Extraembryonic membrane of the polyembryonic parasitoid Macrocentrus cingulum Brischke (Hym., Braconidae) is essential for evasion of encapsulation

Abstract:  The wasp Macrocentrus cingulum (Hymenoptera) is an endoparasitoid that uses larvae of the Asian corn borer Ostrinia furnacalis as one of its hosts. The wasp is polyembryonic and a single wasp egg gives rise to several dozens of embryos. Earlier studies showed that the fibrous layer on the surface of M. cingulum eggs protects them from the immune system of the moth larvae. However, the way in which the embryos of the parasitoid avoid being encapsulated remained unknown. In this paper, we show that the evasion of encapsulation is mediated through the extraembryonic membrane. We also show that M. cingulum embryos developed normally in the larvae of O. furnacalis but were encapsulated when injected into the larvae of Helicoverpa armigera , which is not a host species for the wasp larvae. When the extraembryonic membrane was removed, either chemically using the enzyme dispase or mechanically using a dissecting needle, the 'unprotected' embryos were also encapsulated both in vivo and in vitro by the haemocytes of the normal host O. furnacalis . It was also shown that the extraembryonic membrane was labelled strongly with fluorescein isothiocyanate (FITC)-conjugated Helix pomatia ( H.p. ) lectin. This suggests that a chemical in, or on, the extraembryonic membrane, that helps the embryos of M. cingulum to avoid encapsulation, is possibly a glycodeterminant produced in the haemocoel of the wasp.     

Molecular identification and functional analysis of Niemann-Pick type C2 protein in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae)

Niemann-Pick type C2 (NPC2) proteins in arthropods have been extensively differentiated and possibly duplicated according to environmental conditions and are probable to have different functions. The participation of NPC2 proteins in chemical communication in arthropods brings new objectives in environmental-friendly strategies for pest population control. In this study, NPC2 gene in Macrocentrus cingulum (McinNPC2) was newly identified by rapid amplification cDNA ends (RACE) technology. McinNPC2 amino acid sequence alignment with other representative NPC2 annotates to evaluate the highly conserved consensus amino acids, but with odorant binding proteins in M. cingulum show that only one consensus amino acid. Primary six-cysteine structures that are same to odorant binding proteins in M. cingulum were observed in McinNPC2. Phylogenetic analysis of McinNPC2 indicated that the nearest monophyletic group forming one clade with high posterior probability values clusters as Cyphomyrmex costatus (CcosNPC2) whereas the nearest evolutionary relation group as some odorant binding proteins. Moreover, quantitative real-time PCR (qPCR) measurements show that the McinNPC2 gene expression level in various tissues of the female is significantly and ubiquitously higher than in male, whereas the highest expression level in female antennae. We further explore the binding characterization of recombinant McinNPC2 to candidate odor molecules and did the modeling and docking simulations. The results showed ligands binding specificity and docking tests results indicate that β-ionone, an aroma compound commonly found in essential oils, can strongly bind with McinNPC2. In conclusion, we proposed that McinNPC2 may be involved in chemical communication and play roles in perception of plant volatiles.     

Sense organs on the ovipositor of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae): their probable role in stinging,oviposition and host selection process

Parasitoid wasps from the insect order Hymenoptera can be deployed successfully as biological control agents for a number of pests, and have previously been introduced for the control of corn pest insect species from the Lepidopteran genus Ostrinia. Organs on the ovipositor of parasitoid wasps have mechanical and tactile senses that coordinate the complex movements of egg laying, and the ovipositor of Hymenopteran insects have evolved associated venom glands as part of their stinging defense. The ovipositor of parasitic wasps has evolved an additional function as a piercing organ that is required for the deposition of eggs within suitable host larvae. The morphology and ultrastructure of sense organs on the ovipositor and sheath of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) are described using scanning and transmission electron microscopy. Three types of sensilla trichodea were shown to be abundant on the outer sheath of the ovipositor, with types II and III being most distal, and the inner surface of the ovipositor covered with microtrichia, more densely near the apex. Sensilla coeloconica are distributed on both ventral and dorsal valves, while campaniform sensilla and secretory pores are only located on the dorsal valve. The olistheter-like interlocking mechanism, as well as the morphology of the ventral and dorsal valve tips and the ventral valve seal may be important in stinging, oviposition and in the host selection process.     

Racemization of Amino Acid Residues in Proteins during Roasting

Formation of N-acetylkanamycin amidohydrolase (NAKA) in a strain of Streptomyces kanamyceticus proceeded in parallel with the increase of both kanamycin (KM) and N-acetylkanamycin (NAK) in the initial period. NAKA formation then suddenly began to decrease in NAK but KM continued to increase before reaching a maximum level. The study on conversion of intracellular NAK into KM using washed cells has shown that the endogenous activity of NAKA is most efficiently induced by acetate together with the activity of isocitrate lyase and N-acetyltyrosine amidohydrolase. Sclerin (SCL) stimulated their induction in the same manner, while N-acetyltyrosine repressed NAKA. Glucose effect was relieved by SCL and some amino acids. From these results, change in the chemical composition of cells in idiophase was well elucidated.     

Levels of encapsulation and melanization in two larval instars of Ostrinia furnacalis Guenée (Lep., Pyralidae) during simulation of parasitization by Macrocentrus cingulum Brischke (Hym., Braconidae)

Abstract:  The hymenopteran Macrocentrus cingulum is a polyembryonic endoparasitoid that uses larvae of the lepidopteran Ostrinia furnacalis as one of its host insects. Previous studies indicated that although this parasitoid does not transmit polydnaviruses when it lays its eggs, a layer of fibrous tissue on the surface of the eggs helps them to avoid being encapsulated by the immune system of the host insect. However, as eggs of M. cingulum that are laid into late instar larvae of O . furnacalis often do not survive, there is a tendency for the adults to lay their eggs in earlier instar larvae. We studied the amounts of encapsulation and melanization around beads of DEAE-Sephadex A-25 injected into the haemoceol of fourth and fifth larval instars of O. furnacalis . The beads were injected to simulate the presence of eggs of the parasitoid M. cingulum . We found that the levels of encapsulation and melanization around the beads increased with the age of the O. furnacalis larvae. Likewise, the total counts of the haemocytes circulating within the haemolymph increased with the age of the O. furnacalis larvae and were correlated negatively with the percentage of larvae parasitized by M. cingulum . It appears that young O. furnacalis possess a weak cellular defence, and as a result are more susceptible to being parasitized. Hence, the correlation between the levels of encapsulation and the age of the host insect appears simply to reflect passive evasion.     

Understanding the Functional Roles of Amino Acid Residues in Enzyme Catalysis

The MACiE database contains 223 distinct step-wise enzyme reaction mechanisms and holds representatives from each EC sub-subclass where there is a crystal structure and sufficient evidence in the literature to support a mechanism. Each catalytic step of every reaction sequence in MACiE is fully annotated so that it includes the function of the catalytic residues involved in the reaction and the mechanism by which substrates are transformed into products. Using MACiE as a knowledge base, we have seen that the top 10 most catalytic residues are histidine, aspartate, glutamate, lysine, cysteine, arginine, serine, threonine, tyrosine and tryptophan. Of these only seven (cysteine, histidine, aspartate, lysine, serine, threonine and tyrosine) dominate catalysis and provide essentially five functional roles that are essential. Stabilisation is the most common and essential role for all classes of enzyme, followed by general acid/base (proton acceptor and proton donor) functionality, with nucleophilic addition following closely behind (nucleophile and nucleofuge). We investigated the occurrence of these residues in MACiE and the Catalytic Site Atlas and found that, as expected, certain residue types are associated with each functional role, with some residue types able to perform diverse roles. In addition, it was seen that different EC classes of enzyme have a tendency to employ different residues for catalysis. Further, we show that whilst the differences between EC classes in catalytic residue composition are not immediately obvious from the general classes of Ingold mechanisms, there is some weak correlation between the mechanisms involved in a given EC class and the functions that the catalytic amino acid residues are performing. The analysis presented here provides a valuable insight into the functional roles of catalytic amino acid residues, which may have applications in many aspects of enzymology, from the design of novel enzymes to the prediction and validation of enzyme reaction mechanisms.     

Two Amino Acid Residues of Transposase Contributing to Differential Transposability of IS1 Elements in Escherichia coli

Escherichia coli W3110 contains four types of IS1 elements in the chromosome. Using an insertion element entrapping system, we collected 116 IS1 plasmid insertion mutants, which resulted from a minimum of 26 independent IS1 insertion events. All of them had insertions of IS1 of the IS1A (IS1E and IS1G) type. Inspection of the transposase sequences of the four IS1 types and the IS1 of the resistance plasmid R100 showed that two amino acid residues, His-193 and Leu-217 of transposase, might contribute to differential transposability of IS1 elements in W3110. The two amino acid residues of the transposase in IS1A (IS1E and IS1G) were altered separately by site-directed mutagenesis, and each mutant was found to mediate transposition at a frequency about 30-fold lower than that of IS1A (IS1E and IS1G). Thus, the assumption that His-193 and Leu-217 of transposase contribute to differential transposability of IS1 elements in W3110 was confirmed.     

Pulsed EPR Determination of Water Accessibility to Spin-Labeled Amino Acid Residues in LHCIIb

Membrane proteins reside in a structured environment in which some of their residues are accessible to water, some are in contact with alkyl chains of lipid molecules, and some are buried in the protein. Water accessibility of residues may change during folding or function-related structural dynamics. Several techniques based on the combination of pulsed electron paramagnetic resonance (EPR) with site-directed spin labeling can be used to quantify such water accessibility. Accessibility parameters for different residues in major plant light-harvesting complex IIb are determined by electron spin echo envelope modulation spectroscopy in the presence of deuterated water, deuterium contrast in transversal relaxation rates, analysis of longitudinal relaxation rates, and line shape analysis of electron-spin-echo-detected EPR spectra as well as by the conventional techniques of measuring the maximum hyperfine splitting and progressive saturation in continuous-wave EPR. Systematic comparison of these parameters allows for a more detailed characterization of the environment of the spin-labeled residues. These techniques are applicable independently of protein size and require ∼10-20 nmol of singly spin-labeled protein per sample. For a residue close to the N-terminus, in a domain unresolved in the existing x-ray structures of light-harvesting complex IIb, all methods indicate high water accessibility.     

Phosphate Acceptor Amino Acid Residues in Structural Proteins of Rhabdoviruses

Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses.     

Amino Acid Residues Contributing to the Substrate Specificity of the Influenza A Virus Neuraminidase

Influenza A viruses possess two glycoprotein spikes on the virion surface: hemagglutinin (HA), which binds to oligosaccharides containing terminal sialic acid, and neuraminidase (NA), which removes terminal sialic acid from oligosaccharides. Hence, the interplay between these receptor-binding and receptor-destroying functions assumes major importance in viral replication. In contrast to the well-characterized role of HA in host range restriction of influenza viruses, there is only limited information on the role of NA substrate specificity in viral replication among different animal species. We therefore investigated the substrate specificities of NA for linkages between N-acetyl sialic acid and galactose (NeuAcalpha2-3Gal and NeuAcalpha2-6Gal) and for different molecular species of sialic acids (N-acetyl and N-glycolyl sialic acids) in influenza A viruses isolated from human, avian, and pig hosts. Substrate specificity assays showed that all viruses had similar specificities for NeuAcalpha2-3Gal, while the activities for NeuAcalpha2-6Gal ranged from marginal, as represented by avian and early N2 human viruses, to high (although only one-third the activity for NeuAcalpha2-3Gal), as represented by swine and more recent N2 human viruses. Using site-specific mutagenesis, we identified in the earliest human virus with a detectable increase in NeuAcalpha2-6Gal specificity a change at position 275 (from isoleucine to valine) that enhanced the specificity for this substrate. Valine at position 275 was maintained in all later human viruses as well as swine viruses. A similar examination of N-glycolylneuraminic acid (NeuGc) specificity showed that avian viruses and most human viruses had low to moderate activity for this substrate, with the exception of most human viruses isolated between 1967 and 1969, whose NeuGc specificity was as high as that of swine viruses. The amino acid at position 431 was found to determine the level of NeuGc specificity of NA: lysine conferred high NeuGc specificity, while proline, glutamine, and glutamic acid were associated with lower NeuGc specificity. Both residues 275 and 431 lie close to the enzymatic active site but are not directly involved in the reaction mechanism. This finding suggests that the adaptation of NA to different substrates occurs by a mechanism of amino acid substitutions that subtly alter the conformation of NA in and around the active site to facilitate the binding of different species of sialic acid.     

In Silico and In Vitro Analyses Identified Three Amino Acid Residues Critical to the Catalysis of Two Aphid Farnesyl Diphosphate Synthase

Farnesyl diphosphate synthase (FPPS) plays an essential role in the isoprenoid biosynthetic pathway of microbes, plants and animals. In the present study, we first cloned two FPPSs from the bird cherry-oat aphid (RpFPPS1 and RpFPPS2), and activity assay by gas chromatography-mass spectrometry showed that both RpFPPS1 and RpFPPS2 were active in vitro. They were then subjected to homology modeling and molecular docking. Molecular interaction analysis indicated that three amino acid residues (R120, R121 and K266) might play key roles in the catalysis of the two aphid FPPSs by forming hydrogen bonds with the diphosphate moiety of the allylic substrate. These in silico results were subsequently confirmed by site-directed mutagenesis and in vitro activity assay of the mutant enzymes, in which each of the single mutations R120G, R121G and K266I abolished the activities of the two FPPSs. This study contributes to our understanding of the catalytic mechanism of farnesyl diphosphate synthases.     

FKBP12.6 Activates RyR1: Investigating the Amino Acid Residues Critical for Channel Modulation

We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12_{E31Q/D32N/W59F}) where the residues Glu³¹, Asp³², and Trp⁵⁹ were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12_{E31Q/D32N/W59F} mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12_{E31Q/D32N/W59F} activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu³¹, Asp³², and Trp⁵⁹ in FKBP12 and Gln³¹, Asn³², and Phe⁵⁹ in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues.     

FKBP12.6 Activates RyR1: Investigating the Amino Acid Residues Critical for Channel Modulation

We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12_{E31Q/D32N/W59F}) where the residues Glu³¹, Asp³², and Trp⁵⁹ were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12_{E31Q/D32N/W59F} mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12_{E31Q/D32N/W59F} activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu³¹, Asp³², and Trp⁵⁹ in FKBP12 and Gln³¹, Asn³², and Phe⁵⁹ in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues.     

A New Spatial Representation of Amino Acid Residues in Globular Proteins

Abstract

For the globular proteins with known three-dimensional structures, an ellipsoid model of each protein was constructed with least volume and its dimensions were derived. The spatial arrangements were made for the C_α and side chain atoms of that protein within that ellipsoid. This new spatial representation shows the residue position from the centroid, as well as the depth from the surface. The average spatial parameters were then calculated. The correlations between these new spatial parameters and the existing parameters of the amino acid residues were then derived.     

Identification of Channel-lining Amino Acid Residues in the Hydrophobic Segment of Colicin Ia

Colicin Ia is a bactericidal protein of 626 amino acid residues that kills its target cell by forming a channel in the inner membrane; it can also form voltage-dependent channels in planar lipid bilayer membranes. The channel-forming activity resides in the carboxy-terminal domain of ~177 residues. In the crystal structure of the water-soluble conformation, this domain consists of a bundle of 10 α-helices, with eight mostly amphipathic helices surrounding a hydrophobic helical hairpin (helices H8-H9). We wish to know how this structure changes to form a channel in a lipid bilayer. Although there is evidence that the open channel has four transmembrane segments (H8, H9, and parts of H1 and H6-H7), their arrangement relative to the pore is largely unknown. Given the lack of a detailed structural model, it is imperative to better characterize the channel-lining protein segments. Here, we focus on a segment of 44 residues (573–616), which in the crystal structure comprises the H8-H9 hairpin and flanking regions. We mutated each of these residues to a unique cysteine, added the mutant colicins to the cis side of planar bilayers to form channels, and determined whether sulfhydryl-specific methanethiosulfonate reagents could alter the conduction of ions through the open channel. We found a pattern of reactivity consistent with parts of H8 and H9 lining the channel as α-helices, albeit rather short ones for spanning a lipid bilayer (12 residues). The effects of the reactions on channel conductance and selectivity tend to be greater for residues near the amino terminus of H8 and the carboxy terminus of H9, with particularly large effects for G577C, T581C, and G609C, suggesting that these residues may occupy a relatively constricted region near the cis end of the channel.     

Amino Acid Residues Involved in the Functional Integrity of Escherichia coli Methionine Aminopeptidase

Amino acid residues in the metal-binding and putative substrate-binding sites of Escherichia coli methionine aminopeptidase (MAP) were mutated, and their effects on the function of the enzyme were investigated. Substitution of any amino acid residue at the metal-binding site resulted in complete loss of the two cobalt ions bound to the protein and diminished the enzyme activity. However, only Cys70 and Trp221 at the putative substrate-binding site are involved in the catalytic activity of MAP. Changing either of them caused partial loss of enzyme activity, while mutations at both positions abolished MAP function. Both residues are found to be conserved in type I but not type II MAPs.     

三种鹿茸片中氨基酸质量分数的比较

通过对东北梅花鹿茸片、新西兰鹿茸片以及东北梅花鹿茸骨片中氨基酸质量分数的比较测定,结果表明:东北梅花鹿茸片中的氨基酸质量分数高于其余两种茸片中的氨基酸质量分数,从而为合理选购和使用鹿茸片提供科学理论依据。     

Site Directed Mutagenesis of Amino Acid Residues at the Active Site of Mouse Aldehyde Oxidase AOX1

Mouse aldehyde oxidase (mAOX1) forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Eschericia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.