Molecular characterisation of two Ornithodoros savignyi enzyme isoforms belonging to the 5'-nucleotidase family

• Main Author: Stutzer, Christian
• Other Authors: Prof A W H Neitz
• Published: 2013
• Subjects: Enzymes; Proteins; Immune cells; Platelets; UCTD
• Online Access: http://hdl.handle.net/2263/25634; http://upetd.up.ac.za/thesis/available/etd-01282009-171755/

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. === Dissertation (MSc)--University of Pretoria, 2009. === Biochemistry === unrestricted