Hemolymph protease-5 links the melanization and Toll immune pathways in the tobacco hornworm, Manduca sexta

Abstract

Proteolytic activation of phenoloxidase (PO) and the cytokine Spätzle during immune responses of insects is mediated by a network of hemolymph serine proteases (HPs) and noncatalytic serine protease homologs (SPHs) and inhibited by serpins. However, integration and conservation of the system and its control mechanisms are not fully understood. Here we present biochemical evidence that PO-catalyzed melanin formation, Spätzle-triggered Toll activation, and induced synthesis of antimicrobial peptides are stimulated via hemolymph (serine) protease 5 (HP5) in Manduca sexta Previous studies have demonstrated a protease cascade pathway in which HP14 activates proHP21; HP21 activates proPAP2 and proPAP3, which then activate proPO in the presence of a complex of SPH1 and SPH2. We found that both HP21 and PAP3 activate proHP5 by cleavage at ESDR¹⁷⁶*IIGG. HP5 then cleaves proHP6 at a unique site of LDLH¹¹²*ILGG. HP6, an ortholog of Drosophila Persephone, activates both proHP8 and proPAP1. HP8 activates proSpätzle-1, whereas PAP1 cleaves and activates proPO. HP5 is inhibited by Manduca sexta serpin-4, serpin-1A, and serpin-1J to regulate its activity. In summary, we have elucidated the physiological roles of HP5, a CLIPB with unique cleavage specificity (cutting after His) that coordinates immune responses in the caterpillar.

Keywords: clip domain; hemolymph protein; insect immunity; serine protease cascade; zymogen activation.

Fig. 1.

A simplified model of the serine protease network in M. sexta. The part directly surrounding HP5 (in bold font) is elucidated in the current study. The system outputs (colored red) include PO and Spätzle-1. Black arrows denote proteolytic activation of substrate proteins. Serpin-1A, Serpin-1J, and Serpin-4 inhibit M. sexta HP5.

Fig. 2.

Involvement of M. sexta HP5 in melanization and antimicrobial peptide induction. (A) The 10% SDS/PAGE and immunoblot analysis of the purified proHP5 from Sf9 cells. Aliquots of the protein (1.0 µg for staining; 25 ng for immunodetection) were treated by SDS sample buffer, separated along with protein size markers, and detected by Coomassie brilliant blue (CBB; Left) or using 1:2,000 diluted HP5 antiserum (HP5 Ab; Right). Positions and sizes of the M_r makers are indicated. (B) Concentration-dependent proPO activation caused by proHP5 in the absence of a microbial elicitor. Aliquots of 1:10 diluted IP (5.0 µL) from day 2, fifth instar at 24 h after bacterial injection were incubated with various amounts of proHP5 for 45 min at 25 °C. PO activities were measured and plotted as mean ± SEM (n = 3). (C) Synergistic enhancement of M. luteus-elicited proPO activation by proHP5. Aliquots of 1:10 diluted IP (5.0 µL) were incubated with buffer A, M. luteus (0.1 µg), proHP5 (0.1 µg), or both for 45 min at 25 °C. PO activities were assayed and plotted as the bar graph (mean ± SEM, n = 3). Interactions of plasma with M. luteus and proHP5 both led to proPO activation. (D) Induction of antimicrobial peptide transcript levels. Fifty microliters of buffer A, proHP5 (4 μg), PAP3 (250 ng), or both in buffer A were incubated for 60 min at 25 °C and then injected into individual day 0, fifth instar larvae. At 24 h after injection, fat body RNA samples were prepared from three insects in each group for qRT-PCR analysis of the antimicrobial peptide mRNA levels in these samples (three biological samples, each pooled from three larvae, and three technical replicates per sample). After normalization against rpS3, relative antimicrobial peptide transcript levels after buffer A injection are taken as an internal control (i.e., adjusted to 1.00) for calculating antimicrobial peptide mRNA levels in other samples. Student’s t test is run to reveal statistical significance of the mRNA level changes. *P < 0.05; n.s. P > 0.05.

Fig. 3.

Proteolytic activation of M. sexta proHP5 by PAP3 and HP21. (A) PAP3 cleavage. Purified proHP5 (0.1 µg, 1.0 µL), PAP3 (20 ng, 1.0 µL), and buffer A (to 20 µL) were incubated at 25 °C for 1 h. The reaction mixture and controls lacking one of the proteins were separated by 10% SDS/PAGE under reducing condition and detected by immunoblotting using HP5 (Left) or 6×His (Right) antibodies or staining (Middle gel; lane 1, 0.8 µg proHP5; lane 2, 50 ng PAP3; and lane 3, both proHP5 and PAP3, CBB; lane 4, 800 ng proHP5 and 20 ng PAP3, silver stain, 15% gel, position of the 10 kDa marker in red). (B) HP21 cleavage. Purified proHP5 (25 ng), proHP21 (10 ng), HP14 (10 ng, from a mixture of preincubated 1.0 µg E. coli peptidoglycan with 0.3 µg PGRP1, 0.3 µg MBP, and 0.1 µg proHP14 in 24 µL buffer A at 37 °C for 1 h), and buffer A were incubated at 25 °C for 1 h. The reaction mixture and controls were subjected to 10% SDS/PAGE and immunoblot analysis using HP5 antibodies. Sizes and positions of the M_r markers are indicated on the left (except for the silver stained gel strip). The HP5 precursor, catalytic domain, and light chain are marked with circles, triangles, and asterisk, respectively.

Fig. 4.

Cleavage activation of proHP6 and proSPH2 by M. sexta HP5. (A) HP6. PAP3 (20 ng), proHP5 (25 ng), proHP6 (50 ng), and buffer A (to 12 µL) were incubated at 25 °C for 1 h. The reaction mixture and controls lacking one of the proteins were resolved by 10% SDS/PAGE under the reducing condition and detected by immunoblotting using HP6 antibodies. Sizes and positions of the M_r markers are indicated on the left. (B) SPH2. HP21 was produced in a mixture of 1.0 µg E. coli peptidoglycan with 0.3 µg PGRP1, 0.3 µg MBP, 0.1 µg proHP14, and 0.1 µg proHP21 in 30 µL buffer A at 37 °C for 1 h. HP21 (20 ng) was then incubated with proHP5 (25 µg), proSPH2 (0.1 µg), and buffer A (to 12 µL) at 25 °C for 1 h. This mixture and controls lacking one of the components were subjected to 10% SDS/PAGE and immunoblot analysis using SPH2 antibody. Arrowhead indicates HP6 catalytic domain and SPH2 heavy chain. (C) Function of SPH2 activated by HP5 along with proSPH1 as a PAP cofactor. A mixture of HP21 (20 ng), proHP5 (25 ng), PAP1 (20 ng), proSPH1 (0.1 µg), proSPH2 (0.1 µg), proPO (0.16 µg), and buffer A (to 20 µL) was incubated at 25 °C for 50 min. In addition to the negative controls lacking one of the components, a positive control included the natural complex of SPH1 and SPH2 (20 ng) isolated from hemolymph (30) reacted with PAP1 (20 ng) and proPO (0.16 µg) to generate active PO. PO activities were measured and plotted as mean ± SEM (n = 3). Student’s t test is run to reveal statistical significance of the PO activity differences between the indicated groups. *P < 0.05.

Fig. 5.

Proteolytic processing of proHP8 and proPAP1 by HP5-generated HP6. (A) Activation of proPAP1. PAP3 (20 ng), proHP5 (25 ng), proHP6 (50 ng), proPAP1 (50 ng), and buffer A (to 20 µL) were incubated at 25 °C for 1 h. The reaction mixture and controls were subjected to 10% SDS/PAGE and immunoblot analysis (Left) using PAP1 antibodies. In a duplicated experiment, amidase activities in the reaction and control mixtures were determined using 150 µL of 25 µM of IEARpNA (46) and plotted as mean ± SD (n = 3). (B) Activation of proHP8. PAP3 (20 ng), proHP5 (25 ng), proHP6 (50 ng), proHP8 (60 ng), and buffer (to 20 µL) were incubated at 25 °C for 1 h. Half of the reaction mixture and controls were separated by 10% SDS/PAGE under reducing condition and detected by immunoblotting using HP8 antibodies. Sizes and positions of the M_r markers are indicated. Arrowhead indicates catalytic domain.

Fig. 6.

Formation of an SDS-stable complex of HP5 complex with M. sexta serpins. (A) Composition of the serpin-protease complex (SPC) isolated from induced plasma (IP). Protease cascades in IP were stimulated by addition of M. luteus in the presence of 1-phenyl-2-thiourea to prevent melanization at 25 °C for 30 min. Following phenylmethylsulfonyl fluoride treatment to inhibit remaining protease activity, the HP5-serpin complexes (along with proHP5 and its cleavage products) were isolated by affinity chromatography on an HP5 antibody column. Elution fraction 4 was subjected to 10% SDS/PAGE followed by immunoblot analysis (Left) or CBB staining (Right). Arrowhead indicates SPC. (B) proHP6 activation blocked by inhibition of HP5 by serpin-1J. PAP3 (20 ng), proHP5 (25 ng), proHP6 (50 ng), and serpin-1J (1 µg) were incubated at 25 °C for 10 min. The samples were separated by 10% SDS/PAGE and detected by immunoblot analysis using HP6 antibodies. (C) SPC formation using purified proteins. PAP3-treated proHP5 was reacted with serpin-1J at 25 °C for 10 min. The mixture and controls were subjected to 10% SDS/PAGE and immunoblotting. Sizes of the molecular mass markers are indicated.