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Takao Minamikawa - One of the best experts on this subject based on the ideXlab platform.
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Identification and possible roles of three types of Endopeptidase from germinated wheat seeds
Journal of biochemistry, 1999Co-Authors: Keita Sutoh, Hideki Kato, Takao MinamikawaAbstract:Little or no Endopeptidase activity was detected in extracts of dry mature wheat seeds, but when they were allowed to imbibe water in darkness, the activity expressed per seedling increased notably after d 1, reached a maximum on d 3 and then decreased. Two major Endopeptidases, named WEP-1 and WEP-2, were present in the 50-70% saturated ammonium sulfate fraction of d-3 seedlings, and could be separated by hydrophobic column chromatography. WEP-1 was further purified and identified as a 31-kDa polypeptide that was immunoreactive to antiserum raised against REP-1, a major rice Cysteine Endopeptidase. Experiments with proteinase inhibitors revealed that WEP-1 and WEP-2 are Cysteine and serine Endopeptidases, respectively. The two enzymes differed in substrate specificity, pH dependence, and the ability to digest major wheat seed proteins. Determination of its amino-terminal amino acid sequence indicated the similarity of WEP-1 to other cereal Cysteine Endopeptidases which are involved in the digestion of seed storage proteins. The expression of WEP-1 in de-embryonated seeds was induced in the presence of gibberellic acid and its effect was eliminated by abscisic acid. In addition to WEP-1 and WEP-2, a legumain-like asparaginyl Endopeptidase was identified in the extract of seedlings on hydrophobic chromatography. The asparaginyl Endopeptidase may function in the early step of mobilization of wheat storage proteins in germinated seeds.
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The structure and organization of two Cysteine Endopeptidase genes from rice.
Plant & cell physiology, 1999Co-Authors: Hideki Kato, Ai Shintani, Takao MinamikawaAbstract:REP-1 is a major Cysteine Endopeptidase that digests seed storage glutelin of rice. A cDNA clone (pRP60) for REP-1 and a cDNA clone (pRP80) for a related enzyme were previously isolated. The expression of both mRNAs is regulated by gibberellin. In this study, we revealed the structure and organization of Rep1 and RepA, genes corresponding to pRP60 and pRP80 mRNAs, respectively. Rep1 has no introns whereas RepA consists of five exons and four introns, and both genes exist as one copy gene in the rice genome. The gibberellin-responsive elements conserved in cereal alpha-amylase genes are not included in the 5'-upstream region of Rep1 or RepA. A molecular phylogenetic tree of plant Cysteine Endopeptidases was constructed, and their relationship was discussed.
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molecular cloning and characterization of vigna mungo processing enzyme 1 vmpe 1 an asparaginyl Endopeptidase possibly involved in post translational processing of a vacuolar Cysteine Endopeptidase sh ep
Plant Molecular Biology, 1999Co-Authors: Takashi Okamoto, Takao MinamikawaAbstract:Asparaginyl Endopeptidase is a Cysteine Endopeptidase that has strict substrate specificity toward the carboxy side of asparagine residues. Vigna mungo processing enzyme 1, termed VmPE-1, occurs in the cotyledons of germinated seeds of V. mungo, and is possibly involved in the post-translational processing of a vacuolar Cysteine Endopeptidase, designated SH-EP, which degrades seed storage protein. VmPE-1 also showed a substrate specificity to asparagine residues, and its enzymatic activity was inhibited by NEM but not E-64. In addition, purified VmPE-1 had a potential to process the recombinant SH-EP precursor to its intermediate in vitro. cDNA clones for VmPE-1 and its homologue, named VmPE-1A, were identified and sequenced, and their expressions in the cotyledons of V. mungo seedlings and other organs were investigated. VmPE-1 mRNA and SH-EP mRNA were expressed in germinated seeds at the same stage of germination although the enzymatic activity of VmPE-1 rose prior to that of SH-EP. The level of VmPE-1A mRNA continued increasing as germination proceeded. In roots, stems and leaves of fully grown plants, and in hypocotyls, VmPE-1 and VmPE-1A were little expressed. We discuss possible functions of VmPE-1 and VmPE-1A in the cotyledons of germinated seeds.
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A vacuolar Cysteine Endopeptidase (SH-EP) that digests seed storage globulin: Characterization, regulation of gene expression, and posttranslational processing
Journal of Plant Physiology, 1998Co-Authors: Takashi Okamoto, Takao MinamikawaAbstract:Summary A Cysteine Endopeptidase named SH-EP was isolated as one of the major hydrolytic enzymes involved in the mobilization of storage globulin in the cotyledon of germinated seeds of Vigna mungo . The enzyme, by itself or in combination with a serine Endopeptidase, digests the globulin subunits to smaller peptides in vitro . Protein immunoblot analysis with antiserum raised against SH-EP showed that this enzyme is synthesized in the cotyledon after the onset of imbibition and increases until day 4 and decreases thereafter. Application of plant hormones and growth regulators, as well as amino acids as proteolytic end-products, appears to affect the development of SH-EP to a limited extent in the cotyledon of the germinated seeds. SH-EP of 33 kDa is synthesized on membrane-bound polysomes as a large, inactive 45-kDa precursor, which is cotranslationally processed to a 43-kDa intermediate through cleavage of a signal peptide. The amino-terminal propeptide region of the intermediate is cleaved further to form the 33-kDa mature enzyme by a multistep processing. The 43-kDa intermediate is also subjected to the cleavage of the carboxy-terminal decapeptide containing a Lys-Asp-Glu-Leu tail, which is known as a retention signal for the endoplasmic reticulum lumen. An asparaginyl Endopeptidase, designated VmPE-1, was isolated as one of the processing enzymes responsible for the cleavage of the amino-terminal propeptide region of the precursor to SH-EP.
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Hormonal regulation of expression of two Cysteine Endopeptidase genes in rice seedlings.
Plant & cell physiology, 1997Co-Authors: Ai Shintani, Hideki Kato, Takao MinamikawaAbstract:Two cDNA probes (pRP60 and pRP80) were used to examine the hormonal regulation of expression of two Cysteine Endopeptidase genes in germinated whole seeds and de-embryonated seeds of rice. The pRP60 protein is a major rice Cysteine Endopeptidase named REP-1 that digests rice glutelin, the main seed storage protein. The pRP80 protein has features of a Cysteine Endopeptidase but has not yet been confirmed to be one. Neither mRNA was detectable in dry seeds, and the levels per seed increased sharply upon imbibition, reached peaks at d 6 to d 9, and then decreased. Both mRNAs were expressed in a seed-specific manner, but the accumulation of pRP60 mRNA was about ten times greater than the other. When seeds were de-embryonated and incubated, the amounts of both mRNAs were reduced to very low levels, but in the presence of 0.01 to 1 microM gibberellic acid (GA3) both again reached high levels. Addition of abscisic acid (ABA) or uniconazole, an inhibitor of gibberellin biosynthesis, partly eliminated the effect of GA3. Protein immunoblot analysis showed that the accumulation of the REP-1 protein in seeds was regulated by GA3 and ABA similarly to that of pRP60 mRNA, but the change in pRP60 mRNA with time after the onset of imbibition preceded that of the REP-1 protein.
Christine Gietl - One of the best experts on this subject based on the ideXlab platform.
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Involvement of Arabidopsis thaliana endoplasmic reticulum KDEL-tailed Cysteine Endopeptidase 1 (AtCEP1) in powdery mildew-induced and AtCPR5-controlled cell death.
PloS one, 2017Co-Authors: Timo Höwing, Caroline Hoefle, Ralph Hückelhoven, Marcel Dann, Christine GietlAbstract:Programmed cell death (PCD) is a prerequisite for successful development and it limits the spread of biotrophic pathogens in a rapid hypersensitive response at the site of infection. KDEL-tailed Cysteine Endopeptidases (KDEL CysEP) are a subgroup of papain-type Cysteine Endopeptidases expressed in tissues undergoing PCD. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2, and AtCEP3) are expressed. We have previously shown that AtCEP1 is a factor of basal resistance to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum, and is expressed in spatiotemporal association with the late fungal development on Arabidopsis leaves. The endoplasmic reticulum-localized proenzyme of AtCEP1 was further visualized at the haustorial complex encased with callose. The AtCPR5 gene (CONSTITUTIVE EXPRESSION OF PR GENES 5) is a regulator of expression of pathogenesis related genes. Loss of AtCPR5 leads to spontaneous expression of chlorotic lesions which was associated with enhanced expression of AtCEP1. We used the atcpr5-2 mutant plants and the atcep1 atcpr5-2 double mutants harboring a non-functional reporter (PCEP1::pre-pro-3xHA-EGFP-KDEL) for visualization of AtCEP1 promoter activity. We found the specific up-regulation of AtCEP1 in direct neighborhood of spreading leaf lesions thus likely representing cells undergoing PCD. Furthermore, we found a strong resistance of atcpr5 mutant plants against infection with E. cruciferarum. Loss of AtCEP1 had no obvious influence on the strong resistance of atcpr5-2 mutant plants against infection with E. cruciferarum. However, the area of necrotic leaf lesions associated with E. cruciferarum colonies was significantly larger in atcpr5-2 as compared to atcep1 atcpr5-2 double mutant plants. The presence of AtCEP1 thus contributes to AtCPR5-controlled PCD at the sites of powdery mildew infection.
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Ex vivo processing for maturation of Arabidopsis KDEL-tailed Cysteine Endopeptidase 2 (AtCEP2) pro-enzyme and its storage in endoplasmic reticulum derived organelles
Plant Molecular Biology, 2014Co-Authors: Georg Hierl, Timo Höwing, Erika Isono, Friedrich Lottspeich, Christine GietlAbstract:Ricinosomes are specialized ER-derived organelles that store the inactive pro-forms of KDEL-tailed Cysteine Endopeptidases (KDEL-CysEP) associated with programmed cell death (PCD). The Arabidopsis genome encodes three KDEL-CysEP (AtCEP1, AtCEP2, and AtCEP3) that are differentially expressed in vegetative and generative tissues undergoing PCD. These Arabidopsis proteases have not been characterized at a biochemical level, nor have they been localized intracellularly. In this study, we characterized AtCEP2. A 3xHA-mCherry-AtCEP2 gene fusion including pro-peptide and KDEL targeting sequences expressed under control of the endogenous promoter enabled us to isolate AtCEP2 “ex vivo”. The purified protein was shown to be activated in a pH-dependent manner. After activation, however, protease activity was pH-independent. Analysis of substrate specificity showed that AtCEP2 accepts proline near the cleavage site, which is a rare feature specific for KDEL-CysEPs. mCherry-AtCEP2 was detected in the epidermal layers of leaves, hypocotyls and roots; in the root, it was predominantly found in the elongation zone and root cap. Co-localization with an ER membrane marker showed that mCherry-AtCEP2 was stored in two different types of ER-derived organelles: 10 μm long spindle shaped organelles as well as round vesicles with a diameter of approximately 1 μm. The long organelles appear to be ER bodies, which are found specifically in Brassicacae. The round vesicles strongly resemble the ricinosomes first described in castor bean. This study provides a first evidence for the existence of ricinosomes in Arabidopsis , and may open up new avenues of research in the field of PCD and developmental tissue remodeling.
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ex vivo processing for maturation of arabidopsis kdel tailed Cysteine Endopeptidase 2 atcep2 pro enzyme and its storage in endoplasmic reticulum derived organelles
Plant Molecular Biology, 2014Co-Authors: Georg Hierl, Timo Höwing, Erika Isono, Friedrich Lottspeich, Christine GietlAbstract:Ricinosomes are specialized ER-derived organelles that store the inactive pro-forms of KDEL-tailed Cysteine Endopeptidases (KDEL-CysEP) associated with programmed cell death (PCD). The Arabidopsis genome encodes three KDEL-CysEP (AtCEP1, AtCEP2, and AtCEP3) that are differentially expressed in vegetative and generative tissues undergoing PCD. These Arabidopsis proteases have not been characterized at a biochemical level, nor have they been localized intracellularly. In this study, we characterized AtCEP2. A 3xHA-mCherry-AtCEP2 gene fusion including pro-peptide and KDEL targeting sequences expressed under control of the endogenous promoter enabled us to isolate AtCEP2 “ex vivo”. The purified protein was shown to be activated in a pH-dependent manner. After activation, however, protease activity was pH-independent. Analysis of substrate specificity showed that AtCEP2 accepts proline near the cleavage site, which is a rare feature specific for KDEL-CysEPs. mCherry-AtCEP2 was detected in the epidermal layers of leaves, hypocotyls and roots; in the root, it was predominantly found in the elongation zone and root cap. Co-localization with an ER membrane marker showed that mCherry-AtCEP2 was stored in two different types of ER-derived organelles: 10 μm long spindle shaped organelles as well as round vesicles with a diameter of approximately 1 μm. The long organelles appear to be ER bodies, which are found specifically in Brassicacae. The round vesicles strongly resemble the ricinosomes first described in castor bean. This study provides a first evidence for the existence of ricinosomes in Arabidopsis, and may open up new avenues of research in the field of PCD and developmental tissue remodeling. Electronic supplementary material The online version of this article (doi:10.1007/s11103-013-0157-6) contains supplementary material, which is available to authorized users.
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Endoplasmic reticulum KDEL-tailed Cysteine Endopeptidase 1 of Arabidopsis (AtCEP1) is involved in pathogen defense
Frontiers in plant science, 2014Co-Authors: Timo Höwing, Erika Isono, Christina Huesmann, Caroline Hoefle, Marie-kristin Nagel, Ralph Hückelhoven, Christine GietlAbstract:Programmed cell death (PCD) is a genetically determined process in all multicellular organisms. Plant PCD is effected by a unique group of papain-type Cysteine Endopeptidases (CysEP) with a C-terminal KDEL endoplasmic reticulum (ER) retention signal (KDEL CysEP). KDEL CysEPs can be stored as pro-enzymes in ER-derived endomembrane compartments and are released as mature CysEPs in the final stages of organelle disintegration. KDEL CysEPs accept a wide variety of amino acids at the active site, including the glycosylated hydroxyprolines of the extensins that form the basic scaffold of the cell wall. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2, and AtCEP3) are expressed. Cell- and tissue-specific activities of these three genes suggest that KDEL CysEPs participate in the abscission of flower organs and in the collapse of tissues in the final stage of PCD as well as in developmental tissue remodelling.We observed that AtCEP1 is expressed in response to biotic stress stimuli in the leaf. atcep1 knockout mutants showed enhanced susceptibility to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum. A translational fusion protein of AtCEP1 with a three-fold hemaglutinin-tag and the green fluorescent protein under control of the endogenous AtCEP1 promoter (PCEP1::pre-pro-3xHA-EGFP-AtCEP1-KDEL) rescued the pathogenesis phenotype demonstrating the function of AtCEP1 in restriction of powdery mildew. The spatiotemporal AtCEP1-reporter expression during fungal infection together with microscopic inspection of the interaction phenotype suggested a function of AtCEP1 in controlling late stages of compatible interaction including late epidermal cell death. Additionally, expression of stress response genes appeared to be deregulated in the interaction of atcep1 mutants and E. cruciferarum. Possible functions of AtCEP1 in restricting parasitic success of the obligate biotrophic powdery mildew fungus are discussed.
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ricinosomes and endosperm transfer cell structure in programmed cell death of the nucellus during ricinus seed development
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: John S Greenwood, Michael Helm, Christine GietlAbstract:The ricinosome (precursor protease vesicle) is an organelle found exclusively in plant cells. Ricinosomes contain a 45-kDa pro-Cysteine Endopeptidase (CysEP) with a C-terminal KDEL endoplasmic reticulum retention signal. CysEP is a member of a unique group of papain-type Cysteine peptidases found specifically in senescing and ricinosome-containing tissues. During seed development in the castor oil plant (Ricinus communis L.), the cells of the nucellus are killed as the major seed storage organ, the cellular endosperm, expands and begins to accumulate reserves. The destruction of the maternal seed tissues is a developmentally programmed cell death. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling revealed that nuclear DNA fragmentation occurs in the nucellar cells adjacent to the expanding endosperm. These cells exhibit ultrastructural features consistent with programmed cell death, including vesiculation of the cytosol, development of irregularly shaped nuclei, vacuolar collapse, and shrinkage of the cytoplasm. Ricinosomes containing the CysEP were identified in the nucellar cells by light and electron microscopy and immunocytochemistry. Both proCysEP and mature CysEP are present in protein extracts of the nucellar tissues during seed development. Upon collapse of the nucellar cells, the content of the ricinosomes is released into the cytoplasm, where the activated CysEP digests the remaining proteinaceous cellular debris. Digestion products of the nucellar cells are presumed taken up by the outermost cells of the endosperm, which have labyrinthine ingrowths of the outer walls typical of transfer cells.
Carlos Termignoni - One of the best experts on this subject based on the ideXlab platform.
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multi antigenic vaccine against the cattle tick rhipicephalus boophilus microplus a field evaluation
Vaccine, 2012Co-Authors: Luís Fernando Parizi, Adriana Seixas, Carlos Termignoni, Daiane Patrícia Oldiges, Jose Reck, Melina Garcia Guizzo, Carlos Logullo, Pedro L Oliveira, Joao Ricardo MartinsAbstract:The tick Rhipicephalus (Boophilus) microplus is a blood-sucking ectoparasite of cattle that severely impairs livestock production. Studies on tick immunological control address mostly single-antigen vaccines. However, from the commercial standpoint, so far no single-antigen vaccine has afforded appropriate protection against all R. microplus populations. In this context, multi-antigen cocktails have emerged as a way to enhance vaccine efficacy. In this work, a multi-antigenic vaccine against R. microplus was analyzed under field conditions in naturally infested cattle. The vaccine was composed by three tick recombinant proteins from two tick species that in previous single-vaccination reports provided partial protection of confined cattle against R. microplus infestations: vitellin-degrading Cysteine Endopeptidase (VTDCE) and boophilus yolk pro-cathepsin (BYC) from R. microplus, and glutathione S-transferase from Haemaphysalis longicornis (GST-Hl). Increased antibody levels against three proteins were recorded after immunizations, with a distinct humoral immune response dynamics for each protein. Compared to the control group, a statistically significant lower number of semi-engorged female ticks were observed in vaccinated cattle after two inoculations. This reduction persisted for 3 months, ranging from 35.3 to 61.6%. Furthermore, cattle body weight gain was significantly higher in vaccinated animals when compared to control cattle. Compared to the single-antigen vaccines composed by VTDCE, BYC or GST-Hl, this three-antigen vaccine afforded higher protection levels against R. microplus infestations.
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A Rhipicephalus (Boophilus) microplus cathepsin with dual peptidase and antimicrobial activity.
International journal for parasitology, 2012Co-Authors: Daiane Patrícia Oldiges, Adriana Seixas, Aoi Masuda, Itabajara Da Silva Vaz, Luís Fernando Parizi, Karine Rigon Zimmer, Daniel M. Lorenzini, Carlos TermignoniAbstract:The cattle tick, Rhipicephalus (Boophilus) microplus, is a haematophagous arthropod responsible for considerable losses in the livestock industry. Immunological control with vaccines is a promising alternative to replace chemical acaricides. Due to their importance in parasite physiology, Cysteine Endopeptidases are potential targets. In a previous study, native Vitellin Degrading Cysteine Endopeptidase (VTDCE) was successfully tested as a vaccine antigen for bovines against R. microplus. In this work, nucleotide and amino acid VTDCE sequences were obtained from cDNA databanks, based on data from Edman sequencing and mass spectrometry. Subsequently, cloning and expression, purification, immunological and biochemical characterisation of the recombinant protein were performed to determine the biological importance of VTDCE. By Western blot, polyclonal antibodies produced against recombinant VTDCE recognised native VTDCE. Interestingly, molecular analysis showed that the VTDCE sequence has similarity to antimicrobial peptides. Indeed, experimental results revealed that VTDCE has an antimicrobial activity which is independent of Endopeptidase activity. We believe that this is the first known study to show that an arthropod enzyme has antimicrobial activity.
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Vitellin- and hemoglobin-digesting enzymes in Rhipicephalus (Boophilus) microplus larvae and females.
Comparative biochemistry and physiology. Part B Biochemistry & molecular biology, 2010Co-Authors: Andreia Bergamo Estrela, Adriana Seixas, Vivian De Oliveira Nunes Teixeira, Antônio F.m. Pinto, Carlos TermignoniAbstract:The aim of the present study was to address the involvement of Rhipicephalus microplus larval Cysteine Endopeptidase (RmLCE) in protein digestion in R. microplus larvae and adult females. In this work, an improved purification protocol for native RmLCE was developed. Partial amino acid sequence of the purified enzyme indicates that it is the same enzyme as Boophilus microplus cathepsin-L1 (BmCL1). When vitellin (Vt) degradation by egg and larval enzymes was analyzed, stage-specific differences for RmLCE activity in comparison to vitellin-degrading Cysteine Endopeptidase (VTDCE) were observed. RmLCE is also able to degrade host hemoglobin (Hb). In agreement, an acidic Cysteine Endopeptidase activity was detected in larval gut. It was shown that Cysteine and aspartic Endopeptidases are involved in Vt and Hb digestion in R. microplus larvae and females. Interestingly, we observed that the aspartic Endopeptidase Boophilus yolk cathepsin (BYC) is associated with a Cysteine Endopeptidase activity, in larvae. Synergic hemoglobin digestion by BYC and RmLCE was observed and indicates the presence of an Hb-degrading enzymatic cascade involving these enzymes. Our results suggest that RmLCE/BmCL1 has a continued role in vitellin and hemoglobin digestion during tick development.
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Localization and function of Rhipicephalus (Boophilus) microplus vitellin-degrading Cysteine Endopeptidase
Parasitology, 2010Co-Authors: Adriana Seixas, Andreia Bergamo Estrela, Juliana Casagrande Ceolato, Emerson Guedes Pontes, Flávio Alves Lara, Katia C. Gondim, Carlos TermignoniAbstract:The tick Rhipicephalus (Boophilus) microplus is an important parasite of cattle in many areas of the tropics. Characterization of molecules involved in mechanisms such as vitellogenesis and embryo development may contribute to a better understanding of this parasite's physiology. The vitellin-degrading Cysteine Endopeptidase (VTDCE) is the most active enzyme involved in vitellin hydrolysis in R. microplus eggs. Here we show an association between VTDCE and vitellin in an additional site, apart from the active site. Our data also demonstrate Cysteine Endopeptidase activity in different tissues such as ovary, gut, fat body, salivary gland and female haemolymph, where it is controlled by a physiological inhibitor. In R. microplus female gut, VTDCE is localized in areas of protein synthesis and trafficking with the underlying haemolymph. VTDCE is also localized in the ovary basal region, in vesicle membranes of ovary pedicel cells and in oocyte cytosol. These results suggest that VTDCE plays a role in vitellin digestion during tick development.
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vaccine potential of a tick vitellin degrading enzyme vtdce
Veterinary Immunology and Immunopathology, 2008Co-Authors: Adriana Seixas, Alexandre T Leal, Maria Clara L Nascimentosilva, Aoi Masuda, Carlos Termignoni, Itabajara Da Silva VazAbstract:VTDCE (Vitelin-Degrading Cysteine Endopeptidase) is a peptidase with an active role in Rhipicephalus (Boophilus) microplus embryogenesis. VTDCE is found in the tick's eggs and was shown to be the most active protein in vitellin (VT) hydrolysis of the three peptidases already characterized in R. microplus eggs (Boophilus Yolk pro-cathepsin (BYC), Tick Heme Binding Aspartic Proteinase (THAP) and VTDCE). VTDCE activity was assessed in vitro using the natural substrate and a synthetic substrate (N-Cbz-Phe-Arg-MCA). The activity was inhibited by anti-VTDCE antibodies. In the present study, it was shown that VTDCE acts differently from BYC and THAP in VT hydrolysis and that the vaccination of bovines with VTDCE induces a partial protective immune response against R. microplus infestation. Immunized bovines challenged with R. microplus larvae presented an overall protection of 21%, and a reduction in the weight of fertile eggs of 17.6% was observed. The data obtained indicate that VTDCE seems to be important for tick physiology, and that it induces partial protective immune response when inoculated in bovines. This suggests that VTDCE can be useful to improve the protective capacity observed for other antigens.
Takashi Okamoto - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning and characterization of vigna mungo processing enzyme 1 vmpe 1 an asparaginyl Endopeptidase possibly involved in post translational processing of a vacuolar Cysteine Endopeptidase sh ep
Plant Molecular Biology, 1999Co-Authors: Takashi Okamoto, Takao MinamikawaAbstract:Asparaginyl Endopeptidase is a Cysteine Endopeptidase that has strict substrate specificity toward the carboxy side of asparagine residues. Vigna mungo processing enzyme 1, termed VmPE-1, occurs in the cotyledons of germinated seeds of V. mungo, and is possibly involved in the post-translational processing of a vacuolar Cysteine Endopeptidase, designated SH-EP, which degrades seed storage protein. VmPE-1 also showed a substrate specificity to asparagine residues, and its enzymatic activity was inhibited by NEM but not E-64. In addition, purified VmPE-1 had a potential to process the recombinant SH-EP precursor to its intermediate in vitro. cDNA clones for VmPE-1 and its homologue, named VmPE-1A, were identified and sequenced, and their expressions in the cotyledons of V. mungo seedlings and other organs were investigated. VmPE-1 mRNA and SH-EP mRNA were expressed in germinated seeds at the same stage of germination although the enzymatic activity of VmPE-1 rose prior to that of SH-EP. The level of VmPE-1A mRNA continued increasing as germination proceeded. In roots, stems and leaves of fully grown plants, and in hypocotyls, VmPE-1 and VmPE-1A were little expressed. We discuss possible functions of VmPE-1 and VmPE-1A in the cotyledons of germinated seeds.
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A vacuolar Cysteine Endopeptidase (SH-EP) that digests seed storage globulin: Characterization, regulation of gene expression, and posttranslational processing
Journal of Plant Physiology, 1998Co-Authors: Takashi Okamoto, Takao MinamikawaAbstract:Summary A Cysteine Endopeptidase named SH-EP was isolated as one of the major hydrolytic enzymes involved in the mobilization of storage globulin in the cotyledon of germinated seeds of Vigna mungo . The enzyme, by itself or in combination with a serine Endopeptidase, digests the globulin subunits to smaller peptides in vitro . Protein immunoblot analysis with antiserum raised against SH-EP showed that this enzyme is synthesized in the cotyledon after the onset of imbibition and increases until day 4 and decreases thereafter. Application of plant hormones and growth regulators, as well as amino acids as proteolytic end-products, appears to affect the development of SH-EP to a limited extent in the cotyledon of the germinated seeds. SH-EP of 33 kDa is synthesized on membrane-bound polysomes as a large, inactive 45-kDa precursor, which is cotranslationally processed to a 43-kDa intermediate through cleavage of a signal peptide. The amino-terminal propeptide region of the intermediate is cleaved further to form the 33-kDa mature enzyme by a multistep processing. The 43-kDa intermediate is also subjected to the cleavage of the carboxy-terminal decapeptide containing a Lys-Asp-Glu-Leu tail, which is known as a retention signal for the endoplasmic reticulum lumen. An asparaginyl Endopeptidase, designated VmPE-1, was isolated as one of the processing enzymes responsible for the cleavage of the amino-terminal propeptide region of the precursor to SH-EP.
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A Cysteine Endopeptidase (SH-EP) in Germinated Vigna mungo Seeds: Post-translational Processing and Intracellular Transport
Basic and Applied Aspects of Seed Biology, 1997Co-Authors: Takashi Okamoto, T. MinamikawaAbstract:A plant Cysteine Endopeptidase, designated SH-EP, is a major protease occurring in cotyledons of Vigna mungo seedlings, and acts to degrade seed globulin stored in protein bodies (protein storage vacuoles). SH-EP is synthesized on membrane-bound ribosomes as a 43-kDa intermediate through the cotranslational cleavage of a signal sequence, and the intermediate is processed further to the 33-kDa mature enzyme via 39- and 36-kDa intermediates, (a) N-terminalprocessing— Experiments of in vitro processing of the SH-EP intermediates revealed that two processing enzymes, VmPE-1 and VmPE-2 (V. mungo processing enzymes 1 and 2), are involved in the processing. VmPE-1 was purified from the day-3 cotyledons. The enzyme is the same type of protease as asparaginyl Endopeptidases in terms of the primary structure and substrate specificity, (b) C-terminal processing — The amino acid sequence of SH-EP deduced from the cDNA contains C-terminus with Lys-Asp-Glu-Leu (KDEL) tail, which is known as a retention signal for endoplasmic reticulum (ER) while mature SH-EP is localized in protein bodies. The analysis for C-terminal amino acid residues of SH-EP indicated that the C-terminal propeptide of 10 amino acid residues containing the KDEL tail is processed to form mature SH-EP.
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Promoter regions of Cysteine Endopeptidase genes from legumes confer germination-specific expression in transgenic tobacco seeds.
Plant molecular biology, 1996Co-Authors: Daisuke Yamauchi, Takashi Okamoto, Yoko Terasaki, Takao MinamikawaAbstract:Cysteine Endopeptidases, SH-EP from Vigna mungo and EP-C1 from Phaseolus vulgaris, act to degrade seed storage protein during seed germination. Using transgenic tobacco plants, expression of SH-EP and promoter activity of the EP-C1 gene were analyzed in transgenic tobacco plants. The promoters of the two genes in tobacco seeds showed germination-specific activation, although post-translational processing of SH-EP and regulatory regions of promoter of the gene for EP-C1 were found to differ between leguminous seeds and transgenic tobacco seeds.
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Purification of a Processing Enzyme (VmPE‐1) that is Involved in Post‐Translational Processing of a Plant Cysteine Endopeptidase (SH‐EP)
European journal of biochemistry, 1995Co-Authors: Takashi Okamoto, Takao MinamikawaAbstract:A Cysteine Endopeptidase, designated SH-EP, occurs in the cotyledons of germinated seeds of Vigna mungo and acts to degrade the seed storage protein in protein storage vacuoles. SH-EP is synthesized on membrane-bound ribosomes as an inactive 45-kDa precursor, which is cotranslationally processed to a 43-kDa intermediate through cleavage of the signal sequence; the 43-kDa intermediate of SH-EP is further processed to the 33-kDa mature enzyme via 39-kDa and 36-kDa intermediates [Mitsuhashi, W. & Minamikawa, T. (1989) Plant Physiol. 89, 274–279]. The present in vitro processing experiments indicated that at least two processing enzymes, designated VmPE-1 and VmPE-2 (V. mungo processing enzymes 1 and 2), were involved in post-translational processing, of SH-EP in cotyledons of V. mungo seedlings. VmPE-1 was purified from the cotyledons as a protease that was involved in the processing of the 43-kDa intermediate to the 36-kDa intermediate. The enzyme has a molecular mass of 33 kDa as estimated by SDS/polyacrylamide gel electrophoresis, and showed high similarity to the jackbean asparaginyl Endopeptidase in terms of the primary structure and substrate specificity. We discuss the function of VmPE-1 in the processing of SH-EP and related proteases in the cotyledons of germinated seeds.
Adriana Seixas - One of the best experts on this subject based on the ideXlab platform.
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multi antigenic vaccine against the cattle tick rhipicephalus boophilus microplus a field evaluation
Vaccine, 2012Co-Authors: Luís Fernando Parizi, Adriana Seixas, Carlos Termignoni, Daiane Patrícia Oldiges, Jose Reck, Melina Garcia Guizzo, Carlos Logullo, Pedro L Oliveira, Joao Ricardo MartinsAbstract:The tick Rhipicephalus (Boophilus) microplus is a blood-sucking ectoparasite of cattle that severely impairs livestock production. Studies on tick immunological control address mostly single-antigen vaccines. However, from the commercial standpoint, so far no single-antigen vaccine has afforded appropriate protection against all R. microplus populations. In this context, multi-antigen cocktails have emerged as a way to enhance vaccine efficacy. In this work, a multi-antigenic vaccine against R. microplus was analyzed under field conditions in naturally infested cattle. The vaccine was composed by three tick recombinant proteins from two tick species that in previous single-vaccination reports provided partial protection of confined cattle against R. microplus infestations: vitellin-degrading Cysteine Endopeptidase (VTDCE) and boophilus yolk pro-cathepsin (BYC) from R. microplus, and glutathione S-transferase from Haemaphysalis longicornis (GST-Hl). Increased antibody levels against three proteins were recorded after immunizations, with a distinct humoral immune response dynamics for each protein. Compared to the control group, a statistically significant lower number of semi-engorged female ticks were observed in vaccinated cattle after two inoculations. This reduction persisted for 3 months, ranging from 35.3 to 61.6%. Furthermore, cattle body weight gain was significantly higher in vaccinated animals when compared to control cattle. Compared to the single-antigen vaccines composed by VTDCE, BYC or GST-Hl, this three-antigen vaccine afforded higher protection levels against R. microplus infestations.
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A Rhipicephalus (Boophilus) microplus cathepsin with dual peptidase and antimicrobial activity.
International journal for parasitology, 2012Co-Authors: Daiane Patrícia Oldiges, Adriana Seixas, Aoi Masuda, Itabajara Da Silva Vaz, Luís Fernando Parizi, Karine Rigon Zimmer, Daniel M. Lorenzini, Carlos TermignoniAbstract:The cattle tick, Rhipicephalus (Boophilus) microplus, is a haematophagous arthropod responsible for considerable losses in the livestock industry. Immunological control with vaccines is a promising alternative to replace chemical acaricides. Due to their importance in parasite physiology, Cysteine Endopeptidases are potential targets. In a previous study, native Vitellin Degrading Cysteine Endopeptidase (VTDCE) was successfully tested as a vaccine antigen for bovines against R. microplus. In this work, nucleotide and amino acid VTDCE sequences were obtained from cDNA databanks, based on data from Edman sequencing and mass spectrometry. Subsequently, cloning and expression, purification, immunological and biochemical characterisation of the recombinant protein were performed to determine the biological importance of VTDCE. By Western blot, polyclonal antibodies produced against recombinant VTDCE recognised native VTDCE. Interestingly, molecular analysis showed that the VTDCE sequence has similarity to antimicrobial peptides. Indeed, experimental results revealed that VTDCE has an antimicrobial activity which is independent of Endopeptidase activity. We believe that this is the first known study to show that an arthropod enzyme has antimicrobial activity.
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Vitellin- and hemoglobin-digesting enzymes in Rhipicephalus (Boophilus) microplus larvae and females.
Comparative biochemistry and physiology. Part B Biochemistry & molecular biology, 2010Co-Authors: Andreia Bergamo Estrela, Adriana Seixas, Vivian De Oliveira Nunes Teixeira, Antônio F.m. Pinto, Carlos TermignoniAbstract:The aim of the present study was to address the involvement of Rhipicephalus microplus larval Cysteine Endopeptidase (RmLCE) in protein digestion in R. microplus larvae and adult females. In this work, an improved purification protocol for native RmLCE was developed. Partial amino acid sequence of the purified enzyme indicates that it is the same enzyme as Boophilus microplus cathepsin-L1 (BmCL1). When vitellin (Vt) degradation by egg and larval enzymes was analyzed, stage-specific differences for RmLCE activity in comparison to vitellin-degrading Cysteine Endopeptidase (VTDCE) were observed. RmLCE is also able to degrade host hemoglobin (Hb). In agreement, an acidic Cysteine Endopeptidase activity was detected in larval gut. It was shown that Cysteine and aspartic Endopeptidases are involved in Vt and Hb digestion in R. microplus larvae and females. Interestingly, we observed that the aspartic Endopeptidase Boophilus yolk cathepsin (BYC) is associated with a Cysteine Endopeptidase activity, in larvae. Synergic hemoglobin digestion by BYC and RmLCE was observed and indicates the presence of an Hb-degrading enzymatic cascade involving these enzymes. Our results suggest that RmLCE/BmCL1 has a continued role in vitellin and hemoglobin digestion during tick development.
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Localization and function of Rhipicephalus (Boophilus) microplus vitellin-degrading Cysteine Endopeptidase
Parasitology, 2010Co-Authors: Adriana Seixas, Andreia Bergamo Estrela, Juliana Casagrande Ceolato, Emerson Guedes Pontes, Flávio Alves Lara, Katia C. Gondim, Carlos TermignoniAbstract:The tick Rhipicephalus (Boophilus) microplus is an important parasite of cattle in many areas of the tropics. Characterization of molecules involved in mechanisms such as vitellogenesis and embryo development may contribute to a better understanding of this parasite's physiology. The vitellin-degrading Cysteine Endopeptidase (VTDCE) is the most active enzyme involved in vitellin hydrolysis in R. microplus eggs. Here we show an association between VTDCE and vitellin in an additional site, apart from the active site. Our data also demonstrate Cysteine Endopeptidase activity in different tissues such as ovary, gut, fat body, salivary gland and female haemolymph, where it is controlled by a physiological inhibitor. In R. microplus female gut, VTDCE is localized in areas of protein synthesis and trafficking with the underlying haemolymph. VTDCE is also localized in the ovary basal region, in vesicle membranes of ovary pedicel cells and in oocyte cytosol. These results suggest that VTDCE plays a role in vitellin digestion during tick development.
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vaccine potential of a tick vitellin degrading enzyme vtdce
Veterinary Immunology and Immunopathology, 2008Co-Authors: Adriana Seixas, Alexandre T Leal, Maria Clara L Nascimentosilva, Aoi Masuda, Carlos Termignoni, Itabajara Da Silva VazAbstract:VTDCE (Vitelin-Degrading Cysteine Endopeptidase) is a peptidase with an active role in Rhipicephalus (Boophilus) microplus embryogenesis. VTDCE is found in the tick's eggs and was shown to be the most active protein in vitellin (VT) hydrolysis of the three peptidases already characterized in R. microplus eggs (Boophilus Yolk pro-cathepsin (BYC), Tick Heme Binding Aspartic Proteinase (THAP) and VTDCE). VTDCE activity was assessed in vitro using the natural substrate and a synthetic substrate (N-Cbz-Phe-Arg-MCA). The activity was inhibited by anti-VTDCE antibodies. In the present study, it was shown that VTDCE acts differently from BYC and THAP in VT hydrolysis and that the vaccination of bovines with VTDCE induces a partial protective immune response against R. microplus infestation. Immunized bovines challenged with R. microplus larvae presented an overall protection of 21%, and a reduction in the weight of fertile eggs of 17.6% was observed. The data obtained indicate that VTDCE seems to be important for tick physiology, and that it induces partial protective immune response when inoculated in bovines. This suggests that VTDCE can be useful to improve the protective capacity observed for other antigens.
