烟曲霉Zeta类谷胱甘肽转移酶基因的克隆表达及生物信息学研究
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摘要
烟曲霉(Aspergillus fumigatus)是近年来临床上仅次于白色念珠菌的一种重要的条件致病真菌,致侵袭性曲霉病,严重可危及生命。本文克隆了烟曲霉Zeta类GST基因(AfuGST),该基因全长696bp,编码231个氨基酸,分子量约为25.3Kda,并在大肠杆菌中高效表达了AfuGST蛋白,经分离纯化获得纯化的蛋白,活性测定结果显示AfuGST对底物ECA的活性是0.33μmol/min per mg,对底物NBD-Cl的活性是0.056μmol/min per mg,测活结果显示克隆的AfuGST具有GST酶活性。
对AfuGST进行了生物信息学研究显示该蛋白整个二级结构中存在着大量的α螺旋,含量为48%,在N末端有五段β折叠,含量占18%。同源模拟了烟曲霉Zeta类谷胱甘肽转移酶的三维结构,发现N-末端结构域包括一个βαβαββα的结构基元,其中β折叠是以反平行的方式形成的混合四链β-折叠片。C末端结构域是一个纯α-螺旋,其核心由四个螺旋(α4/α5/α6/α7)形成的束组成。通过AfuGST的三维结构与小鼠Zeta类GST的三维结构进行比对,发现AfuGST的第17位SER是与GSH结合的重要功能氨基酸之一。该研究为进一步研究条件致病真菌的解毒、代谢机制奠定了基础。
Aspergillus fumigatus is a saprophytic fungus that plays an essential role in recycling environmental carbon and nitrogen. Its natural ecological niche is the soil, wherein it survives and grows on organic debris. Although this species is not the most prevalent fungus in the world, it is one of the most ubiquitous of those with airborne conidia. It sporulates abundantly, with every conidial head producing thousands of conidia. The conidia released into the atmosphere have a diameter small enough (2 to 3μm) to reach the lung alveoli. A. fumigatus does not have an elaborate mechanism for releasing its conidia into the air; dissemination simply relies on disturbances of the environment and strong air currents. Once the conidia are in the air, their small size makes them buoyant, tending to keep them airborne both indoors and outdoors. Environmental surveys indicate that all humans will inhale at least several hundred A. fumigatus conidia per day. For most patients, therefore, disease occurs predominantly in the lungs, although dissemination to virtually any organ occurs in the most severely predisposed. Humans and animals constantly inhale numerous conidia of this fungus. The conidia are normally eliminated in the immunocompetent host by innate immune mechanisms, and aspergilloma and allergic bronchopulmonary aspergillosis, uncommon clinical syndromes, are the only infections observed in such hosts. Thus, A. fumigatus was considered for years to be a weak pathogen. With increases in the number of immunosuppressed patients, however, there has been a dramatic increase in severe and usually fatal invasive aspergillosis, now the most common mold infection worldwide.
Glutathione S-transferases (GSTs ; EC 2.5.1.18) are a family of multi-functional enzymes involved in the cellular detoxification and excretion of a variety of xenobiotic substances, representing an integral part of phase II biotransformation enzymes. Until now there are less report on GSTs from fungi. More research on characterization, catalytic mechanism and the evolution status on GSTs in fungi are the focus of the thesis.
By comparison with other major groups, such as mammals, plants and insects, relatively little is known about GSTs from fungi. It is clear that these enzymes are expressed widely in a large number of fungal species, although not in Saccharomyces cerevisiae. As with mammals, plants and insects, the enzymes are expressed in multiple forms which appear to be inducible by xenobiotics. Full-length sequences have only been published for the two GSTs of Issatchenkia orientalis and for the recombinant enzymes of S. cerevisiae. The sequences of I. orientalis GSTs Y-1 and Y-2 are quite distinct from those of the Alpha/Mu/Pi GST classes, but show limited similarity with the N-terminal region of several Theta-class enzymes. GST Y-1 shows conservation of the N-terminal catalytically essential serine of the Theta class, but in GST Y-2 this is replaced with a threonine, which may explain this enzyme's significantly lower catalytic activity compared with GST Y-1. Comparison of partial N-terminal sequences and immunoblotting suggest that GSTs from Phanerochaete chrysosporium, Mucor circinelloides and Yarrowia lipolytica are also similar to those in the Theta class; however, in the absence of full-length sequence data, such allocations cannot be definitive.
Two GST genes have been cloned and sequenced from S. cerevisiae and their recombinant proteins studied. These enzymes show little similarity to mainstream GSTs, although their N-terminal sequence shows moderate similarity to GSTs Y-1 and Y-2 of I. orientalis, to GST-III and GST-IV of maize (see next section) and to other S. cerevisiae proteins, such as the product of URE2, elongation factor 1g] and the a-agglutinin protein. It is noteworthy that a GST purified from Mucor circinelloides also showed some N-terminal sequence similarity to the a-agglutinin protein. While the recombinant S. cerevisiae enzymes are membrane-bound in the endoplasmic reticulum, they function as dimers, suggesting little similarity to microsomal GSTs (which are trimeric).
A zeta glutathione S-transferase (GST) has been cloned from Aspergillus fumigatus by RT-PCR. Open reading frame analysis indicated that the A. fumigatus GST (AfuGST) gene encodes a protein of 231 amino acid residues with a calculated molecular mass of 25.3kDa and a calculated pI of 5.52. The expression vector (pET-GST) was constructed by inserting AfuGST cDNA into pET-30a(+) vector. pET-GST plasmid was transformed to E. coli BL21(DE3), and the recombinant AfuGST protein was induced expression in E. coli BL21(DE3). The recombinant AfuGST was purified by affinity chromatography and characterized. The recombinant AfuGST exhibited GST enzymatic activity towards the substrates ethacrynic acid (ECA) and 4-Chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl).
Finally further research by bioinformatic softwares on the classification and evolution status of the AfuGST is done. Data confirms above result that AfuGST belongs to zeta class GSTs. The AfuGST Three-dimensional structure shows that monomer is formed by two distinct domains; The N-terminal domain (domain I) constitutes roughly one-third of the protein and consists of aβαβαββαstructural motif in whichβ3 is antiparallel with respect to the otherβ-strands. The six helices of the C-terminal domain (domain II) make aα-helical bundle. The core of C-terminal domain is a bundle of four helices (α4/α5/α6/α7). Ser17 in AfuGST are assumed as the most important functional amino acids acting as conjunct site with GSH.
对AfuGST进行了生物信息学研究显示该蛋白整个二级结构中存在着大量的α螺旋,含量为48%,在N末端有五段β折叠,含量占18%。同源模拟了烟曲霉Zeta类谷胱甘肽转移酶的三维结构,发现N-末端结构域包括一个βαβαββα的结构基元,其中β折叠是以反平行的方式形成的混合四链β-折叠片。C末端结构域是一个纯α-螺旋,其核心由四个螺旋(α4/α5/α6/α7)形成的束组成。通过AfuGST的三维结构与小鼠Zeta类GST的三维结构进行比对,发现AfuGST的第17位SER是与GSH结合的重要功能氨基酸之一。该研究为进一步研究条件致病真菌的解毒、代谢机制奠定了基础。
Aspergillus fumigatus is a saprophytic fungus that plays an essential role in recycling environmental carbon and nitrogen. Its natural ecological niche is the soil, wherein it survives and grows on organic debris. Although this species is not the most prevalent fungus in the world, it is one of the most ubiquitous of those with airborne conidia. It sporulates abundantly, with every conidial head producing thousands of conidia. The conidia released into the atmosphere have a diameter small enough (2 to 3μm) to reach the lung alveoli. A. fumigatus does not have an elaborate mechanism for releasing its conidia into the air; dissemination simply relies on disturbances of the environment and strong air currents. Once the conidia are in the air, their small size makes them buoyant, tending to keep them airborne both indoors and outdoors. Environmental surveys indicate that all humans will inhale at least several hundred A. fumigatus conidia per day. For most patients, therefore, disease occurs predominantly in the lungs, although dissemination to virtually any organ occurs in the most severely predisposed. Humans and animals constantly inhale numerous conidia of this fungus. The conidia are normally eliminated in the immunocompetent host by innate immune mechanisms, and aspergilloma and allergic bronchopulmonary aspergillosis, uncommon clinical syndromes, are the only infections observed in such hosts. Thus, A. fumigatus was considered for years to be a weak pathogen. With increases in the number of immunosuppressed patients, however, there has been a dramatic increase in severe and usually fatal invasive aspergillosis, now the most common mold infection worldwide.
Glutathione S-transferases (GSTs ; EC 2.5.1.18) are a family of multi-functional enzymes involved in the cellular detoxification and excretion of a variety of xenobiotic substances, representing an integral part of phase II biotransformation enzymes. Until now there are less report on GSTs from fungi. More research on characterization, catalytic mechanism and the evolution status on GSTs in fungi are the focus of the thesis.
By comparison with other major groups, such as mammals, plants and insects, relatively little is known about GSTs from fungi. It is clear that these enzymes are expressed widely in a large number of fungal species, although not in Saccharomyces cerevisiae. As with mammals, plants and insects, the enzymes are expressed in multiple forms which appear to be inducible by xenobiotics. Full-length sequences have only been published for the two GSTs of Issatchenkia orientalis and for the recombinant enzymes of S. cerevisiae. The sequences of I. orientalis GSTs Y-1 and Y-2 are quite distinct from those of the Alpha/Mu/Pi GST classes, but show limited similarity with the N-terminal region of several Theta-class enzymes. GST Y-1 shows conservation of the N-terminal catalytically essential serine of the Theta class, but in GST Y-2 this is replaced with a threonine, which may explain this enzyme's significantly lower catalytic activity compared with GST Y-1. Comparison of partial N-terminal sequences and immunoblotting suggest that GSTs from Phanerochaete chrysosporium, Mucor circinelloides and Yarrowia lipolytica are also similar to those in the Theta class; however, in the absence of full-length sequence data, such allocations cannot be definitive.
Two GST genes have been cloned and sequenced from S. cerevisiae and their recombinant proteins studied. These enzymes show little similarity to mainstream GSTs, although their N-terminal sequence shows moderate similarity to GSTs Y-1 and Y-2 of I. orientalis, to GST-III and GST-IV of maize (see next section) and to other S. cerevisiae proteins, such as the product of URE2, elongation factor 1g] and the a-agglutinin protein. It is noteworthy that a GST purified from Mucor circinelloides also showed some N-terminal sequence similarity to the a-agglutinin protein. While the recombinant S. cerevisiae enzymes are membrane-bound in the endoplasmic reticulum, they function as dimers, suggesting little similarity to microsomal GSTs (which are trimeric).
A zeta glutathione S-transferase (GST) has been cloned from Aspergillus fumigatus by RT-PCR. Open reading frame analysis indicated that the A. fumigatus GST (AfuGST) gene encodes a protein of 231 amino acid residues with a calculated molecular mass of 25.3kDa and a calculated pI of 5.52. The expression vector (pET-GST) was constructed by inserting AfuGST cDNA into pET-30a(+) vector. pET-GST plasmid was transformed to E. coli BL21(DE3), and the recombinant AfuGST protein was induced expression in E. coli BL21(DE3). The recombinant AfuGST was purified by affinity chromatography and characterized. The recombinant AfuGST exhibited GST enzymatic activity towards the substrates ethacrynic acid (ECA) and 4-Chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl).
Finally further research by bioinformatic softwares on the classification and evolution status of the AfuGST is done. Data confirms above result that AfuGST belongs to zeta class GSTs. The AfuGST Three-dimensional structure shows that monomer is formed by two distinct domains; The N-terminal domain (domain I) constitutes roughly one-third of the protein and consists of aβαβαββαstructural motif in whichβ3 is antiparallel with respect to the otherβ-strands. The six helices of the C-terminal domain (domain II) make aα-helical bundle. The core of C-terminal domain is a bundle of four helices (α4/α5/α6/α7). Ser17 in AfuGST are assumed as the most important functional amino acids acting as conjunct site with GSH.
引文
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[2] Rippon JW. Medical Mycology: the pathogenic fungi and the pathogenic actionmycetes. 3rd Ed. W.B. Saunders Company, Philadelphia, 1988.
[3] Armstrong RN. Structure, catalytic mechanism, and evolution of the glutathione S-transferase. Chem. Res. Toxicol. 1997, 10:2-18.
[4] Frova C. The plant glutathione transferase gene family: genomic structure, functions, expression and evolution. Physiologia Plantarum. 2003, 119:469-479.
[5] Thom R, Dixon PD, Edwards R, Cole JD, Lapthorn JA. The Structure of a Zeta Class Glutathione S-Transferase from Arabidopsis thaliana Characterisation of a GST with Novel Active-site Architecture and a Putative Role in Tyrosine Catabolism. Mol. Biol. 2001, 308:949-962.
[6] Edwards R, Dixon DP, Walbot V. Plant glutathione S-transferases: enzymes with multiple functions in sickness and in health. Trends Plant Sci. 2000, 5:193-198.
[7] 杨海灵,聂力嘉,朱圣庚,周先碗,谷胱甘肽硫转移酶结构与功能研究进展,成都大学学报(自然科学版),2006 年 01 期
[8] Chang LH, Tam MF. Site-directed mutagenesis and chemical modification of histidine residues on an alpha-class chick liver glutathione S-transferase CL 3-3. Histidines are not needed for theactivity of the enzyme and diethylpyrocarbonate modifies both histidine and lysine residue. Eur J Biochem. 1993, 211:805-811.
[9] Verlaan M, te Morsche RH, Roelofs HM, et al. Glutathione S-transferase mu null genotype affords protection against alcohol induced chronic pancreatitis. Am J Med Genet, 2003, 120A(1) : 34-39.
[10] Sheehan D, Meade G, Foley VM, Dowd CA. Structure, function and evolution of glutathione transferases implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem. J. 2001, 360:1-16.
[11] Fukai F, Ohtaki H, Ueda T, Katayama T. A possible role of glutathione S-transferase in rat ovary and testis. J. Clin. Biochem. Nutr. 1992, 12:93-107.
[12] Chen H, Juchau MR. Recombinant human glutathione S-transferases catalyse enzymic isomerization of 13-cis-retinoic acid to all-trans-retinoic acid. Biochem. J. 1998, 336:223-226.
[13] Kanaoka Y, Ago H, Inagaki E, Nanayama T, Miyano M, Kikuno R, Fujii Y, Eguch N, Toh H, Urade Y, Hayaishi O. Cloning and crystal structure of hematopoietic prostaglandin D synthase. Cell. 1997, 90:1085-1095.
[14] Blackburn AC, Woollatt E, Sutherland GR, Board PG. Characterization and chromosome location of the gene GSTZ1 encoding the human Zeta class glutathione transferase and maleylacetoacetate isomerase. Cytogen. Cell Genet, 1998, 83:109-114.
[15] Marrs KA, Alfenito MR, Lloyd AM, Walbot V. A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2. Nature, 1995, 375:397-400.
[16] Alfenito MR, Souer E, Goodman CD, Buell R, Mol J, Koes R, Walbot V. Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases. Plant Cell, 1998, 10:1135-1149
[17] Dixon DP, Lapthorn A, Edwards R. Plant glutathione transferases. Genome Biol. 2002, 3:1-10.
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