Proteome sequencing and analysis of Ophiocordyceps sinensis at different culture periods.

BACKGROUND: Ophiocordyceps sinensis is an important traditional Chinese medicine for its comprehensive active ingredients, such as cordycepin, cordycepic acid, and Cordyceps polysaccharide. O. sinensis zjut, a special strain isolated from O. sinensis, has similar pharmacological functions to wild O. sinensis. Currently, O. sinensis with artificial cultivation has been widely studied, but systematic fundamental research at protein levels has not been determined. RESULTS: Proteomes of O. sinensis zjut at different culture periods (growth period, 3rd day; pre-stable period, 6th day; and stable period, 9th day) were relatively quantified by relative isotope markers and absolute quantitative technology. In total, 4005 proteins were obtained and further annotated with Gene Ontology, Kyoto Encyclopedia of Genes and Genomes database. Based on the result of the annotations, metabolic pathways of active ingredients, amino acids and fatty acid were constructed, and the related enzymes were exhibited. Subsequently, comparative proteomics of O. sinensis zjut identified the differentially expressed proteins (DEPs) by growth in different culture periods, to find the important proteins involved in metabolic pathways of active ingredients. 605 DEPs between 6d-VS-3d, 1188 DEPs between 9d-VS-3d, and 428 DEPs between 9d-VS-6d were obtained, respectively. CONCLUSION: This work provided scientific basis to study protein profile and comparison of protein expression levels of O. sinensis zjut, and it will be helpful for metabolic engineering works to active ingredients for exploration, application and improvement of this fungus.

Identification of microRNA-like RNAs in Ophiocordyceps sinensis.

Ophiocordyceps sinensis is well known as a traditional Chinese medicine and has widely been used for over 2,000 years to stimulate immune system, decrease blood pressure and to inhibit tumor growth. While miRNAs are increasingly recognized for their roles in post-transcriptional regulation of gene expression in animals and plants, miRNAs in fungi were less studied until the discovery of microRNA-like RNA (milRNA). High-throughput sequencing and bioinformatics approaches were used to identify conserved and novel milRNAs in O. sinensis. 40 conserved milRNAs were identified, while 23 pre-miRNA candidates encoding 31 novel milRNAs were predicted. Furthermore, the potential target genes of milRNAs in human were predicted and gene ontology analysis was applied to these genes. Enrichment analysis of GO-represented biological process showed that target genes of both conserved and novel milRNAs are involved in development, metabolic and immune processes, indicating the potential roles of milRNAs of O. sinensis in pharmacological effects as health food and traditional Chinese medicine. This study is the first report on genome-wide analysis of milRNAs in O. sinensis and it provides a useful resource to further study the potential roles of milRNAs as active components of O. sinensis in health food or traditional Chinese medicine.

Proteomic identification of marker proteins and its application to authenticate Ophiocordyceps sinensis.

Ophiocordyceps sinensis (O. sinensis) is a highly valuable fungus because of its nutritious and medicinal properties. The objective of this study was to identify protein markers using a proteomics approach followed by the development of an immunoassay to authenticate O. sinensis. Four authentic O. sinensis samples collected from four production regions and four counterfeit samples were examined individually. Overall 22 characteristic proteins of O. sinensis were identified by two-dimensional electrophoresis (2-DE) coupled with the matrix-assisted laser desorption/ionization-time-of-light mass spectrometry (MALDI-TOF/MS). Three authentic O. sinensis samples and three counterfeit samples were examined by the couple of alkaline native gradient PAGE (AN-PAGE) and electrospray ionization quadrupole-time-of-light mass spectrometry (ESI-Q-TOF/MS). One distinctive protein was identified to be cyanate hydratase, which was also one of the 22 distinctively characteristic proteins of O. sinensis and termed as IP4 in 2-D gel. Due to the abundance and high specificity of IP4, it was isolated and purified. Its purity was evaluated by high performance liquid chromatography (HPLC) and identified by ESI-Q-TOF/MS. Then the purified IP4 was used to produce polyclonal antibodies in BALB/c mice. The specificity of the anti-IP4 antibody was evaluated by an association of double immunodiffusion (DID) and indirect ELISA assay. Then an indirect enzyme linked immunosorbent assay (ELISA) was preliminarily developed to authenticate O. sinensis by detecting IP4. To evaluate the feasibility and accuracy of this method, three authentic O. sinensis samples and three counterfeits were analyzed. The P/N ratios (dividing the sample OD450nm by the OD450nm of negative controls) of three authentic O. sinensis samples were above 8, while, those of three counterfeits were lower than 1. These results indicated that the established ELISA assay based on proteomic protocols detection of protein markers might have a great potential in the authentication and also quality assessment of O.sinensis in those commercial products.

In vitro Stimulation of NK Cells and Lymphocytes Using an Extract Prepared from Mycelial Culture of Ophiocordyceps sinensis.

Ophiocordyceps sinensis is a natural fungus that has been valued as a health food and used in traditional Chinese medicine for centuries. The fungus is parasitic and colonizes insect larva. Naturally occurring O. sinensis thrives at high altitude in cold and grassy alpine meadows on the Himalayan mountain ranges. Wild Ophiocordyceps is becoming increasingly rare in its natural habitat, and its price limits its use in clinical practice. Therefore, the development of a standardized alternative is a great focus of research to allow the use of Ophiocordyceps as a medicine. To develop an alternative for wild Ophiocordyceps, a refined standardized extract, CBG-CS-2, was produced by artificial fermentation and extraction of the mycelial strain Paecilomyces hepiali CBG-CS-1, which originated from wild O. sinensis. In this study, we analyzed the in vitro immune-modulating effect of CBG-CS-2 on natural killer cells and B and T lymphocytes. CBG-CS-2 stimulated splenocyte proliferation and enhanced Th1-type cytokine expression in the mouse splenocytes. Importantly, in vitro CBG-CS-2 treatment enhanced the killing activity of the NK-92MI natural killer cell line. These results indicate that the mycelial culture extract prepared from Ophiocordyceps exhibits immune-modulating activity, as was observed in vivo and this suggests its possible use in the treatment of diseases caused by abnormal immune function.