Fig 1.
Expression patterns of immune-related molecules CLIP2, Spätzle1, and gloverin2 in silkworm immune tissues.
Expression analysis of immune-related molecules in the fat body and hemolymph of silkworm at different developmental stages (A and B). a-tubulin was used as the reference protein. IV-3: day 3 of fourth-instar larvae; IV-M: molting of fourth-instar larvae; V-0~V-6: days 0–6 of fifth-instar larvae; W-1–W-3: days 1–3 after wandering; P-1: day 1 after pupation. The lower bands recognized by CLIP2 antibodies in fat body and hemolymph may be degraded bands resulting from CLIP2 being unspecifically cleaved. The red arrow indicates a covalent complex formed by CLIP2 and its specific inhibitor. (C) Effects of Escherichia coli and Micrococcus luteus on the expression of CLIP2 in silkworm hemolymph. (D) Effects of bacteria, lipopolysaccharide (LPS), peptidoglycan (PGN) and curdlan (CDN) on the expression of CLIP2 in silkworm hemolymph. Error bars represent mean ± SD (n = 3). **P < 0.01; ***P < 0.001. M: protein molecular weight marker.
Fig 2.
In vitro analysis of proSpätzle1 (proSpz1) cleavage by CLIP2.
(A) Purification of recombinant proCLIP2Xa protein by Ni-NTA (nitrilotriacetic acid) affinity chromatography with a stepwise imidazole gradient. CL: crude extract; FT: flow-through; 20–500: elution fractions of a stepwise imidazole gradient. The arrow indicates the recombinant proCLIP2Xa. M: protein molecular weight marker; CBS: commassie blue staining; IB: immunoblotting. The hydrolysis activity of recombinant proCLIP2Xa was detected using polypeptide substrate Ile-Glu-Ala-Arg-p-nitroanilide (IEARpNA) (B) and the activity-based probe desthiobiotin-FP (C). ProCLIP2Xa (0.75 μg) was pre-incubated with factor Xa (1 μg). Subsequently, the catalytic activity of factor Xa (1 μg), proCLIP2Xa (0.75 μg), and pre-incubation solution of both were detected by a multifunctional microplate reader using IEARpNA as a substrate. Desthiobiotin-FP probe can specifically covalently label the active serine site of serine hydrolase. (D) Proteolytic activation of proSpz1 by recombinant CLIP2 (rCLIP2). The black arrow indicates the rCLIP2, the red arrow indicates the proSpz1 and the C-terminal fragment of proSpz1 (Spz1-C). Error bars represent mean ± SD (n = 3). ***P < 0.001.
Fig 3.
Effects of proSpätzle1 (proSpz1) and CLIP2 injection on the humoral immune response.
Day 2 of fifth-instar larvae were injected with buffer, CLIP2, proSpz1, the incubation mixture of CLIP2 and proSpz1. At 12 h after injection, the fat body and hemolymph of each group were collected. The expression levels of antibacterial peptides (AMPs) were determined by RT-qPCR (A) and immunoblotting (B). Error bars show mean ± SD (n = 3). Different letters represent the significant differences (one-way ANOVA followed by Tukey’s test, P < 0.05). Furthermore, larvae at day 1 of the wandering stage were injected with CLIP2 or BSA. At 12 h and 24 h after injection, the fat body and hemolymph of each group were collected. The expression levels of antibacterial peptides (AMPs) were determined by RT-qPCR (C) and immunoblotting (D). Size and positions of molecular mass standards are indicated to the left of each blot. (E) Hemolymph collected 12 h post-injection of CLIP2 was used to determine the antimicrobial activity against Micrococcus luteus (left) and Escherichia coli (right). Error bars represent mean ± SD (n = 3). *** P < 0.001.
Fig 4.
Identification and inhibitory activity analysis of physiological regulators of CLIP2.
(A) Immunoprecipitated purified CLIP2-inhibitor complexes were subjected to SDS-PAGE and were detected by coomassie blue staining (CBS) or immunoblotting (IB). CLIP2 and associated proteins isolated from normal hemolymph (lane 1) or Micrococcus luteus-treated hemolymph (lane 2). The red arrow indicates the potential CLIP2-serpin complex. Coomassie blue stained bands corresponding to potential complexes were excised for trypsin digestion followed LC-MS/MS analysis; the identified CLIP and serpins were listed in (B). iBAQ (intensity-based absolute quantification) intensity was used to estimate the abundance of each protein. The target protease type of serpin is based on previous studies [31], with T for trypsin and C for chymotrypsin. SDS-stable complex formation between rCLIP2 and serpin-1a (C) or serpin-6 (D). rCLIP2 (200 ng) was incubated with corresponding serpins at room temperature for 5 min under a molar mass ratio of 1:5 (rCLIP2: serpins). The samples were subjected to SDS-PAGE and immunoblot analysis using antibodies against serpin-1a (upper), serpin-6(upper), CLIP2 (middle), and His-tag (lower). Red arrows indicate the CLIP2-serpin complex. (E) rCLIP2 (250 ng) was incubated with serpin-1a or serpin-6 at room temperature for 0, 0.5, 1, 1.5, 2, and 2.5 min under a molar mass ratio of 1:5 (rCLIP2: serpins). The reaction mixtures were separated by SDS-PAGE, followed by immunoblot analysis with antibodies against CLIP2. The red arrow indicates the CLIP2-serpin complex. Intensity of the ~80 kDa band corresponding to CLIP2-serpin complex was analyzed using ImageJ, and plotted against incubation time. (F) Serpin-1a (upper) or serpin-6 (lower) inhibits the cleavage of proSpz1 by CLIP2. CLIP2 (100 ng) was incubated at room temperature for 5 min with a 5-fold molar excess of serpin-1a or serpin-6, then incubated with proSpz1 (1 μg) at room temperature for 30 min. The samples were subjected to SDS-PAGE and immunoblotting using anti-His-tag antibodies. Red arrows indicate the CLIP2-serpin complex. Black arrows indicate the proSpz1 or serpin. In addition, the samples from each of the above groups were added to the BmN cell culture medium; the cultured BmN cells were collected 24 h later to detect the expression of the antimicrobial peptide gene gloverin2 by RT-qPCR (G). Error bars represent mean ± SD (n = 3). *P < 0.05; *** P < 0.001.
Fig 5.
Expression patterns and synergistic regulation analysis of serpin-1a and serpin-6.
B. mori serpin-1 and -6 mRNA and protein levels in in fat body (A) and hemolymph (B) from different developmental stages. The fat body and hemolymph from day 3 of the fourth-instar larvae to day 1 of the pupal stage were collected for RNA isolation and immunoblotting. The transcript levels of serpin-1, -3 to -7, and -32 in the fat body were measured by RT-qPCR (A). Error bars represent mean ± SD (n = 3). The protein abundances of serpin-1 and -6 were detected in the fat body and hemolymph by immunoblotting using antibodies against serpin-1a and -6 (B). Since serpin-1a antibodies can recognize all variants of serpin-1, the detection of serpin-1a protein expression in silkworm fat body and hemolymph is labeled as serpin-1. a-tubulin was used as the reference protein. IV-3: day 3 of fourth-instar larvae; IV-M: molting of fourth-instar larvae; V-0~V-6: days 0–6 of fifth-instar larvae; W-1–W-3: days 1–3 after wandering; P-1: day 1 after pupation. The red arrow indicates the potential serpin-protease complex. (C) Effects of Escherichia coli and Micrococcus luteus on the expression of serpin-1 and -6 in silkworm immune-related tissues. The transcript levels of serpin-1a and -6 in the fat body and hemocyte were measured by RT-qPCR (upper and middle). The protein abundances of serpin-1 and -6 in hemolymph detected by immunoblotting using antibodies against serpin-1a and -6 (lower). (D) Immunoprecipitated purified serpin and associated proteins were subjected to SDS-PAGE and detected by immunoblotting (IB) using antibodies against serpin-1a (upper left), serpin-6 (upper right), and CLIP2 (lower). CH: control hemolymph; LPSiH: the hemolymph induced by lipopolysaccharide (LPS); PGNiH: the hemolymph induced by peptidoglycan (PGN); CDNiH: the hemolymph induced by curdlan (CDN). M: protein molecular weight marker. Red arrows indicate the CLIP2-serpin complex. (E) Intensity of the CLIP2-serpin complex detected by antibodies against CLIP2 (D lower) was analyzed using ImageJ. The remaining value after subtracting the gray value of the CH group from the gray value of the PAMPiH (PAMP induced hemolymph) group is used for histogram plotting to compare the intensity of the complex formed between CLIP2 and serpin-1a or -6 under pathological conditions.
Fig 6.
Effect of knockout serpin-1 on the background expression of antimicrobial peptide genes.
(A) Serpin-1, -6, and CLIP2 protein levels in the fat body and hemolymph of wild-type (WT) and serpin-1 knockout (serpin-1-/-) individuals on day 3 of the fifth-instar. The protein levels of serpin-1, -6 and CLIP2 were detected by immunoblotting (IB) using antibodies against serpin-1a (upper), -6 (middle) and CLIP2 (lower). a-tubulin was used as the reference protein. M: protein molecular weight marker. Red arrows indicate the CLIP2-serpin-1a complex. (B) The background expression levels of antibacterial peptide genes in the fat body of WT and serpin-1-/- individuals on day 3 of the fifth-instar and day 1 of the wandering stage detected using RT-qPCR. Error bars represent mean ± SD (n = 3). **P < 0.01; ***P < 0.001.
Fig 7.
Effects of serpin-1 knockout on serpin-6 expression and immune homeostasis in silkworm under immune challenge.
(A) Wild-type (WT) and serpin-1 knockout (serpin-1-/-) individuals were respectively injected with Escherichia coli, Pseudomonas aeruginosa, Micrococcus luteus, or Enterococcus mundtii; survival curves were plotted until 72 h post-infection. Statistical analysis between WT and serpin-1-/- groups were calculated using the log-rank test. (B) The hemolymph of each group was collected at 24 h post-injection to detect the levels of serpin-1, serpin-6, CLIP2 and gloverin2 proteins by immunoblotting (IB). WT and serpin-1-/- individuals were injected with 5 μL PBS (CK), 2.5 μg lipopolysaccharide (LPS) or peptidoglycan (PGN). At 18 h post-injection, the fat body of each group was collected to detect the transcript levels of serpins (C) and antibacterial peptide genes (D) by RT-qPCR; the hemolymph of each group was collected to detect the protein levels of serpins, CLIP2 and gloverin2 by immunoblotting (E). M: protein molecular weight marker. Error bars represent mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 8.
Schematic diagram representing the synergistic mechanism of serpin-1a and serpin-6 in the regulation of silkworm Toll pathway homeostasis under normal and infection conditions.
Activation of the silkworm immune cascade CLIP2-Spz1 is co-regulated by serpin-1a and serpin-6: (1) under normal conditions, the activity of CLIP2 is regulated primarily by the constitutively expressed serpin-1a in hemolymph; Toll pathway activation is maintained at background levels in the fat body. (2) under pathogen infection conditions, the expression and activation of CLIP2 in the hemolymph increases, and serpin-6 expression is induced to assist serpin-1a in regulating CLIP2 activity, effectively avoiding excessive Toll pathway activation, and maintaining immune homeostasis. PAMPs, pathogen-associated molecular patterns; LPS, lipopolysaccharide; PGN, peptidoglycan; AMPs, antimicrobial peptides. The thickness of the lines indicates the intensity of signal transmission or the level of gene expression.