Two types of O-methyltransferase are involved in biosynthesis of anticancer methoxylated 4'-deoxyflavones in Scutellaria baicalensis Georgi.

The medicinal plant Scutellaria baicalensis Georgi is rich in specialized 4'-deoxyflavones, which are reported to have many health-promoting properties. We assayed Scutellaria flavones with different methoxyl groups on human cancer cell lines and found that polymethoxylated 4'-deoxyflavones, like skullcapflavone I and tenaxin I have stronger ability to induce apoptosis compared to unmethylated baicalein, showing that methoxylation enhances bioactivity as well as the physical properties of specialized flavones, while having no side-effects on healthy cells. We investigated the formation of methoxylated flavones and found that two O-methyltransferase (OMT) families are active in the roots of S. baicalensis. The Type II OMTs, SbPFOMT2 and SbPFOMT5, decorate one of two adjacent hydroxyl groups on flavones and are responsible for methylation on the C6, 8 and 3'-hydroxyl positions, to form oroxylin A, tenaxin II and chrysoeriol respectively. The Type I OMTs, SbFOMT3, SbFOMT5 and SbFOMT6 account mainly for C7-methoxylation of flavones, but SbFOMT5 can also methylate baicalein on its C5 and C6-hydroxyl positions. The dimethoxylated flavone, skullcapflavone I (found naturally in roots of S. baicalensis) can be produced in yeast by co-expressing SbPFOMT5 plus SbFOMT6 when the appropriately hydroxylated 4'-deoxyflavone substrates are supplied in the medium. Co-expression of SbPFOMT5 plus SbFOMT5 in yeast produced tenaxin I, also found in Scutellaria roots. This work showed that both type I and type II OMT enzymes are involved in biosynthesis of methoxylated flavones in S. baicalensis.

iCatch: a new strategy for capturing large DNA fragments using homing endonucleases.

Natural genetic materials contain many biosynthetic gene clusters encoding potentially valuable natural products, many of which can be used directly without codon optimization or other manipulations. With the development of synthetic biology, several DNA assembly standards have been proposed, conveniently facilitating the reuse of natural materials. Among these standards, the iBrick assembly standard was developed by our laboratory to manipulate large DNA fragments, employing two homing endonucleases. Considering the difficulty of cloning large iBrick parts using conventional endonuclease-mediated restriction and ligation methods, we herein present a new method, known as iCatch, which readily captures biosynthetic gene clusters. As the clusters cloned by iCatch have the prefix and suffix of the iBrick standard, they serve as new iBrick parts and are therefore conducive to further editing and assembly with the iBrick standard. iCatch employs the natural homologous recombination system to flank the region of interest with I-SceI and PI-PspI recognition sites, after which the genome is digested with I-SceI or PI-PspI and the fragments are then self-ligated to clone the target DNA fragments. We used this method to successfully capture the actinorhodin biosynthetic cluster from Streptomyces coelicolor and then heterologously expressed this cluster in a thermophilic Streptomyces strain. We propose that iCatch can be used for the cloning of DNA sequences that are dozens of kilobases in length, facilitating the heterologous expression of microbial natural products. Moreover, this cloning methodology can be a complementary tool for the iBrick standard, especially in applications requiring the manipulation of large DNA fragments.

Thermolysin damages animal life through degradation of plasma proteins enhanced by rapid cleavage of serpins and activation of proteases.

Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians.

Functional analysis of insect molting fluid proteins on the protection and regulation of ecdysis.

Molting fluid accumulates between the old and new cuticles during periodical ecdysis in Ecdysozoa. Natural defects in insect ecdysis are frequently associated with melanization (an immunity response) occurring primarily in molting fluids, suggesting that molting fluid may impact immunity as well as affect ecdysis. To address this hypothesis, proteomic analysis of molting fluids from Bombyx mori during three different types of ecdysis was performed. Many proteins were newly identified, including immunity-related proteins, in each molting fluid. Molting fluids inhibited the growth of bacteria in vitro. The entomopathogenic fungi Beauveria bassiana, which can escape immune responses in feeding larvae, is quickly recognized by larvae during ecdysis, followed by melanization in molting fluid and old cuticle. Fungal conidia germination was delayed, and no hyphae were detected in the hemocoels of pharate instar insects. Molting fluids protect the delicate pharate instar insects with extremely thin cuticles against microorganisms. To explore the function of molting fluids in ecdysis regulation, based on protein similarity, 32 genes were selected for analysis in ecdysis regulation through RNAi in Tribolium castaneum, a model commonly used to study integument development because RNAi is difficult to achieve in B. mori. We identified 24 molting proteins that affected ecdysis after knockdown, with different physiological functions, including old cuticle protein recycling, molting fluid pressure balance, detoxification, and signal detection and transfer of molting fluids. We report that insects secrete molting fluid for protection and regulation of ecdysis, which indicates a way to develop new pesticides through interrupting insect ecdysis in the future.

Insect prophenoloxidase: the view beyond immunity.

Insect prophenoloxidase (PPO) is an important innate immunity protein due to its involvement in cellular and humoral defense. It belongs to a group of type-3 copper-containing proteins that occurs in almost all organisms. Insect PPO has been studied for over a century, and the PPO activation cascade is becoming clearer. The insect PPO activation pathway incorporates several important proteins, including pattern-recognition receptors (PGRP, ß GRP, and C-type lectins), serine proteases, and serine protease inhibitors (serpins). Due to their complexity, PPO activation mechanisms vary among insect species. Activated phenoloxidase (PO) oxidizes phenolic molecules to produce melanin around invading pathogens and wounds. The crystal structure of Manduca sexta PPO shows that a conserved amino acid, phenylalanine (F), can block the active site pocket. During activation, this blocker must be dislodged or even cleaved at the N-terminal sequence to expose the active site pockets and allow substrates to enter. Thanks to the crystal structure of M. sexta PPO, some domains and specific amino acids that affect PPO activities have been identified. Further studies of the relationship between PPO structure and enzyme activities will provide an opportunity to examine other type-3 copper proteins, and trace when and why their various physiological functions evolved. Recent researches show that insect PPO has a relationship with neuron activity, longevity, feces melanization (phytophagous insects) and development, which suggests that it is time for us to look back on insect PPO beyond the view of immunity in this review.

Recombinant Drosophila prophenoloxidase 1 is sequentially cleaved by α-chymotrypsin during in vitro activation.

Insect prophenoloxidase (PPO) is an essential innate immunity protein to induce pathogen into melanization. In Bombyx mori, pro-phenoloxidase-activating enzyme (PPAE) can directly cleave and activate PPO. However, PPO in Manduca sexta cannot be cleaved into active phenoloxidase (PO) by serine proteases unless cofactors are involved, which indicates that PPO activation is complicated. Here we use recombinant Drosophila melanogaster prophenoloxidase 1 (rPPO1) to study the mechanism of PPO activation induced by a typical serine protease, α-chymotrypsin. Small amounts of α-chymotrypsin cleave rPPO1 at the N- and C-terminus to produce a large fragment rPPO1(N1/C1) that needs further cleavage by α-chymotrypsin to produce a smaller fragment rPO1(60-kD) with PO activity. rPO1(60-kD) oxidizes dopamine without being affected by high temperature, or by having salt and Ethylene diamine tetraacetic acid (EDTA) in the solution. After incubation with dopamine, rPO1(60-kD) cannot be detected using reducing SDS-PAGE due to formation of a large complex. Trypsin, another typical serine protease, cleaves rPPO1 at the N- and C-terminus to produce a small fragment rPPO1(N'/C') without PO activity. Several rPPO1 mutants were created through over-expressing active fragments that have direct PO activity. They are easily cleaved by low amounts of α-chymotrypsin without increasing PO activity. Therefore, rPPO1 can be sequentially cleaved in at least three places by α-chymotrypsin to produce activated rPO1(60-kD).

Formation of disulfide bonds in insect prophenoloxidase enhances immunity through improving enzyme activity and stability.

Type 3 copper proteins, including insect prophenoloxidase (PPO), contain two copper atoms in the active site pocket and can oxidize phenols. Insect PPO plays an important role in immunity. Insects and other invertebrates show limited recovery from pathogen invasion and wounds if phenoloxidase (PO) activity is low. In most insect PPOs, two disulfide bonds are present near the C-terminus. However, in Pimpla hypochondriaca (a parasitoid wasp), each PPO contains one disulfide bond. We thus questioned whether the formation of two sulfide bonds in insect PPOs improved protein stability and/or increased insect innate immunity over time. Using Drosophila melanogaster PPO1 as a model, one or two disulfide bonds were deleted to evaluate the importance of disulfide bonds in insect immunity. rPPO1 and mutants lacking disulfide bonds could be expressed and showed PO activity. However, the PO activities of mutants lacking one or two disulfide bonds significantly decreased. Deletion of disulfide bonds also reduced PPO thermostability. Furthermore, antibacterial activities against Escherichia coli and Bacillus subtilis significantly decreased when disulfide bonds were deleted. Therefore, the formation of two disulfide bond(s) in insect PPO enhances antibacterial activity by increasing PO activity and stability.

Activity of fusion prophenoloxidase-GFP and its potential applications for innate immunity study.

Insect prophenoloxidase (PPO) is essential for physiological functions such as melanization of invading pathogens, wound healing and cuticle sclerotization. The insect PPO activation pathway is well understood. However, it is not very clear how PPO is released from hemocytes and how PPO takes part in cellular immunity. To begin to assess this, three Drosophila melanogaster PPO genes were separately fused with GFP at the C-terminus (rPPO-GFP) and were over-expressed in S2 cells. The results of staining and morphological observation show that rPPO-GFP expressed in S2 cells has green fluorescence and enzyme activity if Cu(2+) was added during transfection. Each rPPO-GFP has similar properties as the corresponding rPPO. However, cells with rPPO-GFP over-expressed are easier to trace without PO activation and staining. Further experiments show that rPPO1-GFP is cleaved and activated by Drosophila serine protease, and rPPO1-GFP binds to Micrococcus luteus and Beauveria bassiana spores as silkworm plasma PPO. The above research indicates that the GFP-tag has no influence on the fusion enzyme activation and PPO-involved innate immunity action in vitro. Thus, rPPO-GFP may be a convenient tool for innate immunity study in the future if it can be expressed in vivo.

Transcriptional profiling of midgut immunity response and degeneration in the wandering silkworm, Bombyx mori.

BACKGROUND: Lepidoptera insects have a novel development process comprising several metamorphic stages during their life cycle compared with vertebrate animals. Unlike most Lepidoptera insects that live on nectar during the adult stage, the Bombyx mori silkworm adults do not eat anything and die after egg-laying. In addition, the midguts of Lepidoptera insects produce antimicrobial proteins during the wandering stage when the larval tissues undergo numerous changes. The exact mechanisms responsible for these phenomena remain unclear. PRINCIPAL FINDINGS: We used the silkworm as a model and performed genome-wide transcriptional profiling of the midgut between the feeding stage and the wandering stage. Many genes concerned with metabolism, digestion, and ion and small molecule transportation were down-regulated during the wandering stage, indicating that the wandering stage midgut loses its normal functions. Microarray profiling, qRT-PCR and western blot proved the production of antimicrobial proteins (peptides) in the midgut during the wandering stage. Different genes of the immune deficiency (Imd) pathway were up-regulated during the wandering stage. However, some key genes belonging to the Toll pathway showed no change in their transcription levels. Unlike butterfly (Pachliopta aristolochiae), the midgut of silkworm moth has a layer of cells, indicating that the development of midgut since the wandering stage is not usual. Cell division in the midgut was observed only for a short time during the wandering stage. However, there was extensive cell apoptosis before pupation. The imbalance of cell division and apoptosis probably drives the continuous degeneration of the midgut in the silkworm since the wandering stage. CONCLUSIONS: This study provided an insight into the mechanism of the degeneration of the silkworm midgut and the production of innate immunity-related proteins during the wandering stage. The imbalance of cell division and apoptosis induces irreversible degeneration of the midgut. The Imd pathway probably regulates the production of antimicrobial peptides in the midgut during the wandering stage.

Existence of prophenoloxidase in wing discs: a source of plasma prophenoloxidase in the silkworm, Bombyx mori.

In insects, hemocytes are considered as the only source of plasma prophenoloxidase (PPO). PPO also exists in the hemocytes of the hematopoietic organ that is connected to the wing disc of Bombyx mori. It is unknown whether there are other cells or tissues that can produce PPO and release it into the hemolymph besides circulating hemocytes. In this study, we use the silkworm as a model to explore this possibility. Through tissue staining and biochemical assays, we found that wing discs contain PPO that can be released into the culture medium in vitro. An in situ assay showed that some cells in the cavity of wing discs have PPO1 and PPO2 mRNA. We conclude that the hematopoietic organ may wrongly release hemocytes into wing discs since they are connected through many tubes as repost in previous paper. In wing discs, the infiltrating hemocytes produce and release PPO probably through cell lysis and the PPO is later transported into hemolymph. Therefore, this might be another source of plasma PPO in the silkworm: some infiltrated hemocytes sourced from the hematopoietic organ release PPO via wing discs.

Specific amino acids affecting Drosophila melanogaster prophenoloxidase activity in vitro.

Insect prophenoloxidase (PPO) is a key enzyme that induces melanization around invading pathogens and at wounds to prevent further infection. Drosophila melanogaster has three PPO genes which have different biochemical properties following over-expression in S2 cells. As shown by automatic melanization of S2 cells, recombinant PPO3 (rPPO3) became activated upon Cu(2+) addition (Cu(2+)-aided cells melanization without ethanol activation and substrate addition: +Cu(2+); -DOPA, -Ethanol). The exact reasons for this phenomenon are still unknown. In this study, using site-directed mutagenesis and over-expression methods, we found that the place holder, two independent amino acids (equal to Manduca sexta amino acid residues: F218 and S393 in MsPPO1, F224 and E395 in MsPPO2) in the active site pocket and a missing fragment (similar to (565)RPGDPGT(571) in MsPPO1 and (571)QGSDPRR(577) in MsPPO2) at the C-terminus of PPO3, affect rPPO3-S2 cells Cu(2+)-aided auto-melanization. Some mutations nearly rescued rPPO3 Cu(2+)-aided auto-activation, which suggests that the auto-activation of wild type rPPO3 was not due to cleavage by serine proteases. We also found that the corresponding amino acids in the active site pocket have similar effect on PPO1 as on PPO3. PPO1 staining activity (Cu(2+) added or not during PPO transfection; cells melanized after ethanol activation and substrate addition: ±Cu(2+); +DOPA, +Ethanol) has a positive relationship with the active site pocket size as does rPPO3. The fragment of rPPO1 corresponding to the one missing from the C-terminus of PPO3 has no influence on rPPO1 staining activity after it is deleted. However, the staining activities of rPPO2 mutants decreased after deletion of those corresponding amino acid sequences. When the corresponding fragments from PPO1 or PPO2 were inserted into PPO3, the mutant rPPO3 had no influence on staining activity, but had a significantly lowered Cu(2+)-aided auto-activation. Thus, we found that some amino acids are important for rPPO3 Cu(2+)-aided auto-activation as well as PPO staining activity in vitro.

Properties of Drosophila melanogaster prophenoloxidases expressed in Escherichia coli.

Insect prophenoloxidases (PPOs) are a group of important innate immunity proteins. Although there have been numerous studies dealing with the PPO activation cascade, the detailed biochemical behaviors of the PPO family proteins remain to be clearly established. This is due primarily to the difficulty in obtaining adequate amounts of PPO proteins for comprehensive characterization. In this study, we expressed three Drosophila melanogaster PPO genes in Escherichia coli, and extensively evaluated expression conditions for obtaining soluble proteins. Through the manipulation of expression conditions, particularly the culture temperature of PPO-transformed E. coli cells, we were able to obtain large quantities of soluble recombinant PPO proteins. Additional Cu(2+), either added into the culture medium during PPO induction or directly mixed with the purified rPPO preparations, was necessary to produce Cu(2+) associated proenzymes. Cu(2+) associated PPOs showed obvious enzyme activities after activation by either ethanol or cetylpyridinium chloride, or by AMM1 (a pupal protein fraction containing native serine proteases for PPO activation). Dose responses for association of individual purified Drosophila rPPOs with Cu(2+) showed that Drosophila rPPO1 and rPPO3 had relatively higher affinity for Cu(2+) than rPPO2 did. Surprisingly, however, high concentration of Cu(2+) (2 mM) completely inhibited PPO activity. Each rPPO had similar activity when dopamine or l-DOPA was the substrate. However, rPPO1 alone had very high activity if l-tyrosine was used as a substrate. After activation by ethanol or 2-propanol, Km and Vmax of the three rPPOs changed as shown in the following: rPPO2

A systematic study on hemocyte identification and plasma prophenoloxidase from Culex pipiens quinquefasciatus at different developmental stages.

Culexpipiens quinquefasciatus (C. quinquefasciatus) is an important vector that can transmit human diseases such as West Nile virus, lymphatic filariasis, Japanese encephalitis and St. Louis encephalitis. However, very limited research concerning the humoral and cellular immune defenses of C. quinquefasciatus has been done. Here we present the research on hemocyte identification and plasma including hemocyte prophenoloxidase from C. quinquefasciatus at all developmental stages in order to obtain a complete picture of C. quinquefasciatus innate immunity. We identified hemocytes into four types: prohemocytes, oenocytoids, plasmatocytes and granulocytes. Prophenoloxidase (PPO) is an essential enzyme to induce melanization after encapsulation. PPO-positive hemocytes and plasma PPO were observed at all developmental stages. As for specific hemocyte types, prophenoloxidase was found in the plasmatocytes at larval stage alone and in the smallest prohemocytes during almost all developmental stages. Moreover, the granulocytes were PPO-positive from blood-fed female mosquitoes and oenocytoids were observed PPO-positive in pupae and in adult females after blood-feeding. As for plasma, there were different patterns of PPO in C. quinquefasciatus at different developmental stages. These results are forming a basis for further studies on the function of C. quinquefasciatus hemocytes and prophenoloxidase as well as their involvement in fighting against mosquito-borne pathogens.