The Manduca sexta serpinome: Analysis of serpin genes and proteins in the tobacco hornworm

Li, Miao; Christen, Jayne M.; Dittmer, Neal T.; Cao, Xiaolong; Zhang, Xiufeng; Jiang, Haobo; Kanost, Michael R.

doi:10.1016/j.ibmb.2018.09.008

Cited by 26 publications

(30 citation statements)

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“…We have shown that HP5 cleaves proHP6 after a His residue, a very unusual protease specificity. None of the M. sexta serpins has a His residue at the predicted P1 residue of the scissile bond of reactive center loop that determines primary selectivity of serpins in protease inhibition (30). Serpin-4 has an Arg residue at the P1 site (31).…”

Section: Resultsmentioning

confidence: 99%

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

Wang

Yang

Cao

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Resultsmentioning

confidence: 99%

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

Wang

Yang

Cao

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…In a previous study, we found that full length serpin-3 expressed in E. coli was not fully active 13 . Therefore, we produced a new construct with deletion of the amino-terminal 30 residues of serpin-3, not present in most other serpins 11 and found in preliminary experiments that this construct, serpin-3ΔN, retained inhibitory activity. The serpin-3ΔN sequence encoding 31-435 residues of the mature protein was amplified from full-length serpin-3 in vector H6pQE60 13 by PCR, using a forward primer.…”

Section: Expression and Purification Of Pap3 And Serpin-3∆n Recombinmentioning

confidence: 99%

“…Immune responses mediated by hemolymph serine proteases and their regulation by serpins have been studied intensively in a model species, Manduca sexta , the tobacco hornworm 10 . The M. sexta genome contains 32 serpin genes, and only 10 of these have been studied with regard to their function 11 , 12 . M. sexta serpin-3 expression increases during immune challenge, and it inhibits key proteases in two immune cascades.…”

Section: Introductionmentioning

confidence: 99%

Peptides based on the reactive center loop of Manduca sexta serpin-3 block its protease inhibitory function

Takahashi

Kanost

2020

Sci Rep

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one innate immune response in insects is the proteolytic activation of hemolymph prophenoloxidase (propo), regulated by protease inhibitors called serpins. in the inhibition reaction of serpins, a protease cleaves a peptide bond in a solvent-exposed reactive center loop (RcL) of the serpin, and the serpin undergoes a conformational change, incorporating the amino-terminal segment of the RcL into serpin β-sheet A as a new strand. This results in an irreversible inhibitory complex of the serpin with the protease. We synthesized four peptides with sequences from the hinge region in the RCL of Manduca sexta serpin-3 and found they were able to block serpin-3 inhibitory activity, resulting in suppression of inhibitory protease-serpin complex formation. An RCL-derived peptide with the sequence Ser-Val-Ala-Phe-Ser (SVAFS) displayed robust blocking activity against serpin-3. Addition of acetyl-SVAFS-amide to hemolymph led to unregulated proPO activation. Serpin-3 associated with Ac-SVAFS-COO − had an altered circular dichroism spectrum and enhanced thermal resistance to change in secondary structure, indicating that these two molecules formed a binary complex, most likely by insertion of the peptide into β-sheet A. The interference of RCL-derived peptides with serpin activity may lead to new possibilities of "silencing" arthropod serpins with unknown functions for investigation of their physiological roles. Serpins have been extensively studied since the name was coined in 1985 to define a family of serine protease inhibitors with conserved tertiary structure 1. Broadly distributed throughout vertebrates, invertebrates, plants, unicellular organisms and even viruses, most serpins act as serine protease inhibitors, although some have other functions, including acting as hormone transporters and molecular chaperones 2,3,4,5,6,7. The tertiary structure of serpins is highly conserved, comprised of three β-sheets, 8-9 α-helices, and a solvent-exposed reactive center loop (RCL) that is near the carboxyl-terminal end of the serpin sequence 2. When a serpin encounters a target protease, the protease cleaves a specific peptide bond of the RCL (designated P1-P1′, the "scissile bond") but the second half of the hydrolysis reaction does not occur, leaving the protease and serpin in an acyl-intermediate state. The amino-terminal end of the RCL rapidly inserts into serpin β-sheet A as a new strand (strand 4A) (Fig. 1a), and the protease is translocated to the other side of serpin molecule, resulting in formation of an irreversible inhibitory complex, with the active site of the protease distorted 7. Alternatively, the cleavage of the scissile bond can proceed as a typical protease hydrolysis reaction, producing a shortened and disabled serpin protein, with no inhibition of the protease. The balance of these two types of serpin-protease interactions determines the efficiency of a serpin as an inhibitor of a given protease 8. Rapidly expanding sequence data from insect genomes and transcriptomes has resulted in discovery of thousands of...

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“…The number of serpin genes documented in insects seems to be dominated by lepidopteron insects as compared to other insect orders. For instance, in B. mori , 34 serpin genes have been reported (Zou, Picheng, Weng, Mita, & Jiang, ) followed by 32 in M. sexta (M. Li et al, ). Though dipteran insects contain a fair number of serpin genes, however, the number varies from 31 reported in Culex quinquefasciatus to 18 in Anopheles gambiae (Gulley et al, ).…”

Section: Serpins In Insects and Their Role In Host‐pathogen Interactionmentioning

confidence: 99%

Role of serine protease inhibitors in insect‐host‐pathogen interactions

Shakeel

Mandal

et al. 2019

Arch Insect Biochem Physiol

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Serine protease inhibitors (serpins), evolutionary old, structurally conserved molecules, are a superfamily of proteins found in almost all living organisms. Serpins are relatively large, typically 350–500 amino acids in length, with three β‐sheets and seven to nine α‐helices folding into a conserved tertiary structure with a reactive center loop. Serpins perform various physiological functions in insects, including development, digestion, host‐pathogen interactions, and innate immune response. In insects, the innate immune system is characterized as the first and major defense system against the invasion of microorganisms. Serine protease cascades play a critical role in the initiation of innate immune responses, such as melanization and the production of antimicrobial peptides, and are strictly and precisely regulated by serpins. Herein, we provide a microreview on the role of serpins in the insect‐host‐pathogen interactions, emphasizing their role in immune responses, particularly in diamondback moth (Plutella xylostella), highlighting the important discoveries and also the gaps that remain to be explored in future studies.

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The Manduca sexta serpinome: Analysis of serpin genes and proteins in the tobacco hornworm

Cited by 26 publications

References 86 publications

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

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

Peptides based on the reactive center loop of Manduca sexta serpin-3 block its protease inhibitory function

Role of serine protease inhibitors in insect‐host‐pathogen interactions

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