Plant lectins as versatile tools to fight coronavirus outbreaks.

The S protein forming the homotrimeric spikes of pathogenic beta-coronaviruses, such as MERS-CoV, SARS-CoV and SARS-CoV-2, is a highly glycosylated protein containing mainly N-glycans of the complex and high-mannose type, as well as O-glycans. Similarly, the host cell receptors DPP4 for MERS-CoV and ACE2 for SARS-CoV and SARS-CoV-2, also represent N- and O-glycosylated proteins. All these glycoproteins share common glycosylation patterns, suggesting that plant lectins with different carbohydrate-binding specificities could be used as carbohydrate-binding agents for the spikes and their receptors, to combat COVID19 pandemics. The binding of plant lectins to the spikes and their receptors could mask the non-glycosylated receptor binding domain of the virus and the corresponding region of the receptor, thus preventing a proper interaction of the spike proteins with their receptors. In this review, we analyze (1) the ability of plant lectins to interact with the N- and O-glycans present on the spike proteins and their receptors, (2) the in vitro and in vivo anti-COVID19 activity already reported for plant lectins and, (3) the possible ways for delivery of lectins to block the spikes and/or their receptors.

Heliothis virescens chymotrypsin is translationally controlled by AeaTMOF binding ABC putative receptor.

Heliothis virescens larval chymotrypsin (GenBank accession number AF43709) was cloned, sequenced and its three dimensional (3D) conformation modeled. The enzyme's transcript was first detected 6 days after larval emergence and the transcript level was shown to fall between larval ecdysis periods. Comparisons between the activities of larval gut chymotrypsin and trypsin shows that chymotrypsin activity is only 16% of the total trypsin activity and the pH optimum of the larval chymotrypsin is between pH 9-10, however the enzyme also exhibited a broad activity between pH 4-6. Injections of AeaTMOF and several shorter analogues into 3rd instar larvae followed by Northern blot analyses showed that although the chymotrypsins activities were inhibited by 60%-80% the transcript level of the sequenced chymotrypsin was not reduced and was similar to controls in which the chymotrypsin activity was not inhibited, indicating that AeaTMOF and its analogues exert a translational control. Based on these observations a putative AeaTMOF receptor (ABCC4) homologous to the Ae. aegypti ABC receptor sequence was found in the H. virescens genome. 3D molecular modeling and docking of the AeaTMOF and several of its analogues to the ABCC4 receptor showed that it can bind AeaTMOF and its analogues as was shown before for the Ae. aegypti receptor.

Cloning and characterization of Aedes aegypti blood downregulated chymotrypsin II.

Aedes aegypti adult and larval blood downregulated chymotrypsin II was cloned, sequenced and its 3D conformation modeled. Cloning of the enzymes from adult and larval guts indicated that both genes sit at the same location on Chromosome 2. Genomic analyses showed that larval and adult genes are the same and both have four exons and three introns that are located on an 8.32 Kb DNA in direction with the Ae. aegypti genome. The adult and larval transcript synthesis is controlled by alternative splicing explaining small difference in the amino acids sequences. Chymotrypsin II that was extracted from guts of sugar-fed and at 48 after blood feeding showed a pH optimum of 4-5 with a broad shoulder of activity from pH 6 to 10. Dot blot analyses show that the enzyme's transcript is downregulated after females take a blood meal and upregulated at 48 h after the blood meal. A Chymotrypsin II transcript was also detected in the larval gut during different times of larval developmental stages, indication that Ae. aegypti chymotrypsin II is synthesized by adults and larval guts. The possibility that JH III and 20HE play an active role in the regulation is discussed.

Cloning and characterization of Aedes aegypti juvenile hormone epoxide hydrolases (JHEHs).

Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of juvenile hormone III (JH III) in insects. To study the role that JHEH plays in female Aedes aegypti JHEH 1, 2, and 3 complementary DNA (cDNAs) were cloned and sequenced. Northern blot analyses show that the three transcripts are expressed in the head thorax, the gut, the ovaries, and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds JH III acid (JH IIIA) at the catalytic groove better than JH III. The cDNA of JHEH 1 and 2 are very similar indicating close relationship. Knocking down of jheh 1, 2, and 3 in adult female and larval Ae. aegypti using double-stranded RNA (dsRNA) did not affect egg development or caused adult mortality. Larvae that were fed bacterial cells expressing dsRNA against jheh 1, 2, and 3 grew normally. Treating blood-fed female Ae. aegypti with [12-3 H](10R) JH III and analyzing the metabolites by C18 reversed phase chromatography showed that JHEH preferred substrate is not JH III but JH IIIA. Genomic analysis of jheh 1, 2, and 3 indicate that jheh 1 and 2 are transcribed from a 1.53 kb DNA whereas jheh 3 is transcribed from a 10.9 kb DNA. All three genes are found on chromosome two at distinct locations. JHEH 2 was expressed in bacterial cells and purified by Ni affinity chromatography. Sequencing of the recombinant protein by MS/MS identified JHEH 2 as the expressed recombinant protein.

Man-Specific, GalNAc/T/Tn-Specific and Neu5Ac-Specific Seaweed Lectins as Glycan Probes for the SARS-CoV-2 (COVID-19) Coronavirus.

Seaweed lectins, especially high-mannose-specific lectins from red algae, have been identified as potential antiviral agents that are capable of blocking the replication of various enveloped viruses like influenza virus, herpes virus, and HIV-1 in vitro. Their antiviral activity depends on the recognition of glycoprotein receptors on the surface of sensitive host cells-in particular, hemagglutinin for influenza virus or gp120 for HIV-1, which in turn triggers fusion events, allowing the entry of the viral genome into the cells and its subsequent replication. The diversity of glycans present on the S-glycoproteins forming the spikes covering the SARS-CoV-2 envelope, essentially complex type N-glycans and high-mannose type N-glycans, suggests that high-mannose-specific seaweed lectins are particularly well adapted as glycan probes for coronaviruses. This review presents a detailed study of the carbohydrate-binding specificity of high-mannose-specific seaweed lectins, demonstrating their potential to be used as specific glycan probes for coronaviruses, as well as the biomedical interest for both the detection and immobilization of SARS-CoV-2 to avoid shedding of the virus into the environment. The use of these seaweed lectins as replication blockers for SARS-CoV-2 is also discussed.

Mannose-Specific Lectins from Marine Algae: Diverse Structural Scaffolds Associated to Common Virucidal and Anti-Cancer Properties.

To date, a number of mannose-specific lectins have been isolated and characterized from seaweeds, especially from red algae. In fact, man-specific seaweed lectins consist of different structural scaffolds harboring a single or a few carbohydrate-binding sites which specifically recognize mannose-containing glycans. Depending on the structural scaffold, man-specific seaweed lectins belong to five distinct structurally-related lectin families, namely (1) the griffithsin lectin family (ß-prism I scaffold); (2) the Oscillatoria agardhii agglutinin homolog (OAAH) lectin family (ß-barrel scaffold); (3) the legume lectin-like lectin family (ß-sandwich scaffold); (4) the Galanthus nivalis agglutinin (GNA)-like lectin family (ß-prism II scaffold); and, (5) the MFP2-like lectin family (MFP2-like scaffold). Another algal lectin from Ulva pertusa, has been inferred to the methanol dehydrogenase related lectin family, because it displays a rather different GlcNAc-specificity. In spite of these structural discrepancies, all members from the five lectin families share a common ability to specifically recognize man-containing glycans and, especially, high-mannose type glycans. Because of their mannose-binding specificity, these lectins have been used as valuable tools for deciphering and characterizing the complex mannose-containing glycans from the glycocalyx covering both normal and transformed cells, and as diagnostic tools and therapeutic drugs that specifically recognize the altered high-mannose N-glycans occurring at the surface of various cancer cells. In addition to these anti-cancer properties, man-specific seaweed lectins have been widely used as potent human immunodeficiency virus (HIV-1)-inactivating proteins, due to their capacity to specifically interact with the envelope glycoprotein gp120 and prevent the virion infectivity of HIV-1 towards the host CD4+ T-lymphocyte cells in vitro.

Overview of the Structure⁻Function Relationships of Mannose-Specific Lectins from Plants, Algae and Fungi.

To date, a number of mannose-binding lectins have been isolated and characterized from plants and fungi. These proteins are composed of different structural scaffold structures which harbor a single or multiple carbohydrate-binding sites involved in the specific recognition of mannose-containing glycans. Generally, the mannose-binding site consists of a small, central, carbohydrate-binding pocket responsible for the "broad sugar-binding specificity" toward a single mannose molecule, surrounded by a more extended binding area responsible for the specific recognition of larger mannose-containing N-glycan chains. Accordingly, the mannose-binding specificity of the so-called mannose-binding lectins towards complex mannose-containing N-glycans depends largely on the topography of their mannose-binding site(s). This structure⁻function relationship introduces a high degree of specificity in the apparently homogeneous group of mannose-binding lectins, with respect to the specific recognition of high-mannose and complex N-glycans. Because of the high specificity towards mannose these lectins are valuable tools for deciphering and characterizing the complex mannose-containing glycans that decorate both normal and transformed cells, e.g., the altered high-mannose N-glycans that often occur at the surface of various cancer cells.

Morniga-G, a T/Tn-Specific Lectin, Induces Leukemic Cell Death via Caspase and DR5 Receptor-Dependent Pathways.

Morniga-G, the Gal-specific black mulberry (Morus nigra) lectin, displays high affinity for T (CD176) and Tn (CD175) antigens, frequently expressed at the cancer cell surface. The effects of Morniga-G were investigated on a Tn-positive leukemic Jurkat cell line. The lectin, used in a concentration range between 5⁻20 µg/mL, induced cell death in leukemic Jurkat cells. Microscopic and cytofluorometric analyses indicated that Jurkat cell death was essentially apoptotic, associated with an increase in the ceramide content and a depolarization of the mitochondrial transmembrane potential. This lectin-mediated cell death was inhibited by the pan caspase-inhibitor zVAD. In addition, cleavage of caspases 8, 9, and 3 was observed in Morniga-G-treated Jurkat cells whereas Jurkat cell lines that are deficient in caspase 8⁻10, caspase 9, or FADD, survived to the lectin-mediated toxicity. Furthermore, in the presence of TRAIL- or DR5-blocking mononoclonal antibodies, Jurkat cells became resistant to Morniga-G, suggesting that the lectin triggers cell death via the TRAIL/DR5 pathway. In silico computer simulations suggest that Morniga-G might facilitate both the DR5 dimerization and the building of TRAIL/DR5 complexes. Finally, upon treatment of Jurkat cells with benzyl-GalNAc, an O-glycosylation inhibitor, a decrease in Tn antigen expression associating with a reduced Morniga-G toxicity, was observed. Taken together, these results suggest that Morniga-G induces the cell death of Tn-positive leukemic cells via concomitant O-glycosylation-, caspase-, and TRAIL/DR5-dependent pathways.

Juvenile hormone affects the splicing of Culex quinquefasciatus early trypsin messenger RNA.

The full length of Culex quiquefasciatus early trypsin has been cloned and sequenced and a three-dimensional (3D) model of the enzyme was built showing that the enzyme has the canonical trypsin's active pocket containing H78, D123, S129, and D128. The biosynthesis of juvenile hormone (JH) III by the corpora allata (CA) in female Cx. quiquefasciatus is sugar-dependent. Females that were maintained on water after emergence synthesize very little JH III, JH III bisepoxide, and methyl farnesoate (MF) (3.8, 1.1, and 0.8 fmol/4 hr/CA, respectively). One hour after sugar feeding, the synthesis of JH III and JH III bisepoxide reached a maximum (11.3 and 5.9 fmol/4 hr/CA, respectively) whereas MF biosynthesis reached a maximum at 24 hr (5.2 fmol/4 hr/CA). The early trypsin is transcribed with a short intron (51 nt) is spliced when JH III biosynthesis is high in sugar fed and at 1 hr after the blood meal (22 and 15 fmol/4 hr/CA, respectively). We investigated the transcriptional and posttranscriptional regulation of the early trypsin gene showing that JH III concentrations influence splicing. In the absence JH III the unspliced transcript is linked by a phosphoamide bond at the 5'-end to RNA ribonuleoprotein (RNP). The biosynthesis of the early trypsin was followed in ligated abdomens (without CA) of newly emerged females that fed blood by enema. Our results show that the early trypsin biosynthesis depends on sugar and blood feeding, whereas the late trypsin biosynthesis does not depend on sugar feeding, or JH III biosynthesis. Downregulating the early trypsin transcript does not affect the late trypsin.

Evolution and structural diversification of Nictaba-like lectin genes in food crops with a focus on soybean (Glycine max).

Background and Aims: The Nictaba family groups all proteins that show homology to Nictaba, the tobacco lectin. So far, Nictaba and an Arabidopsis thaliana homologue have been shown to be implicated in the plant stress response. The availability of more than 50 sequenced plant genomes provided the opportunity for a genome-wide identification of Nictaba -like genes in 15 species, representing members of the Fabaceae, Poaceae, Solanaceae, Musaceae, Arecaceae, Malvaceae and Rubiaceae. Additionally, phylogenetic relationships between the different species were explored. Furthermore, this study included domain organization analysis, searching for orthologous genes in the legume family and transcript profiling of the Nictaba -like lectin genes in soybean. Methods: Using a combination of BLASTp, InterPro analysis and hidden Markov models, the genomes of Medicago truncatula , Cicer arietinum , Lotus japonicus , Glycine max , Cajanus cajan , Phaseolus vulgaris , Theobroma cacao , Solanum lycopersicum , Solanum tuberosum , Coffea canephora , Oryza sativa , Zea mays, Sorghum bicolor , Musa acuminata and Elaeis guineensis were searched for Nictaba -like genes. Phylogenetic analysis was performed using RAxML and additional protein domains in the Nictaba-like sequences were identified using InterPro. Expression analysis of the soybean Nictaba -like genes was investigated using microarray data. Key Results: Nictaba -like genes were identified in all studied species and analysis of the duplication events demonstrated that both tandem and segmental duplication contributed to the expansion of the Nictaba gene family in angiosperms. The single-domain Nictaba protein and the multi-domain F-box Nictaba architectures are ubiquitous among all analysed species and microarray analysis revealed differential expression patterns for all soybean Nictaba-like genes. Conclusions: Taken together, the comparative genomics data contributes to our understanding of the Nictaba -like gene family in species for which the occurrence of Nictaba domains had not yet been investigated. Given the ubiquitous nature of these genes, they have probably acquired new functions over time and are expected to take on various roles in plant development and defence.

Expression of Bacillus thuringiensis cytolytic toxin (Cyt2Ca1) in citrus roots to control Diaprepes abbreviatus larvae.

Diaprepes abbreviatus (L.) is an important pest of citrus in the USA. Currently, no effective management strategies of D. abbreviatus exist in citriculture, and new methods of control are desperately sought. To protect citrus against D. abbreviatus a transgenic citrus rootstock expressing Bacillus thuringiensis Cyt2Ca1, an insect toxin protein, was developed using Agrobacterium-mediated transformation of 'Carrizo' citrange [Citrus sinensis (L) Osbeck Poncirus trifoliate (L) Raf]. The transgenic citrus root stock expressed the cytolytic toxin Cyt2Ca1 constitutively under the control of a 35S promoter in the transgenic Carrizo citrange trifoliate hybrid including the roots that are the food source of larval D. abbreviatus. The engineered citrus was screened by Western blot and RT-qPCR analyses for cyt2Ca1 and positive citrus identified. Citrus trees expressing different levels of cyt2Ca1 transcripts were identified (Groups A-C). High expression of the toxin in the leaves (109 transcripts/ng RNA), however, retarded plant growth. The transgenic plants were grown in pots and the roots exposed to 3week old D. abbreviatus larvae using no-choice plant bioassays. Three cyt2Ca1 transgenic plants were identified that sustained less root damage belonging to Group B and C. One plant caused death to 43% of the larvae that fed on its roots expressed 8×106cyt2Ca1 transcripts/ng RNA. These results show, for the first time, that Cyt2Ca1 expressed in moderate amounts by the roots of citrus does not retard citrus growth and can protect it from larval D. abbreviatus.

Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy.

Aberrant O-glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O-glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola, and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O-glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors.

Ribosome Inactivating Proteins from Rosaceae.

Ribosome-inactivating proteins (RIPs) are widespread among higher plants of different taxonomic orders. In this study, we report on the RIP sequences found in the genome/transcriptome of several important Rosaceae species, including many economically important edible fruits such as apple, pear, peach, apricot, and strawberry. All RIP domains from Rosaceae share high sequence similarity with conserved residues in the catalytic site and the carbohydrate binding sites. The genomes of Malus domestica and Pyrus communis contain both type 1 and type 2 RIP sequences, whereas for Prunus mume, Prunus persica, Pyrus bretschneideri, and Pyrus communis a complex set of type 1 RIP sequences was retrieved. Heterologous expression and purification of the type 1 as well as the type 2 RIP from apple allowed to characterize the biological activity of the proteins. Both RIPs from Malus domestica can inhibit protein synthesis. Furthermore, molecular modelling suggests that RIPs from Rosaceae possess three-dimensional structures that are highly similar to the model proteins and can bind to RIP substrates. Screening of the recombinant type 2 RIP from apple on a glycan array revealed that this type 2 RIP interacts with terminal sialic acid residues. Our data suggest that the RIPs from Rosaceae are biologically active proteins.

A cell-based reporter assay for screening for EcR agonist/antagonist activity of natural ecdysteroids in Lepidoptera (Bm5) and Diptera (S2) cell cultures, followed by modeling of ecdysteroid-EcR interactions and normal mode analysis.

Ecdysteroid signal transduction is a key process in insect development and therefore an important target for insecticide development. We employed an in vitro cell-based reporter bioassay for the screening of potential ecdysone receptor (EcR) agonistic and antagonistic compounds. Natural ecdysteroids were assayed with ecdysteroid-responsive cell line cultures that were transiently transfected with the reporter plasmid ERE-b.act.luc. We used the dipteran Schneider S2 cells of Drosophila melanogaster and the lepidopteran Bm5 cells of Bombyx mori, representing important pest insects in medicine and agriculture. Measurements showed an EcR agonistic activity only for cyasterone both in S2 (EC50=3.3µM) and Bm5 cells (EC50=5.3µM), which was low compared to that of the commercial dibenzoylhydrazine-based insecticide tebufenozide (EC50=0.71µM and 0.00089µM, respectively). Interestingly, a strong antagonistic activity was found for castasterone in S2 cells with an IC50 of 0.039µM; in Bm5 cells this effect only became visible at much higher concentrations (IC50=18µM). To gain more insight in the EcR interaction, three-dimensional modeling of dipteran and lepidopteran EcR-LBD was performed. In conclusion, we showed that the EcR cell-based reporter bioassay tested here is a useful and practical tool for the screening of candidate EcR agonists and antagonists. The docking experiments as well as the normal mode analysis provided evidence that the antagonist activity of castasterone may be through direct binding with the receptor with specific changes in protein flexibility. The search for new ecdysteroid-like compounds may be particularly relevant for dipterans because the activity of dibenzoylhydrazines appears to be correlated with an extension of the EcR-LBD binding pocket that is prominent in lepidopteran receptors but less so in the modeled dipteran structure.

Modeling and hexahedral meshing of cerebral arterial networks from centerlines.

Computational fluid dynamics (CFD) simulation provides valuable information on blood flow from the vascular geometry. However, it requires extracting precise models of arteries from low-resolution medical images, which remains challenging. Centerline-based representation is widely used to model large vascular networks with small vessels, as it encodes both the geometric and topological information and facilitates manual editing. In this work, we propose an automatic method to generate a structured hexahedral mesh suitable for CFD directly from centerlines. We addressed both the modeling and meshing tasks. We proposed a vessel model based on penalized splines to overcome the limitations inherent to the centerline representation, such as noise and sparsity. The bifurcations are reconstructed using a parametric model based on the anatomy that we extended to planar n-furcations. Finally, we developed a method to produce a volume mesh with structured, hexahedral, and flow-oriented cells from the proposed vascular network model. The proposed method offers better robustness to the common defects of centerlines and increases the mesh quality compared to state-of-the-art methods. As it relies on centerlines alone, it can be applied to edit the vascular model effortlessly to study the impact of vascular geometry and topology on hemodynamics. We demonstrate the efficiency of our method by entirely meshing a dataset of 60 cerebral vascular networks. 92% of the vessels and 83% of the bifurcations were meshed without defects needing manual intervention, despite the challenging aspect of the input data. The source code is released publicly.

The unique architecture and function of cellulose-interacting proteins in oomycetes revealed by genomic and structural analyses.

BACKGROUND: Oomycetes are fungal-like microorganisms evolutionary distinct from true fungi, belonging to the Stramenopile lineage and comprising major plant pathogens. Both oomycetes and fungi express proteins able to interact with cellulose, a major component of plant and oomycete cell walls, through the presence of carbohydrate-binding module belonging to the family 1 (CBM1). Fungal CBM1-containing proteins were implicated in cellulose degradation whereas in oomycetes, the Cellulose Binding Elicitor Lectin (CBEL), a well-characterized CBM1-protein from Phytophthora parasitica, was implicated in cell wall integrity, adhesion to cellulosic substrates and induction of plant immunity. RESULTS: To extend our knowledge on CBM1-containing proteins in oomycetes, we have conducted a comprehensive analysis on 60 fungi and 7 oomycetes genomes leading to the identification of 518 CBM1-containing proteins. In plant-interacting microorganisms, the larger number of CBM1-protein coding genes is expressed by necrotroph and hemibiotrophic pathogens, whereas a strong reduction of these genes is observed in symbionts and biotrophs. In fungi, more than 70% of CBM1-containing proteins correspond to enzymatic proteins in which CBM1 is associated with a catalytic unit involved in cellulose degradation. In oomycetes more than 90% of proteins are similar to CBEL in which CBM1 is associated with a non-catalytic PAN/Apple domain, known to interact with specific carbohydrates or proteins. Distinct Stramenopile genomes like diatoms and brown algae are devoid of CBM1 coding genes. A CBM1-PAN/Apple association 3D structural modeling was built allowing the identification of amino acid residues interacting with cellulose and suggesting the putative interaction of the PAN/Apple domain with another type of glucan. By Surface Plasmon Resonance experiments, we showed that CBEL binds to glycoproteins through galactose or N-acetyl-galactosamine motifs. CONCLUSIONS: This study provides insight into the evolution and biological roles of CBM1-containing proteins from oomycetes. We show that while CBM1s from fungi and oomycetes are similar, they team up with different protein domains, either in proteins implicated in the degradation of plant cell wall components in the case of fungi or in proteins involved in adhesion to polysaccharidic substrates in the case of oomycetes. This work highlighted the unique role and evolution of CBM1 proteins in oomycete among the Stramenopile lineage.

Expression analysis of a type S2 EUL-related lectin from rice in Pichia pastoris.

Rice (Oryza sativa) expresses different putative carbohydrate-binding proteins belonging to the class of lectins containing an Euonymus lectin (EUL)-related domain, one of them being OrysaEULS2. The OrysaEULS2 sequence consists of a 56 amino acid N-terminal domain followed by the EUL sequence. In this paper the original sequence of the EUL domain of OrysaEULS2 and some mutant forms have been expressed in Pichia pastoris. Subsequently, the recombinant proteins were purified and their carbohydrate binding properties determined. Analysis of the original protein on the glycan array revealed interaction with mannose containing structures and to a lesser extent with glycans containing lactosamine related structures. It was shown that mutation of tryptophan residue 134 into leucine resulted in an almost complete loss of carbohydrate binding activity of OrysaEULS2. Our results show that the EUL domain in OrysaEULS2 interacts with glycan structures, and hence can be considered as a lectin. However, the binding of the protein with the array is much weaker than that of other EUL-related lectins. Furthermore, our results indicate that gene divergence within the family of EUL-related lectins lead to changes in carbohydrate binding specificity.

Sequencing and structural homology modeling of the ecdysone receptor in two chrysopids used in biological control of pest insects.

In insects, the process of molting and metamorphosis are mainly regulated by a steroidal hormone 20-hydroxyecdysone (20E) and its analogs (ecdysteroids) that specifically bind to the ecdysone receptor ligand-binding domain (EcR-LBD). Currently, several synthetic non-steroidal ecdysone agonists, including tebufenozide, are commercially available as insecticides. Tebufenozide exerts its activity by binding to the 20E-binding site and thus activating EcR permanently. It appears that subtle differences in the architecture among LBDs may underpin the differential binding affinity of tebufenozide across taxonomic orders. In brief, first we demonstrated the harmlessness of tebufenozide towards Chrysoperla externa (Ce). Then, a molecular analysis of EcR-LBD of two neuropteran insects Chrysoperla carnea and Ce was presented. Finally, we constructed a chrysopid in silico homology model docked ponasterone A (PonA) and tebufenozide into the binding pocket and analyzed the amino acids indentified as critical for binding to PonA and tebufenozide. Due to a restrict extent in the cavity at the bottom of the ecdysone-binding pocket a steric clash occurred upon docking of tebufenozide. The absence of harm biological effect and the docking results suggest that tebufenozide is prevented of any deleterious effects on chrysopids.

Bactericidal Properties of Proline-Rich Aedes aegypti Trypsin Modulating Oostatic Factor (AeaTMOF).

The antimicrobial properties of proline-rich Aedes aegypti decapeptide TMOF (AeaTMOF) and oncocin112 (1-13) were compared. Incubations with multidrug-resistant Escherichia coli cells showed that AeaTMOF (5 mM) was able to completely inhibit bacterial cell growth, whereas oncocin112 (1-13) (20 mM) partially inhibited bacterial growth as compared with bacterial cells that were not multidrug-resistant cells. AeaTMOF (5 mM) was very effective against Acinetobacter baumannii and Pseudomonas aeruginosa, completely inhibiting cell growth during 15 h incubations. AeaTMOF (5 mM) completely inhibited the Gram-positive bacteria Staphylococcus aureus and Bacillus thurengiensis sups. Israelensis cell growth, whereas oncocin112 (1-13) (10 and 20 mM) failed to affect bacterial cell growth. E. coli cells that lack the SbmA transporter were inhibited by AeaTMOF (5 mM) and not by oncocin112 (1-13) (10 to 20 mM), indicating that AeaTMOF can use other bacterial transporters than SbmA that is mainly used by proline-rich antimicrobial peptides. Incubation of E. coli cells with NaAzide showed that AeaTMOF does not use ABC-like transporters that use ATP hydrolysis to import molecules into bacterial cells. Three-dimensional modeling and docking of AeaTMOF to SbmA and MdtM transporters showed that AeaTMOF can bind these proteins, and the binding location of AeaTMOF inside these protein transporters allows AeaTMOF to be transported into the bacterial cytosol. These results show that AeaTMOF can be used as a future antibacterial agent against both multidrug-resistant Gram-positive and -negative bacteria.

The Ribosome Is the Ultimate Receptor for Trypsin Modulating Oostatic Factor (TMOF).

Aedes aegypti Trypsin Modulating Oostatic Factor (AeaTMOF). a mosquito decapeptide that controls trypsin biosynthesis in female and larval mosquitoes. enters the gut epithelial cells of female mosquitoes using ABC-tmfA receptor/importer. To study the ultimate targeted receptor after AeaTMOF enters the cell, AeaTMOF was incubated in vitro with either Escherichia coli or Spodoptera frugiperda protein-expressing extracts containing 70S and 80S ribosomes, respectively. The effect of AeaTMOF on luciferase biosynthesis in vitro using 70S ribosomes was compared with that of oncocin112 (1-13), a ribosome-binding antibacterial peptide. The IC50 of 1 µM and 2 µM, respectively, for both peptides was determined. Incubation with a protein-expressing system and S. frugiperda 80S ribosomes determined an IC50 of 1.8 µM for Aedes aegypti larval late trypsin biosynthesis. Incubation of purified E. coli ribosome with increasing concentration of AeaTMOF shows that the binding of AeaTMOF to the bacterial ribosome exhibits a high affinity (KD = 23 ± 3.4 nM, Bmax = 0.553 ± 0.023 pmol/µg ribosome and Kassoc = 4.3 × 107 M-1). Molecular modeling and docking experiments show that AeaTMOF binds bacterial and Drosophila ribosome (50S and 60S, respectively) at the entrance of the ribosome exit tunnel, blocking the tRNA entrance and preventing protein biosynthesis. Recombinant E. coli cells that express only ABC-tmfA importer are inhibited by AeaTMOF but not by oncocin112 (1-13). These results suggest that the ribosome is the ultimate targeted receptor of AeaTMOF.