Cloning and expression of a functionally active truncated N-glycosylated KSHV complement regulatory protein and immunohistochemical studies with the anti-KCP peptide antibody

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dc.contributor.advisorKotwal, Girish Jen_ZA
dc.contributor.authorGomes Pereira, Neuza Alexandraen_ZA
dc.date.accessioned2017-10-13T07:47:36Z
dc.date.available2017-10-13T07:47:36Z
dc.date.issued2005en_ZA
dc.date.updated2017-07-14T08:23:38Z
dc.description.abstractKaposi sarcoma herpes virus (KSHV) is a typical DNA virus that is associated with a number of proliferative diseases including Kaposi's sarcoma. The KSHV open reading frame (ORF) 4 encodes a complement regulatory protein (Kaposi complement-binding protein, KCP) that binds complement proteins and inhibits the complement-mediated lysis of cells infected by the virus, thus providing a strategy for evasion of the host complement system. Kaposi's sarcoma is an angiogenic skin lesion that has been recognized as one of the most abundant tumours found in many parts of Southern Africa and which can occasionally become highly invasive, aggressive and capable of causing death, particularly amongst AIDS patients. It is of major significance to understand how complement control proteins (CCPs) such as KCP perform their biological functions at the molecular and structural levels, because of their potentials as therapeutic agents, their implications in the pathology and importance in the etiology of many disease conditions. This study was therefore undertaken to characterise the structure-function relationship of KCP. Based on primary sequence analysis and comparison to other functionally and structurally similar proteins, oligonucleotide primers were designed to amplify by PCR, three regions of the predicted ORF 4 from human herpes virus-8 (llliV-8) DNA isolated from a primary effusion lymphoma cell line. The PCR products were inserted by ligation into the expression vector pPIC9 to generate three recombinant plasmids for heterologous expression in the yeast, Pichia pastoris and to produce separately, the 4 N-terminal Sushi domains (KCP-S, small), KCP protein lacking the putative transmembrane binding domain (KCP-M, medium) and the full-length protein (KCPF, full). Expression of the viral proteins was confirmed by SDS-PAGE and Western blot analyses using a rabbit polyclonal antibody directed against a selected peptide region that is common to all three recombinant KCPs. All the KCP proteins migrated electrophoretically as higher bands compared to their expected sizes. The lower mobilities of the proteins may be due to g1ycosy1ation since there are potential N-and O-glycosylation sites in the protein's primary sequence. Also, diffused bands were obtained in all the electrophoretic gels and Western blots carried out, which is characteristic of glycoproteins. Furthermore, the antibody recognized several larger and smaller bands that may represent aggregates and/or degradation products respectively. Both partially purified KCP-S and KCP-S directly from expression media were able to inhibit complement-mediated lysis of sensitized sheep erythrocytes by approximately 60% in a hemolysis assay. This result confirms previous reports that recombinant KCP is twice more efficient in inhibiting the classical pathway-mediated lysis of erythrocytes than the vaccinia virus complement control protein (VCP), which also contains 4 Sushi domains. The KCP-F and KCP-M proteins did not show any significant complement inhibitory activities. Preliminary immunohistochemical studies using the same antibody were carried out to determine the expression and distribution of KCP proteins in Kaposi's sarcoma.en_ZA
dc.identifier.apacitationGomes Pereira, N. A. (2005). <i>Cloning and expression of a functionally active truncated N-glycosylated KSHV complement regulatory protein and immunohistochemical studies with the anti-KCP peptide antibody</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Virology. Retrieved from http://hdl.handle.net/11427/25664en_ZA
dc.identifier.chicagocitationGomes Pereira, Neuza Alexandra. <i>"Cloning and expression of a functionally active truncated N-glycosylated KSHV complement regulatory protein and immunohistochemical studies with the anti-KCP peptide antibody."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Virology, 2005. http://hdl.handle.net/11427/25664en_ZA
dc.identifier.citationGomes Pereira, N. 2005. Cloning and expression of a functionally active truncated N-glycosylated KSHV complement regulatory protein and immunohistochemical studies with the anti-KCP peptide antibody. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Gomes Pereira, Neuza Alexandra AB - Kaposi sarcoma herpes virus (KSHV) is a typical DNA virus that is associated with a number of proliferative diseases including Kaposi's sarcoma. The KSHV open reading frame (ORF) 4 encodes a complement regulatory protein (Kaposi complement-binding protein, KCP) that binds complement proteins and inhibits the complement-mediated lysis of cells infected by the virus, thus providing a strategy for evasion of the host complement system. Kaposi's sarcoma is an angiogenic skin lesion that has been recognized as one of the most abundant tumours found in many parts of Southern Africa and which can occasionally become highly invasive, aggressive and capable of causing death, particularly amongst AIDS patients. It is of major significance to understand how complement control proteins (CCPs) such as KCP perform their biological functions at the molecular and structural levels, because of their potentials as therapeutic agents, their implications in the pathology and importance in the etiology of many disease conditions. This study was therefore undertaken to characterise the structure-function relationship of KCP. Based on primary sequence analysis and comparison to other functionally and structurally similar proteins, oligonucleotide primers were designed to amplify by PCR, three regions of the predicted ORF 4 from human herpes virus-8 (llliV-8) DNA isolated from a primary effusion lymphoma cell line. The PCR products were inserted by ligation into the expression vector pPIC9 to generate three recombinant plasmids for heterologous expression in the yeast, Pichia pastoris and to produce separately, the 4 N-terminal Sushi domains (KCP-S, small), KCP protein lacking the putative transmembrane binding domain (KCP-M, medium) and the full-length protein (KCPF, full). Expression of the viral proteins was confirmed by SDS-PAGE and Western blot analyses using a rabbit polyclonal antibody directed against a selected peptide region that is common to all three recombinant KCPs. All the KCP proteins migrated electrophoretically as higher bands compared to their expected sizes. The lower mobilities of the proteins may be due to g1ycosy1ation since there are potential N-and O-glycosylation sites in the protein's primary sequence. Also, diffused bands were obtained in all the electrophoretic gels and Western blots carried out, which is characteristic of glycoproteins. Furthermore, the antibody recognized several larger and smaller bands that may represent aggregates and/or degradation products respectively. Both partially purified KCP-S and KCP-S directly from expression media were able to inhibit complement-mediated lysis of sensitized sheep erythrocytes by approximately 60% in a hemolysis assay. This result confirms previous reports that recombinant KCP is twice more efficient in inhibiting the classical pathway-mediated lysis of erythrocytes than the vaccinia virus complement control protein (VCP), which also contains 4 Sushi domains. The KCP-F and KCP-M proteins did not show any significant complement inhibitory activities. Preliminary immunohistochemical studies using the same antibody were carried out to determine the expression and distribution of KCP proteins in Kaposi's sarcoma. DA - 2005 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2005 T1 - Cloning and expression of a functionally active truncated N-glycosylated KSHV complement regulatory protein and immunohistochemical studies with the anti-KCP peptide antibody TI - Cloning and expression of a functionally active truncated N-glycosylated KSHV complement regulatory protein and immunohistochemical studies with the anti-KCP peptide antibody UR - http://hdl.handle.net/11427/25664 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/25664
dc.identifier.vancouvercitationGomes Pereira NA. Cloning and expression of a functionally active truncated N-glycosylated KSHV complement regulatory protein and immunohistochemical studies with the anti-KCP peptide antibody. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Virology, 2005 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/25664en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDivision of Virologyen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherMedical Virologyen_ZA
dc.titleCloning and expression of a functionally active truncated N-glycosylated KSHV complement regulatory protein and immunohistochemical studies with the anti-KCP peptide antibodyen_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMSc (Med)en_ZA
uct.type.filetype
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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