Self-association of an Insect β-1,3-Glucan Recognition Protein Upon Binding Laminarin Stimulates Prophenoloxidase Activation as an Innate Immune Response

Takahashi, Daisuke; Dai, Hui; Hiromasa, Yasuaki; Krishnamoorthi, Ramaswamy; Kanost, Michael R.

doi:10.1074/jbc.m114.583971

Cited by 31 publications

(27 citation statements)

References 47 publications

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“…Combining the results presented here with our previous studies on βGRPs (18,19) provides further insight into the molecular basis for the initiation of the protease cascade triggered by the autoactivation of HP14 (Fig. 7).…”

Section: Discussionsupporting

confidence: 76%

“…autoactivation, resulting from direct interaction with βGRP2. These results, together with our previous studies showing the selfassociation of insect βGRPs upon binding β-1,3-glucan (18,19), provide an improved model for the molecular basis underlying the initiation of serine protease cascades for insect immune responses.…”

supporting

confidence: 60%

“…2A, lanes 6-8). These results demonstrate that N-terminal LAs are required for the autoactivation cleavage of proHP14 and suggest that HP14 LAs mediate the interaction with the macromolecular complexes of βGRP2 with β-1,3-glucan (18,19).…”

Section: Resultsmentioning

confidence: 73%

“…Fungal infections in hemolymph are first detected by binding of βGRP2 to fungal β-1,3-glucans (Fig. 7A), which induces self-association of βGRP2s through protein-protein interaction between their N-terminal carbohydrate binding modules (18,19) (Fig. 7B).…”

Section: Discussionmentioning

confidence: 99%

“…Such interactions may have broader relevance leading to more detailed understanding of the molecular control of protease cascades that function in innate immune responses. (18,19). (C) The C-terminal glucanase-like domain of βGRP2 interacts with LA domains of HP14, recruiting multiple HP14 zymogens (proHP14) to the β-1,3-glucan/βGRP2 platform.…”

Section: Discussionmentioning

confidence: 99%

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Initiating protease with modular domains interacts with β-glucan recognition protein to trigger innate immune response in insects

Takahashi

Garcia

Kanost

2015

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

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The autoactivation of an initiating serine protease upon binding of pattern recognition proteins to pathogen surfaces is a crucial step in eliciting insect immune responses such as the activation of Toll and prophenoloxidase pathways. However, the molecular mechanisms responsible for autoactivation of the initiating protease remains poorly understood. Here, we investigated the molecular basis for the autoactivation of hemolymph protease 14 (HP14), an initiating protease in hemolymph of Manduca sexta, upon the binding of β-1,3-glucan by its recognition protein, βGRP2. Biochemical analysis using HP14 zymogen (proHP14), βGRP2, and the recombinant proteins as truncated forms showed that the amino-terminal modular low-density lipoprotein receptor class A (LA) domains within HP14 are required for proHP14 autoactivation that is stimulated by its interaction with βGRP2. Consistent with this result, recombinant LA domains inhibit the activation of proHP14 and prophenoloxidase, likely by competing with the interaction between βGRP2 and LA domains within proHP14. Using surface plasmon resonance, we demonstrated that immobilized LA domains directly interact with βGRP2 in a calcium-dependent manner and that high-affinity interaction requires the C-terminal glucanase-like domain of βGRP2. Importantly, the affinity of LA domains for βGRP2 increases nearly 100-fold in the presence of β-1,3-glucan. Taken together, these results present the first experimental evidence to our knowledge that LA domains of an insect modular protease and glucanase-like domains of a βGRP mediate their interaction, and that this binding is essential for the protease autoactivation. Thus, our study provides important insight into the molecular basis underlying the initiation of protease cascade in insect immune responses.innate immunity | insect immunity | modular serine protease | pattern recognition receptor | hemolymph E xtracellular protease cascades play an essential role in a variety of biological processes including embryonic development (1) and host defenses such as blood coagulation and the complement system (2, 3). Protease cascades also function in insect innate immune responses such as melanization and antimicrobial peptide synthesis by inducing the prophenoloxidase (proPO) and Toll pathways, respectively (3-5).In protease cascades, inactive protease zymogens that circulate in an extracellular milieu become sequentially activated in pathways beginning with an initiating modular serine protease, which autoactivates in response to internal or external signals. In hemolymph (insect blood), pathogen-associated molecular patterns (PAMPs) such as peptidoglycans in bacteria and β-1,3-glucan in fungi are first detected by pathogen recognition receptors (PRRs) such as peptidoglycan recognition proteins (PGRPs) and β-1,3-glucan recognition proteins (βGRPs) (4-8). As demonstrated by genetic and biochemical studies of protease cascades in insect immune responses, those recognition signals are integrated by an initiating protease (9-12). The initiating p...

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Section: Discussionsupporting

confidence: 76%

supporting

confidence: 60%

Section: Resultsmentioning

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

Initiating protease with modular domains interacts with β-glucan recognition protein to trigger innate immune response in insects

Takahashi

Garcia

Kanost

2015

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

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Transcriptomic analysis provides insights into the immune responses and nutrition in Ostrinia furnacalis larvae parasitized by Macrocentrus cingulum

Chen

Song

et al. 2021

Arch Insect Biochem Physiol

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Macrocentrus cingulum is a principal endoparasite of Ostrinia furnacalis larvae. M. cingulum larvae repress host immune responses for survival and ingest host nutrients for development until emerging. However, most investigations focused on the mechanisms of how wasps repress the host immunity, the triggered immune responses and nutrient status altered by wasps in host are neglected. In this study, we found that parasitized O. furnacalis larvae activated fast recognition responses and produced some effectors such as lysozyme and antimicrobial peptides, along with more consumption of trehalose, glucose, and even lipid to defend against the invading M. cingulum. However, the expression of peroxidase 6 and superoxide dismutase 2 (SOD 2) was upregulated, and the messenger RNA (mRNA) levels of cellular immunity-related genes such as thioester-containing protein 2 (TEP 2) and hemocytin were also reduced, suggesting that some immune responses were selectively shut down by wasp parasitization. Taken together, all the results indicated that parasitized O. furnacalis larvae selectively activate the immune recognition response, and upregulate effector genes, but suppress ROS reaction and cellular

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Silencing β‐1,3‐glucan binding protein enhances the susceptibility of Plutella xylostella to entomopathogenic fungus Isaria cicadae

Hao

et al. 2022

Pest Management Science

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BACKGROUND The diamondback moth, Plutella xylostella is a notorious pest of brassicaceae crops globally and has developed serious resistance to insecticide. Insects primarily rely on their innate immunity to defense against various pathogens. In this study, we investigated the immunological functions of a β‐1,3‐glucan binding protein from P. xylostella (PxβGBP) and evaluated its potential for biocontrolling P. xylostella. RESULTS The open reading frame of PxβGBP is 1422 bp encoding 473 amino acids residues. PxβGBP contained a CBM39 domain, a PAC domain and a GH16 domain and shared evolutionary conservation with other lepidoptera βGRPs. PxβGBP was strongly expressed in the third instar larvae and fat body. PxβGBP transcript levels increased significantly after the challenge with microbes, including Isaria cicadae, Escherichia coli and Staphylococcus aureus. PxβGBP was identified in P. xylostella larvae challenged by I cicadae, but not in the naïve insects. Recombinant PxβGBP can directly bind fungal and bacterial cells, and also agglutinate the cells of I cicadae, S. aureus and E coli in a zinc‐dependent manner. Knockdown of PxβGBP via RNA interference significantly down‐regulated the expression of antimicrobial peptide gene gloverin, and enhanced the susceptibility of P. xylostella to I. cicadae infection, leading to high mortality. CONCLUSION These results indicated that PxβGBP plays an important role in the immune response of P. xylostella against I. cicadae infection, and could serve as a potential novel target for pest control. © 2022 Society of Chemical Industry.

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Self-association of an Insect β-1,3-Glucan Recognition Protein Upon Binding Laminarin Stimulates Prophenoloxidase Activation as an Innate Immune Response

Cited by 31 publications

References 47 publications

Initiating protease with modular domains interacts with β-glucan recognition protein to trigger innate immune response in insects

Initiating protease with modular domains interacts with β-glucan recognition protein to trigger innate immune response in insects

Transcriptomic analysis provides insights into the immune responses and nutrition in Ostrinia furnacalis larvae parasitized by Macrocentrus cingulum

Silencing β‐1,3‐glucan binding protein enhances the susceptibility of Plutella xylostella to entomopathogenic fungus Isaria cicadae

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