Description of a novel eukaryotic deoxyuridine 5′-triphosphate nucleotidohydrolase in Leishmania major

A Leishmaniamajor full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expression library by genetic complementation of dUTPase deficiency in Escherichiacoli. The cDNA contained an open reading frame that encoded a protein of 269 amino acid residues with a calculated molecular mass of 30.3 kDa. Although eukaryotic dUTPases exhibit extensive similarity and there are five amino acid motifs that are common to all currently known dUTPase enzymes, the sequence of the protozoan gene differs significantly from its eukaryotic counterparts. None of the characteristic motifs were readily identifiable and the sequence encoded a larger polypeptide. However, the products of the reaction were dUMP and PPi, competition experiments with other deoxyribonucleoside triphosphates showed that the reaction is specific for dUTP, and the protozoan gene complemented dUTPase deficiency in Escherichiacoli. The gene is of single copy; Northern blots indicated a transcript of the same size as the cDNA isolated in the screening procedure. The enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. The availability of recombinant enzyme in large quantities will now permit detailed mechanistic and structural studies, which might contribute to a rational design of specifically targeted inhibitors against dUTPase from L. major.

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A member of the HSP90 family from ovineBabesiain China: molecular characterization, phylogenetic analysis and antigenicity

Parasitology ◽

10.1017/s0031182015000797 ◽

2015 ◽

Vol 142 (11) ◽

pp. 1387-1397 ◽

Cited By ~ 5

Author(s):

GUIQUAN GUAN ◽

JUNLONG LIU ◽

AIHONG LIU ◽

YOUQUAN LI ◽

QINGLI NIU ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Amino Acid ◽

Structural Characteristics ◽

Heat Shock Protein 90 ◽

Cellular Transformation ◽

Amino Acid Sequences ◽

Enzyme Linked Immunosorbent Assay ◽

Cdna Expression Library ◽

Expression Library ◽

Reading Frame

SUMMARYHeat shock protein 90 (HSP90) is a key component of the molecular chaperone complex essential for activating many signalling proteins involved in the development and progression of pathogenic cellular transformation. AHsp90gene (BQHsp90) was cloned and characterized fromBabesiasp. BQ1 (Lintan), an ovineBabesiaisolate belonging toBabesia motasi-like group, by screening a cDNA expression library and performing rapid amplification of cDNA ends. The full-length cDNA ofBQHsp90is 2399 bp with an open reading frame of 2154 bp encoding a predicted 83 kDa polypeptide with 717 amino acid residues. It shows significant homology and similar structural characteristics toHsp90of other apicomplex organisms. Phylogenetic analysis, based on the HSP90 amino acid sequences, showed that theBabesiagenus is clearly separated from other apicomplexa genera. Five Chinese ovineBabesiaisolates were divided into 2 phylogenetic clusters, namelyBabesiasp. Xinjiang (previously designated a new species) cluster andB. motasi-like cluster which could be further divided into 2 subclusters (Babesiasp. BQ1 (Lintan)/Babesiasp. Tianzhu andBabesiasp. BQ1 (Ningxian)/Babesiasp. Hebei). Finally, the antigenicity of rBQHSP90 protein from prokaryotic expression was also evaluated using western blot and enzyme-linked immunosorbent assay (ELISA).

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Subunit VII of ubiquinol:cytochrome-c oxidoreductase from Neurospora crassa is functional in yeast and has an N-terminal extension that is not essential for mitochondrial targeting

Biochemical Journal ◽

10.1042/bj3200769 ◽

1996 ◽

Vol 320 (3) ◽

pp. 769-775 ◽

Cited By ~ 3

Author(s):

Gisele LOBO-HAJDU ◽

Hans-Peter BRAUN ◽

Nancy ROMP ◽

Leslie A. GRIVELL ◽

Jan A. BERDEN ◽

...

Keyword(s):

Amino Acid ◽

Neurospora Crassa ◽

Direct Sequencing ◽

Single Copy ◽

Cdna Clones ◽

Mitochondrial Targeting ◽

Calculated Molecular Mass ◽

Reading Frame ◽

Isolated Mitochondria ◽

Mitochondrial Intermediate Peptidase

cDNA clones encoding subunit VII of the Neurospora crassa bc1 complex (ubiquinol:cytochrome-c oxidoreductase), which is homologous with subunit VIII of the complex from yeast (encoded by QCR8), were identified on the basis of functional complementation of a yeast QCR8 deletion strain. The clones contain an open reading frame encoding a protein with a calculated molecular mass of 11.8 kDa. The N-terminal eight residues of the amino acid sequence deduced from the cDNA clones are absent from the mature protein, as revealed by direct sequencing of the isolated protein. To investigate the potential role of the N-terminal octapeptide in mitochondrial targeting, constructs were made encoding the precursor and the mature form of subunit VII from Neurospora. Incubation of isolated mitochondria with the two proteins revealed that the N-terminal extension of the precursor is removed on import. However, the presequence does not encode information for targeting, as the proteins encoded by both constructs can be imported into isolated mitochondria with equal efficiency. In contrast, the octapeptide seems to have functional importance: the defect in the yeast qcr8-null mutant is not complemented on transformation with the construct encoding mature subunit VII from N. crassa in a single-copy plasmid. We therefore speculate that the N-terminal extension plays a role in intramitochondrial sorting of N. crassa subunit VII. This is supported by the fact that the subunit VII precursor is processed by a protease other than the general mitochondrial processing peptidase. Interestingly, the presequence of N. crassa subunit VII has an amino acid composition similar to the octapeptides cleaved off by the mitochondrial intermediate peptidase.

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Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens.

Acta Biochimica Polonica ◽

10.18388/abp.2003_3736 ◽

2003 ◽

Vol 50 (1) ◽

pp. 269-278

Author(s):

Amr M Shabaan ◽

Magdy M Mohamed ◽

Mohga S Abdallah ◽

Hayat M Ibrahim ◽

Amr M Karim

Keyword(s):

Amino Acid ◽

Fusion Protein ◽

Schistosoma Mansoni ◽

Molecular Mass ◽

Vaccine Development ◽

Complete Sequence ◽

Cdna Clones ◽

Western Blots ◽

Calculated Molecular Mass ◽

Reading Frame

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.

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Proteophosphoglycans of Leishmania mexicana: Molecular cloning and characterization of the Leishmania mexicana ppg2 gene encoding the proteophosphoglycans aPPG and pPPG2 that are secreted by amastigotes and promastigotes

Biochemical Journal ◽

10.1042/bj3440787 ◽

1999 ◽

Vol 344 (3) ◽

pp. 787-795 ◽

Cited By ~ 9

Author(s):

Ulrich GÖPFERT ◽

Nathan GOEHRING ◽

Christian KLEIN ◽

Thomas ILG

Keyword(s):

Mrna Level ◽

Single Copy ◽

Gene Replacement ◽

Mammalian Host ◽

Cdna Expression Library ◽

Dna Repeats ◽

The Novel ◽

Leishmania Mexicana ◽

Expression Library ◽

Gene Encoding

Intracellular amastigotes of the pathogenic protozoon Leishmania mexicana secrete an extensively phosphoglycosylated proteophosphoglycan (aPPG) into the phagolysosome of mammalian host macrophages, that appears to fulfil important functions for the parasites. Promastigotes (the sandfly vector forms) of the same species secrete a proteophosphoglycan with identical protein backbone but exhibiting stage-specific phosphoglycosylation patterns [Klein, Göpfert, Goehring, Stierhof and Ilg (1999) Biochem. J. 344, 775-786]. In this study we report the cloning of the novel repeat-containing proteophosphoglycan gene ppg2 by antibody screening of a Leishmania mexicana amastigote cDNA expression library. ppg2 is equally expressed in promastigotes and amastigotes at the mRNA level. Targeted gene replacement of both alleles of the single copy gene ppg2 results in the loss of pPPG2 expression in promastigotes. Antisera against Escherichia coli-expressedppg2 recognize the deglycosylated forms of aPPG as well as pPPG2. These results confirm that ppg2 encodes the protein backbones of aPPG and pPPG2. An unusual finding is that ppg2 exhibits two stable allelic forms, ppg2a and ppg2b. Their main difference lies in the number of central 72 bp DNA repeats (7 versus 8). ppg2a and ppg2b encode polypeptide chains of 574 and 598 amino acids, respectively, that show no homology to known proteins. The novel 24 amino acid Ser-rich peptide repeats encoded by the 72 bp DNA repeats are targets for Ser phosphoglycosylation in Leishmania mexicana.

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Structural features of the low-molecular-mass human salivary mucin

Biochemical Journal ◽

10.1042/bj2870639 ◽

1992 ◽

Vol 287 (2) ◽

pp. 639-643 ◽

Cited By ~ 27

Author(s):

M S Reddy ◽

L A Bobek ◽

G G Haraszthy ◽

A R Biesbrock ◽

M J Levine

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Molecular Mass ◽

Gel Filtration ◽

Structural Features ◽

Cdna Expression Library ◽

Cdna Clones ◽

Expression Library ◽

Mrna Species ◽

Salivary Mucin

The low-molecular-mass human salivary mucin has at least two isoforms, MG2a and MG2b, that differ primarily in their sialic acid and fucose content. In this study, we characterize further these isoforms, particularly their peptide moieties. Trypsin digests of MG2a and MG2b yielded high- and low-molecular-mass glycopeptides following gel filtration on Sephacryl S-300. The larger glycopeptides from MG2a and MG2b had similar amino acid compositions and identical N-terminal sequences, suggesting common structural features between their peptides. An oligonucleotide probe generated from the amino acid sequence of the smaller glycopeptide from MG2a was employed in Northern-blot analysis. This probe specifically hybridized to two mRNA species from human submandibular and sublingual glands. A cDNA clone selected from a human submandibular gland cDNA expression library with antibody generated against deglycosylated MG2a also hybridized to these two mRNA species. In both cases, the larger mRNA was polydisperse, and the hybridization signal was more intense in the sublingual gland. In addition, the N-terminal amino acid sequence of the larger glycopeptide was found to be part of one of the selected MG2 cDNA clones.

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NuMA: an unusually long coiled-coil related protein in the mammalian nucleus.

The Journal of Cell Biology ◽

10.1083/jcb.116.6.1303 ◽

1992 ◽

Vol 116 (6) ◽

pp. 1303-1317 ◽

Cited By ~ 132

Author(s):

C H Yang ◽

E J Lambie ◽

M Snyder

Keyword(s):

Mammalian Cells ◽

Coiled Coil ◽

Nuclear Lamina ◽

Early Prophase ◽

Cdna Expression Library ◽

Cdna Clones ◽

Expression Library ◽

Mitotic Apparatus ◽

Reading Frame ◽

Nuclear Lamins

A bank of 892 autoimmune sera was screened by indirect immunofluorescence on mammalian cells. Six sera were identified that recognize an antigen(s) with a cell cycle-dependent localization pattern. In interphase cells, the antibodies stained the nucleus and in mitotic cells the spindle apparatus was recognized. Immunological criteria indicate that the antigen recognized by at least one of these sera corresponds to a previously identified protein called the nuclear mitotic apparatus protein (NuMA). A cDNA which partially encodes NuMA was cloned from a lambda gt11 human placental cDNA expression library, and overlapping cDNA clones that encode the entire gene were isolated. DNA sequence analysis of the clones has identified a long open reading frame capable of encoding a protein of 238 kD. Analysis of the predicted protein sequence suggests that NuMA contains an unusually large central alpha-helical domain of 1,485 amino acids flanked by nonhelical terminal domains. The central domain is similar to coiled-coil regions in structural proteins such as myosin heavy chains, cytokeratins, and nuclear lamins which are capable of forming filaments. Double immunofluorescence experiments performed with anti-NuMA and antilamin antibodies indicate that NuMA dissociates from condensing chromosomes during early prophase, before the complete disintegration of the nuclear lamina. As mitosis progresses, NuMA reassociates with telophase chromosomes very early during nuclear reformation, before substantial accumulation of lamins on chromosomal surfaces is evident. These results indicate that the NuMA proteins may be a structural component of the nucleus and may be involved in the early steps of nuclear reformation during telophase.

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A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi

Biochemical Journal ◽

10.1042/bj3240619 ◽

1997 ◽

Vol 324 (2) ◽

pp. 619-626 ◽

Cited By ~ 14

Author(s):

Javier PEÑA-DÍAZ ◽

Andrea MONTALVETTI ◽

Ana CAMACHO ◽

Claribel GALLEGO ◽

Luis M. RUIZ-PEREZ ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Reductase Activity ◽

Active Form ◽

Single Copy ◽

Enzymic Activity ◽

Highly Active ◽

Frame Sequence ◽

Isolation And Characterization ◽

Cell Extracts ◽

Coa Reductase

We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5′ flanking region by cDNA analysis and establishment of the splice acceptor sites clearly indicated that the corresponding mRNA apparently lacks sequences encoding a membrane N-terminal domain. The reductase gene is a single copy and is located on a chromosome of 1.36 Mb as determined by contour-clamped homogeneous electric field electrophoresis. The overall cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly sensitive to proteolytic inactivation. Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. Thus Trypanosoma cruziHMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG-CoA reductases so far characterized.

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Cloning and Expression of a Novel Protease with Fibrinolytic Activity from Arenicola cristata

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.998-999.210 ◽

2014 ◽

Vol 998-999 ◽

pp. 210-213

Author(s):

Chun Ling Zhao ◽

Wen Jing Yu ◽

Ji Yu Ju

Keyword(s):

Amino Acid ◽

Recombinant Protein ◽

Active Form ◽

Cloning And Expression ◽

Amino Acid Residues ◽

Gene Encoding ◽

Reading Frame ◽

Arenicola Cristata ◽

Fibrin Plate ◽

Serine Protease Family

cDNA of a novel protease, designated as AFEI, was cloned from digestive tract of Arenicola cristata by RACE. The cDNA of AFEIcomprised 897bp and an open reading frame that encoded polypeptides of 264 amino acid residues. AFEIshowed similarity to serine protease family and contained the conserved catalytic amino acid residues. The gene encoding the active form of AFEIwas expressed in E.coli and the purified recombinant protein could dissolve an artificial fibrin plate with plasminogen, which indicated the recombinant protein might be a plasminogen activator for thrombosis therapy.

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Identification and Molecular Characterization of a Gene Encoding a Protective Leishmania amazonensis Trp-Asp (WD) Protein

Infection and Immunity ◽

10.1128/iai.72.4.2194-2202.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 2194-2202 ◽

Cited By ~ 3

Author(s):

Kimberly Campbell ◽

Vsevolod Popov ◽

Lynn Soong

Keyword(s):

Protein Interactions ◽

Protein Complexes ◽

Single Copy ◽

Interleukin 10 ◽

Leishmania Amazonensis ◽

Cdna Expression Library ◽

Protein Protein Interactions ◽

Expression Library ◽

Gene Encoding ◽

Mucocutaneous Leishmaniasis

ABSTRACT Several Leishmania proteins have been identified and characterized in pursuit of understanding pathogenesis and protection in cutaneous leishmaniasis. In the present study, we utilized sera from infected BALB/c mice to screen a Leishmania amazonensis amastigote cDNA expression library and obtained the full-length gene that encodes a novel Trp-Asp (WD) protein designated LAWD (for Leishmania antigenic WD protein). The WD family of proteins mediates protein-protein interactions and coordinates the formation of protein complexes. The single-copy LAWD gene is transcribed as a ∼3.1-kb mRNA in both promastigotes and amastigotes, with homologues being detected in several other Leishmania species. Immunoelectron microscopy revealed a predominant localization of the LAWD protein in the flagellar pocket. Analyses of sera from human patients with cutaneous and mucocutaneous leishmaniasis indicated that these individuals mounted significant humoral responses against LAWD. Given that recombinant LAWD protein elicited the production of high levels of gamma interferon, but no detectable levels of interleukin-10 (IL-10), in CD4+ cells of L. amazonensis-infected mice, we further examined whether LAWD could elicit protective immunity. DNA vaccination with the LAWD and IL-12 genes significantly delayed lesion development, which correlated with a dramatic reduction in parasite burdens. Thus, we have successfully identified a promising vaccine candidate and antigenic vehicle to aid in the dissection of the complicated pathogenic immune response of L. amazonensis.

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Human and Murine Immune Responses to a NovelLeishmania major Recombinant Protein Encoded by Members of a Multicopy Gene Family

Infection and Immunity ◽

10.1128/iai.66.7.3279-3289.1998 ◽

1998 ◽

Vol 66 (7) ◽

pp. 3279-3289 ◽

Cited By ~ 112

Author(s):

John R. Webb ◽

Antonio Campos-Neto ◽

Pamela J. Ovendale ◽

Tricia I. Martin ◽

Erika J. Stromberg ◽

...

Keyword(s):

Immune Responses ◽

Leishmania Major ◽

Mononuclear Cells ◽

Blot Hybridization ◽

Interleukin 12 ◽

Predicted Amino Acid Sequence ◽

Cdna Expression Library ◽

Expression Library ◽

Peripheral Blood Mononuclear ◽

Amino Terminal

ABSTRACT Vaccination of BALB/c mice with Leishmania majorpromastigote culture filtrate proteins plus Corynebacterium parvum confers resistance to infection with L. major. To define immunogenic components of this protein mixture, we used sera from vaccinated mice to screen an L. major amastigote cDNA expression library. One of the immunoreactive clones thus obtained encoded a novel protein of L. major with a molecular mass of 22.1 kDa. The predicted amino acid sequence of this clone exhibited significant homology to eukaryotic thiol-specific-antioxidant (TSA) proteins. Therefore, we have designated this protein L. major TSA protein. Southern blot hybridization analyses indicate that there are multiple copies of the TSA gene in all species ofLeishmania analyzed. Northern blot analyses demonstrated that the TSA gene is constitutively expressed in L. majorpromastigotes and amastigotes. Recombinant TSA protein containing an amino-terminal six-histidine tag was expressed in Escherichia coli with the pET17b system and was purified to homogeneity by affinity chromatography. Immunization of BALB/c mice with recombinant TSA protein resulted in the development of strong cellular immune responses and conferred protective immune responses against infection with L. major when the protein was combined with interleukin 12. In addition, recombinant TSA protein elicited in vitro proliferative responses from peripheral blood mononuclear cells of human leishmaniasis patients and significant TSA protein-specific antibody titers were detected in sera of both cutaneous-leishmaniasis and visceral-leishmaniasis patients. Together, these data suggest that the TSA protein may be useful as a component of a subunit vaccine against leishmaniasis.

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