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Insect β-glucan recognition protein (βGRP), a pathogen recognition receptor for innate immune responses, detects β-1,3-glucan on fungal surfaces via its N-terminal carbohydrate-binding domain (N-βGRP) and triggers serine protease cascades for the activation of prophenoloxidase (pro-PO) or Toll pathways. Using biophysical and biochemical methods, we characterized the interaction of the N-terminal domain from Manduca sexta βGRP2 (N-βGRP2) with laminarin, a soluble form of β-1,3-glucan. We found that carbohydrate binding by N-βGRP2 induces the formation of two types of protein-carbohydrate complexes, depending on the molar ratio of carbohydrate to protein ([C]/[P]). Precipitation, analytical ultracentrifugation, and chemical cross-linking experiments have shown that an insoluble aggregate forms when the molar ratio of carbohydrate to protein is low ([C]/[P] ∼ 1). In contrast, a soluble complex, containing at least five N-βGRP2 molecules forms at a higher molar ratio of carbohydrate/protein ([C]/[P] >5). A hypothesis that this complex is assembled partly due to protein-protein interactions was supported by chemical cross-linking experiments combined with LC-MS/MS spectrometry analysis, which permitted identification of a specific intermolecular cross-link site between N-βGRP molecules in the soluble complex. The pro-PO activation in naive plasma was strongly stimulated by addition of the insoluble aggregates of N-βGRP2. The soluble complex with laminarin formed in the plasma also stimulated pro-PO activation, but at a lower level. Taken together, these results provide experimental evidence for novel mechanisms in which associations of βGRP with microbial polysaccharide promotes assembly of βGRP oligomers, which may form a platform needed to trigger the pro-PO pathway activation cascade.

The reactions of chemical warfare agent simulants, 2-chloroethyl ethyl sulfide (2-CEES) and di-i-propyl fluoro phosphate (DFP), in fluids have been investigated. Data analyses confirm the major degradation pathway to be hydrolysis of 2-CEES to 2-hydroxyethyl ethyl sulfide, along with minor self-condensation products. Among the three fluids examined, 2-CEES degradation was the fastest in Gamble’s fluid during a 96 h period. Upon addition of Exceptional Hazard Attenuation Materials (EHAMs) to 2-CEES containing Gamble’s fluid, degradation was generally improved during the first 24 h period. The 96 h outcome was similar for fluid samples with or without EHAM 2 and EHAM 4. EHAM 1-added fluid contained only one degradation product, 2-nitroethyl ethyl sulfide. DFP degradation was the slowest in Gamble’s fluid, but was enhanced by the addition of EHAMs. FTIR and solid state31P NMR confirm the destructive adsorption of 2-CEES and DFP by the EHAMs. The results collectively demonstrate that 2-CEES and DFP decompose to various extents in Gamble’s fluid over a 96 h period but the fluid still contains a considerable amount of intact simulant. EHAM 1 appears to be promising for 2-CEES and DFP mitigation while EHAM 2 and EHAM 4 work well for early on concentration reduction of 2-CEES and DFP.

In response to invading microorganisms, insect β-1,3-glucan recognition protein (βGRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Here we report on the nuclear magnetic resonance (NMR) solution structure of the N-terminal domain of βGRP (N-βGRP) from Indian meal moth (Plodia interpunctella), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. NMR and isothermal calorimetric titrations of N-βGRP with laminarihexaose, a glucose hexamer containing β-1,3 links, suggest a weak binding of the ligand. However, addition of laminarin, a glucose polysaccharide (~6 kDa) containing β-1,3 and β-1,6 links that activates the proPO pathway, to N-βGRP results in the loss of NMR cross-peaks from the backbone (15)N-(1)H groups of the protein, suggesting the formation of a large complex. Analytical ultracentrifugation (AUC) studies of formation of the N-βGRP-laminarin complex show that ligand binding induces self-association of the protein-carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (~102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to submicromolar concentrations. The structural model thus derived from this study for the N-βGRP-laminarin complex in solution differs from the one in which a single N-βGRP molecule has been proposed to bind to a triple-helical form of laminarin on the basis of an X-ray crystallographic structure of the N-βGRP-laminarihexaose complex [Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N., and Yamaguchi, Y. (2011) J. Biol. Chem. 286, 29158-29165]. AUC studies and phenoloxidase activation measurements conducted with the designed mutants of N-βGRP indicate that electrostatic interactions involving Asp45, Arg54, and Asp68 between the ligand-bound protein molecules contribute in part to the stability of the N-βGRP-laminarin macro complex and that a decreased stability is accompanied by a reduced level of activation of the proPO pathway. An increased level of β-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of the βGRP-β-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the initial signal of pathogen recognition for the activation of the proPO pathway.

The region (101-112) of C1B domain in PKCγ plays a crucial role in the activation of the enzyme and subsequent gap junction inhibition. Substitution studies on peptides correlating to the C1B region show that a flexible structure and ability to be phosphorylated on serine 109 are critical for this purpose.

In response to wounding or infection, insects produce a battery of antimicrobial peptides (AMPs) and other defense molecules to kill the invading pathogens. To study their structures, functions, and transcriptional regulation, we synthesized Manduca sexta moricin, a 42-residue peptide (GKIPVKAIKQAGKVIGKGLRAINIAGTTHDVVSFFRPKKKKH, 4539 Da). The compound exhibited potent antimicrobial activities against a broad spectrum of Gram-positive and Gram-negative bacteria with a minimum inhibitory concentration of 1.4 µM. The mRNA levels of M. sexta moricin increased substantially in fat body and hemocytes after the larvae were challenged with bacterial cells. We determined the solution structure of this AMP by two-dimensional 1H1H -nuclear magnetic resonance spectroscopy. The tertiary structure is composed of an eight-turn α-helix spanning almost the entire peptide. Insights of relationships between the structure and function are also presented. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.

Clip domains are structural modules found in arthropod serine proteinases and some proteolytically inactive homologues, which mediate extracellular signaling pathways of development and immunity. While little is known about their structures or functions, clip domains are proposed to be sites for interactions of proteinases with their activators, cofactors, and substrates. Here we report the solution structure of dual clip domains from Manduca sexta prophenoloxidase activating proteinase-2. Each domain adopts a new mixed alpha/beta fold (a three-stranded antiparallel beta-sheet flanked by two alpha-helices), and the architecture provides structural information on clip domains from a catalytically active proteinase for the first time. Examination of the structure in conjunction with a multiple sequence alignment of the clip domains from different groups suggests a substrate-binding site, a bacteria-interacting region, and a surface for specific interactions. In summary, our results provide insights into the structural basis of clip domain functions and this structure may represent the prototype of group-2 clip domains.

Cathelicidins are an important family of cationic host defense peptides in vertebrates with both antimicrobial and immunomodulatory activities. Fowlicidin-1 and fowlicidin-2 are two newly identified chicken cathelicidins with potent antibacterial activities. Here we report structural and functional characterization of the putatively mature form of the third chicken cathelicidin, fowlicidin-3, for exploration of its therapeutic potential. NMR spectroscopy revealed that fowlicidin-3 comprises 27 amino-acid residues and adopts a predominantly α-helical structure extending from residue 9 to 25 with a slight kink induced by a glycine at position 17. It is highly potent against a broad range of Gram-negative and Gram-positive bacteria in vitro, including antibiotic-resistant strains, with minimum inhibitory concentrations in the range 1–2 µm. It kills bacteria quickly, permeabilizing cytoplasmic membranes immediately on coming into contact with them. Unlike many other host defense peptides with antimicrobial activities that are diminished by serum or salt, fowlicidin-3 retains bacteria-killing activities in the presence of 50% serum or physiological concentrations of salt. Furthermore, it is capable of suppressing lipopolysaccharide-induced expression of proinflammatory genes in mouse macrophage RAW264.7 cells, with nearly complete blockage at 10 µm. Fowlicidin-3 appears to be an excellent candidate for future development as a novel antimicrobial and antisepsis agent, particularly against antibiotic-resistant pathogens.

Cationic antimicrobial peptides are naturally occurring antibiotics that are actively being explored as a new class of anti-infective agents. We recently identified three cathelicidin antimicrobial peptides from chicken, which have potent and broad-spectrum antibacterial activities in vitro (Xiao Y, Cai Y, Bommineni YR, Fernando SC, Prakash O, Gilliland SE & Zhang G (2006) J Biol Chem281, 2858-2867). Here we report that fowlicidin-1 mainly adopts an alpha-helical conformation with a slight kink induced by glycine close to the center, in addition to a short flexible unstructured region near the N terminus. To gain further insight into the structural requirements for function, a series of truncation and substitution mutants of fowlicidin-1 were synthesized and tested separately for their antibacterial, cytolytic and lipopolysaccharide (LPS)-binding activities. The short C-terminal helical segment after the kink, consisting of a stretch of eight amino acids (residues 16-23), was shown to be critically involved in all three functions, suggesting that this region may be required for the peptide to interact with LPS and lipid membranes and to permeabilize both prokaryotic and eukaryotic cells. We also identified a second segment, comprising three amino acids (residues 5-7) in the N-terminal flexible region, that participates in LPS binding and cytotoxicity but is less important in bacterial killing. The fowlicidin-1 analog, with deletion of the second N-terminal segment (residues 5-7), was found to retain substantial antibacterial potency with a significant reduction in cytotoxicity. Such a peptide analog may have considerable potential for development as an anti-infective agent.

Aphid saliva is predicted to contain proteins that modulate plant defenses and facilitate feeding. Armet is a well-characterized bifunctional protein in mammalian systems. Here we report a new role of Armet, namely as an effector protein in the pea aphid, Acyrthosiphon pisum. Pea aphid Armet's physical and chemical properties and its intracellular role are comparable to those reported for mammalian Armets. Uniquely, we detected Armet in aphid watery saliva and in the phloem sap of fava beans fed on by aphids. Armet's transcript level is several times higher in the salivary gland when aphids feed on bean plants than when they feed on an artificial diet. Knockdown of the Armet transcript by RNA interference disturbs aphid feeding behavior on fava beans measured by the electrical penetration graph technique and leads to a shortened life span. Inoculation of pea aphid Armet protein into tobacco leaves induced a transcriptional response that included pathogen-responsive genes. The data suggest that Armet is an effector protein mediating aphid-plant interactions.

Insect β-glucan recognition protein (βGRP), a pathogen recognition receptor for innate immune responses, detects β-1,3-glucan on fungal surfaces via its N-terminal carbohydrate binding domain (N-βGRP) and triggers serine protease cascades leading to the activation of prophenoloxidase (proPO) or Toll pathway. Here we use biochemical and biophysical techniques to probe the molecular interaction of N-βGRP from Manduca sexta with laminarin, soluble β-1,3-glucan. Intriguingly, the carbohydrate binding induces the formation of two types of protein self-association of N-βGRPs depending on the molar ratio of carbohydrate to protein (C/P). Insoluble aggregation occurs at the lower molar ratio (C/P ~1), and a soluble oligomer, likely containing at least six N-βGRP molecules, forms at the higher molar ratio (C/P >5). Further, the PO activation is significantly enhanced in the naive plasma supplemented with the insoluble aggregate of N-βGRP. The soluble complex formed in plasma with laminarin also stimulates the PO a...

Laccase-2 is a highly conserved multicopper oxidase that functions in insect cuticle pigmentation and tanning. In many species, alternative splicing gives rise to two laccase-2 isoforms. A comparison of laccase-2 sequences from three orders of insects revealed eleven positions at which there are conserved differences between the A and B isoforms. Homology modeling suggested that these eleven residues are not part of the substrate binding pocket. To determine whether the isoforms have different kinetic properties, we compared the activity of laccase-2 isoforms from Tribolium castaneum and Anopheles gambiae . We partially purified the four laccases as recombinant enzymes and analyzed their ability to oxidize a range of laccase substrates. The predicted endogenous substrates tested were dopamine, N -acetyldopamine (NADA), N -β-alanyldopamine (NBAD) and dopa, which were detected in T. castaneum previously and in A. gambiae as part of this study. Two additional diphenols (catechol and hydroquinone) and one non-phenolic substrate (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) were also tested. We observed no major differences in substrate specificity between the A and B isoforms. Dopamine, NADA and NBAD were oxidized with catalytic efficiencies ranging from 51 to 550 min −1 mM −1 . These results support the hypothesis that dopamine, NADA and NBAD are endogenous substrates for both isoforms of laccase-2. Catalytic efficiencies associated with dopa oxidation were low, ranging from 8 to 30 min −1 mM −1 ; in comparison, insect tyrosinase oxidized dopa with a catalytic efficiency of 201 min −1 mM −1 . We found that dopa had the highest redox potential of the four endogenous substrates, and this property of dopa may explain its poor oxidation by laccase-2. We conclude that laccase-2 splice isoforms are likely to oxidize the same substrates in vivo , and additional experiments will be required to discover any isoform-specific functions.

Cathelicidins are an important family of cationic host defense peptides in vertebrates with both antimicrobial and immunomodulatory activities. Fowlicidin-1 and fowlicidin-2 are two newly identified chicken cathelicidins with potent antibacterial activities. Here we report structural and functional characterization of the putatively mature form of the third chicken cathelicidin, fowlicidin-3, for exploration of its therapeutic potential. NMR spectroscopy revealed that fowlicidin-3 comprises 27 amino-acid residues and adopts a predominantly alpha-helical structure extending from residue 9 to 25 with a slight kink induced by a glycine at position 17. It is highly potent against a broad range of Gram-negative and Gram-positive bacteria in vitro, including antibiotic-resistant strains, with minimum inhibitory concentrations in the range 1-2 microM. It kills bacteria quickly, permeabilizing cytoplasmic membranes immediately on coming into contact with them. Unlike many other host defense peptides with antimicrobial activities that are diminished by serum or salt, fowlicidin-3 retains bacteria-killing activities in the presence of 50% serum or physiological concentrations of salt. Furthermore, it is capable of suppressing lipopolysaccharide-induced expression of proinflammatory genes in mouse macrophage RAW264.7 cells, with nearly complete blockage at 10 microM. Fowlicidin-3 appears to be an excellent candidate for future development as a novel antimicrobial and antisepsis agent, particularly against antibiotic-resistant pathogens.

Cathelicidins are a group of cationic host defense peptides with pluripotent immunomodulatory activities. We recently identified fowlicidin-1, a chicken cathelicidin possessing broad-spectrum antibacterial and LPS-neutralizing activities but with considerable toxicity toward mammalian cells. NMR spectroscopy revealed that fowlicidin-1 mainly adopts an α-helical conformation with glycine-induced kink in the center and a short flexible unstructured region at the N-terminal end. To reveal functional significance of different structural regions and identify peptide analog(s) with enhanced therapeutic potential, a series of deletion and substitution analogs of fowlicidin -1 were synthesized. Eight amino acids (residues 16–23) at the C-terminal end were found to be critically involved in bacterial killing, LPS binding and cytotoxicity. We also revealed that the N-terminal unstructured segment (residues 1–5) plays a minimum role in antibacterial and LPS-neutralizing activities. Removal of valine at position 5 reduced the cytotoxicity by 4-fold while retaining a similar antibacterial potency to the parent peptide. However, omission of tryptophan at position 6 resulted in at least 4-fold reduction in bacteria-killing efficiency, indicating the critical involvement of tryptophan. Taken together, fowlicidin-1(6–26) represents a short peptide analog with excellent potential to be explored as a novel antimicrobial. This project was supported by Oklahoma Center for the Advancement of Science and Technology, Oklahoma Agricultural Experiment Station and HHMI-Undergraduate Research Scholarship.