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

Daisuke Takahashi, Brandon L. Garcia, and Michael R. Kanost1

Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506

Edited by Alexander S. Raikhel, University of California, Riverside, CA, and approved September 25, 2015 (received for review August 28, 2015) The autoactivation of an initiating serine protease upon binding of (HP14) is one such initiating protease. Biochemical studies have pattern recognition proteins to pathogen surfaces is a crucial step in shown that HP14 zymogen (proHP14) autoactivates in the presence eliciting insect immune responses such as the activation of Toll and of peptidoglycan (9) or β-1,3-glucan and a β-1,3-glucan recognition prophenoloxidase pathways. However, the molecular mechanisms protein (βGRP1 or βGRP2)(10,13).Themolecularbasisun- responsible for autoactivation of the initiating protease remains derlying HP14 autoactivation, however, still remains to be explored. poorly understood. Here, we investigated the molecular basis for the HP14 has a carboxyl-terminal serine protease domain (SP) and a autoactivation of hemolymph protease 14 (HP14), an initiating series of amino-terminal modular domains that presumably play an protease in hemolymph of Manduca sexta, upon the binding of important role in autoactivation and/or proteolytic cleavage of β-1,3-glucan by its recognition protein, βGRP2. Biochemical analysis downstream protease zymogens (9). These modules include low- using HP14 zymogen (proHP14), βGRP2, and the recombinant pro- density lipoprotein receptor class A repeats (LAs, also called teins as truncated forms showed that the amino-terminal modular complement-type repeats, CRs), a complement control protein low-density lipoprotein receptor class A (LA) domains within HP14 module (CCP), and a unique cysteine-rich domain (7C) (Fig. 1). are required for proHP14 autoactivation that is stimulated by its in- HP14 orthologs in other insects, Tenebrio molitor (MSP) (11) and teraction with βGRP2. Consistent with this result, recombinant LA Drosophila melanogaster (ModSP) (12), share similar domain or-

domains inhibit the activation of proHP14 and prophenoloxidase, ganizations. Although little is known about the role of those BIOCHEMISTRY likely by competing with the interaction between βGRP2 and LA modular domains in initiating proteases, both LA and CCP mod- domains within proHP14. Using surface plasmon resonance, we dem- ules have been shown to mediate protein–protein/carbohydrate onstrated that immobilized LA domains directly interact with βGRP2 interactions in physiological and immune processes of mammals. in a calcium-dependent manner and that high-affinity interaction For instance, LA domains represent building blocks of the low- requires the C-terminal glucanase-like domain of βGRP2. Importantly, density lipoprotein receptor (LDLr) family and its related proteins the affinity of LA domains for βGRP2 increases nearly 100-fold in the (LRP) (14, 15), and they interact with many extracellular ligands presence of β-1,3-glucan. Taken together, these results present the (16, 17). CCP modules are also found in a number of proteins in first experimental evidence to our knowledge that LA domains of an the vertebrate complement system including serine proteases that insect modular protease and glucanase-like domains of a βGRP me- initiate the classical and lectin pathways (C1r and MASPs). diate their interaction, and that this binding is essential for the pro- Here, we directly investigated the role of modular domains of tease autoactivation. Thus, our study provides important insight into HP14 to better understand the initial event of the immune prote- the molecular basis underlying the initiation of protease cascade in ase cascade at a molecular level. Our biochemical and biophys- insect immune responses. ical analyses reveal an essential role for LA domains in HP14

innate immunity | insect immunity | modular serine protease | Significance pattern recognition receptor | hemolymph Serine protease cascades in innate immune responses require xtracellular protease cascades play an essential role in a va- autoactivation of initiating modular proteases in response to Eriety of biological processes including embryonic development recognition of nonself/altered self. Molecular mechanisms link- (1) and host defenses such as blood coagulation and the com- ing pathogen recognition and protease autoactivation remain plement system (2, 3). Protease cascades also function in insect unclear. Here, we report biochemical interactions between innate immune responses such as melanization and antimicrobial Manduca sexta hemolymph protease 14 (HP14), an initiating peptide synthesis by inducing the prophenoloxidase (proPO) and protease in an immune cascade, with recognition protein β-1,3- Toll pathways, respectively (3–5). glucan recognition protein (βGRP2). The amino-terminal LA do- In protease cascades, inactive protease zymogens that circulate mains of HP14 bind to βGRP2, and this interaction is essential for in an extracellular milieu become sequentially activated in path- HP14 autoactivation. Binding of β-1,3-glucan to βGRP2 leads to a ways beginning with an initiating modular serine protease, which dramatic increase in affinity between HP14 LA domains and autoactivates in response to internal or external signals. In hemo- βGRP2, likely representing a mechanism for promoting HP14 lymph (insect blood), pathogen-associated molecular patterns activation upon infection. Our study provides an important in- (PAMPs) such as peptidoglycans in bacteria and β-1,3-glucan in sight into a previously unexplored mechanism for the initiation fungi are first detected by pathogen recognition receptors (PRRs) of an insect immune protease cascade. such as peptidoglycan recognition proteins (PGRPs) and β-1,3- β – Author contributions: D.T., B.L.G., and M.R.K. designed research; D.T. and B.L.G. per- glucan recognition proteins ( GRPs) (4 8). As demonstrated by formed research; D.T., B.L.G., and M.R.K. analyzed data; and D.T., B.L.G., and M.R.K. genetic and biochemical studies of protease cascades in insect wrote the paper. immune responses, those recognition signals are integrated by an The authors declare no conflict of interest. initiating protease (9–12). The initiating protease then undergoes This article is a PNAS Direct Submission. autoactivation, and the activated form of the protease subsequently 1To whom correspondence should be addressed. Email: [email protected]. activates downstream serine protease zymogens in the proPO and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Toll activation pathways. Hemolymph protease 14 in Manduca sexta 1073/pnas.1517236112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1517236112 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 fragment was catalytically active (10). The C-terminal SP domain was not detected here because of much lower sensitivity of the anti-HP14 antibody for that domain. Activated HP14 migrated at ∼60 kDa position under nonreducing conditions, indicating that N-terminal modular domains and C-terminal SP domain remain connected by a disulfide bond after the autoactivation (Fig. 2B). In contrast, CCP-7C-SP, which lacks the N-terminal LAs, was not cleaved when mixed with β-1,3-glucan and/or βGRP2 (Fig. 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). LAs from mammalian proteins contain a conserved calcium- binding site, and calcium binding is critical to maintain the structural integrity required for ligand binding (15, 16). We therefore predicted that calcium ions are necessary for the proHP14 autoactivation and tested this hypothesis. Chelation of calcium ions by EDTA resulted in significantly reduced activation of proHP14 (Fig. 2C), further supporting an essential role of the N-terminal LAs in the autoactivation. βGRP2 consists of an N-terminal carbohydrate binding module (N-βGRP2) and a C-terminal glucanase-like domain (Fig. 1). We tested whether proHP14 is processed when incubated with N-βGRP2 and β-1,3-glucan (Fig. 2D). ProHP14 did not undergo autoactivation β under this condition, suggesting that the C-terminal glucanase-like Fig. 1. Domain architecture of M. sexta GRP2, HP14, and their recombi- β nant truncated forms. βGRP2 consists of an amino-terminal carbohydrate- domain of GRP2 is necessary for the proHP14 autoactivation. binding module (CBM) and a carboxyl-terminal glucanase-like domain. No β-1,3-glucanase activity was observed because of the lack of conserved catalytic residues responsible for activity (8). For HP14, four low-density lipoprotein receptor class A repeats (LA2–LA5), complement control protein domain (CCP), a cysteine-rich domain (7C), and catalytic serine protease domain (SP) are indicated. LA1 is not shown in the diagram, because it was removed before purification of proHP14 from hemolymph and from the recombinant LA1–LA5 before purification from cell culture medium, perhaps due to intracellular processing (10). The disulfide bond between 7C and SP domains and the L404-V405 cleavage site for autoactivation are also indicated. Also shown with βGRP2 and HP14 are the recombinant constructs used in this study (N-βGRP2, rHP14-LA, rHP14-CCP, and CCP-7C-SP).

autoactivation, resulting from direct interaction with βGRP2. These results, together with our previous studies showing the self- association 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. Results The Autoactivation of HP14 Requires HP14 LA Domains and βGRP2 Glucanase-Like Domain. To investigate the role of the modular domains of HP14, we first performed in vitro reconstitution experiments for proHP14 autoactivation in the presence of β-1,3- glucan (curdlan) and βGRP2. These experiments used endogenous proHP14 and βGRP2 purified from hemolymph and a set of truncated recombinant forms of these two proteins (Fig. 1). HP14 was purified in its zymogen form (proHP14), as shown by SDS/PAGE under reducing conditions followed by immunoblot β β analysis (Fig. 2A, lane 1) and migrated to the ∼65 kDa position Fig. 2. Autoactivation assay of proHP14 and CCP-7C-SP with GRP2 and -1,3- glucan. (A) Purified proHP14 (200 ng) or the recombinant truncated form of as a single band. Likewise, the recombinant CCP-7C-SP frag- HP14-lacking LA domains, CCP-7C-SP, were incubated with 150 ng of βGRP2 ment did not undergo autoactivation during expression and pu- and 100 μg of curdlan for 1 h at 37 °C. Molar concentrations of proHP14, rification (Fig. 2A, lane 5). Consistent with a previous study (10), CCP-7C-SP, and βGRP2 were 65 nM, 95 nM, and 60 nM, respectively. Samples proHP14 was cleaved when incubated with insoluble β-1,3-glu- were subjected to SDS/PAGE under reducing conditions, followed by immu- can and βGRP2 (Fig. 2A, lane 4), but it was not cleaved when noblot analysis using anti-HP14 polyclonal antibody as the primary antibody. incubated with β-1,3-glucan or βGRP2 alone (Fig. 2A, lanes 2 The band labeled LA-CCP-7C indicates the N-terminal fragment of HP14 pro- ∼ and 3). The processed fragment that migrated to the ∼40 kDa duced after activation cleavage. The C-terminal SP ( 30 kDa) was not clearly position corresponds to the molecular mass for the N-terminal detected because of much lower sensitivity of the anti-HP14 antibody for this domain. (B) The proHP14 autoactivation in the presence of βGRP2 and β-1,3- fragment of HP14, containing LA-CCP-7C domains (10). These glucan analyzed by SDS/PAGE under both reducing (R) and nonreducing (NR) results are consistent with the previous study demonstrating that conditions followed by immunoblot analysis. (C) Autoactivation assay of the cleavage of proHP14 under the same condition occurred at proHP14 in the presence of excess EDTA (30 mM). (D) Analysis of proHP14 the predicted proteolytic activation site and the cleaved SP autoactivation in the presence of βGRP2 or N-βGRP2.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1517236112 Takahashi et al. Downloaded by guest on September 26, 2021 Fig. 3. Primary structure and calcium binding of HP14 LA domains. (A) Sequence alignments of LAs (LA1–LA5) in HP14. Consensus residues found in the seven LAs in the human LDLr (14, 15) are marked as follows. The conserved cysteine residues are highlighted in yellow, other conserved residues in green, and the residues identified by Edman degradation analysis of purified rHP14-LA in red. The three conserved disulfide linkages (I-III, II-V, and IV-VI) are also indicated above the alignment. Arrows below the alignment indicate the aspartate and glutamate residues coordinating calcium with their side chains, as demonstrated by the crystal structure of LA5 of the LDLr (15). (B) ITC analysis of calcium binding to the recombinant LA domains (rHP14-LA). The solid line in the

lower graph represents the fit of the data to an N-identical binding site model. KD = 3.18 μM and the stoichiometry (N) = 3.2, respectively.

Molecular Characterization and Calcium Binding Activity of HP14 LA the autoactivation in a concentration-dependent manner, and Domains. Each LA domain is ∼40 residues long and contains no activation was detected when rHP14-LA was added at more three disulfide bonds (14, 15). Five LA domains are encoded in μ

than 0.3 M (Fig. 4). These results suggest that rHP14-LA BIOCHEMISTRY the HP14 cDNA, and consensus residues responsible for disul- competes with and abrogates the binding of LA domains within fide linkages and calcium binding are well conserved (Fig. 3A). A proHP14 to the βGRP2/β-1,3-glucan complex. We also pro- notable exception exists for LA4, which lacks two cysteine resi- duced the recombinant CCP domain (rHP14-CCP) (Fig. S1) dues required to form a putative I-III disulfide linkage. We used a and tested its effect on proHP14 autoactivation. In contrast to baculovirus-insect cell expression system to produce a recombi- rHP14-LA, the addition of rHP14-CCP had no detectable ef- nant construct for HP14 LAs, predicted to have a total of 14 fect (Fig. S2). disulfide bonds. Although our expression construct (rHP14-LA) was designed to contain all five LAs (LA1–LA5), purified rHP14- The Recombinant LA Domains Inhibit proPO Activation. We then LA migrated at a smaller molecular mass (22 kDa) than LA1–LA5 tested a hypothesis that rHP14-LA would block proPO activation (26 kDa). Subsequent Edman degradation analysis revealed that in plasma of M. sexta due to competition with binding of proHP14 the amino-terminal sequence of the purified protein was Gln- to the βGRP2/β-1,3-glucan complex. PO activity increased in Leu-Ser-Asn positioned in the linker region between LA1 and plasma supplemented with insoluble β-1,3-glucan from Saccharo- LA2 (Fig. 3A), indicating that LA1 was cleaved off before the myces cerevisiae (zymosan), but the addition of rHP14-LA at more construct was purified. Cleavageatthissiteisconsistentwith than 0.2 μM significantly inhibited proPO activation (Fig. 5A). the previous observation that proHP14 purified from hemo- This inhibitory effect is consistent with the effect of rHP14-LA on lymph lacked LA1, indicating that this processing also occurs in proHP14 autoactivation described above (Fig. 4). Furthermore, the natural endogenous proHP14 (10). Calcium binding to rHP14-LA was monitored by using isothermal titration calorimetry (ITC). Analysis of the binding isotherm with an N-identical binding sites model showed that rHP14-LA bound calcium with an apparent affinity of KD ∼ 3 μM, and thermodynamic parameters of ΔH = −18 kcal/mol, and ΔS = −34.3 cal/mol deg. These values are comparable to those obtained for LAs (CR17 and 18) from the human LDLR family (20). The calcium concentration in hemolymph is ∼5 mM (21) and, thus, likely sufficient to saturate HP14 LAs. Although the construct contains four LAs (LA2–LA5), the resulting binding stoichiometry was 3.20 ± 0.08 (Fig. 3B), suggesting that only three of the domains possess a functional calcium-binding site. LA4 is devoid of two conserved cysteine residues forming a disulfide bond I–III that maintains the structural integrity of LA domains. Furthermore, LA3 and LA5 lack one or two aspartate residues predicted to participate + in Ca2 coordination through the acidic side chain; these residues are replaced by asparagine or glutamate (Fig. 3A). Thus, further study is required to identify which LA domain is deficient in calcium Fig. 4. Effects of rHP14-LA on the autoactivation of proHP14. Purified binding activity. proHP14 (150 ng) was incubated with 150 ng of βGRP2 and 100 μg of curdlan for 1 h at 37 °C. rHP14-LA was added to the reaction mixture at concen- The Recombinant LA Domains Inhibit the Autoactivation of proHP14. trations indicated below the immunoblot. Samples were subjected to SDS/ Using rHP14-LA, we investigated its effect on proHP14 autoacti- PAGE under reducing conditions followed by immunoblot analysis using vation. Addition of increasing amounts of rHP14-LA suppressed anti-HP14 antibody.

Takahashi et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 the surface of immobilized rHP14-LA (Fig. 6A). Steady-state affinity analysis revealed an apparent dissociation constant (KD,ss)of ∼190 nM (Fig. 6A, Inset) and a comparable affinity constant of KD,kin = 38 nM was calculated when kinetic analyses were performed (red traces). No interaction was observed for injections of 1 mM β-1,3-glucan alone. In the absence of β-1,3-glucan, βGRP2 failed to induce proHP14 autoactivation (Fig. 1A,lane2),therefore, we sought to understand whether β-1,3-glucan might influ- ence the βGRP2/rHP14-LA interaction. To this end, we injected various concentrations of βGRP2 over rHP14-LA in the absence of β-1,3-glucan (Fig. 6B). The resulting sensorgrams show a dose- dependent response characterized by rapid association and dissociation rates and KD,ss of 16 μM (Fig. 6B, Inset). These results indicate that the affinity between βGRP2 and rHP14-LA is sig- Fig. 5. Inhibition of the prophenoloxidase (proPO) activation by rHP14-LA. nificantly enhanced in the presence of β-1,3-glucan. LAs in the (A) Ten microliters of plasma from the day two fifth instar larvae of M. sexta LDLR family proteins share a characteristic ligand recognition μ were incubated with 3 g of zymosan and increasing amounts of rHP14-LA mode in which lysine residues from ligand molecules interact with – μ + (0.05 2.3 M) for 1 h at 25 °C, and then 2 mM dopamine was added as a the Ca2 -coordinating residues of LAs, including acidic (Asp/Glu) phenoloxidase substrate. One unit of PO activity was defined as the amount of enzyme producing an increase in absorbance at 470 nm of 0.001 per min. and tryptophan residues (16, 22, 23). We therefore tested whether Error bars represent SD from three technical replicates. The control con- calcium binding by HP14 LAs (Fig. 3B) was critical for the tained no carbohydrate or protein added to plasma. (B) rHP14-LA was added βGRP2/rHP14-LA interaction (Fig. 6C). The binding response before or after incubation of plasma with zymosan. was greatly diminished in the presence of 10 mM EDTA, suggesting that the calcium maintains the structural integrity of HP14 LA domains necessary for the interaction with βGRP2. To further when rHP14-LA was added to the reaction mixture after plasma localize the βGRP2/HP14-LA interaction sites, we next measured and β-1,3-glucan were incubated for 1 h, there was no substantial the ability of N-βGRP2 to interact with immobilized rHP14-LA in inhibition of proPO activation (Fig. 5B), indicating that rHP14-LA the presence of β-1,3-glucan (Fig. 6D). N-βGRP2 bound rHP14- has no direct effect on phenoloxidase itself. Thus, it appears that LA significantly weaker than full-length βGRP2, implicating a rHP14-LA disrupts the interaction between the βGRP2/β-1,3- critical role for the C-terminal glucanase-like domain in mediating glucan complex and proHP14, preventing proHP14 autoactivation the βGRP2/rHP14-LA interaction. HP14 did not autoactivate in and, thereby, proPO activation. the presence of N-βGRP2 and β-1,3-glucan (Fig. 2D), further supporting the critical role of βGRP2 glucanase-like domain. HP14 LA Domains Physically Interact with βGRP2 Glucanase-Like Taken together, these results present the first experimental evi- Domain. Our biochemical experiments suggested that HP14 LAs dence to our knowledge that HP14-LA domains and the βGRP2- interact with βGRP2 and/or β-1,3-glucan. We tested rHP14-LA glucanase-like domain mediate βGRP2/HP14 interaction leading for its ability to bind β-1,3-glucan by using a pull-down assay, but to HP14 zymogen autoactivation. there were no detectable interactions between rHP14-LA and β-1,3-glucan (Fig. S3). To test a hypothesis that HP14 LAs me- Discussion diate a direct interaction with βGRP2, we used surface plasmon Insects rely on innate immune responses, such as melanization resonance (SPR). A concentration series of βGRP2 was coinjected and antimicrobial peptide synthesis, to combat invading patho- with saturating amounts of soluble β-1,3-glucan (laminarin) over gens. Therefore, the efficient initiation of the immune protease

Fig. 6. βGRP2 directly interacts with HP14 LA domains, and this interaction is significantly enhanced in the presence of β-1,3-glucan. (A) Binding of βGRP2 to rHP14-LA in the presence of soluble β-1,3-glucan (laminarin) was measured by coinjecting a fixed 1 mM concentration of β-1,3-glucan with a concentration series of βGRP2 consisting of 5.9, 12, 23, 47, 94, 180, 380, and 750 nM over a rHP14-LA biosensor in a running buffer of 20 mM Hepes, pH 7.4, with 150 mM NaCl, 0.005%

Tween 20, and 5 mM CaCl2. Steady-state affinity analysis (Inset) and kinetic analysis (red traces) yielded dissociation constants of KD,ss = 0.19 μMandKD, kin = 0.038 μM, respectively. (B) Binding of βGRP2 to rHP14-LA was measured in the absence of β-1,3-glucan by injecting βGRP2 in a concentration series consisting of

0.31, 0.63, 1.3, 2.5, 5.0, 10, and 15 μM. Steady-state affinity analysis was conducted (Inset) and resulted in a calculated KD,ss of 16 μM. (C)Toevaluatetheeffectof calcium on the βGRP2/HP14-LA interaction, a fixed concentration of 750 nM βGRP2 was coinjected with 1 mM β-1,3-glucan in a buffer containing either 5 mM CaCl2 (black) or 10 mM EDTA (blue). (D) To determine whether the N-terminal domain of βGRP2 (N-βGRP2) participates in the βGRP2/HP14-LA interaction, full-length βGRP2 (black) or N-βGRP2 (blue) at 750 nM was coinjected with 1 mM β-1,3-glucan over immobilized rHP14-LA. Resulting sensorgrams for this experiment were corrected for the molecular mass of each species. All injections were performed a minimum of twice and representative sensorgrams are shown in each graph.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1517236112 Takahashi et al. Downloaded by guest on September 26, 2021 cascades is a crucial step in successful host defense. These ini- autoactivation, HP14 converts a downstream hemolymph protease tiation signals are subsequently amplified through the activation zymogen, proHP21, into its active form (HP21), which further of multiple downstream proteases. Furthermore, the proPO ac- drives the protease cascade for proPO activation (Fig. 7D). tivation system needs to be locally controlled because its prod- Initiating proteases have long been studied for the complement ucts including active proteases, cytotoxic quinones, and reactive classical and lectin pathways (25–27). Initiating proteases, C1r and + oxygen species are potentially dangerous to host insects (24). MASPs, are composed of Ca2 -binding CUB domains separated Combining the results presented here with our previous studies by EGF domain, two CCP domains, and a catalytic SP domain, on βGRPs (18, 19) provides further insight into the molecular sharing similar organization with the initiating proteases in insects basis for the initiation of the protease cascade triggered by the (HP14, MSP, and ModSP). The proteases form oligomers (het- autoactivation of HP14 (Fig. 7). Fungal infections in hemolymph erotetramer consisting of C1s-C1r-C1r-C1s or dimer of MASPs) are first detected by binding of βGRP2 to fungal β-1,3-glucans within a cage (bouquet-like structure) formed by recognition β (Fig. 7A), which induces self-association of GRP2s through proteins, C1q and mannose binding lectins/ficolins. It has been – protein protein interaction between their N-terminal carbohy- proposed that, in such initiation complexes, the binding of C1q to drate binding modules (18, 19) (Fig. 7B). We have suggested that ligands induces substantial conformational changes of the C1s- β β such macromolecular assembly of GRP2 and -1,3-glucan serves C1r-C1r-C1s tetramer that triggers the autoactivation of C1r (28). as a platform to trigger the activation of the protease cascade for Such a complex formation before nonself recognition is unlikely the proPO activation. In support of this model, we note that the β ∼ β for GRP2 and HP14 because of the relatively low affinity (KD affinity between GRP2 and rHP14-LA is relatively weak in the 16 μM) between HP14 LAs and βGRP2 in the absence of β-1,3- absence of β-1,3-glucan (KD of 16 μM) (Fig. 6B). Considering β ∼ μ glucan. However, it is likely that the binding of multiple proHP14 the concentration of GRP in M. sexta hemolymph ( 0.5 M) (8), molecules to the platform of βGRP2/β-1,3-glucan triggers con- such a weak affinity is unlikely to lead to a direct interaction of formational changes, with pairs of proHP14 interacting to trigger βGRP2 with HP14 LAs in the absence of infection. Interestingly, the autoactivation (Fig. 7D). the affinity is significantly enhanced in the presence of β-1,3- A previous study showed that calcium was required to activate glucan, as evidenced by a nearly 100-fold decrease in K from D the protease cascade for proPO activation in plasma of Bombyx 16 μMto0.19μM(Fig.6A). These results strongly suggest that mori, another lepidopteran insect, but not to activate proPO itself βGRP can effectively interact with HP14 only in the presence of by hPPAE, a proPO-activating enzyme (29), suggesting that cal- β-1,3-glucan, i.e., upon recognition of fungal invasion (Fig. 7C), providing compelling evidence for the molecular platform cium is necessary for earlier steps in the proPO activation cascade. BIOCHEMISTRY β β Here, we showed that, in the absence of calcium, both the proHP14 formed by GRP2 and -1,3-glucans that allows for the recruitment β and localized activation of proHP14. After the recruitment and autoactivation and the rHP14-LA/ GRP2 interaction were significantly reduced (Figs. 2C and 6C). This result provides a molecular explanation for calcium requirement in proPO activation. ModSP and MSP, apparent orthologs of HP14, also function in the protease cascade for Toll pathway activation that leads to antimicrobial peptide synthesis (11, 12, 30). Detection of Gram- positive bacteria is mediated by the recognition of lysine-type peptidoglycans by two PRRs, PGRP-SAs and Gram-negative bacteria binding protein 1 (GNBP1) (11, 31). It has been suggested that PGRP-SAs form molecular clusters on binding to peptidoglycan and recruit GNBP1, and subsequently the initiating protease, MSP, to form an initial peptidoglycan recognition complex for the cascade (11). Biochemical study has shown that, in the recognition complex, + MSP autoactivates in the presence of Ca2 (30), implying the involvement of LA domains in the autoactivation. Moreover, GNBP1 and βGRP2 (GNBP3) belong to the same protein family and share the C-terminal glucanase-like domain (32). These findings collec- tively suggest that, as demonstrated for βGRP2 and HP14, GNBP1 and the initiating protease (ModSP and MSP) interact with one another via GNBP1 glucanase-like domain and the protease LA domains, resulting in the autoactivation of the initiating protease for the Toll pathway activation. In conclusion, this study provides an important insight into the molecular basis underlying the initiation of protease cascades for insect immune responses. Upon fungal infection in hemolymph, HP14 zymogens are directly recruited to the molecular platform formed by βGRP2 and β-1,3-glucan through interactions between Fig. 7. A molecular model for the initiation of the protease cascade for HP14 LA domains and βGRP2 glucanase-like domain. We suggest proPO activation. (A) β-1,3-glucans on a fungal cell surface are detected by βGRP2. (B) Binding of βGRP2 to β-1,3-glucans induces the formation of mo- such initiation complex formation triggers the autoactivation of lecular platform in which multiple βGRP2s and β-1,3-glucans are associated HP14 possibly induced by conformational changes in HP14. Active through protein–protein interactions between N-terminal CBM (N-βGRP2) HP14 then initiates the protease cascade leading to activation of (18, 19). (C) The C-terminal glucanase-like domain of βGRP2 interacts with LA phenoloxidase. To our knowledge, this is the first study that eluci- domains of HP14, recruiting multiple HP14 zymogens (proHP14) to the β-1,3- dates functional protein–protein interactions between a PRR and β glucan/ GRP2 platform. (D) Multiple proHP14s are held in proximity within an initiating protease in insect immune responses, and clearly de- the platform, and protein–protein interaction between βGRP2 and proHP14 may induce a conformational change in proHP14, enabling the proHP14 fines an essential role for LA domains in the autoactivation of the (oval shape in light green) to cleave another zymogen molecule. Activated initiating protease. Such interactions may have broader relevance HP14 (diamond shape in dark green) then activates proHP21, a downstream leading to more detailed understanding of the molecular control of serine protease in proPO activation pathway. protease cascades that function in innate immune responses.

Takahashi et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 Materials and Methods Pull-Down Assay To Study Protein–Carbohydrate Interaction. Pull-down assay Protein Expression and Purification. βGRP2 and HP14 zymogen were purified was performed to study interaction of rHP14-LA or CCP domain with insoluble from hemolymph of M. sexta larvae by following described methods (33, 34). carbohydrates as detailed in SI Materials and Methods. HP14 LA domains (rHP14-LA) and CCP-7C-SP fragments were expressed in – insect cells by using baculovirus expression vector system. The N-terminal Protein Protein Interaction by SPR. All SPR experiments were conducted by μ domain of βGRP2 (N-βGRP2) was expressed in Escherichia coli BL21(DE3) cells using a Biacore 3000 (GE Healthcare) at 25 °C and a flow rate of 20 L/min. A − β and purified as described (19). HP14 CCP domain (rHP14-CCP) was expressed twofold dilution series (6 750 nM) of GRP2 was coinjected with a fixed 1 mM β as inclusion bodies in E. coli BL21(DE3) cells, refolded in vitro. Detailed de- concentration of -1,3-glucan for 3 min followed by 5 min of dissociation scriptions for expression and purification are provided in SI Materials phase in a running buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM CaCl2, and Methods. and 0.005% Tween-20). Duplicate injections over rHP14-LA surface densities of 120, 220, and 450 resonance units (RU) were performed. Regeneration was Isothermal Titration Calorimetry Measurements of Calcium Binding to rHP14- achieved by two 30-s pulses of 7.5 mM glycine (pH 2.2), 1.25 M NaCl. The β β LA. Calcium binding titrations to rHP14-LA were performed with a MicroCal GRP2/rHP14-LA interaction was measured in the absence of -1,3-glucan by β − μ ITC200 (GE Healthcare). rHP14-LA (40 μΜ) in 20 mM Hepes, pH 7.2, 100 mM using a GRP2 concentration series (0.31 15 M), which was injected for 1 min followed by 2 min of dissociation with no regeneration required. Duplicate NaCl was titrated with 1.5 mM CaCl2 at 30 °C. A total of 20 injections were performed. Data were fit to an N-identical binding sites model with Origin 7 injections over an rHP14-LA surface density of 680 RU were performed. To β (OriginLab). Further details are available in SI Materials and Methods. determine the effect of calcium on the GRP2/rHP14-LA interaction, a fixed concentration (750 nM) of βGRP2 was injected in a buffer containing EDTA (20 mM Hepes, pH 7.4, 150 mM NaCl, 0.005% Tween-20, and 10 mM EDTA). To In Vitro Reconstitution Experiment for proHP14 Autoactivation. Purified evaluate relative binding of βGRP2 vs. N-βGRP2 to rHP14-LA, injections were proHP14 or the recombinant CCP-7C-SP construct (∼200 ng) was incubated performed at a fixed 750 nM concentration. Response was corrected for with curdlan (100 μg), βGRP2, or N-βGRP2 (150 ng) in 45 μL of 50 mM Tris·HCl, the molecular mass of each analyte by using βGRP2 = 52.5 kDa and N-βGRP2 = pH 7.5, 50 mM NaCl, 5 mM CaCl at 37 °C for 1 h. rHP14-LA and CCP domains 2 13.8 kDa. To account for bulk changes in refractive index, 1 mM β-1,3-glucan– were also added at various concentrations to examine their effects on only injections were subtracted from all sensorgrams where β-1,3-glucan was proHP14 autoactivation. The reaction mixtures (12 μL) were analyzed by SDS/ coinjected. All injections were performed a minimum of two times. Additional PAGE followed by immunoblot analysis using 1:2,000 dilution of an anti- details for SPR experiments are in SI Materials and Methods. HP14 antibody to reveal possible proteolytic cleavage.

ACKNOWLEDGMENTS. We thank Maureen Gorman, Neal Dittmer, and Brian ProPO Activation Assay. Phenoloxidase activation assay was conducted μ Geisbrecht for helpful suggestions regarding this work; Haobo Jiang and Yang by using the method described (18). rHP14-LA, ranging from 0.05 Mto Wang for cDNA and antiserum for Manduca sexta HP14 and Subbaratnam μ 2.3 M, were added with zymosan to the plasma sample to examine its Muthukrishnan for chitin; and Lisa Brummett for technical assistance. This work effect on proPO activation. Further details are described in SI Materials was supported by NIH Grants GM41247 and AI113552 and is contribution and Methods. 16-004-J from the Kansas Agricultural Experiment Station.

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