Shotgun analysis on the peritrophic membrane of the silkworm Bombyx mori

The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. We used gel electrophoresis and mass spectrometry to identify the extracted proteins from the silkworm PM to obtain an in-depth understanding of the biological function of the silkworm PM components. A total of 305 proteins, with molecular weights ranging from 8.02 kDa to 788.52 kDa and the isoelectric points ranging from 3.39 to 12.91, were successfully identified. We also found several major classes of PM proteins, i.e. PM chitin-binding protein, invertebrate intestinal mucin, and chitin deacetylase. The protein profile provides a basis for further study of the physiological events in the PM of Bombyx mori. [BMB Reports 2012; 45(11): 665-670]     

Molecular structure of the peritrophic membrane (PM): identification of potential PM target sites for insect control

Peritrophic membranes (PMs) are an invertebrate-unique structure that lines the digestive tract, playing important roles in facilitating food digestion and providing protection to the gut epithelium. The importance of PMs in insects has been recognized ever since its presence was identified 200 years ago. In the last 5 years, significant progress towards understanding the PM molecular structure and the mechanism for PM formation has been made. Recent studies on Type 1 PMs from lepidopteran larvae have suggested a model for the PM molecular structure and formation. The important physiological functions of the PM suggest that PMs can be a significant structural target for insect control and the current understanding of the structure of lepidopteran larval PMs has provided us with potential opportunities for targeting the PM by various mechanisms.     

棉铃虫围食膜的蛋白质组鉴定

【目的】围食膜(peritrophic membrane, PM)是昆虫抵御随食物摄入的病原微生物入侵的第一道天然屏障。本研究旨在鉴定出农业重大害虫棉铃虫Helicoverpa armigera围食膜的总蛋白成分,为进一步揭示昆虫围食膜的形成机制及研发新颖的害虫控制策略奠定基础。【方法】剥离棉铃虫5龄幼虫PM,用三氟甲磺酸(trifluoromethane sulfonic acid, TFMS)处理,采用液质联用技术(LC-MS/MS)鉴定围食膜蛋白质组,然后对鉴定结果进行生物信息学分析。【结果】本研究共鉴定出棉铃虫幼虫围食膜蛋白质169个,是目前鉴定最多的棉铃虫围食膜蛋白。通过GO分析,可以将这些鉴定的蛋白分为细胞组分、分子功能和生物学过程三大类;KEGG富集结果显示,鉴定蛋白可以富集在12条代谢通路中;蛋白互作分析(protein protein interaction, PPI)结果表明,以ACC和CG3011等蛋白为核心可以形成蛋白互作网络。【结论】本研究鉴定了169个棉铃虫幼虫围食膜蛋白质,并对其进行了GO, KEGG和PPI分析,结果有助于人们全面理解昆虫围食膜的分子结构和功能。     

Shotgun strategy-based proteome profiling analysis on the head of silkworm Bombyx mori

Insect head is comprised of important sensory systems to communicate with internal and external environment and endocrine organs such as brain and corpus allatum to regulate insect growth and development. To comprehensively understand how all these components act and interact within the head, it is necessary to investigate their molecular basis at protein level. Here, the spectra of peptides digested from silkworm larval heads were obtained from liquid chromatography tandem mass spectrometry (LC–MS/MS) and were analyzed by bioinformatics methods. Totally, 539 proteins with a low false discovery rate (FDR) were identified by searching against an in-house database with SEQUEST and X!Tandem algorithms followed by trans-proteomic pipeline (TPP) validation. Forty-three proteins had the theoretical isoelectric point (pI) greater than 10 which were too difficult to separate by two-dimensional gel electrophoresis (2-DE). Four chemosensory proteins, one odorant-binding protein, two diapause-related proteins, and a lot of cuticle proteins, interestingly including pupal cuticle proteins were identified. The proteins involved in nervous system development, stress response, apoptosis and so forth were related to the physiological status of head. Pathway analysis revealed that many proteins were highly homologous with the human proteins which involved in human neurodegenerative disease pathways, probably implying a symptom of the forthcoming metamorphosis of silkworm. These data and the analysis methods were expected to be of benefit to the proteomics research of silkworm and other insects.     

Shotgun proteomic analysis of Bombyx mori brain: emphasis on regulation of behavior and development of the nervous system

The insect brain plays crucial roles in the regulation of growth and development and in all types of behavior. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and high‐performance liquid chromatography ? electron spray ionization tandem mass spectrometry (ESI‐MS/MS) shotgun to identify the proteome of the silkworm brain, to investigate its protein composition and to understand their biological functions. A total of 2210 proteins with molecular weights in the range of 5.64–1539.82 kDa and isoelectric points in the range of 3.78–12.55 were identified. These proteins were annotated according to Gene Ontology Annotation into the categories of molecular function, biological process and cellular component. We characterized two categories of proteins: one includes behavior‐related proteins involved in the regulation of behaviors, such as locomotion, reproduction and learning; the other consists of proteins related to the development or function of the nervous system. The identified proteins were classified into 283 different pathways according to KEGG analysis, including the PI3K‐Akt signaling pathway which plays a crucial role in mediating survival signals in a wide range of neuronal cell types. This extensive protein profile provides a basis for further understanding of the physiological functions in the silkworm brain.     

The scanning electron microscopic study of the infection and conidial development of Aspergillus tamarii Kita on its host, the silkworm, Bombyx mori Linn.

Development of Aspergillosis on the integument of the silkworm, Bombyx mori Linn., was examined by scanning electron microscopy. Aspergillosis is a fungal disease caused by an insect mycopathogen Aspergillus tamarii Kita, which infects the silkworms in countries where sericulture (the rearing of silkworms)is prevalent. The present study showed the course of infection and the conidial development of A. tamarii on the integument of B. mori. Five different strains (KA, NB₁₈, NB₄ D₂, NB₇ and PM) of B. mori were inoculated on their body surface with ca. 1 × 10⁶ conidia/ml. Among the five breeds tested, the conidial germination was greatest on the larval surface of KA breed, and least on PM. Most of the conidia germinated on the cuticle approximately 8–12 hours after inoculation, forming a suctorial appressorium within 24 hours. The hyphae reached the hemocoel, where they grew and multiplied extensively, forming a mycelial complex and causing death of the host larva in about 5–6 days. The death of the host was followed by growth of the fungus through mesodermal and epidermal tissues, leading to larval mummification about 6–7 days post-inoculation. Extensive aerial outgrowths of the fungus followed, mostly through the intersegmental regions of larvae. Abundant branched conidiophores developed, forming a confluent yellow brown mat over the entire host body 7 days after inoculation. Each conidiophore had an apical vesicle bearing numerous phialides from which conidia were developed in long chains.     

Protein Profile of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Spore

Natural wild-type strains of Bacillus subtilis spore is regarded as a non-pathogenic for both human and animal, and has been classified as a novel food which is currently being used as probiotics added in the consumption. To identify B. subtilis spore proteins, we have accomplished a preliminary proteomic analysis of B. subtilis spore, with a combination of two-dimensional electrophoretic separations and matrix-assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI–TOF–MS). In this article, we presented a reference map of 158 B. subtilis spore proteins with an isoelectric point (pI) between 4 and 7. Followed by mass spectrometry (MS) analysis, we identified 71 B. subtilis spore proteins with high level of confidence. Database searches, combined with hydropathy analysis and GO analysis revealed that most of the B. subtilis spore proteins were hydrophilic proteins related to catalytic function. These results should accelerate efforts to understand the resistance of spore to harsh conditions.     

A proteomic approach analysing the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> response to virulent and avirulent <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> strains

The present work is directed at studying changes at the proteome level in Arabidopsis thaliana leaves in response to Pseudomonas syringae virulent (Pst) and avirulent (Pst avrRpt2) strains. Arabidopsis leaves were sampled from challenged plants at 4, 8 and 24 h post inoculation. Proteins were TCA–acetone–phenol extracted and subjected to 2-DE (5–8 pH range) and MS/MS (MALDI–TOF–TOF) analysis. Out of 800 matched spots on each of the 36 gels analysed, 147 spots were either absent in at least one of the conditions studied (time or treatments; qualitative variable spots) or differentially accumulated between time and treatments (quantitative variable spots). Out of the 24 proteins successfully identified over TAIR10 database, 23 have not been reported previously in similar proteomics studies of the Arabidopsis thaliana–Pseudomonas syringae interaction. The exhaustive statistical analysis performed, including principal component and heat map, showed that 24 h post inoculation can clearly discriminate the challenged plants from the control. The protein change occurred early (4 h post inoculation) following the virulent pathogen infection, whereas the change occurred later (24 h post inoculation) following the avirulent pathogen inoculation. Concerning the variable proteins, three behavioural groups can be observed: group 1 (common protein changes in response to virulent and avirulent pathogen infection), group 2 (protein changes in response to virulent pathogen infection) and group 3 (protein changes in response to avirulent pathogen infection). Differential identified proteins following the pathogen infection belonged to different groups including those of oxidative stress defence, enzymes of metabolic pathways and molecular chaperones.     

Proteomic Analysis of Interactions Between the Generalist Herbivore <Emphasis Type="Italic">Spodoptera exigua</Emphasis> (Lepidoptera: Noctuidae) and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In this study, comparative proteomics was used to investigate the interaction of Spodoptera exigua and Arabidopsis thaliana. By using 2-D electrophoresis of differentially expressed proteins, combined with high-throughput matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF/TOF MS, the changes in the abundance of proteins induced by insect feeding were studied in A. thaliana. More than 1,100 protein spots were reproducibly detected on each gel. The intensities of 30 protein spots in particular changed significantly, showing differences in volume of at least twofold. Among these, 17 protein spots were upregulated, and 13 were downregulated following an 8-h insect feeding period. Nineteen insect-feeding-responsive proteins were identified, all of which were involved in metabolic regulation, binding functions or cofactor requirement of protein, cell rescue, and defense and virulence, as assessed by Munich Information Center for Protein Sequences function category. About 50% of these were involved in metabolism, including transketolase, S-adenosylmethionine synthase 3, 2,3-biphosphoglycerate-independent phosphoglycerate mutase, beta-ureidopropionase, GDP-d-mannose 3′,5′-epimerase, and fatty acid synthase. The identification of insect-feeding-responsive proteins on Arabidopsis provides not only new insights into insect stress but also a good start for further investigation of their functions. Understanding how the plant responses to insects in the proteomic level will provide tools for a better management of insect pest in the field.     

A proteomics approach for the analysis of hemolymph proteins involved in the immediate defense response of the soft tick, <Emphasis Type="Italic">Ornithodoros savignyi</Emphasis>, when challenged with <Emphasis Type="Italic">Candida albicans</Emphasis>

A proteomics approach was employed to identify proteins secreted into the hemolymph of Ornithodorus savignyi ticks 2 h after immune-challenge with the yeast, Candida albicans. Profiling of the proteins present in hemolymph of unchallenged ticks versus ticks challenged with heat-killed yeast revealed five proteins to be differentially expressed. The modulated protein spots were subjected to tandem mass spectrometry (MS/MS) analysis, but could not be positively identified. These proteins can be assigned to the immune response as they were not induced after aseptic injury. In an attempt to identify hemolymph proteins that recognize and bind to yeast cells, hemolymph obtained from both unchallenged and challenged ticks was incubated with C. albicans. Elution of the bound proteins followed by SDS–PAGE analysis indicated that three proteins (97, 88 and 26 kDa) present in both unchallenged and challenged hemolymph samples bind to yeast cells. The constant presence of these three proteins in tick hemolymph leads us to believe that they may be involved in non-self recognition and participate in yeast clearance from tick plasma. The analyzed yeast-binding proteins could also not be positively identified, suggesting that all the tick immune proteins investigated in this study are novel.     

Proteomic analysis of egg white proteins during the early phase of embryonic development

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development.     

The MALDI mass spectrometry in the identification of new proteins in snake venoms

By MALDI MS, we searched cobra venoms for new low-content polypeptides. A number of new proteins with molecular masses 7–25 kDa, characteristic of the known snake protein toxins, were identified, with the content of one of them less than 0.02%.     

Proteomic Analysis of the Effect of Cyanide on Klebsiella oxytoca

Cyanide has been proved to be degraded by Klebsiella oxytoca. In order to examine the physiological responses of cyanide degradation by this bacterium, two-dimensional (2-DE) electrophoresis approach and MALDI–TOF–MS allow us to identify 106 proteins spots that were significantly altered in the presence of 1 mM cyanide in relative to that in 1 mM ammonia when K. oxytoca grown at the late-log phase. Among them, 27 proteins were successfully identified. These proteins were involved in carbohydrate metabolism, nucleotide metabolism, amino acid metabolism, nitrogen metabolism, stress responses, oxidation–reduction reactions, transporters, and miscellaneous function. Some proteins related with regulation of nitrogen assimilation pathways (glutamine synthetase), oxidative stress repairing (catalase), and protection (neutral trehalase and glycosyltransferase) could improve the effectiveness of cyanide biodegradation. Although the nitrogenase was suggested to participate in cyanide degradation in our previous study, this enzyme induction was not observed as expected. These findings could provide new insights into the inducible mechanisms underlying the capacity of K. oxytoca to tolerate cyanide stress.     

Comparative temporal analyses of the <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L. var. <Emphasis Type="Italic">mongolica litv.</Emphasis> apical bud proteome from dormancy to growth

Bud dormancy in perennial plants adapts to environmental and seasonal changes. Bud dormancy is of ecological interest because it affects forest population growth characteristics and is of economical interest because it impacts wood production levels. To understand Pinus sylvestris L. var. mongolica litv. bud-dormancy and bud-burst mechanisms, we characterized the proteomes of their apical buds at the four critical stages that occur during the dormancy-to-growth transition. Ninety-six proteins with altered expression patterns were identified using NanoLC–ESI-MS/MS. The majority of these proteins (57%) are involved in metabolic and other cellular processes. For 28% of the proteins, a function could not be assigned. However, because their expression levels changed, they may be potential candidate bud development- or dormancy-related proteins. Of the 75 non-redundant bud proteins identified, ascorbate peroxidase, pathogenesis-related protein PR-10, and heat shock proteins dramatically increased during August and November, suggesting that they may involved in the initiation of bud dormancy. Conversely, S-adenosylmethionine synthetase, abscisic acid/stress-induced proteins, superoxide dismutase (SOD), caffeoyl-CoA O-methyltransferase, actin, and type IIIa membrane protein cp-wap13 had greater expression levels during April, suggesting that they may be involved in the initiation of bud dormancy-release. Cell division cycle protein 48 and eukaryotic initiation factors 4A-15 and 4A had greater expression levels during May, suggesting that they may regulate cell proliferate and differentiation in the shoot apical meristem. These observations provide insights into the molecular mechanisms that induce or break bud dormancy.     

Proteome analysis of microtubule-associated proteins and their interacting partners from mammalian brain

The microtubule (MT) cytoskeleton is essential for a variety of cellular processes. MTs are finely regulated by distinct classes of MT-associated proteins (MAPs), which themselves bind to and are regulated by a large number of additional proteins. We have carried out proteome analyses of tubulin-rich and tubulin-depleted MAPs and their interacting partners isolated from bovine brain. In total, 573 proteins were identified giving us unprecedented access to brain-specific MT-associated proteins from mammalian brain. Most of the standard MAPs were identified and at least 500 proteins have been reported as being associated with MTs. We identified protein complexes with a large number of subunits such as brain-specific motor/adaptor/cargo complexes for kinesins, dynein, and dynactin, and proteins of an RNA-transporting granule. About 25% of the identified proteins were also found in the synaptic vesicle proteome. Analysis of the MS/MS data revealed many posttranslational modifications, amino acid changes, and alternative splice variants, particularly in tau, a key protein implicated in Alzheimer’s disease. Bioinformatic analysis of known protein–protein interactions of the identified proteins indicated that the number of MAPs and their associated proteins is larger than previously anticipated and that our database will be a useful resource to identify novel binding partners.     

Question 3: The Worlds of the Prebiotic and Never Born Proteins

Starting from the statement that no reliable methods are known to produce high molecular weight polypeptides under prebiotic conditions, a possible approach, at least to understand the differences between extant proteins and the possible large number of never born proteins, could be biological. Using the phage display method a large library of totally random amino acidic sequences was obtained. Consequently, different experiments to directly consider the frequency of stable folds were performed, and the interesting results obtained from such new approach are discussed in terms of contingency, contributing to the discussion on the selection mechanism of extant proteins. Presented at the International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Purification and characterization of trypsin-like enzymes from North Pacific krill (<Emphasis Type="Italic">Euphausia pacifica</Emphasis>)

Two trypsin-like enzymes (TLEs) were purified from North Pacific krill (Euphausia pacifica) by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography. The purified enzymes were identified as trypsins by LC-ESI-MS/MS analysis. The relative molecular mass of TLE I and TLE II were 33 and 32.3 kDa, respectively, with isoelectric points of 4.5 and 4.3, respectively. The TLEs showed excellent thermal stable in the crude extract and the purified TLEs were active over a wide pH (6.0–11.0) and temperature (10–70°C) range. Compared with trypsins from other organisms, the purified TLEs had physiological efficiencies of 1.6–6.7-fold. The difference in Arg, Ile and Asp content might explain why E. pacifica TLEs have good thermal stability and physiological efficiency.     

昆虫围食膜的研究进展

围食膜是大多数昆虫中肠内的半透性薄膜 ,主要由几丁质、蛋白质构成。依据其形成的方式分 :Ⅰ型围食膜 ,由整个中肠细胞分泌形成多层管状膜 ;Ⅱ型围食膜由中肠前端特殊的细胞分泌成连续的套筒管状膜。由于位于食物与中肠上皮细胞间而在中肠生理中起重要作用 ,围食膜保护中肠上皮免于机械损伤以及病原菌、毒素的入侵 ;作为半透膜以及将中肠分为不同的区室而在营养物质的消化和吸收中具有重要作用。该文综述了有关围食膜结构、组分、功能、通透性以及与害虫防治的关系等方面的研究进展。