Title page for ETD etd-01282009-171755


Document Type Master's Dissertation
Author Stutzer, Christian
Email s99073732@tuks.co.za
URN etd-01282009-171755
Document Title Molecular characterisation of two Ornithodoros savignyi enzyme isoforms belonging to the 5'-nucleotidase family
Degree MSc
Department Biochemistry
Supervisor
Advisor Name Title
Dr A R M Gaspar Co-Supervisor
Dr C Maritz-Olivier Co-Supervisor
Prof A W H Neitz Supervisor
Keywords
  • enzymes
  • proteins
  • immune cells
  • platelets
Date 2008-09-03
Availability unrestricted
Abstract

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.

©University of Pretoria 2008

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