8 research outputs found
An initial event in insect innate immune response: structural and biological studies of interactions between β-1,3-glucan and the N-terminal domain of β-1,3-glucan recognition protein
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 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 15N-1H groups of the protein, suggesting the formation of a large complex. Analytical ultra centrifugation (AUC) studies of formation of N-βGRP:laminarin complex show that ligand-binding induces sel-fassociation 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 sub-micromolar concentrations. The structural model thus derived from the present studies for 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 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 carried out 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 N-βGRP:laminarin macro complex and that a decreased stability is accompanied by a reduced activation of the proPO pathway. Increased β-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of 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
Structural and functional studies of interactions between [beta]-1,3-glucan and the N-terminal domains of [beta]-1,3-glucan recognition proteins involved in insect innate immunity
Doctor of PhilosophyDepartment of BiochemistryRamaswamy KrishnamoorthiInsect [beta]-1,3-glucan recognition protein ([beta]GRP), 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. Delineation of mechanistic details of these processes may help develop strategies to control insect-borne diseases and economic losses. Multi-dimensional nuclear magnetic resonance (NMR) techniques were employed to solve the solution structure of the Indian meal moth (Plodia interpunctella) [beta]GRP N-terminal domain (N-[beta]GRP), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. This is the first determined three-dimensional structure of N-[beta]GRP, which adopts an immunoglobulin fold. Addition of laminarin, a [beta]-1,3 and [beta]-1,6 link-containing glucose polysaccharide (âź6 kDa) that activates the proPO pathway, to N-[beta]GRP results in the loss of NMR cross-peaks from the backbone [subscript]1[subscript]5N-[subscript]1H groups of the protein, suggesting the formation of a large complex. Analytical ultracentrifugation (AUC) studies of formation of the N-[beta]GRP: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 (âź102 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-[beta]GRP:laminarin complex in solution differs from the one in which a single N-[beta]GRP molecule has been proposed to bind to a triple-helical form of laminarin on the basis of a X-ray crystal structure of the N-[beta]GRP:laminarihexaose complex. AUC studies and phenoloxidase activation measurements made with designed mutants of N-[beta]GRP indicate that electrostatic interactions between the ligand-bound protein molecules contribute to the stability of the N-[beta]GRP:laminarin complex and that a decreased stability results in a reduction of proPO activation. These novel findings suggest that ligand-induced self-association of the [beta]GRP:[beta]-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the pathogen recognition signal. In the case of the homolog of GNBPA2 from Anopheles gambiae, the malaria-causing Plasmodium carrier, multiligand specificity was characterized, suggesting a functional diversity of the immunoglobulin domain structure
Chemical warfare agent simulants in Gambleâs fluid: Is the fluid toxic? Can it be made safer by inclusion of solid nanocrystalline metal oxides?
Citation: Karote, Dennis, Brandon Walker, Huaien Dai, Ramaswamy Krishnamoorthi, Janis Voo, and Shyamala Rajagopalan. âChemical Warfare Agent Simulants in Gambleâs Fluid: Is the Fluid Toxic? Can It Be Made Safer by Inclusion of Solid Nanocrystalline Metal Oxides?â Edited by Meehir Palit. Journal of Chemistry 2013 (December 5, 2012): 641620. https://doi.org/10.1155/2013/641620.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 Gambleâs fluid during a 96âh period. Upon addition of Exceptional Hazard Attenuation Materials (EHAMs) to 2-CEES containing Gambleâs fluid, degradation was generally improved during the first 24âh period. The 96âh 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 Gambleâs fluid, but was enhanced by the addition of EHAMs. FTIR and solid state 31P 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 Gambleâs fluid over a 96âh 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
An Initial Event in the Insect Innate Immune Response: Structural and Biological Studies of Interactions between βâ1,3-Glucan and the NâTerminal Domain of βâ1,3-Glucan Recognition Protein
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 (<i>Plodia
interpunctella</i>), 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 (âź6
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 <sup>15</sup>Nâ<sup>1</sup>H groups of the
protein, suggesting the formation of a large complex. Analytical ultracentrifugation
(AUC) studies of formation of the N-βGRPâlaminarin complex
show that ligand binding induces self-association of the proteinâcarbohydrate
complex into a macro structure, likely containing six protein and
three laminarin molecules (âź102 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-βGRPâlaminarin 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-βGRPâlaminarihexaose
complex [Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N.,
and Yamaguchi, Y. (2011) <i>J. Biol. Chem</i>. <i>286</i>, 29158â29165]. 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-βGRPâlaminarin 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 βGRPâβ-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
Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae
Citation: Gorman, M.J., Sullivan, L.I., Nguyen, T.D.T., Dai,H., Arakane,Y., Dittmer,
N.T.,âŚKanost, M.R. (2012). Kinetic properties of alternatively spliced isoforms of
laccase-2 from Tribolium castaneum and Anopheles gambiae. Insect Biochemistry and
Molecular Biology, 42(3), 193-202.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, Nacetyldopamine (NADA), N-b-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 (2,20-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