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Pascal Boireau - One of the best experts on this subject based on the ideXlab platform.
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immunoproteomic analysis of the excretory secretory products of trichinella pseudospiralis adult worms and newborn larvae
Parasites & Vectors, 2017Co-Authors: Yang Wang, Xue Lin Wang, Pascal Boireau, Xue Bai, Haichao Zhu, Haining Shi, Bin Tang, Xuepeng Cai, Xuenong LuoAbstract:The nematode Trichinella pseudospiralis is an intracellular parasite of mammalian skeletal muscle cells and exists in a non-encapsulated form. Previous studies demonstrated that T. pseudospiralis could induce a lower host inflammatory response. Excretory-secretory (ES) proteins as the most important products of host-parasite interaction may play the main functional role in alleviating host inflammation. However, the ES products of T. pseudospiralis early stage are still unknown. The identification of the ES products of the early stage facilitates the understanding of the molecular mechanisms of the immunomodulation and may help finding early diagnostic markers. In this study, we used two-dimensional gel electrophoresis (2-DE)-based western blotting coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS/MS) to separate and identify the T. pseudospiralis adult worms ES products immunoreaction-positive proteins. In total, 400 protein spots were separated by 2-DE. Twenty-eight protein spots were successfully identified using the sera from infected pigs and were characterized to correlate with 12 different proteins of T. pseudospiralis, including adult-specific DNase II-10, poly-cysteine and histidine-tailed protein isoform 2, serine protease, serine/threonine-protein kinase ULK3, enolase, putative venom allergen 5, chymotrypsin-like elastase family member 1, uncharacterized protein, peptidase inhibitor 16, death-associated protein 1, Deoxyribonuclease II superfamily and golgin-45. Bioinformatic analyses showed that the identified proteins have a wide diversity of molecular functions, especially Deoxyribonuclease II (DNase II) activity and serine-type endopeptidase activity. Early candidate antigens from the ES proteins of T. pseudospiralis have been screened and identified. Our results suggest these proteins may play key roles during the T. pseudospiralis infection and suppress the host immune response. Further, they are the most likely antigen for early diagnosis and the development of a vaccine against the parasite.
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Immunoproteomic analysis of the excretory-secretory products of Trichinella pseudospiralis adult worms and newborn larvae
Parasites and Vectors, 2017Co-Authors: Yang Wang, Xue Lin Wang, Pascal Boireau, Xue Bai, Haichao Zhu, Haining Shi, Bin Tang, Xuepeng Cai, Xuenong Luo, Ming Yuan LiuAbstract:Background: The nematode Trichinella pseudospiralis is an intracellular parasite of mammalian skeletal muscle cells and exists in a non-encapsulated form. Previous studies demonstrated that T. pseudospiralis could induce a lower host inflammatory response. Excretory-secretory (ES) proteins as the most important products of host-parasite interaction may play the main functional role in alleviating host inflammation. However, the ES products of T. pseudospiralis early stage are still unknown. The identification of the ES products of the early stage facilitates the understanding of the molecular mechanisms of the immunomodulation and may help finding early diagnostic markers. Results: In this study, we used two-dimensional gel electrophoresis (2-DE)-based western blotting coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS/MS) to separate and identify the T. pseudospiralis adult worms ES products immunoreaction-positive proteins. In total, 400 protein spots were separated by 2-DE. Twenty-eight protein spots were successfully identified using the sera from infected pigs and were characterized to correlate with 12 different proteins of T. pseudospiralis, including adult-specific DNase II-10,poly-cysteine and histidine-tailed protein isoform 2, serine protease, serine/threonine-protein kinase ULK3, enolase, putative venom allergen 5, chymotrypsin-like elastase family member 1, uncharacterized protein, peptidase inhibitor 16, death-associated protein 1, Deoxyribonuclease II superfamily and golgin-45. Bioinformatic analyses showed that the identified proteins have a wide diversity of molecular functions, especially Deoxyribonuclease II (DNase II) activity and serine-type endopeptidase activity. Conclusions: Early candidate antigens from the ES proteins of T. pseudospiralis have been screened and identified. Our results suggest these proteins may play key roles during the T. pseudospiralis infection and suppress the host immune response. Further, they are the most likely antigen for early diagnosis and the development of a vaccine against the parasite.
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The functions of Deoxyribonuclease II in immunity and development
DNA and Cell Biology, 2008Co-Authors: Ma Feng Liu, Xue Lin Wang, Wei Fang Wang, Qi Jun Chen, Pascal Boireau, Ming Yuan LiuAbstract:Apoptosis, which is usually accompanied by DNA degradation, is important not only for the homeostasis of metazoans but also for mammalian development. If DNA is not properly degraded in these processes, it can cause diverse diseases, such as anemia, cataracts, and some autoimmune diseases. A large effort has been made to identify these nucleases that are responsible for these effects. In contrast to Deoxyribonuclease I (DNase I), Deoxyribonuclease II (DNase II) has been less well characterized in these processes. Additionally, enzymes of DNase II family in Trichinella spiralis, which is an intracellular parasitic nematode, are also considered involved in the development of the nematode. We have compiled information from studies on DNase II from various organisms and found some nonclassic features in these enzymes of T. spiralis. Here we have reviewed the characterization and functions of DNase II in these processes and predicted the functions of these enzymes in T. spiralis during host invasion and development.
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Identification of stage-specifically expressed genes of Trichinella spiralis by suppression subtractive hybridization
Parasitology, 2007Co-Authors: M.y. Liu, X.l. Wang, Danielle Le Rhun, Q.j. Chen, Pascal BoireauAbstract:Newborn larvae (NBL) and adult (Ad) stage-specifically expressed genes or members of gene families of Trichinella spiralis were identified by suppression subtractive hybridization (SSH). Six cDNA clones were identified as NBL stage-specific, including 1 member of the T. spiralis gene family encoding glutamic acid-rich proteins, 2 clones encoding novel serine proteases, 2 closely related clones encoding proteins that are members of a Deoxyribonuclease II (DNase II)-like family and 1 clone with no similarity to known genes. Four stage-specific clones encoding homologues of retinoid X receptor, caveolin, C2H2 type zinc finger protein and a putative protein with no homology to known sequences were obtained from 3-day-old adult worms. One gene specifically up-regulated in the 5-day-old adult worms encoding a putative cuticle collagen was also identified.
Alan Eastman - One of the best experts on this subject based on the ideXlab platform.
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Deoxyribonuclease II is a lysosomal barrier to transfection
Molecular Therapy, 2003Co-Authors: Dasein Pintogonzalez Howell, Alan Eastman, Ronald J Krieser, Michael A. BarryAbstract:DNA delivered in nonviral vectors or as naked DNA must overcome a number of extracellular and intracellular barriers to transfection. Since many vectors deliver DNA into cells by the endocytic route, DNA degradation by lysosomal nucleases has been proposed as a significant barrier to transfection, despite the fact that this has not yet been formally demonstrated to occur. To test this hypothesis, we have investigated the role of Deoxyribonuclease II (DNase II), the primary acidic endonuclease active in the lysosome, in transfection. Two genetic systems were engineered in which mammalian cells either overexpressed DNase II or were knocked out for the enzyme. In both models, higher levels of DNase II correlated with decreased transfection efficiency by nonviral DNA delivery vectors. These data provide direct evidence implicating lysosomal DNase II as a barrier to transfection.
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A family history of Deoxyribonuclease II: surprises from Trichinella spiralis and Burkholderia pseudomallei.
Gene, 2003Co-Authors: Kyle S. Maclea, Ronald J Krieser, Alan EastmanAbstract:Deoxyribonuclease IIα (DNase IIα) is an acidic endonuclease found in lysosomes and nuclei, and it is also secreted. Though its Caenorhabditis elegans homolog, NUC-1, is required for digesting DNA of apoptotic cell corpses and dietary DNA, it is not required for viability. However, DNase IIα is required in mice for correct development and viability, because undigested cell corpses lead to lesions throughout the body. Recently, we showed that, in contrast to previous reports, active DNase IIα consists of one contiguous polypeptide. To better analyze DNase II protein structure and determine residues important for activity, extensive database searches were conducted to find distantly related family members. We report 29 new partial or complete homologs from 21 species. Four homologs with differences at the purported active site histidine residue were detected in the parasitic nematodes Trichinella spiralis and Trichinella pseudospiralis. When these mutations were reconstructed in human DNase IIα, the expressed proteins were inactive. DNase II homologs were also identified in non-metazoan species. In particular, the slime-mold Dictyostelium, the protozoan Trichomonas vaginalis, and the bacterium Burkholderia pseudomallei all contain sequences with significant similarity and identity to previously cloned DNase II family members. We report an analysis of their sequences and implications for DNase II protein structure and evolution.
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Cleavage and nuclear translocation of the caspase 3 substrate Rho GDP-dissociation inhibitor, D4-GDI, during apoptosis
Cell Death & Differentiation, 1999Co-Authors: Ronald J Krieser, Alan EastmanAbstract:While investigating endonucleases potentially involved in apoptosis, an antisera was raised to bovine Deoxyribonuclease II, but it recognized a smaller protein of 26 kDa protein in a variety of cell lines. The 26 kDa protein underwent proteolytic cleavage to 22 kDa concomitantly with DNA digestion in cells induced to undergo apoptosis. Sequencing of the 26 kDa protein identified it as the Rho GDP-dissociation inhibitor D4-GDI. Zinc, okadaic acid, calyculin A, cantharidin, and the caspase inhibitor z-VAD-fmk, all prevented the cleavage of D4-GDI, DNA digestion, and apoptosis. The 26 kDa protein resided in the cytoplasm of undamaged cells, whereas following cleavage, the 22 kDa form translocated to the nucleus. Human D4-GDI, and D4-GDI mutated at the caspase 1 or caspase 3 sites, were expressed in Chinese hamster ovary cells which show no detectable endogenous D4-GDI. Mutation at the caspase 3 site prevented D4-GDI cleavage but did not inhibit apoptosis induced by staurosporine. The cleavage of D4-GDI could lead to activation of Jun N-terminal kinase which has been implicated as an upstream regulator of apoptosis in some systems. However, the results show that the cleavage of D4-GDI and translocation to the nucleus do not impact on the demise of the cell.
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the cloning and expression of human Deoxyribonuclease II a possible role in apoptosis
Journal of Biological Chemistry, 1998Co-Authors: Ronald J Krieser, Alan EastmanAbstract:We have previously implicated Deoxyribonuclease II (DNase II) as an endonuclease responsible for DNA digestion during apoptosis. The full-length human cDNA has now been cloned. The cDNA contains an open reading frame of 1078 bases coding for a 40-kDa protein. This protein is 10 kDa larger than commercially supplied enzyme, which has been proteolytically cleaved at an internal aspartate residue. The gene is located at chromosome 19p13.2, and has no significant homology to other human proteins, but has >30% identity to three predicted genes in Caenorhabditis elegans. To determine whether overexpression of DNase II induces apoptosis in Chinese hamster ovary cells, the cDNA was cotransfected with a plasmid encoding green fluorescent protein. Within 24 h, a significant proportion of green fluorescent protein-positive cells contained condensed chromatin, whereas vector-only controls remained viable. Considering that DNase II is normally active only at low pH, it was surprising that transfection induced chromatin condensation. To confirm that transfection was not activating another endonuclease, cells were incubated with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-fluoromethylketone; this failed to inhibit chromatin condensation induced by DNase II. These results demonstrate that DNase II acts downstream of caspase activation and that it may be activated by an as yet unknown mechanism to induce DNA digestion during apoptosis.
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Intracellular acidification is associated with, but not required for caspase activation, DNA fragmentation or apoptosis.
International journal of oncology, 1997Co-Authors: Jason E. Reynolds, Chad M. Wolf, Alan EastmanAbstract:Apoptosis is characterized by DNA digestion mediated by either a Ca2+/Mg2+-dependent endonuclease or the acid-activated Deoxyribonuclease II (DNase II). However, DNA digestion frequently does not correlate with changes in Ca2+ whereas intracellular acidification is a consistent marker of apoptosis. To confirm the role of low pH in regulating DNA digestion, ML-I cells were damaged with etoposide then incubated at various extracellular pH (pH,). When pH, was 8.1, DNA digestion still occurred, and intracellular pH still decreased but only to 7.2, a pH at which DNase LT is inactive. In contrast, low pH, inhibited the DNA digestion and apoptosis induced by etoposide. An upstream event in apoptosis is the activation of proteases known as caspases. The activity of caspases was inhibited at low pH, demonstrating that the pH-sensitive step is upstream of caspase action. Similar results have been obtained in other models of apoptosis. Hence, both DNase II and Ca2+/Mg2+-dependent endonuclease appear unlikely to cause DNA digestion in apoptosis, unless their ion dependence is modified by, for example, proteolytic cleavage.
Koichiro Kishi - One of the best experts on this subject based on the ideXlab platform.
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Production of murine monoclonal anti-human DNase II antibodies, and their use for immunoaffinity purification of DNase II from human liver and urine
Legal Medicine, 2003Co-Authors: Tamiko Nakajima, Toshihiro Yasuda, Haruo Takeshita, Kouichi Mogi, Yasushi Kaneko, Koichiro KishiAbstract:Abstract Murine monoclonal anti-human Deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion and DNA-cast polyacrylamide gel electrophoresis were very useful screening methods for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.
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Production and characterization of murine monoclonal anti-human DNase II antibodies, and their use for immunoaffinity purification of DNase II from human liver and urine
Biochimica et biophysica acta, 2002Co-Authors: Tamiko Nakajima, Toshihiro Yasuda, Haruo Takeshita, Shinjiro Mori, Emiko Nakazato, Kouichi Mogi, Yasushi Kaneko, Koichiro KishiAbstract:Four murine monoclonal anti-human Deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion (SRED) and DNA-cast polyacrylamide gel electrophoresis (DNA-cast PAGE) were very useful for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining of the 32-kDa enzyme band, which is one of the three non-identical subunits of human DNase II molecule separated by sodium dodecyl sulfate (SDS)-PAGE followed by blotting on a transfer membrane. A formyl-cellulofine resin conjugated with each antibody specifically adsorbed and efficiently desorbed the active DNase II enzyme. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.
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The molecular basis for genetic polymorphism of human Deoxyribonuclease II (DNase II): a single nucleotide substitution in the promoter region of human DNase II changes the promoter activity.
FEBS Letters, 2000Co-Authors: Toshihiro Yasuda, Haruo Takeshita, Tamiko Nakajima, Yoshimitsu Nakashima, Shinjiro Mori, Emiko Nakazato, Kouichi Mogi, Koichiro KishiAbstract:Deoxyribonuclease II (DNase II) levels in human vary depending on whether the individual has the DNASE2*H (high) allele or the DNASE2*L (low) allele. We examined the promoter activity of the 5'-flanking region of each of these alleles by transient transfection luciferase assay. DNASE2*H had 5-fold higher promoter activity than DNASE2*L in human hepatoma HepG2 cell. Comparison of the nucleotide sequences of the proximal promoter regions revealed a G to A transition at position -75; G and A residues were assigned to DNASE2*H and *L, respectively. Since no differences were found between the open reading frame sequences of these alleles, it is likely that the A-75G transition causes the allelic difference in the promoter activity of the gene, underlying the genetic polymorphism.
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Structural requirements of a human Deoxyribonuclease II for the development of the active enzyme form, revealed by site-directed mutagenesis.
Biochemical and biophysical research communications, 1999Co-Authors: Toshihiro Yasuda, Haruo Takeshita, Reiko Iida, Tamiko Nakajima, Osamu Hosomi, Yoshimitsu Nakashima, Shinjiro Mori, Koichiro KishiAbstract:Abstract Using site-directed mutagenesis, we eliminated three potential N-glycosylation sites (N 86 , N 212 , and N 266 ) of human Deoxyribonuclease II (DNase II), conserved in mammalian enzymes, and a proteolytic processing site (Q 46 –R 47 ), forming a propeptide subunit of the enzyme. We expressed a series of these mutant DNase II constructs in COS-7 and Hep G2 cells. Liberation of each glycosylation site at N 86 and N 266 and the cleavage site interfered dramatically with expression of the intracellular and secreted DNase II activities, irrespective of cell line transfected. A chimeric mutant in which the signal peptide of the DNase II was replaced with that of human DNase I had no intracellular or secreted enzyme activity. Therefore, a simultaneous attachment of a carbohydrate moiety to N 86 and N 266 , cleavage of the propeptide from the single DNase II precursor, and the inherent signal peptide might be required for subcellular sorting and proteolytic maturation of the enzyme.
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Chromosomal localization of a human Deoxyribonuclease II gene (DNASE2) to 19p13.2-p13.1 using both the polymerase chain reaction and fluorescence in situ hybridization analysis.
Biochemical and biophysical research communications, 1998Co-Authors: Toshihiro Yasuda, Haruo Takeshita, Reiko Iida, Tamiko Nakajima, Osamu Hosomi, Yoshimitsu Nakashima, Koichi Mogi, Koichiro KishiAbstract:Recently obtained information on the cDNA encoding human Deoxyribonuclease II (DNase II) (T. Yasuda et al., 1998, J. Biol. Chem. 273, 2610-2616) has made it possible to demonstrate the precise position of the the human DNase II gene (DNASE2) on human chromosomes. Two different sets of oligonucleotide primers specific for human DNase II cDNA sequences were used to amplify unique DNA fragments in the human DNase II gene from a panel of human x rodent hybrid cell lines carrying different human chromosomes. Based on this analysis, DNASE2 was assigned to human chromosome 19. Furthermore, regional localization of the gene to 19p13.2-p13.1 was achieved by fluorescence in situ hybridization analysis using a full-length cDNA probe corresponding to the entire open reading frame.
Hui-ling Chen - One of the best experts on this subject based on the ideXlab platform.
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sp1 and sp3 are involved in up regulation of human Deoxyribonuclease II transcription during differentiation of hl 60 cells
FEBS Journal, 2003Co-Authors: San-fang Chou, Hui-ling ChenAbstract:Expression of DNase II in macrophages is potentially crucially important in the removal of unwanted DNA. We have previously shown that DNase II expression is up-regulated at the transcriptional level during the phorbol 12-myristate-13-acetate (PMA)-induced differentiation of HL-60 and THP-1 cells. In this study, we investigated the cis-regulatory elements and transcription factors involved in this process in HL-60 cells. cis-Regulatory elements in the DNase II promoter were located by 5′ deletion and site-directed mutagenesis of promoter-luciferase constructs and transient transfection of HL-60 cells. Furthermore, the binding proteins were identified by electrophoretic mobility shift assay (EMSA) in the presence of specific antibodies. In the DNase II promoter, 249 base pairs upstream of the transcription start site were essential for maximal promoter activity in both untreated and PMA-treated HL-60 cells and, within this region, three Sp1 and Sp3 binding sites were identified as essential for transcriptional regulation and PMA induction. Western blot analysis showed that PMA treatment resulted in increased levels of Sp1 and Sp3 proteins. Furthermore, cotransfection analysis in Drosophila SL2 cells showed that Sp1 was more potent than Sp3 in activating the DNase II promoter. We therefore conclude that Sp1 and/or Sp3 are involved in the up-regulation of DNase II expression during the differentiation of HL-60 cells.
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Sp1 and Sp3 are involved in up‐regulation of human Deoxyribonuclease II transcription during differentiation of HL‐60 cells
European journal of biochemistry, 2003Co-Authors: San-fang Chou, Hui-ling ChenAbstract:Expression of DNase II in macrophages is potentially crucially important in the removal of unwanted DNA. We have previously shown that DNase II expression is up-regulated at the transcriptional level during the phorbol 12-myristate-13-acetate (PMA)-induced differentiation of HL-60 and THP-1 cells. In this study, we investigated the cis-regulatory elements and transcription factors involved in this process in HL-60 cells. cis-Regulatory elements in the DNase II promoter were located by 5′ deletion and site-directed mutagenesis of promoter-luciferase constructs and transient transfection of HL-60 cells. Furthermore, the binding proteins were identified by electrophoretic mobility shift assay (EMSA) in the presence of specific antibodies. In the DNase II promoter, 249 base pairs upstream of the transcription start site were essential for maximal promoter activity in both untreated and PMA-treated HL-60 cells and, within this region, three Sp1 and Sp3 binding sites were identified as essential for transcriptional regulation and PMA induction. Western blot analysis showed that PMA treatment resulted in increased levels of Sp1 and Sp3 proteins. Furthermore, cotransfection analysis in Drosophila SL2 cells showed that Sp1 was more potent than Sp3 in activating the DNase II promoter. We therefore conclude that Sp1 and/or Sp3 are involved in the up-regulation of DNase II expression during the differentiation of HL-60 cells.
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Up-regulation of human Deoxyribonuclease II gene expression during myelomonocytic differentiation of HL-60 and THP-1 cells.
Biochemical and biophysical research communications, 2002Co-Authors: San Fang Chou, Hui-ling ChenAbstract:Several recent studies have suggested that intracellular Deoxyribonuclease II (DNase II) is responsible for the degradation of DNA from apoptotic cells that are engulfed by macrophages. In this study, we studied DNase II expression during the phorbol 12-myristate-13-acetate (PMA)-induced differentiation of HL-60 and THP-1 cells. Basal levels of DNase II mRNA and protein were low, with expression being up-regulated approximately 15- and 7-fold, respectively, in HL-60 and THP-1 cells 72 h after PMA treatment. Nuclear run-on and luciferase reporter assays showed that transcription of DNase II gene was increased in PMA-treated cells. Together, these results demonstrate that DNase II gene transcription is increased during myelomonocytic differentiation, resulting in increased levels of mRNA and protein. This increase in DNase II levels in differentiated HL-60 and THP-1 cells suggests that it may play an important role in macrophages.
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porcine spleen Deoxyribonuclease II covalent structure cdna sequence molecular cloning and gene expression
Journal of Biological Chemistry, 1998Co-Authors: Chengching Wang, Hui-ling Chen, Ta-hsiu LiaoAbstract:Abstract Porcine spleen DNase II, a lysosomal acid hydrolase, is a noncovalently linked α·β heterodimer (Liao, T.-H. (1985) J. Biol. Chem. 260, 10708–10713). The α subunit, after disulfide cleavage, yields two chains, α1 and α2. The complete amino acid sequences of the α1, β, and α2 chains were elucidated by protein sequencing, and the pairings of one interchain disulfide between α1 and α2 and of three intrachain disulfides in α2 were assigned. Six carbohydrate attachment sites, two in β and four in α2, were detected by sugar analyses. The cDNA of DNase II was amplified using primers synthesized on the basis of the amino acid sequences determined. The amplified fragments shown to be a cDNA sequence of 1,292 bases. This cDNA sequence has an open reading frame encoding a 364-amino acid polypeptide containing a putative transmembrane peptide at the NH2-end, two small connecting peptides in the middle, and a peptide at the COOH terminus. These are evidently removed to form mature DNase II. Thus, all three chains in the sequence α1, β, and α2 are coded by the same cDNA. When Chinese hamster ovary cells were transfected with a cloned plasmid with an inserted cDNA fragment encoding the entire reading frame, the expressed protein was released into the growth medium as an active form of DNase II.
Ming Yuan Liu - One of the best experts on this subject based on the ideXlab platform.
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Immunoproteomic analysis of the excretory-secretory products of Trichinella pseudospiralis adult worms and newborn larvae
Parasites and Vectors, 2017Co-Authors: Yang Wang, Xue Lin Wang, Pascal Boireau, Xue Bai, Haichao Zhu, Haining Shi, Bin Tang, Xuepeng Cai, Xuenong Luo, Ming Yuan LiuAbstract:Background: The nematode Trichinella pseudospiralis is an intracellular parasite of mammalian skeletal muscle cells and exists in a non-encapsulated form. Previous studies demonstrated that T. pseudospiralis could induce a lower host inflammatory response. Excretory-secretory (ES) proteins as the most important products of host-parasite interaction may play the main functional role in alleviating host inflammation. However, the ES products of T. pseudospiralis early stage are still unknown. The identification of the ES products of the early stage facilitates the understanding of the molecular mechanisms of the immunomodulation and may help finding early diagnostic markers. Results: In this study, we used two-dimensional gel electrophoresis (2-DE)-based western blotting coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS/MS) to separate and identify the T. pseudospiralis adult worms ES products immunoreaction-positive proteins. In total, 400 protein spots were separated by 2-DE. Twenty-eight protein spots were successfully identified using the sera from infected pigs and were characterized to correlate with 12 different proteins of T. pseudospiralis, including adult-specific DNase II-10,poly-cysteine and histidine-tailed protein isoform 2, serine protease, serine/threonine-protein kinase ULK3, enolase, putative venom allergen 5, chymotrypsin-like elastase family member 1, uncharacterized protein, peptidase inhibitor 16, death-associated protein 1, Deoxyribonuclease II superfamily and golgin-45. Bioinformatic analyses showed that the identified proteins have a wide diversity of molecular functions, especially Deoxyribonuclease II (DNase II) activity and serine-type endopeptidase activity. Conclusions: Early candidate antigens from the ES proteins of T. pseudospiralis have been screened and identified. Our results suggest these proteins may play key roles during the T. pseudospiralis infection and suppress the host immune response. Further, they are the most likely antigen for early diagnosis and the development of a vaccine against the parasite.
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The functions of Deoxyribonuclease II in immunity and development
DNA and Cell Biology, 2008Co-Authors: Ma Feng Liu, Xue Lin Wang, Wei Fang Wang, Qi Jun Chen, Pascal Boireau, Ming Yuan LiuAbstract:Apoptosis, which is usually accompanied by DNA degradation, is important not only for the homeostasis of metazoans but also for mammalian development. If DNA is not properly degraded in these processes, it can cause diverse diseases, such as anemia, cataracts, and some autoimmune diseases. A large effort has been made to identify these nucleases that are responsible for these effects. In contrast to Deoxyribonuclease I (DNase I), Deoxyribonuclease II (DNase II) has been less well characterized in these processes. Additionally, enzymes of DNase II family in Trichinella spiralis, which is an intracellular parasitic nematode, are also considered involved in the development of the nematode. We have compiled information from studies on DNase II from various organisms and found some nonclassic features in these enzymes of T. spiralis. Here we have reviewed the characterization and functions of DNase II in these processes and predicted the functions of these enzymes in T. spiralis during host invasion and development.