Expression and characterization of an iron-containing superoxide dismutase from Burkholderia pseudomallei

A superoxide dismutase (SOD) gene from Burkholderia pseudomallei, the causative agent of melioidosis, was cloned and expressed in Escherichia coli, and its product was functionally and physically characterized. The gene has an open-reading frame of 579 bp. The deduced amino acid sequence has 192 residues with a calculated molecular mass of ～22 kDa. Sequence comparison with other bacterial SODs showed that the protein contains typical metal-binding motifs and other Fe-SOD-conserved residues. The sequence has substantial similarity with other bacterial Fe-SOD sequences. The enzymatic activity of the expressed protein was inhibited by hydrogen peroxide but not by sodium azide or potassium cyanide, attributes that indeed are characteristic of typical bacterial Fe-SODs. Western blotting with antiserum against the recombinant Fe-SOD revealed that it is expressed in B. pseudomallei. Transformed E. coli that expressed the Fe-SOD had significantly increased SOD activity and was highly tolerant to paraquat-mediated replication inhibition, compared to transformed cells carrying an empty vector. Our results provide a basis for further biochemical characterization of the enzyme and elucidation of its role in the pathogenesis of B. pseudomallei.     

Molecular cloning and characterization of a cDNA for an iron-superoxide dismutase in rice (Oryza sativa L.)

We have isolated a cDNA encoding Fe-SOD from rice (Oryza sativa L.). The deduced amino acid sequence consists of a polypeptide with 255 amino acids, including a putative transit peptide (40 a.a.) in amino-terminal residues. This sequence is similar to the known plant Fe-SODs but not classified in the group of known Fe-SODs. The metal analysis and SOD assays of the partial purified recombinant protein expressed in E. coli showed that this cDNA encodes an iron-containing SOD. However this SOD activity was not inhibited by the treatment with hydrogen peroxide, which was expected to inhibit known Fe-SOD activity. mRNA of rice Fe-SOD was detected in all vegetative tissues examined, being especially abundant in calli, and strongly increased by light induction. These results suggested that this cDNA encodes rice Fe-SOD, which is apparently distinct from known plant Fe-SODs.     

The superoxide dismutase-encoding gene of the obligately anaerobic bacterium Bacteroides gingivalis

The gene (sod) encoding the superoxide dismutase (SOD) of the obligately anaerobic bacterium Bacteroides gingivalis was cloned. The amino acid (aa) sequence of the SOD, deduced from the nucleotide sequence of the sod gene, basically resembled that of known Fe-SODs. However, the aa sequence of the B. gingivalis SOD was found to be intermediate between those of Fe-SOD and Mn-SOD in a limited region around the putative second ligand, where major differences between the aa sequences of Fe-SOD and Mn-SOD are known to exist.     

A cambialistic SOD in a strictly aerobic hyperthermophilic archaeon, Aeropyrum pernix.

The superoxide dismutase (SOD) gene of Aeropyrum pernix, a strictly aerobic hyperthermophilic archaeon, was cloned and expressed in Escherichia coli, and its gene product was characterized. The molecular mass of the protein, based on the deduced amino acid sequence, was 24.6 kDa. The sequence showed overall similarity to the sequences of known Mn- and Fe-SODs. The metal binding residues conserved in Mn- and Fe-SODs were also found in A. pernix SOD. When the SOD gene was expressed in E. coli cells, the product formed a homodimer, and contained both Mn and Fe. Metal reconstitution experiments showed that A. pernix SOD is cambialistic, i.e. active with either Fe or Mn. The specific activities were 906 U/mg with Mn and 175 U/mg with Fe. No loss of activity of Mn-reconstituted SOD was observed at 105 degrees C even after 5 h incubation. Sodium azide, an inhibitor of SODs, did not inhibit the Mn-reconstituted SOD from A. pernix even at concentrations up to 400 mM. This SOD from an aerobic hyperthermophilic archaeon, Aeropyrum pernix, was extremely thermostable and active with either Mn or Fe. With Mn as a metal cofactor, it was more thermostable, and less sensitive to sodium azide and sodium fluoride than with Fe.     

Identification and characterization of a novel Neospora caninum immune mapped protein 1

Immune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42.9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.     

Characterization of an iron-containing superoxide dismutase from a higher plant, Citrus limonum

An iron-containing superoxide dismutase (SOD, EC 1.15.1.1) was fully characterized from leaves of the higher plant Citrus limonum R. cv. Verna. This enzyme is the first iron-containing SOD to be characterized in the plant family Rutaceae . The purified Fe-SOD has a molecular mass of about 47 kDa and is composed of two non-covalently joined equal subunits. The amino acid composition determined for the enzyme was compared with that of a wide range of SODs and had highest degree of homology with the Fe-SODs from Brassica campestris and Nuphar luteum . The enzyme was more labile at high temperatures than some eucaryotic and procaryotic Fe-SODs. It showed a maximum stability at pH 7.8. The sensitivity of the enzyme to cyanide, hydrogen peroxide and o -phenanthroline was similar to those reported for other Fe-SODs. but the lemon enzyme was comparatively resistant to H₂O₂. By kinetic competition experiments, the rate constant for the disproportionation of superoxide radicals by lemon Fe-SOD was found to be 1.9 × 10⁹ M ⁻¹ s⁻¹ at pH 7.8 and 25°C. A comparative study between the molecular properties of this higher plant Fe-SOD and SODs from different origins is presented.     

Characterization of a superoxide dismutase gene from the archaebacterium Methanobacterium thermoautotrophicum

A gene encoding superoxide dismutase (SOD) was cloned from the archaebacterium Methanobacterium thermoautotrophicum, the first example from an anaerobic bacterium. The deduced amino acid sequence showed overall similarity to sequences of known Mn- and Fe-SODs from aerobic organisms. Judging from a detailed sequence comparison, the cloned SOD gene is classified as Mn-SOD. By comparison of Mn-SOD sequences among various species it was suggested that archaebacterial superoxide dismutase is a direct descendant of a primordial enzyme. Between a putative promoter and the start codon there is an inverted repeat sequence which is also found in the counterpart of Halobacterium halobium.     

特异种质烟草HZNH的Fe-SOD基因的克隆与表达

超氧化物歧化酶(superoxidedismutase,SOD)是一种广泛存在于动物、植物、微生物体内的金属酶,按其结合的金属性离子可分为Fe SOD、Mn SOD和CuZn SOD三种,它们通过催化超氧阴离子自由基O·-2发生歧化反应,达到清除O·-2的效果,具有防御氧毒性、增强机体抗辐射损伤能力、防衰老,治疗某些肿瘤、炎症、自身免疫疾病等功效,在农业、医药、食品、化工等产业中的应用前景广阔,因此广受国内外科研工作者的关注和重视[1].而试图通过转SOD基因技术来培育高抗逆农作物新品种和基因克隆与表达技术来实现SOD的大规模发酵生产,已成为国内外SOD…     

The primary structure of superoxide dismutase purified from anaerobically maintained Bacteroides gingivalis

The superoxide dismutase (SOD) of Bacteroides gingivalis can use either iron or manganese as a cofactor in its catalytic activity. In this study, the complete amino acid sequence of this SOD purified from anaerobically maintained B. gingivalis cells was determined. The proteins consisted of 191 amino acid residues and had a molecular mass of 21,500. The sequence of B. gingivalis SOD showed 44-51% homology with those for iron-specific SODs (Fe-SODs) and 40-45% homology with manganese-specific SODs (Mn-SODs) from several bacteria. However, this sequence homology was considerably less than that seen among the Fe-SOD (65-74%) or Mn-SOD family (42-60%). This indicates that B. gingivalis SOD, which accepts either iron or manganese as metal cofactor, is a structural intermediate between the Fe-SOD and Mn-SOD families.     

Fe-superoxide dismutase and 2-hydroxy-1,4-benzoquinone reductase preclude the auto-oxidation step in 4-aminophenol metabolism by <Emphasis Type="Italic">Burkholderia</Emphasis> sp. strain AK-5

Burkholderia sp. strain AK-5 converts 4-aminophenol to maleylacetic acid via 1,2,4-trihydroxybenzene, which is unstable in vitro and non-enzymatically auto-oxidized to 2-hydroxy-1,4-benzoquinone. Crude extract of strain AK-5 retarded the auto-oxidation and reduced the substrate analogue, 2,6-dimethoxy-1,4-benzoquinone, in the presence of NADH. The two enzymes responsible were purified to homogeneity. The deduced amino acid sequence of the enzyme that inhibited the auto-oxidation showed a high level of identity to sequences of iron-containing superoxide dismutases (Fe-SODs) and contained a conserved metal-ion-binding site; the purified enzyme showed superoxide dismutase activity and contained 1 mol of Fe per mol of enzyme, identifying it as Fe-SOD. Among three type SODs tested, Fe-SOD purified here inhibited the auto-oxidation most efficiently. The other purified enzyme showed a broad substrate specificity toward benzoquinones, including 2-hydroxy-1,4-benzoquinone, converting them to the corresponding 1,4-benzenediols; the enzyme was identified as 2-hydroxy-1,4-benzoquinone reductase. The deduced amino acid sequence did not show a high level of identity to that of benzoquinone reductases from bacteria and fungi that degrade chlorinated phenols or nitrophenols. The indirect role of Fe-SOD in 1,2,4-trihydroxybenzene metabolism is probably to scavenge and detoxify reactive species that promote the auto-oxidation of 1,2,4-trihydroxybenzene in vivo. The direct role of benzoquinone reductase would be to convert the auto-oxidation product back to 1,2,4-trihydroxybenzene. These two enzymes together with 1,2,4-trihydroxybenzene 1,2-dioxygenase convert 1,2,4-trihydroxybenzene to maleylacetic acid.     

GRA12, a novel dense granule protein from Neospora caninum

Neospora caninum, an obligate intracellular parasite of the phylum Apicomplexa, is a major cause of abortion in cattle. After invasion, tachyzoites can reside in the parasitophorous vacuole (PV) and ingest nutrition through the intravacuolar network (IVN). Secreted dense granule proteins of N. caninum (NcGRAs) may play important roles in maintaining the structures of the PV and IVN. In this study, we predicted a NcGRA12 gene; aligned it with Toxoplasma gondii GRA12 for homology analysis; and analyzed the ORF, signal peptide and transmembrane domain. Then, we cloned the NcGRA12 gene, expressed the NcGRA12 protein, prepared polyclonal antibodies, and carried out colocalization analysis of NcGRA12 with NcGRA6 in extracellular tachyzoites and intracellular PVs using an immunofluorescence assay (IFA). Finally, we determined the solubility of the NcGRA12 protein. The results showed that NcGRA12 shared 59.13% nucleotide homology and 44.9% amino acid homology with TgGRA12. There was no predicted signal peptide or transmembrane domain. IFA data of extracellular tachyzoites showed that the NcGRA12 protein was secreted by the apical organ and located at the posterior end of tachyzoites, which was consistent with TgGRA12. IFA data of intracellular PVs identified NcGRA12 in the IVN membranes. Moreover, NcGRA12 could colocalize with NcGRA6 in intracellular PVs but not extracellular tachyzoites. Solubility analysis showed that NcGRA12 existed in soluble and membrane-related forms in the PV. Overall, we provide the first report of the novel NcGRA12 protein and verify that it is associated with the IVN membranes of PVs in N. caninum. These data lay a foundation for further research into the function of NcGRA12.     

The Selenophosphate synthetase gene from Leishmania major.

Selenophosphate synthetase coding sequence was cloned from Leishmania major by RT-PCR amplification. The DNA sequence was found to have an open reading frame encoding protein with 398 amino acids and does not have in-frame UGA codon. The deduced amino acid sequence indicates that it has cysteine residue instead of selenocysteine at the active site of enzyme. Amino acid sequence alignment of Selenophosphate synthetase from parasite with the human enzyme showed approximately 45% homology. The sequences also indicated presence of conserved amino acid residues and motifs that are present in mammalian Selenophosphate synthetase. Southern analysis done with restriction enzyme digested genomic DNA and pulse filed separated chromosome suggests that L. major genome contain a single copy of Selenophosphate synthetase sequence. Expression analysis by Northern analysis and RT-PCR indicated Selenophosphate synthetase mRNA is present in promastigote and amastigote stages of parasite.     

褐飞虱乙酰胆碱酯酶基因全长cDNA的克隆及序列分析

利用反转录聚合酶链式反应(RT_PCR)结合快速扩增cDNA末端(RACE)技术克隆了褐飞虱Nilaparvata lugens 乙酰胆碱酯酶基因cDNA。该cDNA全长2 467 bp,包含一个1 938 bp的开放阅读框(GenBank登录号AJ852420); 在推导出的646个氨基酸残基的前体蛋白中, N端的前30个氨基酸残基为信号肽,随后的616个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的分子量为69 418 D。在一级结构中,形成催化活性中心的3个氨基酸残基(Ser242,Glu371和His485),以及在亚基内形成二硫键的6个半胱氨酸完全保守; 位于催化功能域的14个芳香族氨基酸中有10 个完全保守。该酶的氨基酸序列与黑尾叶蝉的同源性最高,达83%。对来自23种昆虫（包括褐飞虱）的30个乙酰胆碱酯酶的聚类分析显示,褐飞虱的乙酰胆碱酯酶与其中6个Ⅱ型乙酰胆碱酯酶（AChE2）同属一个支系; 此外,只存在于昆虫AChE2中的超变区及特异的氨基酸残基,也存在于褐飞虱的乙酰胆碱酯酶中。以上结果表明,所克隆的褐飞虱的乙酰胆碱酯酶基因是一个与黑腹果蝇的orthologous型基因同源的AChE2基因。     

Infected dendritic cells facilitate systemic dissemination and transplacental passage of the obligate intracellular parasite Neospora caninum in mice

The obligate intracellular parasite Neospora caninum disseminates across the placenta and the blood-brain barrier, to reach sites where it causes severe pathology or establishes chronic persistent infections. The mechanisms used by N. caninum to breach restrictive biological barriers remain elusive. To examine the cellular basis of these processes, migration of different N. caninum isolates (Nc-1, Nc-Liverpool, Nc-SweB1 and the Spanish isolates: Nc-Spain 3H, Nc-Spain 4H, Nc-Spain 6, Nc-Spain 7 and Nc-Spain 9) was studied in an in vitro model based on a placental trophoblast-derived BeWo cell line. Here, we describe that infection of dendritic cells (DC) by N. caninum tachyzoites potentiated translocation of parasites across polarized cellular monolayers. In addition, powered by the parasite's own gliding motility, extracellular N. caninum tachyzoites were able to transmigrate across cellular monolayers. Altogether, the presented data provides evidence of two putative complementary pathways utilized by N. caninum, in an isolate-specific fashion, for passage of restrictive cellular barriers. Interestingly, adoptive transfer of tachyzoite-infected DC in mice resulted in increased parasitic loads in various organs, e.g. the central nervous system, compared to infections with free parasites. Inoculation of pregnant mice with infected DC resulted in an accentuated vertical transmission to the offspring with increased parasitic loads and neonatal mortality. These findings reveal that N. caninum exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues. The findings are indicative of conserved dissemination strategies among coccidian apicomplexan parasites.     

Molecular cloning, expression, and characterization of the Cu,Zn superoxide dismutase (SOD1) gene from the entomopathogenic fungus Cordyceps militaris

We describe the molecular characterization of the Cu,Zn superoxide dismutase (SOD1) gene of Cordyceps militaris, which is one of the entomopathogenic fungi called a vegetable wasp and plant worm. The SOD1 gene of C. militaris spans 922 bp and consisted of three introns and four exons coding for 154 amino acid residues. The deduced amino acid sequence of the C. militaris SOD1 cDNA showed 88% identity to Claviceps purpurea SOD1, 82% to Neurospora crassa SOD1, and 75-64% to SOD1 sequences from other fungi. The C. militaris SOD1 possesses the typical metal binding ligands of six histidines and one aspartic acid common to fungal SOD1s. The cDNA encoding C. militaris SOD1 was expressed as a 17-kDa polypeptide in the baculovirus-infected insect Sf9 cells. The enzyme activity of the purified recombinant C. militaris SOD1 was approximately 568 U per mg(-1) . Southern blot analysis of the genomic DNA suggested the C. militaris SOD1 was a single gene. Northern and Western blot analysis and enzyme activity assays indicated SOD1 was expressed constitutively. This is the first report of an SOD1 gene from any entomopathogenic fungus.     

Functional expression of a recombinant copper/zinc superoxide dismutase of filarial nematode, Brugia malayi

A gene encoding a copper/zinc superoxide dismutase (Cu/ Zn-SOD) of a filarial nematode, Brugia malayi, has been isolated and the biochemical properties of a functionally expressed recombinant enzyme were investigated. The cloned complementary DNA contained a single open reading frame of 477 bp encoding 158 amino acids (aa), which conserved metal-binding residues as well as residues specific for Cu/Zn-SODs. Comparison of the deduced aa sequence of the enzyme with that of other helminthes species, including filarial worms, exhibited high degree of similarities (49-98%). Recombinant enzyme of 32 kDa had an isoelectric point of 6.6 and was shown to consist of 2 subunits linked by interchain disulfide bonds. Enzyme activity of the recombinant protein was inhibited by potassium cyanide and hydrogen peroxide but not by sodium azide. It showed a wide range of pH optima, i.e., 7.0-11.0 and was highly resistant to heat inactivation.     

Genetic characterization and expression of the novel fungal protease, EPg222 active in dry-cured meat products

EPg222 protease is a novel extracellular enzyme produced by Penicillium chrysogenum (Pg222) isolated from dry-cured hams that has the potential for use over a broad range of applications in industries that produce dry-cured meat products. The gene encoding EPg222 protease has been identified. Peptide sequences of EPg222 were obtained by de novo sequencing of tryptic peptides using mass spectrometry. The corresponding gene was amplified by PCR using degenerated primers based on a combination of conserved serine protease-encoding sequences and reverse translation of the peptide sequences. EPg222 is encoded as a gene of 1,361 bp interrupted by two introns. The deduced amino acid sequence indicated that the enzyme is synthesized as a preproenzyme with a putative signal sequence of 19 amino acids (aa), a prosequence of 96 aa and a mature protein of 283 aa. A cDNA encoding EPg222 has been cloned and expressed as a functionally active enzyme in Pichia pastoris. The recombinant enzyme exhibits similar activities to the native enzyme against a wide range of protein substrates including muscle myofibrillar protein. The mature sequence contains conserved aa residues characteristic of those forming the catalytic triad of serine proteases (Asp42, His76 and Ser228) but notably the food enzyme exhibits specific aa substitutions in the immunoglobulin-E recognition regions that have been identified in protein homologues that are allergenic.     

Direct evidence for cyanide-insensitive quinol oxidase (alternative oxidase) in apicomplexan parasite Cryptosporidium parvum: phylogenetic and therapeutic implications

Cryptosporidium parvum is a parasitic protozoan that causes the diarrheal disease cryptosporidiosis, for which no satisfactory chemotherapy is currently available. Although the presence of mitochondria in this parasite has been suggested, its respiratory system is poorly understood due to difficulties in performing biochemical analyses. In order to better understand the respiratory chain of C. parvum, we surveyed its genomic DNA database in GenBank and identified a partial sequence encoding cyanide-insensitive alternative oxidase (AOX). Based on this sequence, we cloned C. parvum AOX (CpAOX) cDNA from the phylum apicomplexa for the first time. The deduced amino acid sequence (335 a.a.) of CpAOX contains diiron coordination motifs (-E-, -EXXH-) that are conserved among AOXs. Phylogenetic analysis suggested that CpAOX is a mitochondrial-type AOX, possibly derived from mitochondrial endosymbiont gene transfer. The recombinant enzyme expressed in Escherichia coli showed quinol oxidase activity. This activity was insensitive to cyanide and highly sensitive to ascofuranone, a specific inhibitor of trypanosome AOX.     

Cloning and sequencing of the triacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichia coli

Aspergillus oryzae produces at least three extracellular lipolytic enzymes, L1, L2 and L3 (cutinase, mono- and diacylglycerol lipase, and triacylglycerol lipase, respectively). We cloned the triacylglycerol lipase gene (provisionally designated tglA) by screening a genomic library using a PCR product obtained with two degenerate oligonucleotide primers corresponding to amino acid sequences of L3 as probes. Nucleotide sequencing of the genomic DNA and cDNA revealed that the L3 gene (tglA) has an open reading frame comprising 954 nucleotides, which contains three introns of 47, 83 and 62 bp. The deduced amino acid sequence of the tglA gene corresponds to 254 amino acid residues including a signal sequence of 30 amino acids and, in spite of the difference in substrate specificity, it is homologous to those of cutinases from fungi. Three residues presumed to form the catalytic triad, Ser, Asp and His, are conserved. The cloned cDNA of the tglA gene was expressed in Escherichia coli, and enzyme assaying and zymography revealed that the cloned cDNA encodes a functional triacylglycerol lipase.     

Isolation and identification of an actin gene from Babesia gibsoni

Actin is a ubiquitous and highly conserved microfilament protein that is hypothesized to play a mechanical force-generating role in the unusual gliding motility of sporozoan zoites and their active penetration of host cells. We have identified and isolated an actin gene from a Babesia gibsoni cDNA library by random sequencing. The complete nucleotide sequence of the actin gene is 1,243 bp; a single open reading frame encodes a polypeptide of 377 amino acid residues. The deduced amino acid sequence showed a high homology with actins from other species, especially with reported apicomplexan protozoans. The antiserum against recombinant actin expressed in Escherichia coli recognizes a 42-kDa native protein, which is consistent with its expected size. Immunofluorescence and confocal microscopic observation revealed that the protein is diffusely distributed throughout the B. gibsoni parasites.