食药用真菌多糖及复合多糖生物活性研究

食药用真菌多糖有多种生物学功能,在抗肿瘤、免疫调节作用、抗衰老、降血脂等方面发挥着重要的生物活性.对正常细胞无毒副作用是食药用真菌多糖的突出优点.合适剂量食药用真菌多糖配伍使用时,各多糖间药理作用呈现协同性,可以提高在抗肿瘤、免疫调节等方面的药效.     

灵芝免疫调节蛋白LZ-8的制备和晶体分析

LZ-8蛋白是从灵芝菌丝中分离到的真菌免疫调节蛋白,具有多种免疫调节功能,然而这一蛋白的作用机制尚不清楚。通过蛋白质晶体结构的解析,能够得到蛋白质空间结构特点,从而阐述蛋白质功能的机制。旨在得到LZ-8蛋白的晶体,并获得空间结构数据。以pET21a为表达载体,获得诱导表达的rLZ-8,通过亲和层析、分子筛凝胶层析和阴离子交换层析纯化,蛋白纯度在98%以上,采用悬滴气相扩散法得到蛋白晶体,并获得3.2?数据,为进一步对真菌免疫调节蛋白功能和结构的研究奠定了基础。     

金针菇功能性蛋白的研究进展

主要综述了近年关于金针菇功能性蛋白,即核糖体失活蛋白、真菌免疫调节蛋白、金针菇毒素(一种成孔溶细胞素)的分离纯化及其性质,它们的生物学作用与作用机制和它们可能的应用及展望。     

食药用真菌多糖构效关系研究进展

真菌多糖具有抗病毒、抗凝血、抗肿瘤、免疫调节、降血脂、延缓衰老等多种生物活性,而多糖的功能与结构密切相关。多糖结构与功能关系的研究已经成为人们关注的一个热点。综述了食药用真菌多糖一级结构、高级结构及多糖的理化性质与其生物学活性之间的关系。     

真菌多糖的活性研究进展

真菌多糖是一类从真菌的子实体或菌丝体分离出来的天然高分子化合物。研究表明,真菌多糖具有抗病毒、抗凝血、降血脂、抗肿瘤、免疫调节、延缓衰老等多种生物活性,现已被开发成多种药物和功能性食品添加剂。随着天然药物化学、药理研究的不断深入,真菌多糖已引起国内外学者的极大兴趣,其重要生物学效应成为当今研究的热点。     

紫芝免疫调节蛋白基因的原核表达与功能分析

以紫芝(Ganodermasinense)真菌免疫调节蛋白基因(FIP-gsi)为材料,采用原核表达技术进行蛋白表达,利用基质辅助激光解吸附质谱技术(MALDI-MS)鉴定表达的蛋白,通过体外诱导细胞因子表达技术分析细胞因子基因的表达模式,为真菌免疫调节蛋白生物活性及作用机制的研究奠定基础。结果表明:紫芝真菌免疫调节蛋白基因(FIP-gsi)可在原核细胞中表达,表达出的重组蛋白FIP-gsi约占大肠杆菌总蛋白的46.1%;基质辅助激光解吸附质谱技术鉴定显示表达的蛋白为FIP-gsi,与灵芝(G.lucidum)真菌免疫调节蛋白LZ-8有88.6%的一致性;重组蛋白FIP-gsi能够诱导细胞因子IL(interleukin)-2、IL-4、IFN(interferon)-γ,TNF(tumor necrosis factor)-α,LT(lymphotoxin)及IL-2 R(IL-2 receptor)表达,并且呈现一定的剂量关系。     

真菌多糖抗肿瘤及免疫调节作用研究进展

真菌多糖被认为是目前最有开发前途的保健食品和药品的新资源之一,对真菌多糖的生物活性研究也是保健食品功能因子中最为炽热的研究热点。本文综述了近年来国内外真菌多糖抗肿瘤及免疫调节作用的研究进展。     

钙调磷酸酶信号调控真菌生长代谢、毒力及抗逆性能

钙调磷酸酶是一种丝氨酸/苏氨酸(Ser/Thr)蛋白磷酸酶,在真菌中普遍保守,上游信号途径由Ca2+通道(Cch1)、转运蛋白(Mid1)、钙离子感应蛋白(CaM)、钙调蛋白依赖性磷酸酶等组成。钙调磷酸酶受钙离子和钙调蛋白调节,在调控真菌Ca2+稳态的钙信号级联途径中发挥着中心作用,通过钙信号级联途径参与生物学过程,调控真菌生长、发育和毒力形成来响应外界环境因素的变化,使真菌能够适应不同环境,维持正常的生命活动。本文综述了真菌钙调磷酸酶信号的组成和上下游信号转导途径、调控细胞生长代谢、毒力形成以及抗逆性能调控的研究进展;结合对真菌代谢产物合成的调控作用,对钙调磷酸酶信号作为重要合成生物学元件及调控开关进行了展望。     

真菌G蛋白信号调控蛋白的功能研究进展

G蛋白信号途径是真菌细胞信号转导网络的枢纽,在细胞的各种生物学调控过程中具有重要作用。G蛋白信号调控蛋白(Rgulators of G protein signaling,RGS)是一类重要的G蛋白信号调控因子,能通过促进G蛋白α亚基(Gα)偶联的GTP水解,使Gα和Gβγ亚基发生聚合,导致G蛋白失活,从而迅速关闭与G蛋白偶联的信号途径。自从第一个RGS蛋白在酿酒酵母中被鉴定以来,目前已经有30多个RGS蛋白在重要的模式真菌中被报道,包括构巢曲霉、绿僵菌、稻瘟病菌、玉米赤霉菌、轮枝镰孢菌、新型隐球菌和白色念珠菌等。RGS蛋白在真菌的营养菌丝生长、产孢、毒素和色素生产、致病性和有性生殖等过程中发挥着重要作用。本文对真菌中已报道RGS蛋白的功能进行了总结,对真菌RGS蛋白的结构特征和调控机制进行了评述。     

不同营养方式真菌中分泌蛋白数量及其功能对比研究

【目的】分泌蛋白在植物病原真菌致病过程中发挥着重要的作用,前人多以单种菌株分泌蛋白预测分析,尚未见多种类型真菌中分泌蛋白的预测及对比研究报道。【方法】本研究基于多种不同营养方式真菌的全基因组序列,根据分泌蛋白所具有的基本特征,采用在线分析程序(主要包括SignalP v5.0、ProtComp v9.0等)对模式生物、死体营养型真菌、半活体营养型真菌及活体营养型真菌共19种菌株的分泌蛋白开展预测及功能分析。【结果】在上述真菌约13万条蛋白序列中,分泌蛋白所占比例为0.74%–4.83%,其中,活体营养型真菌所具有的分泌蛋白数量占比最高,平均为3.51%,而死体营养型真菌和模式生物平均比例最低,平均为1.36%。同时,活体营养型真菌具有的功能种类最多,为433种,其次为半活体营养型真菌,为266种,而模式生物具有的功能种类最少,为100种,其中,功能为假设蛋白和非特征蛋白的蛋白数量最多。【结论】该研究为深入解析分泌蛋白在实现不同营养方式真菌的致病机制方面提供理论依据。     

Recent status and prospects of the fungal immunomodulatory protein family

With the exception of polysaccharides and triterpenes/triterpenoids compounds, fungal immunomodulatory protein (FIP), a small molecule protein, is also an important bioactive component with immune regulating activity. It plays a significant role in immunomodulating. The objective of this paper was to review the latest advances in various aspects of research on FIPs, including their basic components and structural character, characters of diversity, gene cloning and expression, and their biological function, etc. In addition, prospects of utilization value and the exploitation foreground of FIPs were also discussed. The review will provide a useful reference for further research, development, and utilizations of FIPs.     

Recent status and prospects of the fungal immunomodulatory protein family

With the exception of polysaccharides and triterpenes/triterpenoids compounds, fungal immunomodulatory protein (FIP), a small molecule protein, is also an important bioactive component with immune regulating activity. It plays a significant role in immunomodulating. The objective of this paper was to review the latest advances in various aspects of research on FIPs, including their basic components and structural character, characters of diversity, gene cloning and expression, and their biological function, etc. In addition, prospects of utilization value and the exploitation foreground of FIPs were also discussed. The review will provide a useful reference for further research, development, and utilizations of FIPs.     

In vitro rapid evolution of fungal immunomodulatory proteins by DNA family shuffling

Fungal immunomodulatory proteins (FIPs) found in a wide variety of mushrooms hold significant therapeutic potential. Despite much research, the structural determinants for their immunomodulatory functions remain unknown. In this study, a DNA shuffling technique was used to create two shuffled FIP protein libraries: an intrageneric group containing products of shuffling between FIP-glu (FIP gene isolated from Ganoderma lucidum) and FIP-gsi (FIP gene isolated from Ganoderma sinense) genes and an intergeneric group containing the products of shuffling between FIP-glu, FIP-fve (FIP gene isolated from Flammulina velutipes), and FIP-vvo (FIP gene isolated from Volvariella volvacea) genes. The gene shuffling generated 426 and 412 recombinant clones, respectively. Using colony blot analysis, we selected clones that expressed relatively high levels of shuffled gene products recognized by specific polyclonal antibodies. We analyzed the DNA sequences of the selected shuffled genes, and testing of their protein products revealed that they maintained functional abilities to agglutinate blood cells and induce cytokine production by splenocytes from Kunming mice in vitro. Meanwhile, the relationships between protein structure and the hemagglutination activity and between the changed nucleotide sites and expression levels were explored by bioinformatic analysis. These combined analyses identified the nucleotide changes involved in regulating the expression levels and hemagglutination activities of the FIPs. Therefore, we were able to generate recombinant FIPs with improved biological activities and expression levels by using DNA shuffling, a powerful tool for the generation of novel therapeutic proteins and for their structural and functional studies.     

Formation of mutually exclusive Rab11 complexes with members of the family of Rab11-interacting proteins regulates Rab11 endocytic targeting and function

Several Rabs, including Rab11, regulate the traffic and sorting of proteins in the endosomal pathway. Recently, six novel Rab11 family interacting proteins (FIPs) were identified. Although they share little overall sequence homology, all FIPs contain a conserved Rab11-binding domain. Here we investigate the role of FIPs as Rab11-targeting proteins and show that the Rab11-binding domain assumes an alpha-helical structure, with the conserved residues forming a hydrophobic Rab11-binding patch. This hydrophobic patch mediates the formation of mutually exclusive complexes between Rab11 and various members of FIP protein family. Furthermore, the formation of Rab11/FIP complexes regulates Rab11 localization by recruiting it to distinct endocytic compartments. Thus, we propose that Rab11/FIP complexes serve as targeting patches, regulating Rab11 localization and recruitment of additional cellular factors to different endocytic compartments.     

Class I Rab11‐family interacting proteins are binding targets for the Rab14 GTPase

Background information. Rab11 and Rab14 are two related Rab GTPases that are believed to function in endosomal recycling and Golgi/endosome transport processes. We, and others, have identified a group of proteins that interact with Rab11 and function as Rab11 effectors, known as the Rab11‐FIPs (family interacting proteins). This protein family has been sub‐classified into two groups—class I FIPs [FIP2, RCP (Rab coupling protein) and Rip11 (Rab11‐interacting protein)] and class II FIPs (FIP3 and FIP4). Results. In the present study we identify the class I FIPs as dual Rab‐binding proteins by demonstrating that they also interact with Rab14 in a GTP‐dependent manner. We show that these interactions are specific for the class I FIPs and that they occur via their C‐terminal regions, which encompass the previously described RBD (Rab11‐binding domain). Furthermore, we show that Rab14 significantly co‐localizes with the TfnR (transferrin receptor) and that Rab14 Q70L co‐localizes with Rab11a and with the class I FIPs on the ERC (endosomal recycling compartment) during interphase. Additionally, we show that during cytokinesis Rab14 localizes to the cleavage furrow/midbody. Conclusions. The data presented in the present study, which identifies the class I FIPs as the first putative effector proteins for the Rab14 GTPase, indicates greater complexity in the Rab‐binding specificity of the class I FIP proteins.     

Molecular cloning and recombinant expression of a gene encoding a fungal immunomodulatory protein from <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The fungal immunomodulatory proteins (FIPs) are a new protein family identified from several edible and medical mushrooms and play an important role in anti-tumor, anti-allergy and immunomodulating activities. A gene encoding the FIP was cloned from the mycelia of Changbai Lingzhi (Ganoderma lucidum) and recombinant expressed in the Pichia pastoris expression system. SDS-PAGE, amino acid composition and circular dichroism analyses of the recombinant FIP (reFIP) indicated that the gene was correctly and successfully expressed. In vitro assays of biological activities revealed that the reFIP exhibited similar immunomodulating capacities as native FIPs. The reFIP significantly stimulated the proliferation of mouse spleen lymphocytes and apparently enhanced the expression level of interleukin-2 released from the mouse splenocytes. In addition, anti-tumor activity assay showed that the reFIP could inhibit the proliferation of human leukemia-NB4 by inducing the cell apoptosis to a degree of about 32.4%. Taken together, the FIP gene from Changbai G. lucidum has been integrated into the yeast genome and expressed effectively at a high level (about 191.2 mg l⁻¹). The reFIP possessed very similar biological activities to native FIPs, suggesting its potential application as a food supplement or immunomodulating agent in pharmaceuticals and even medical studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Functional proteomic analysis reveals that fungal immunomodulatory protein reduced expressions of heat shock proteins correlates to apoptosis in lung cancer cells

BackgroundLing Zhi-8 (LZ-8) and GMI are two fungal immunomodulatory proteins (FIPs) with a similar structure and amino acid sequence and are respectively obtained from the medicinal mushroom Ganoderma lucidum and Ganoderma microsporum. They present the anti-cancer progression and metastasis. We previously demonstrated that LZ-8 reduces the tumor progression in lung cancer LLC1 cell-bearing mouse. However, it is unclear whether these FIPs induce changes in the protein expression profile in cancer cells and the mechanism for such a process is not defined.PurposeThis study determines the changes in the proteomic profile for tumor lesions of LLC1 cell-bearing mouse received with LZ-8 and the potential mechanism for FIPs in anti-lung cancer cells.MethodsThe proteomic profile of tumor lesions was determined using two-dimensional electrophoresis and a LTQ-OrbitrapXL mass spectrometer (LC-MS/MS). The biological processes and the signaling pathway enrichment analysis were performed using Ingenuity Pathway Analysis (IPA). The differentially expressed proteins were verified by Western blot. Cell viability was determined by MTT assay. Cell morphology was characterized using electron microscopy. Migration was detected using the Transwell assay. The apoptotic response was determined using Western blot and flow cytometry.ResultsObtained results showed that 21 proteins in the tumor lesions exhibited differential (2-fold change, p < 0.05) expression between PBS and LZ-8 treatment groups. LZ-8-induced changes in the proteomic profile that may relate to protein degradation pathways. Specifically, three heat shock proteins (HSPs), HSP60, 70 and 90, were significantly downregulated in tumor lesions of LLC1-bearing mouse received with LZ-8. Both LZ-8 and GMI reduced the protein levels for these HSPs in lung cancer cells. Functional studies showed that they inhibited cell migration but effectively induced apoptotic response in LLC1 cells in vitro. In addition, the inhibitors of HSP60 and HSP70 effectively inhibited cell migration and decreased cell viability of LLC1 cells.ConclusionsLZ-8 induced changes in the proteomic profile of tumor lesions which may regulate the HSPs-related cell viability. Moreover, inhibition of HSPs may be related to the anti-lung cancer activity.     

Molecular characterization of Rab11 interactions with members of the family of Rab11-interacting proteins

The Rab11 subfamily of GTPases plays an important role in vesicle trafficking from endosomes to the plasma membrane. At least six Rab11 effectors (family of Rab11-interacting proteins (FIPs)) have been shown to interact with Rab11 and are hypothesized to regulate various membrane trafficking pathways such as transferrin recycling, cytokinesis, and epidermal growth factor trafficking. In this study, we characterized interactions of FIPs with the Rab11 GTPase using isothermal titration calorimetric studies and mutational analysis. Our data suggest that FIPs cannot differentiate between GTP-bound Rab11a and Rab11b in vitro (50-100 nm affinity) and in vivo. We also show that, although FIPs interact with the GDP-bound form of Rab11 in vitro, the binding affinity (>1000 nm) is not sufficient for FIP and GDP-bound Rab11 interactions to occur in vivo. Mutational analysis revealed that both the conserved hydrophobic patch and Tyr628 are important for the GTP-dependent binding of Rab11 to FIPs. The entropy and enthalpy analyses suggest that binding to Rab11a/b may induce conformational changes in FIPs.     

Recombinant Expression and Bioactivity Comparison of Four Typical Fungal Immunomodulatory Proteins from Three Main <Emphasis Type="Italic">Ganoderma</Emphasis> Species

Background

More than a dozen of fungal immunomodulatory proteins (FIPs) have been identified to date, most of which are from Ganoderma species. However, little is known about the similarities and differences between different Ganoderma FIPs’ bioactivities. In the current study, two FIP genes termed FIP-gap1 and FIP-gap2 from G. applanatum, along with LZ-8 and FIP-gsi, another two representative Ganoderma FIP genes from G. lucidum and G. sinense were functionally expressed in Pichia. Subsequently, bioactivities of four recombinant Ganoderma FIPs were demonstrated and compared.

Results

All the four Ganoderma FIP genes could be effectively expressed in P. pastoris GS115 at expression levels ranging from 197.5 to 264.3?mg?L^??1 and simply purified by one step chromatography using HisTrap? FF prepack columns. Amino acid sequence analysis showed that they all possessed the FIP conserved fragments. The homologies of different Ganoderma FIPs were from 72.6 to 86.4%. In vitro haemagglutination exhibited that FIP-gap1, FIP-gsi and LZ-8 could agglutinate human, sheep and mouse red blood cells but FIP-gap2 agglutinated none. Besides, the immunomodulation activities of these Ganoderma FIPs were as: rFIP-gap2?>?rFIP-gap1?>?rLZ-8 and rFIP-gsi in terms of proliferation stimulation and cytokine induction on murine splenocytes. Additionally, the cytotoxic activity of different FIPs was: rFIP-gap1?>?rLZ-8?>?rFIP-gsi?>?rFIP-gap2, examined by their inhibition of three human carcinomas A549, Hela and MCF-7.

Conclusions

Taken together, four typical Ganoderma FIP genes could be functionally expressed in P. pastoris, which might supply as feasible efficient resources for further study and application. Both similarities and differences were indeed observed between Ganoderma FIPs in their amino acid sequences and bioactivities. Comprehensively, rFIP-gaps from G. applanatum proved to be more effective in immunomodulation and cytotoxic assays in vitro than rLZ-8 (G. lucidum) and rFIP-gsi (G. sinense).