistical significance was calculated with One-way ANOVA with Tukey's multiple comparisons test.***p < 0.001, n.s.p > 0.05.

Fig. S12 .
Fig. S12.Formation of covalent complex of Nec and ModSP.This result was obtained by adjusting greyscale of the entire image (Fig. 8A) obtained using the anti-Nec antibody, better revealing the distinct bands at approximately 75 kDa and 80 kDa.The ModSP zymogen, its catalytic domain, ModSP-Nec complex, and cleaved Nec are marked with dot, arrowhead, squares, and asterisk, respectively.

Fig. S14 .
Fig. S14.Mass spectrometric analysis of peptides released from the Nec-protease reactions.(A) MALDI-TOF mass spectrometric analysis.The samples were prepared in the same way as described in fig.S13.(B) Second MS spectra of the N-terminal peptide of Nec, with an observed Mr of 5,223 Da, directly identified by LC-MS/MS in the Grass and Nec reaction.

Fig. S15 .
Fig. S15.Nec does not form complexes with cSP48 (A) or Psh (B).(A)Active cSP48 was produced in a mixture of proModSP* (500 ng), procSP48 (1 μg), and buffer A (to 23 μL) for 1 h at 37°C.Then Nec (800 ng, 2 μL) was added to the mixture and incubated for 1 h at 37°C.The reaction mixture and controls were subjected to 10% SDS-PAGE under reducing condition and detected by immunoblotting using anti-FLAG (left) or anti-Nec (right) antibodies.(B) Active Psh was produced in a mixture of proModSP* (500 ng), procSP48 (100 ng), proGrass (500 ng), proPsh (500 ng), and buffer A (to 23 μL) for 1 h at 37°C.Then, Nec treatment, SDS-PAGE and immunoblot analyses were performed using anti-Myc (left) or anti-Nec (right) antibodies.The cSP48/Psh precursor, its catalytic domain, and cleaved Nec are marked with dot, arrowhead, and asterisk, respectively.The complexes marked with squares were formed between ModSP and Nec, and also between Grass and Nec in panel B. Sizes and positions of the Mr markers are indicated.