杨树菇凝集素AAVP具有抗病毒和促进菌丝分化功能

杨树菇凝集素 (AAVP)是一种新的真菌凝集素 .用半叶法检测证明 ,AAVP具有抑制烟草花叶病毒 (TMV)侵染的活性 .等电聚焦法证实了AAVP可以与TMV的外壳蛋白结合 .用滤纸圆片法检测 ,AAVP对 3种植物病原真菌没有抑制作用 .AAVP滴加在菌丝的表面 ,显著地促进了杨树菇和毛木耳的菌丝分化 ,促进子实体的形成 .Western印迹表明 ,AAVP存在于菌丝、菌柄和菌盖等组织中 ;假猴头 ,香菇 ,草菇 ,杏孢菇 ,灵芝等大型真菌的子实体中存在着多种与AAVP的抗血清有交叉反应的蛋白质 .大多数大型真菌中可能存在着与AAVP具有相似血清学特征的蛋白质家族 ,在防御反应及菌丝分化等多种生理过程中发挥重要的作用     

Opposing developmental functions of Agrocybe aegerita galectin (AAL) during mycelia differentiation

Mycelia of basidiomycetes differentiating into fruiting body is a controlled developmental process, however the underlying molecular mechanism remains unknown. In previous work, a novel fungal Agrocybe aegerita galectin (AAL) was isolated from A. aegerita in our laboratory. AAL was shown to promote mycelial differentiation in A. aegerita and Auricularia polytricha, indicating that AAL might function as a conserved fruiting initiator during basidiomycete mycelia development. In the current work, we investigate the role of AAL in mycelia differentiation and fruiting body formation. First, the expression and localization of AAL in mycelia, primordium and fruiting body were assessed by Western blotting and immunohistochemistry. AAL was found to be ubiquitously expressed in the primordium and fruiting body but not in the mycelia. AAL facilitated mycelia congregation and promoted fruiting body production when AAL was applied on mycelia. At the same time, when AAL was spread on potato dextrose agar (PDA) medium prior to mycelia inoculation, mycelia exhibited slowed growth rates, resulting in mycelia cords formation and inhibition of fruiting body formation. The 5' regulatory sequence of aal was cloned by 'genome walking'. Here, we show that aal lack introns in the coding region and the upstream 740 bp sequence was characterized by the existence of core promoter elements, which included: two CCAAT boxes (-535/-280), a GC box (-145), a TATA box (-30) and a fungal leader intron within the 5' UTR. The identification of regulatory expression elements may provide an explanation to the stage-specific and high-level expression of aal during fruiting development.     

Crystallization and preliminary crystallographic studies of an antitumour lectin from the edible mushroom Agrocybe aegerita

An antitumour lectin named AAL has been purified from the fruiting body of edible mushroom Agrocybe aegerita. In addition to having a distinct bioactivity, AAL shows strong inhibition effects on human and mouse tumour cells. It has been shown that AAL exerts its antitumour effects via apoptosis-induction. AAL and AAL-lactose complex have been crystallized and their diffraction data were collected with resolution of 2.6 A and 3.0 A, respectively. Both crystals belong to space group P 6122 with unit cell parameters a = 123.98 A, b = 123.98 A, c = 56.86 A, alpha= beta=90 degrees, gamma = 120 degrees and a = 123.69 A, b = 123.69 A, c = 56.64 A, alpha = beta = 90 degrees, gamma = 120 degrees, respectively.     

Molecular character of the recombinant antitumor lectin from the edible mushroom Agrocybe aegerita

The lectin from Agrocybe aegerita (AAL) has been found to possess potent tumor-suppressing function and tumor cell apoptosis-inducing activity. In this paper, we report the full sequence, the active expression of the gene encoding AAL at a high level and bioassay of the binding property with lactose, apoptosis-inducing activity and DNase activity of recombinant AAL (rAAL). The results reveal that AAL is a member of the galectin family and the dimeric form is the active unit for the functional performance. The rAAL showed comparable tumor cell apoptosis-inducing activity with the wild AAL but no DNase activity at all. The molecular characters revealed by this study are significant for the in-depth investigation of the functional mechanism of this interesting protein.     

Structural Basis of Specific Recognition of Non-Reducing Terminal N-Acetylglucosamine by an Agrocybe aegerita Lectin

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification that plays essential roles in many cellular pathways. Research in this field, however, is hampered by the lack of suitable probes to identify, accumulate, and purify the O-GlcNAcylated proteins. We have previously reported the identification of a lectin from the mushroom Agrocybe aegerita, i.e., Agrocybe aegerita lectin 2, or AAL2, that could bind terminal N-acetylglucosamine with higher affinities and specificity than other currently used probes. In this paper, we report the crystal structures of AAL2 and its complexes with GlcNAc and GlcNAcβ1-3Galβ1-4GlcNAc and reveal the structural basis of GlcNAc recognition by AAL2 and residues essential for the binding of terminal N-acetylglucosamine. Study on AAL2 may enable us to design a protein probe that can be used to identify and purify O-GlcNAcylated proteins more efficiently.     

抗肿瘤蛋白AAL与靶蛋白MRG15的共定位研究

一种从食用真菌Agrocybe aegerita中提取的凝集素AAL具有显著的抗肿瘤效果。该蛋白的基因序列已经通过RT-PCR得到。T7噬菌体展示库筛选发现该蛋白可以在体外和细胞核内的细胞周期调控蛋白mortality factor related gene on chro-mosome 15(MRG15)发生相互作用。本实验目的是证实AAL和MRG15在细胞核内的共定位,为AAL与MRG15的体内的互作提供证据。构建质粒,将aal与mrg15分别连接到pEGFP-C1和pDsRed-C1上,共转染Hela细胞。将pEGFP-C1和pD-sRed-C1空载体共转染Hela细胞作为对照。利用激光共聚焦显微镜研究AAL和MRG15在细胞内的共定位。AAL和MRG15均定位在细胞核内,荧光图片的重叠表明AAL和MRG15在核内共定位。而对照组中的EGFP和DS-RED在细胞内呈弥散分布。这些为AAL和MRG15在细胞核内的相互作用提供了证据。     

Crystallization and preliminary crystallographic studies of the recombinant antitumour lectin from the edible mushroom Agrocybe aegerita

The antitumour lectin from Agrocybe aegerita, named AAL, shows strong inhibition effects on human and mouse tumour cells via apoptosis induction activity. Recombinant AAL (rAAL) has been expressed and purified. Both rAAL and rAAL-lactose complex have been crystallized and their X-ray diffraction data were collected to resolutions of 1.9 A and 1.6 A, respectively. Both crystals belong to space group P2(1) with unit cell parameters a = 53.20 A, b = 66.01 A, c = 57.86 A, beta = 109.38 and a = 53.38 A, b = 66.29 A, c = 58.02 A, beta = 109.03, respectively.     

一种新杨树菇(Agrocybe aegerita)凝集素的纯化及生化特性

用硫酸铵分级沉淀、离子交换和分子筛等方法 ,从食用菌杨树菇子实体中分离纯化了一种凝集素 ,称作为AAVP(Agrocybeaegeritaantiviralprotein) .经SDS PAGE测定其亚基的相对分子质量为15 8kD ,凝胶过滤分析分子量为 32kD .IEF PAGE计算其等电点为 3 8.AAVP不含糖 ,是一种N端焦谷氨酰环化封闭的蛋白质 ,经N端去封闭后测得N端氨基酸序列为QGVNIYNIVAGA ,用胰蛋白酶消化后得到一大片段 ,测定的氨基酸序列为PDGPWLVEK .AAVP可以凝集供试的 12种动物血和3种血型人血的血红细胞 ,但对各种血红细胞凝集滴度不同 .糖抑制实验表明 ,在供试的 18种单糖和 3种糖蛋白中 ,只有猪胃粘蛋白强烈抑制AAVP的凝血活性 .AAVP具有较好的热稳定性 ,能够忍受极端的酸碱条件 .AAVP的凝血活性不受Ca2 + 、Mg2 + 、Zn2 + 等二价阳离子的影响 .抗肿瘤活性检测表明 ,AAVP对胃癌细胞株SGC 790 1,MGC 80 3,BGC82 3及人急性白血病细胞株HL 6 0有明显的抑制作用 .AAVP对小鼠腹腔注射的半致死剂量为 15 85mg kg .     

Cyclooxygenase inhibitory and antioxidant compounds from the fruiting body of an edible mushroom, Agrocybe aegerita.

In the search for bioactive natural products from edible mushrooms, we have investigated the fruiting body of Agrocybe aegerita. The methanol extract of this mushroom yielded a fatty acid fraction (FAF), along with palmitic acid (1), ergosterol (2), 5,8-epidioxy-ergosta-6,22-dien-3beta-ol (3), mannitol (4) and trehalose (5). The composition of FAF was confirmed by GC-MS and by comparison to the retention values of authentic samples of palmitic, stearic, oleic and linoleic acids. The structures of 1-5 were established using spectroscopic methods. FAF and compounds 1-3 showed cyclooxygenase (COX) enzyme inhibitory and antioxidant activities. The inhibition values of liposome peroxidation by FAF, compounds 1 and 2 at 100 microg/ml were 75, 45, and 43%, respectively. The inhibition values of COX-I enzyme by FAF and 1-3 at 100 microg/ml were 80, 39, 19, and 57%, respectively. Similarly, COX-II enzyme activity was reduced by FAF and 1-3 at 100 microg/ml with values of 88, 45, 28, and 22%, respectively. Compounds 1, 3 and fatty acids were isolated here for the first time from the fruiting body of A. aegerita.     

Pleurotus and Agrocybe hemolysins,new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.     

A nuclear ligand MRG15 involved in the proapoptotic activity of medicinal fungal galectin AAL (Agrocybe aegerita lectin)

Background

We have previously reported a novel fungal galectin Agrocybe aegerita lectin (AAL) with apoptosis-induced activity and nuclear migration activity. The importance of nuclear localization for AAL's apoptosis-induced activity has been established by mutant study. However, the mechanism remains unclear.

Methods

We further investigated the mechanism using a previously reported carbohydrate recognition domain (CRD) mutant protein H59Q, which retained its nuclear localization activity but lost most of its apoptotic activity. The cell membrane-binding ability of recombinant AAL (rAAL) and H59Q was analyzed by FACS, and their cellular partners were identified by affinity chromatography and mass spectroscopy. Furthermore, the interaction of AAL and ligand was proved by mammalian two-hybrid and pull down assays. A knockdown assay was used to confirm the role of the ligand.

Results

The apoptotic activity of AAL could be blocked by lactose. Mutant H59Q retained comparable cell membrane-binding ability to rAAL. Four cellular binding partners of AAL in HeLa cells were identified: glucose-regulated protein 78 (GRP78); mortality factor 4-like protein 1 (MRG15); elongation factor 2 (EEF2); and heat shock protein 70 (Hsp70). CRD region of AAL was required for the interaction between AAL/mutant AAL and MRG15. MRG15 knockdown increased the cells' resistance to AAL treatment.

Conclusion

MRG15 was a nuclear ligand for AAL in HeLa cells. These data implied the existence of a novel nuclear pathway for the antitumor activity of fungal galectin AAL.

General significance

These findings provide a novel explanation of AAL bioactivity and contribute to the understanding of mushroom lectins' antitumor activity.     

柱状田头菇生长发育中9种胞外酶活性的测定

柱状田头菇Agrocybe aegerita作为一种新开发的食用菌,越来越受到消费者的喜爱和研究者的关注,但在其栽培生产中遇到的产量相对较低的问题,一直未能得到很好的解决,致使柱状田头菇的栽培难以得到大规模的推广。本研究对不同来源的柱状田头菇在其6个不同生长发育阶段中,9种与培养料中主要组分分解相关的胞外酶活性的变化进行了测定,结果表明：柱状田头菇属褐腐菌,对非木质纤维素的利用能力最强,对纤维素的利用能力较强,对木质素的利用能力较差,但柱状田头菇具有漆酶活性。     

Primary structure of a fucose-specific lectin obtained from a mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) is a protein composed of two identical subunits having no carbohydrate chain and shows sugar-binding specificity for L-fucose. Full-length cDNA encoding for the lectin has been isolated from a lambda gt11 library, screened with an antiserum directed against AAL. The cDNA clone contained 1,370 nucleotides and an open reading frame of 939 nucleotides encoding 313 amino acids. The amino-terminal sequence (residues 1-30) of the lectin isolated from the mushroom coincided with the deduced amino acid sequence starting from proline at the 2nd residue, indicating that the mature AAL consists of 312 amino acids. Its molecular weight is calculated to be 33,398. The deduced amino acid sequence shows that AAL includes six internal homologous regions, and has considerable homology with a hemagglutinin from a Gram-negative bacterium, Myxococcus xanthus, which forms a fruiting body. No significant homology was observed with higher plant or animal lectins. The recombinant AAL produced by Escherichia coli JM109 carrying the AAL expression plasmid pKA-1 [Fukumori, F. et al. (1989) FEBS Lett. 250, 153-156] was purified from the cell lysate by affinity chromatography using a fucose-starch column, and hundreds of milligrams of the lectin was obtained. The recombinant lectin showed the same biochemical characteristics and sugar binding specificity as did the natural AAL.     

绣球菌蛋白质的营养评价

采用国际上通用的营养评价方法,对绣球菌[Sparassis crispa(Wulf.)Fr.]子实体进行了蛋白质营养价值评价。结果为绣球菌子实体的粗蛋白质含量为12.9%,氨基酸总量为9.33%,其中必需氨基酸含量占氨基酸总量的33.9%。绣球菌的赖氨酸为第一限制氨基酸,苏氨酸相对过剩。绣球菌子实体蛋白质的化学评分(CS)、氨基酸评价(AAS)、必需氨基酸指数(EAAI)、生物价(BV)、营养指数(NI)和氨基酸比值系数分(SRCAA)分别为80.9,87.3,78.3,73.6,10.1和87.9。绣球菌的营养价值高于松口蘑(Tricholoma matsutake)和茶树菇(A-grocybe aegerita)。因此,绣球菌子实体是良好的菌类蛋白源。     

从杨树菇中开发新型食品防腐剂的初步研究

本实验室通过对14株食用真菌的抑菌试验筛选,发现杨树菇具有极强的抑菌作用,且抑菌谱广,可以有效抑制细菌、酵母和部分霉菌。与不同浓度(0.05%、0.1%、0.2%、0.3% g/ml)山梨酸、苯甲酸抑菌能力相比,抑菌效果更为明显。以金黄色葡萄球菌为指示菌研究了其最适发酵条件及有效成分的热稳定性,结果表明:杨树菇的发酵周期为7-9d,发酵温度25℃,发酵的起始pH 6.0~7.0,发酵液装量为80~140 ml/500 ml三角瓶,接种量10%~15%(v/v);发酵液浓缩液经121℃、30 min湿热灭菌处理后仍具有很强的抑菌活性。杨树菇是一种具有开发前途的食用真菌。     

Importance of nuclear localization for the apoptosis-induced activity of a fungal galectin AAL (Agrocybe aegerita lectin)

Agrocybe aegerita lectin (AAL) was identified previously in our group as a novel galectin from medicinal fungi Agrocybe aegerita, and has been shown to effectively induce cancer cell cycle arrest and apoptosis in vitro and tumor regression in vivo. Here, AAL was observed to translocate into the HeLa cell nucleus and induce cell apoptosis when it was predominantly in the nucleus. The N-terminus and C-terminus of AAL were required for nuclear localization. Site mutated proteins were generated based on AAL structure. Dimer interface mutant I25G, carbohydrate recognition domain (CRD) mutant R63H, and loop region mutant L33A could not enter the nucleus and lost the ability to induce apoptosis. CRD mutant H59Q and loop region mutant I144G maintained nuclear localization activity, and H59Q retained residual bioability but I144G had no activity, indicating that nuclear localization is important but not sufficient for AAL to become apoptotically active. Our findings provide a novel antitumor mechanism of fungal galectin.     

Unusual accumulation of polymorphic microsatellite loci in a specific region of the mitochondrial genome of two mushroom-forming Agrocybe species

The cob/tRNA(Tyr) mitochondrial regions of Agrocybe aegerita and of the related species Agrocybe chaxingu display an unusual clustering of four microsatellite loci constituted by motifs of one to six nucleotides whose number of repeats varied from three to 18. In A. chaxingu, these microsatellite loci are followed by a small region bearing one additional microsatelite and one minisatellite locus constituted by an octanucleotide motif repeated 13-18 times. In A. aegerita, this latter region is deleted. This is the first evidence of such an accumulation of microsatellites in mitochondrial genomes. The analyses of the microsatellite loci in 11 A. aegerita and in four A. chaxingu wild strains have shown extensive intraspecific and interspecific variations in the number of tandem repeats (VNTRs), suggesting that these loci could represent powerful molecular markers for strain fingerprinting. Up to 23 different alleles were present in the 15 Agrocybe studied strains, allowing the definition of 12 different haplotypes.     

A lectin from an ascomycete mushroom, Melastiza chateri: no synthesis of the lectin in mycelial isolate

Using an affinity adsorbent prepared from L-fucose and starch, a lectin was isolated from fruit bodies of an ascomycete mushroom, Melastiza chateri. The lectin was found to cross-react with antiserum against Aleuria aurantia lectin (AAL), that had been obtained from another ascomycete mushroom. The N-terminal amino acid sequence was analyzed, and among 20 residues 12 were the same as AAL. The molecular mass of the lectin estimated by SDS-PAGE was approximately 40 kDa, which is larger than that of AAL. Mycelial isolate was obtained from M. chateri by germinating ascospores, and identified by analyzing restriction fragment length polymorphisms (RFLP) of DNA. The isolate from M. chateri did not synthesize the lectin, although the isolate from A. aurantia had been known to synthesize AAL as much as the fruit body.     

A Lectin from an Ascomycete Mushroom,Melastiza chateri: No Synthesis of the Lectin in Mycelial Isolate

Using an affinity adsorbent prepared from L-fucose and starch, a lectin was isolated from fruit bodies of an ascomycete mushroom, Melastiza chateri. The lectin was found to cross-react with antiserum against Aleuria aurantia lectin (AAL), that had been obtained from another ascomycete mushroom. The N-terminal amino acid sequence was analyzed, and among 20 residues 12 were the same as AAL. The molecular mass of the lectin estimated by SDS-PAGE was approximately 40 kDa, which is larger than that of AAL. Mycelial isolate was obtained from M. chateri by germinating ascospores, and identified by analyzing restriction fragment length polymorphisms (RFLP) of DNA. The isolate from M. chateri did not synthesize the lectin, although the isolate from A. aurantia had been known to synthesize AAL as much as the fruit body.