Biochemical studies on trypanosomal prolyl oligopeptidase family pathogenic factors.

Abstract

African Animal trypanosomosis, also known as Nagana, is a parasitic disease which affects many mammalian species, mainly livestock such as cattle, sheep and goats. The disease also affects humans (Human African Trypanosomosis) and in this case is referred to as sleeping sickness. Nagana is caused by the Trypanosoma parasite, which is transmitted to the host by a bite from the tsetse fly (Glossina spp). The Trypanosoma causing trypanosomosis in animals are Trypanosoma congolense, T. vivax and T. brucei brucei. Vaccine development has been unsuccessful, due to the presence of the variant surface glycoprotein on the surface of parasites which undergoes antigenic variation therefore enabling the parasite to avoid detection by vaccines. A chemotherapeutic drug such as isometamidium chloride combined with diminazene and suramin have also had little success due to the increase in drug resistance. During infection of the host, trypanosomal parasites utilise various proteolytic enzymes such as the oligopeptidases, which hydrolyse important host factors such as peptide hormones. These proteolytic enzymes are thus considered to be pathogenic factors which contribute to the manifestation of various trypanosomosis symptoms such as anaemia, fever, paralysis and disturbances in sleep cycle patterns. It is these pathogenic factors which are now being considered as drug targets in the hope to eradicate the spread or continuous advancement of trypanosomosis. Three trypanosomal pathogenic factors, which are serine oligopeptidases which belong to the prolyl oligopeptidase family of serine proteases (Clan SC in subfamily S9) were the focus of this study, namely, prolyl oligopeptidase (POP) from T. b. brucei (TbPOP) and T. congolense (TcoPOP) as well as oligopeptidase B (OPB) from T. congolense (TcoOPB). The full length TbPOP gene was cloned into pTZ57R/T cloning vector and successfully sub-cloned into pET32a expression vector and recombinantly expressed in its insoluble form at a size of approximately 100 kDa using the Escherichia coli BL21 DE3 expression system. TbPOP expression was confirmed by western blot probed with anti-His tag antibodies. Expression of TbPOP was optimised under varying temperatures and IPTG concentrations in an attempt to solubilise the inclusion bodies. However, the protein was expressed as part of inclusion bodies. Therefore, urea denaturation was used for its solubilisation. Following solubilisation, recombinant TbPOP was partially purified on a Ni2+ affinity resin. Further attempts to purify TbPOP by molecular exclusion chromatography (MEC) were unsuccessful, this could be due to aggregation of the protein during the refolding step. Therefore refolding by a Sephadex G-25 desalting

column was attempted as it removes some impurities. However, further purification by MEC and ion exchange chromatography (IEC) were unsuccessful. The full-length TcoPOP gene was successfully cloned into pGEM-T® cloning vector and subsequently sub-cloned into pET32a expression vector. However, upon sequencing of the plasmid DNA, it was discovered that a mutation had occurred in the recombinant TcoPOP DNA sequence forming the stop codon “TAG” which resulted in the termination of protein expression, therefore, further work on TcoPOP was not pursued. TcoOPB was successfully recombinantly expressed in pET28a using the E. coli BL21 DE3 expression system. The protein had a size of approximately 80 kDa. The protein was affinity purified using a Ni2+ affinity resin. Expression of TcoOPB was confirmed by western blot using chicken raised anti-TcoOPB antibodies. Cross-reactivity of chicken anti-TcoOPB antibodies with TbPOP was also assessed and no cross-reactivity was found which was expected as POP and OPB only share 25% sequence identity. In order to determine the biochemical characteristics of TbPOP and TcoOPB, various activity assays and kinetics studies were conducted. It was found that TbPOP was able to hydrolyse type I collagen from rat tail. In contrast however, TbPOP was unable to digest gelatin which is a denatured form of collagen. Upon further analysis of TbPOP with the synthetic peptide substrate Z-Gly-Pro-AMC, it was found not to have activity as it was unable to hydrolyse the substrate, this is thought to be due to the misfolding of the protein during the refolding step. TcoOPB on the other hand was unable to hydrolyse either collagen or gelatin. Further biochemical analysis of TcoOPB was conducted using synthetic peptide substrates, the kinetic parameters of TcoOPB Km, kcat/Km were determined and it was found that OPB had a high affinity for the substrates Z-Arg-Arg-AMC, Z-Gly-Gly-Arg-AMC, H-Ala-Phe-Lys-AMC and Z-Pro-Arg-AMC and lower affinity for the substrates H-Pro-Phe-Arg-AMC, H-D-Val-Leu-Lys-AMC, Z-Gly-Pro-AMC, Suc-Ala-Phe-Lys-AMC,Boc-Leu-Gly-Arg-AMC. OPB was also found to have an optimal pH of 8 – 9 and retained 79% of its optimal activity at the physiological pH of 7.4. TcoOPB was found not to have good diagnostic potential as an indirect ELISA revealed that the antigen was unable to detect antibodies in T. congolense infected cattle sera. This study laid the foundation to conduct further studies on TbPOP, TcoPOP and TcoOPB as chemotherapeutic and diagnostic targets for Nagana.