Publicação
Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response
| Resumo: | Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor a vaccine to be used in Europe is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neospora caninum and Toxoplasma gondii, which are closely related to B. besnoiti, PDI plays an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we presented the nucleotide sequence of the B. besnoiti PDI gene and a 3D theoretical model was built by comparative homology using Swiss-Model server. B. besnoiti expresses a PDI with 471 amino acids, structurally similar to human and yeast PDIs, with four thioredoxin-like domains a, b, b’, a’ and a C-terminal extension c. The a and a’ domains present the characteristic active site pattern CxxC, in this case CGHC and CGYC, respectively. Analysis of the phylogenetic tree for PDI within the phylum Apicomplexa reinforced the close relationship among B. besnoiti, N. caninum and T. gondii. Recombinant B. besnoiti PDI (recBbPDI) and truncated versions corresponding to domains a, b, b’ and a’c were expressed in a heterologous system. Mice were immunized with recBbPDI for the production of monoclonal antibodies (mAbs) by hybridoma technology and four mAbs were produced and characterized. RecBbPDI and domain a’c (recBb-a’c) were functionally active and exhibited a dose dependent cross-linking activity of insulin. In the presence of bacitracin, tocinoic acid, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and 4-chloromercuribenzoic acid (pCMBA) activity of both enzymes was inhibited, in a dose dependent manner. The same happened with recBbPDI in the presence of mAbs (with the exception of T8a), but not with recBb-a’c, whose activity was not sensitive to the presence of mAbs. In vitro proliferation of B. besnoiti tachyzoites was diminished in the presence of PDI inhibitors and anti-PDI mAbs, indicating that this enzyme seems to intervene in the process of host cell adhesion/invasion. In this way, considering the inhibitions obtained, both in the host cell invasion ability and in the enzyme catalytic activity, PDI can represent a potential target for addressing the treatment and/or prevention of besnoitiosis. The panel of monoclonal antibodies here developed represents an important tool for future studies. |
|---|---|
| Autores principais: | Marcelino, Eduardo Miguel Baptista Ferreira |
| Assunto: | Besnoitia besnoiti protein disulfide isomerase PDI monoclonal antibody host cell invasion enzima isomerase de dissulfureto anticorpos monoclonais invasão célula hospedeira |
| Ano: | 2016 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| _version_ | 1865920848888594432 |
|---|---|
| author | Marcelino, Eduardo Miguel Baptista Ferreira |
| author_facet | Marcelino, Eduardo Miguel Baptista Ferreira Marcelino, Eduardo Miguel Baptista Ferreira |
| author_role | author |
| contributor_name_str_mv | Leitão, José Alexandre da Costa Perdigão e Cameira Novo, Carlos Manuel Mendes Repositório Científico de Acesso Aberto da ULisboa |
| country_str | PT |
| creators_json_str | [{\"Person.name\":\"Marcelino, Eduardo Miguel Baptista Ferreira\"}] |
| datacite.contributors.contributor.contributorName.fl_str_mv | Leitão, José Alexandre da Costa Perdigão e Cameira Novo, Carlos Manuel Mendes Repositório Científico de Acesso Aberto da ULisboa |
| datacite.creators.creator.creatorName.fl_str_mv | Marcelino, Eduardo Miguel Baptista Ferreira |
| datacite.date.Accepted.fl_str_mv | 2016-06-07T00:00:00Z |
| datacite.date.available.fl_str_mv | 2017-06-30T00:30:11Z |
| datacite.date.embargoed.fl_str_mv | 2017-06-30T00:30:11Z |
| datacite.rights.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| datacite.subjects.subject.fl_str_mv | Besnoitia besnoiti protein disulfide isomerase PDI monoclonal antibody host cell invasion enzima isomerase de dissulfureto anticorpos monoclonais invasão célula hospedeira |
| datacite.titles.title.fl_str_mv | Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response |
| dc.contributor.none.fl_str_mv | Leitão, José Alexandre da Costa Perdigão e Cameira Novo, Carlos Manuel Mendes Repositório Científico de Acesso Aberto da ULisboa |
| dc.creator.none.fl_str_mv | Marcelino, Eduardo Miguel Baptista Ferreira |
| dc.date.Accepted.fl_str_mv | 2016-06-07T00:00:00Z |
| dc.date.available.fl_str_mv | 2017-06-30T00:30:11Z |
| dc.date.embargoed.fl_str_mv | 2017-06-30T00:30:11Z |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.none.fl_str_mv | http://hdl.handle.net/10400.5/11693 |
| dc.language.none.fl_str_mv | eng |
| dc.publisher.none.fl_str_mv | Universidade de Lisboa. Faculdade de Medicina Veterinária |
| dc.rights.cclincense.fl_str_mv | http://creativecommons.org/licenses/by/4.0/ |
| dc.rights.none.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| dc.subject.none.fl_str_mv | Besnoitia besnoiti protein disulfide isomerase PDI monoclonal antibody host cell invasion enzima isomerase de dissulfureto anticorpos monoclonais invasão célula hospedeira |
| dc.title.fl_str_mv | Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response |
| dc.type.none.fl_str_mv | http://purl.org/coar/resource_type/c_db06 |
| description | Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor a vaccine to be used in Europe is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neospora caninum and Toxoplasma gondii, which are closely related to B. besnoiti, PDI plays an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we presented the nucleotide sequence of the B. besnoiti PDI gene and a 3D theoretical model was built by comparative homology using Swiss-Model server. B. besnoiti expresses a PDI with 471 amino acids, structurally similar to human and yeast PDIs, with four thioredoxin-like domains a, b, b’, a’ and a C-terminal extension c. The a and a’ domains present the characteristic active site pattern CxxC, in this case CGHC and CGYC, respectively. Analysis of the phylogenetic tree for PDI within the phylum Apicomplexa reinforced the close relationship among B. besnoiti, N. caninum and T. gondii. Recombinant B. besnoiti PDI (recBbPDI) and truncated versions corresponding to domains a, b, b’ and a’c were expressed in a heterologous system. Mice were immunized with recBbPDI for the production of monoclonal antibodies (mAbs) by hybridoma technology and four mAbs were produced and characterized. RecBbPDI and domain a’c (recBb-a’c) were functionally active and exhibited a dose dependent cross-linking activity of insulin. In the presence of bacitracin, tocinoic acid, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and 4-chloromercuribenzoic acid (pCMBA) activity of both enzymes was inhibited, in a dose dependent manner. The same happened with recBbPDI in the presence of mAbs (with the exception of T8a), but not with recBb-a’c, whose activity was not sensitive to the presence of mAbs. In vitro proliferation of B. besnoiti tachyzoites was diminished in the presence of PDI inhibitors and anti-PDI mAbs, indicating that this enzyme seems to intervene in the process of host cell adhesion/invasion. In this way, considering the inhibitions obtained, both in the host cell invasion ability and in the enzyme catalytic activity, PDI can represent a potential target for addressing the treatment and/or prevention of besnoitiosis. The panel of monoclonal antibodies here developed represents an important tool for future studies. |
| dirty | 0 |
| eu_rights_str_mv | openAccess |
| format | doctoralThesis |
| fulltext.url.fl_str_mv | https://repositorio.ulisboa.pt/bitstreams/a8aff77d-fb06-4a8b-b12a-bdef2f658400/download |
| id | ul_ea2c2cc06f483e9793984eda7bda2f45 |
| identifier.url.fl_str_mv | http://hdl.handle.net/10400.5/11693 |
| instacron_str | ul |
| institution | Universidade de Lisboa |
| instname_str | Universidade de Lisboa |
| language | eng |
| network_acronym_str | ul |
| network_name_str | Repositório da Universidade de Lisboa |
| oai_identifier_str | oai:repositorio.ulisboa.pt:10400.5/11693 |
| organization_str_mv | urn:organizationAcronym:ul |
| person_str_mv | Marcelino, Eduardo Miguel Baptista Ferreira |
| publishDate | 2016 |
| publisher.none.fl_str_mv | Universidade de Lisboa. Faculdade de Medicina Veterinária |
| reponame_str | Repositório da Universidade de Lisboa |
| repository_id_str | urn:repositoryAcronym:ul |
| service_str_mv | urn:repositoryAcronym:ul |
| spelling | engUniversidade de Lisboa. Faculdade de Medicina Veterináriapt_PTBesnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor a vaccine to be used in Europe is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neospora caninum and Toxoplasma gondii, which are closely related to B. besnoiti, PDI plays an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we presented the nucleotide sequence of the B. besnoiti PDI gene and a 3D theoretical model was built by comparative homology using Swiss-Model server. B. besnoiti expresses a PDI with 471 amino acids, structurally similar to human and yeast PDIs, with four thioredoxin-like domains a, b, b’, a’ and a C-terminal extension c. The a and a’ domains present the characteristic active site pattern CxxC, in this case CGHC and CGYC, respectively. Analysis of the phylogenetic tree for PDI within the phylum Apicomplexa reinforced the close relationship among B. besnoiti, N. caninum and T. gondii. Recombinant B. besnoiti PDI (recBbPDI) and truncated versions corresponding to domains a, b, b’ and a’c were expressed in a heterologous system. Mice were immunized with recBbPDI for the production of monoclonal antibodies (mAbs) by hybridoma technology and four mAbs were produced and characterized. RecBbPDI and domain a’c (recBb-a’c) were functionally active and exhibited a dose dependent cross-linking activity of insulin. In the presence of bacitracin, tocinoic acid, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and 4-chloromercuribenzoic acid (pCMBA) activity of both enzymes was inhibited, in a dose dependent manner. The same happened with recBbPDI in the presence of mAbs (with the exception of T8a), but not with recBb-a’c, whose activity was not sensitive to the presence of mAbs. In vitro proliferation of B. besnoiti tachyzoites was diminished in the presence of PDI inhibitors and anti-PDI mAbs, indicating that this enzyme seems to intervene in the process of host cell adhesion/invasion. In this way, considering the inhibitions obtained, both in the host cell invasion ability and in the enzyme catalytic activity, PDI can represent a potential target for addressing the treatment and/or prevention of besnoitiosis. The panel of monoclonal antibodies here developed represents an important tool for future studies.application/pdfpt_PTProtein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune responseMarcelino, Eduardo Miguel Baptista FerreiraLeitão, José Alexandre da Costa Perdigão e CameiraNovo, Carlos Manuel MendesHostingInstitutionOrganizationalRepositório Científico de Acesso Aberto da ULisboae-mailmailto:repositorio@reitoria.ulisboa.ptrepositorio@reitoria.ulisboa.ptURNurn:tid:1012629732017-06-30T00:30:11Z2016-06-072016-06-07T00:00:00ZHandlehttp://hdl.handle.net/10400.5/11693http://purl.org/coar/access_right/c_abf2open accessBesnoitia besnoitiprotein disulfide isomerasePDImonoclonal antibodyhost cell invasionenzima isomerase de dissulfuretoanticorpos monoclonaisinvasão célula hospedeira4980136 bytesliteraturehttp://purl.org/coar/resource_type/c_db06doctoral thesis2016-06-07http://creativecommons.org/licenses/by/4.0/http://purl.org/coar/access_right/c_abf2application/pdffulltexthttps://repositorio.ulisboa.pt/bitstreams/a8aff77d-fb06-4a8b-b12a-bdef2f658400/download |
| spellingShingle | Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response Marcelino, Eduardo Miguel Baptista Ferreira Besnoitia besnoiti protein disulfide isomerase PDI monoclonal antibody host cell invasion enzima isomerase de dissulfureto anticorpos monoclonais invasão célula hospedeira Marcelino, Eduardo Miguel Baptista Ferreira Besnoitia besnoiti protein disulfide isomerase PDI monoclonal antibody host cell invasion enzima isomerase de dissulfureto anticorpos monoclonais invasão célula hospedeira |
| status | SINGLETON |
| subject.fl_str_mv | Besnoitia besnoiti protein disulfide isomerase PDI monoclonal antibody host cell invasion enzima isomerase de dissulfureto anticorpos monoclonais invasão célula hospedeira |
| title | Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response |
| title_full | Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response |
| title_fullStr | Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response |
| title_full_unstemmed | Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response |
| title_short | Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response |
| title_sort | Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response |
| topic | Besnoitia besnoiti protein disulfide isomerase PDI monoclonal antibody host cell invasion enzima isomerase de dissulfureto anticorpos monoclonais invasão célula hospedeira |
| topic_facet | Besnoitia besnoiti protein disulfide isomerase PDI monoclonal antibody host cell invasion enzima isomerase de dissulfureto anticorpos monoclonais invasão célula hospedeira |
| url | http://hdl.handle.net/10400.5/11693 |
| visible | 1 |