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Molecular characterisation of two Ornithodoros savignyi enzyme isoforms belonging to the 5'-nucleotidase family
Dissertation (MSc)--University of Pretoria, 2009.
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| Format: | Thesis |
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University of Pretoria
2013
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| _version_ | 1867613589464940544 |
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| access_status_str | Open Access |
| author2 | Neitz, A.W.H. (Albert Walter Herman) |
| author_browse | Neitz, A.W.H. (Albert Walter Herman) |
| author_facet | Neitz, A.W.H. (Albert Walter Herman) |
| collection | Thesis |
| dc_rights_str_mv | ©University of Pretoria 2008 E1224/ |
| description | Dissertation (MSc)--University of Pretoria, 2009. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/25634 |
| institution | University of Pretoria (South Africa) |
| last_indexed | 2026-06-10T12:38:33.011Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2013 |
| publishDateRange | 2013 |
| publishDateSort | 2013 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/25634 Molecular characterisation of two Ornithodoros savignyi enzyme isoforms belonging to the 5'-nucleotidase family Neitz, A.W.H. (Albert Walter Herman) Gaspar, A.R.M. (Anabella Regina Marques) Maritz-Olivier, Christine s99073732@tuks.co.za Stutzer, Christian Enzymes Proteins Immune cells Platelets UCTD Dissertation (MSc)--University of Pretoria, 2009. Haemostasis is a highly regulated system, involving a myriad of cell types (endothelium, immune cells, platelets, etc.), proteins (enzymes, receptors, etc.) and signalling molecules (sterols, nucleotides, etc.). Haematophagous organisms, such as ticks, have evolved a number of strategies to overcome host haemostatic responses to feed effectively. Salivary apyrases are a class of nucleotide-metabolising enzymes that blood-feeding parasites utilise to modulate extracellular nucleotides, like ATP and ADP, to prevent platelet activation and aggregation. This specific enzyme function has evolved in blood-feeding parasites from the ecto-ATPdase/CD39 (E-NTPDases)-, Cimex-type- and 5’-nucleotidase/CD73 enzyme families. Furthermore, most arthropod apyrases are ascribed to the 5’-nucleotidase/CD73 enzyme family. The salivary apyrase from Ornithodoros savignyi has not been characterised to a specific enzyme family and the presence of 5’-nucleotidase homologs have not been demonstrated. Therefore, in this study 5’-nucleotidase homologous transcripts were identified from O. savignyi salivary gland DNA, using a 5’-nucleotidase specific degenerate primer and RACE protocols. Two full-length putative 5’-nucleotidase isoforms were identified that shared significant sequence identity and similarity to a 5’-nucleotidase from R. (B.) microplus and putative apyrases from I. scapularis and R. appendiculatus. Utilising computational tools, iso-electric points, molecular weights and cellular localisation were determined. The isoforms were predicted to be soluble secreted proteins, which correlated with the trend observed for parasitic apyrases in the 5’-nucleotidase family. Phylogenetic analysis of the 5’-nucleotidase family revealed that the O. savignyi 5’-nucleotidase isoforms claded monophyletically with the putative apyrases from I. scapularis and R. appendiculatus, excluding the 5’-nucleotidase from R. (B.) microplus. Molecular modelling of these two proteins showed a similar protein structure to a periplasmic ecto-5’-nucleotidase from E. coli. The similar architecture revealed a high conservation of key residues involved in dimetal coordination, catalysis and substrate binding, therefore a similar catalytic mechanism was proposed. It was hypothesised that the isoforms identified may be putative apyrases. To test this hypothesis, the 5’-nucleotidase isoform I was recombinantly expressed in yeast. Cross-reactivity was demonstrated with a polyclonal anti-apyrase antibody produced from O. savignyi native apyrase. The latter implied that the native apyrase may be a member of the 5’-nucleotidase enzyme family. However, no sequence information for native apyrase was available for comparison and therefore native enzyme was purified with ion exchange chromatography. Subsequent, Edman N-terminal sequencing and MS/MS analysis with purified enzyme identified peptide sequence fragments that shared a high degree of sequence identity with both 5’-nucleotidase isoforms. It was concluded that native apyrase is a mixture of the isoforms identified from O. savignyi salivary gland DNA. These results represent the first confirmation of a tick apyrase that belongs to the 5’-nucleotidase family of enzymes. Further confirmation will be achieved by testing activity of the recombinant protein and future experiments may assess the potential of this protein as a vaccine candidate. Biochemistry unrestricted 2013-09-06T22:45:11Z 2009-04-16 2013-09-06T22:45:11Z 2008-09-03 2009-04-16 2009-01-28 Dissertation 2008 E1224/gm http://hdl.handle.net/2263/25634 http://upetd.up.ac.za/thesis/available/etd-01282009-171755/ ©University of Pretoria 2008 E1224/ application/pdf University of Pretoria |
| spellingShingle | Enzymes Proteins Immune cells Platelets UCTD Molecular characterisation of two Ornithodoros savignyi enzyme isoforms belonging to the 5'-nucleotidase family |
| title | Molecular characterisation of two Ornithodoros savignyi enzyme isoforms belonging to the 5'-nucleotidase family |
| title_full | Molecular characterisation of two Ornithodoros savignyi enzyme isoforms belonging to the 5'-nucleotidase family |
| title_fullStr | Molecular characterisation of two Ornithodoros savignyi enzyme isoforms belonging to the 5'-nucleotidase family |
| title_full_unstemmed | Molecular characterisation of two Ornithodoros savignyi enzyme isoforms belonging to the 5'-nucleotidase family |
| title_short | Molecular characterisation of two Ornithodoros savignyi enzyme isoforms belonging to the 5'-nucleotidase family |
| title_sort | molecular characterisation of two ornithodoros savignyi enzyme isoforms belonging to the 5 nucleotidase family |
| topic | Enzymes Proteins Immune cells Platelets UCTD |
| url | http://hdl.handle.net/2263/25634 http://upetd.up.ac.za/thesis/available/etd-01282009-171755/ |