Doctoral Degrees (Biochemistry)

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Antibody-mediated inhibition of proteases of African trypanosomes.
(2006) Huson, Laura.; Coetzer, Theresa Helen Taillefer.
The protozoan parasites Trypanosoma congolense and T. vivax cause trypanosomosis in cattle. The major lysosomal cysteine proteinase of T. congolense, congopain, may contribute to pathogenesis of the disease, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. Oligopeptidase B, a trypanosomal serine peptidase, is also a potential virulence factor in African trypanosomes because it is released into the host circulation by dead or dying parasites, where it retains catalytic activity due to the enzyme's insensitivity to serum protease inhibitors. The vaccine potential of the catalytic domain of congopain, C2, and oligopeptidase B complexed with 0'2-macroglobulin (0'2M) was evaluated by producing antibodies in rabbits. Inhibition of congopain and oligopeptidase B activity by these antibodies was assessed. The oligopeptidase B open reading frame from T. congolense and T. vivax was cloned and expressed in Escherichia coli, from which active recombinant enzymes were purified. These recombinant enzymes exhibited trypsin-like specificity for peptide substrates, cleaving on the carboxy side of basic amino acid residues such as arginine and lysine. Enzymes were found to be optimally active between pH 8 and 10, optimally stable at pH 6, and showed activation by reducing agents and sensitivity to ionic strength. The enzymes showed typical oligopeptidase B-like inhibitor profiles, except that they were not inhibited by thiol sensitive inhibitors such as iodoacetamide and Nethylmaleimide. High yields of bovine and rabbit 0'2M were isolated by a three-step procedure of fractionation by PEG 6000, and zinc chelate and Sephacryl S-300 HR chromatography. Congopain, its catalytic domain C2, papain and cathepsin L all cleaved the bait region of bovine 0'2M and became trapped inside the 0'2M molecule, where their activity against large molecular weight substrates was inhibited. C2 could thus be complexed with 0'2M directly or used to form C2-0'2M-oligopeptidaseB complexes for immunisation purposes. iv The catalytic domain of congopain, C2, was used to immunise rabbits either without adjuvant, as a water-in-oil emulsion with Freund's adjuvant, or in a complex with either bovine or rabbit U2M. Freund's adjuvant elicited the highest anti-C2 antibody response. However, the greatest inhibition, 65%, of C2 activity against Z-Phe-Arg-AMC was obtained with antibodies produced by rabbits receiving C2-U2Mcomplexes. In a second study, C2 and oligopeptidase B were used to immunise rabbits , either in alum, or complexed to bovine U2M. Anti-C2 antibody levels were highest in rabbits immunised with the free proteins in alum, whereas anti-oligopeptidase B antibody levels were comparable for each adjuvant system. Anti-oligopeptidase antibodies produced with alum gave 100% inhibition of oligopeptidase B activity. In contrast, antibodies produced against C2-u2M-oligopeptidase B complexes had little effect on oligopeptidase B activity. However, these antibodies inhibited 55% of C2 activity. Alum was a slightly less efficient adjuvant for C2 and 50% inhibition of C2 activity was observed. It appeared that immunisation of rabbits with C2 complexed to U2M resulted in the production of antibodies that were better able to neutralise the proteolytic activity of C2 and congopain in vitro than that with conventional adjuvants . The immunisation of C2 complexed to bovine u2-macroglobulin therefore has the potential to neutralise parasite congopain in vivo, and may contribute to an anti-disease vaccine against African trypanosomosis. Complexation of oligopeptidase B to u2M offers no benefit, since antibodies produced against this complex are not able to inhibit the activity of oligopeptidase B. Immunisation with oligopeptidase B in alum is sufficient to produce efficient enzyme-inhibiting antibodies in the context of an anti-disease vaccine against African trypanosomosis.
Characterisation of infectious bursal disease virus (IBDV) polyprotein processing.
(2011) Vukea, Phillia Rixongile.; Coetzer, Theresa Helen Taillefer.
Infectious bursal disease virus (IBDV) is a birnavirus that infects the B-cells in the bursa of Fabricius of young chickens, causing Gumboro disease. The IBDV 114 kDa polyprotein (NH2-pVP2-VP4-VP3-COOH) is thought to be processed at 512Ala-Ala513 and 755Ala-Ala756 through the proteolytic activity of VP4, a serine protease which uses a Ser/Lys catalytic dyad, to release pVP2, VP4 and VP3. Precursor VP2 (pVP2) is further processed at its C-terminus to generate VP2 and structural peptides through the cleavage of the 441Ala-Phe442, 487Ala-Ala488, 494Ala-Ala495 and 501Ala-Ala502 peptide bonds to release VP2 and four structural peptides, pep46, pep7a, pep7b and pep11. While the processing at the 441Ala-Phe442 site was shown to be mediated by the endopeptidase activity of VP2, the processing at the other two sites is not well understood. The products resulting from the processing of the IBDV polyprotein were previously identified by anti-VP2 and anti-VP3 antibodies. The present study used anti-VP4 peptide antibodies to identify products resulting from the IBDV polyprotein processing. It was hypothesised that VP4 exists in two forms, the embedded form which exists as an integral part of the polyprotein and a mature form which is released after the processing. In order to characterise the two forms of VP4, six different fragments i.e. full-length polyprotein (Met1-Glu1012), truncated polyprotein (Ile227-Trp891), VP4-RA (Arg453-Ala755), VP4-RK (Arg453-Lys722), VP4-ΔVP3 (Ala513-Trp891, called VP4-AW for the sake of simplicity) and VP4-AA (Ala513-Ala755) were amplified from the IBDV dsRNA, cloned into a T-vector and sub-cloned into several expression vectors. The constructs were sequenced prior to expression. The sequence of the polyprotein coding region was used to determine the pathotype of the isolate used for viral dsRNA isolation. This isolate was from IBDV-infected bursae harvested from commercial chickens during an IBD outbreak in KwaZulu-Natal, South Africa in 1995, thus naming the isolate SA-KZN95. The comparison of the deduced amino acid sequence of SA-KZN95 polyprotein with 52 sequences of other IBDV strains highlighted 21 residues which could be molecular markers of different IBDV pathotypes. The residues of SA-KZN95 were identical to those of the Malaysian very virulent UPM94/273 strain. The constructs representing the embedded and mature forms of VP4 were recombinantly expressed. Processing was observed from the expression of the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, but not from VP4-AA expression. The mutation of the Ser/Lys catalytic dyad in the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, prevented processing thus verifying that the proteolytic activity was due to VP4. Anti-VP4 peptide antibodies were raised in chickens for the identification of the polyprotein cleavage products. The anti-VP4 peptide antibodies detected more cleavage products than expected from the polyprotein, suggesting that additional or different cleavage sites may be used. The characterisation of the cleavage products suggested that the processing for the release of VP4 occurs either at the 487Ala-Ala488 or the 512Ala-Ala513 site in a single polyprotein molecule. Ultimately, an IBDV polyprotein processing strategy that would explain the release of the additional products was proposed in the present study. The present study also illustrated the importance of Pro377 in the processing of the polyprotein where its replacement with Leu induced a prominent change in polyprotein processing. The mutation seemed to induce structural changes that may possibly affect the cleavage sites. Although no autocatalytic activity was observed during the expression of VP4-AA (mature form), it cleaved mutant VP4-RK in trans. It seemed to be active as a dimer on a gelatine gel but no activity was observed against a dialanyl fluorogenic peptide substrate. It also appeared to form peptidase-inhibitor complexes with anti-thrombin III. The present study also describes attempts to detect native VP4 in IBDV-infected bursa homogenates by anti-VP4 peptide antibodies on a western blot and by proteolytic activity determination on gelatine-containing SDS-PAGE gels. The findings of the study provide new information that may contribute to the development of anti-viral agents. These anti-viral agents may target polyprotein processing, capsid assembly and thus prevent virus replication during IBDV infection.
Gene disruption of TcoCATL (Congopain) and oligopeptidase B, pathogenic factors of African trypanosomes.
(2011) Kangethe, Richard Thiga.; Coetzer, Theresa Helen Taillefer.
African trypanosomosis is a parasitic disease in man and animals caused by protozoan parasites of the genus Trypanosoma. T. congolense, T. vivax and T. brucei brucei cause nagana in cattle. The variable nature of the parasite surface coat has hindered the development of an effective vaccine. An option for developing vaccines and chemotherapeutic agents against trypanosomosis is to target pathogenic factors released by the parasite during infection, namely an “anti-disease” approach. Two pathogenic factors released during infection are oligopeptidase B (OPB) and TcoCATL (congopain). TcoCATL, a major lysosomal cysteine peptidase, is a member of the papain family C1 cysteine peptidases. RNA interference (RNAi) was used to down-regulate the expression of TcoCATL in T. congolense IL3000 TRUM183:29-13 parasites in vivo during mouse infections. TcoCATL RNAi was monitored in infected mouse blood by comparing the hydrolysis of Z-Phe-Arg-AMC and parasitaemia between mice in which RNAi was induced and control mice. Mice infected with parasites induced for TcoCATL RNAi had lower parasitaemia when compared to control mice. An attempt was also made at deleting the entire CATL gene array in both T. congolense IL3000 and T. brucei 427 Lister strains. The second pathogenic factor studied, OPB, is a cytosolic trypanosomal peptidase that hydrolyses peptides smaller than 30 amino acid residues, C-terminal to basic residues. In order to evaluate the role that OPB play during disease, RNAi was also applied to knock-down the expression levels of OPB in T. brucei T7T and T. congolense IL3000 TRUM183:29-13 strains (TbOPB and TcoOPB respectively). Oligopeptidase B null mutant strains (Δopb) were also generated in T. brucei brucei Lister 427. An attempt was also made to generate OPB null mutants in T. congolense IL3000 parasites. Western blot analysis of the knock-down experiments using chicken anti-TcoOPB peptide IgY showed that only TbOPB levels were reduced in T. brucei T7T parasites induced for RNAi when compared to TcoOPB RNAi induced cultures. Quantitative assessment of a fourteen day induction experiment for OPB RNAi in T. brucei showed an 87% reduction in TbOPB levels when compared to levels on day one. There was no growth effect observed in T. brucei parasites cultured in vitro and induced for TbOPB RNAi. It was concluded that TbOPB is not necessary for the in vitro survival of T. brucei parasites, thus making the generation of OPB null mutants possible. Δopb T. brucei parasites were successfully generated and grew normally in vitro and were as virulent as wild type strains during infection in mice. Immunohistopatholgy of infected mouse testes revealed Δopb parasites in extra vascular regions showing that T. brucei OPB (TbOPB) is not involved in assisting T. brucei parasites to cross microvascular endothelial cells. Gelatin gel analysis of Δopb null mutants and wild type strains showed an increase in cysteine peptidase activity. Enzymatic activity assays were carried out to identify how closely related oligopeptidases are affected by knocking out TbOPB, and a significant increase of T. brucei prolyl oligopeptidase (TbPOP) activity was observed. However, western blot analysis did not show any increase of TbPOP protein levels in Δopb parasites, suggesting that either TbOPB is responsible for generating an endogenous inhibitor for TbPOP or that another POP-like enzyme might compensate for a loss in OPB activity in Δopb null mutants. This study made a significant contribution to an understanding of the interplay between different trypanosomal peptidases that are important pathogenic factors in trypanosomosis. It highlights the need to simultaneously target several trypanosomal peptidases to develop an effective vaccine or chemotherapeutic agents for African animal trypanosomosis.
Identification and characterisation of novel pathogenic factors of Trypanosoma congolense.
(2010) Pillay, Davita.; Coetzer, Theresa Helen Taillefer.
Trypanosoma congolense is a major causative agent of the bovine disease trypanosomosis which has a considerable economic impact on sub-Saharan Africa. Current control methods for trypanosomosis are unsatisfactory and vaccine development has been hampered by antigenic variation. An anti-disease vaccine is based on the idea that disease is caused by the pathogenic factors released by the parasite, rather than by the parasite itself. Therefore, if these pathogenic factors could be neutralised by antibodies produced by vaccination, the disease could be circumvented. The method used here for identification of novel pathogenic factors is based on the concept that trypanotolerant cattle are able to mitigate the disease by generating a specific immune response against a few key antigens (pathogenic factors). Two immuno-affinity columns were therefore prepared: one containing IgG from noninfected sera and a second column containing IgG from trypanotolerant N’Dama cattle serially infected with T. congolense. The differential binding of antigens to the two columns allowed identification of antigens specifically recognised by the immune system of a trypanotolerant animal, i.e. potential pathogenic factors. The most promising antigens identified included several variant cathepsin L-like cysteine peptidases (CPs) and the Family M1 Clan MA aminopeptidases (APs). For the CPs, a study of the genetic organisation was conducted in order to further understand the variability present in this gene family. To this end, two different mini-libraries of cathepsin L-like genes were prepared: one in which genes as different as possible from congopain (the major CP of T. congolense) were selected, and a second which contained all possible genes present in the congopain array. Analysis of the sequences obtained in these two mini-libraries showed that there was significant variability of the genes within the congopain array. Two variants of CPs, chosen for differences in their catalytic triads, were cloned for expression. The recombinantly expressed CP variants differed in substrate preferences from one another and from C2 (the recombinant truncated form of congopain), and surprisingly, all enzymes were active at physiological pH. The two APs were cloned and expressed as insoluble inclusion bodies in an E. coli system, and subsequently refolded. The refolded APs showed a substrate preference for H-Ala-AMC, an optimum pH of 8.0, localisation to the cytoplasm and inhibition by puromycin. The two APs were not developmentally regulated and present in procyclic, metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro. Immunisation of BALB/c mice with the APs did not provide protection when challenged with T. congolense. For an anti-disease vaccine to be protective, it would possibly have to include all pathogenic factors, including the two APs and at least one CP described in the present study.
Molecular characterisation of metacaspase 5 and the production of oligopeptidase b-specific single chain variable fragment antibodies for potential animal African trypanosomosis chemotherapies and diagnostics.
(2018) Eyssen, Lauren Elizabeth-Ann.; Coetzer, Theresa Helen Taillefer.
African trypanosomosis (AT) is a major obstacle in the establishment of agriculture and economic sustainability in Africa. Animal AT is responsible for large numbers of livestock succumbing to the tsetse transmitted kinetoplastid parasites, Trypanosoma congolense and T. vivax, and as a result, losses in further downstream sectors are experienced. Due to the ability of the trypanosomal parasites to undergo antigenic variation, vaccine candidates are highly unlikely. Peptidases have been identified as virulence factors and are the focus of the development of novel chemotherapies and diagnostics. The metacaspases (MCAs) are a prime example of a chemotherapeutic target and oligopeptidase B (OPB), that of a diagnostic target. Towards the validation of a chemotherapeutic target, recombinant expression was used to obtain an active peptidase which could be enzymatically characterised. Various inhibitors were investigated and their effect on the parasite, analysed. Current diagnostics are based on antibody detection, but an antigen detection format would be preferable as it could differentiate between active and cured infections as anti-trypanosomal antibodies can persist for years. Given the rural, resource-poor locations in the areas of AT incidence, an ideal rapid diagnostic test (RDT) would be robust, affordable, sensitive and specific and requiring minimal training, such as a dipstick test. The MCAs are cysteine peptidases which are found in all kingdoms other than the metazoa, and share a secondary structure fold and catalytic dyad with the metazoan caspases. Since the caspases play a role in apoptosis, it is thought that the MCAs may function in a similar manner. The single copy MCAs of Trypanosoma spp. and Leishmania spp. differ from the multicopy MCAs in that they possess a Pro-, Gln-, Tyr-rich C-terminal domain which is thought to mediate protein-protein interactions. The activity of the single copy MCAs from T. cruzi and L. major has been implicated in the cell cycle of the kinetoplastid parasite. The aim of the project was to express, purify and enzymatically characterise the recombinant and native MCA5 from T. congolense and T. vivax. Using the 3D structures, solved by X-ray diffraction, of MCA2 from T. b. brucei, molecular docking studies were used to validate the inhibition potential of a published library of inhibitors, designed based on the, then, hypothetical structure of TbbMCA2. Since the elucidation of the 3D structure of TbbMCA2 by X-ray diffraction, the inhibitory power of the library of inhibitors against TbbMCA2 and the MCA5s was investigated. The serine peptidase, OPB, has been shown to be released into the host bloodstream by dead and dying parasites. The use of phage displayed scFv (single chain fragment variable) antibodies for the detection of OPB in serum from infected cattle is reported, towards the development of a RDT. Recombinantly expressed TcoMCA5 was shown to autoprocess and over autoprocess when purified using nickel affinity chromatography. Mutagenesis of the catalytic dyad residues reduced the over autoprocessing and the mutated form was enzymatically active at a pH between 6 and 9. This active mutant and purified TcoMCA5 showed a prefence for Arg over Lys at the P1 substrate position and were able to hydrolyse gelatin. Possible novel inhibitors of TbbMCA2 and the MCA5s of T. congolense and T. vivax were identified using a library of ligands (Berg library) based on the P1 specificity of TbbMCA2 and molecular docking. Commercial fluorogenic peptide substrates and inhibitors reported in literature for the characterisation of various MCAs, revealed interactions with the MCAs which should be taken into consideration when modifying the Berg ligands to achieve higher affinity for the MCAs. The application of scFv antibodies, derived from the Nkuku® phagemid library, for the diagnosis of current AAT infections by the detection of OPB, released in the bloodstream of the infected mammalian host, was investigated. After the successful isolation and production of OPB-specific scFv, MCA-specific scFv antibodies can be pursued using the Nkuku® phagemid library. The resulting OPB-specific scFv identified a conserved peptide between T. congolense and T. vivax and was able to detect native OPB in a western blot format. It was predicted that the scFv interacted with OPB in such a way that it would restrict the hinge motion between the C-terminal catalytic and N-terminal regulatory domains of the enzyme and limit access to the active site pocket. The ability of scFv and rabbit-anti-OPB polyclonal antibody in an antigen detection ELISA with sera from T. congolense infected cattle indicated that detection of OPB fluctuated with parasitaemia.
A serine oligopeptidase from African Trypanosomes.
(1998) Morty, Rory Edward.; Lonsdale-Eccles, John David.; Coetzer, Theresa Helen Taillefer.
Protozoan parasites of the genus Trypanosoma are responsible for chronic and widespread disease in livestock and humans in Africa. This study describes the purification and characterisation of a serine oligopeptidase from Trypanosoma brucei brucei and from T. congolense. Serine peptidase activity has previously been described for T. b. brucei although the responsible enzyme was not purified to electrophoretic homogeneity. In the present study this enzyme was purified from bloodstream-form T. b. brucei by a combination of three-phase partitioning, ion-exchange, affinity and molecular exclusion chromatography. Characterisation of the enzyme revealed that it closely resembled a bacterial serine oligopeptidase, Escherichia coli oligopeptidase B, in terms of cleavage-site specificity, inhibition characteristics and molecular mass. Its overall properties indicate that it is probably a serine oligopeptidase and we have called it OP-Tb (oligopeptidase from Trypanosoma brucei). Antibodies to OP-Tb were prepared in chickens. These antibodies were used in the purification of a similar enzyme, designated OP-Tc, from T. congolense. OP-Tc closely resembled OP-Tb in its enzymatic properties. OP-Tb appears to be monomeric, with an apparent molecular mass of 80 kDa. Activity is optimal between pH 8.0 and 10.0, and is enhanced in the presence of reducing agents. Inhibition by 4-(2-aminoethyl)benzenesulfonylfluoride, 3,4-dichloroisocoumarin and diisopropylfluorophosphate indicates that the enzyme may be classified as a serine protease. While various natural and synthetic fluorogenic peptide substrates were hydrolysed by OP-Tb, larger potential substrates (proteins) were not. Studies of the digestion of naturally occurring bioactive peptides suggested that substrates were restricted to peptides smaller than approximately 4 or 5 kDa. These peptides were cleaved at the carboxy side of basic amino acid residues such as arginine and lysine. This is characteristic of a trypsin-like specificity. Because the enzyme is known to be readily released from the parasites, and because it was possible to detect OP-Tb-like activity in the blood of T. b. brucei-infected mammalian hosts, it appears that the enzyme is released into the host bloodstream where it remains uninhibited by endogenous protease inhibitors. Indeed, OP-Tb was not inhibited by mammalian plasma serpins or 012-macroglobulin in vitro. This, and the degradation of host peptide regulatory hormones in vitro, suggests that OP-Tb may have secondary, but important, extracellular roles in the pathogenesis of African trypanosomiasis. A variety of serine protease inhibitors, including inhibitors of OP-Tb were tested for their potential as trypanocidal agents. The results from both in vitro and in vivo studies, suggest that inhibitors of trypanosome oligopeptidases are promising new lead targets for drug development. Furthermore, data presented here also shows that OP-Tb is efficiently inhibited by several of the currently employed trypanocidal drugs. Thus, OP-Tb may already be a cellular target for trypanocidal drugs. If correct, this may represent an important step towards understanding the biochemical mechanisms of the trypanocidal activity of these drugs, as well as providing valuable clues as to how to improve their efficacy.
Studies on a multicatalytic, protease complex from Trypanosoma brucei brucei.
(1999) Lomo, Peter Onyimbo.; Coetzer, Theresa Helen Taillefer.; Lonsdale-Eccles, John David.
Subcellular fractionation (together with immunocytochemical localisation studies) showed that the parasite Trypanosoma brucei brucei possesses a multicatalytic protease complex (MCPTb). This complex is predominantly cytosolic but some activity is also present in the nuclear fraction. MCP-Tb was isolated from T. b. brucei and compared to the properties of other proteasomes reported in the literature and to the 20S MCP isolated from bovine red blood cells (MCP-rbc). The isolation procedure employed four-steps: anion exchange chromatography on Q-Sepharose, adsorption chromatography on HA-Ultrogel, molecular exclusion chromatography on Sephacryl S-300 and glycerol density gradient sedimentation. The molecular mass of intact MCP-Tb was shown to be smaller than that of MCP-rbc. Separation of the different proteasome subunits by 2D-PAGE showed that MCP-Tb has 12 different polypeptide components compared to the 28 different polypeptide components of MCP-rbc. The N-terminal sequence of an MCP-Tb subunit showed that this subunit did not have any obvious sequence homology with the subunits of proteasomes from other cells. Furthermore, anti-MCP-Tb antibodies (which exhibited the in vitro inhibitory activity of MCP-Tb) did not cross-react with MCP-rbc showing that MCP-Tb and MCP-rbc are antigenically distinct. The basic enzymatic properties of MCP-Tb were fairly typical of other 20S proteasomes. MCP-Tb had multiple peptidase activities (identified as chymotrypsin-like, trypsin-like and peptidyl glutamylpeptide hydrolase activities) that are characteristic of proteasomes. Furthermore, the characteristics of inhibition by a variety of inhibitors were similar to those of other proteasomes, including MCP-rbc. The activities of 20S proteasomes from most cell types are activated by endogenous high molecular mass complexes such as the bovine 19S complex called PA700. These complexes form end-on associations with the 20S proteasome. However, no endogenous MCP-activator was found in T. b. brucei. Nevertheless, MCP-Tb was activated in an ATP-dependent manner by bovine PA700. Inhibition of the intrinsic phosphatase activity of PA700 inhibited the protease enhancing effect of PA700. Electron microscopic examination of negatively stained MCP-Tb and MCP-rbc showed particles that were morphologically indistinguishable. However, the MCP-Tb also exhibited unique end-on associations between individual units forming long (up to 200 nm) ribbon-like chains. Since access to the active sites of proteasomes occurs through the pores at the end of the complexes, this end-on association, when coupled to our observation of an apparent lack of an endogenous activator, suggests that T. b. brucei may have evolved an alternate mechanism for controlling their proteasome activity.
Studies on acid phosphatases of Trypanosoma congolense.
(1997) Tosomba, Omalokoho Médard.; Coetzer, Theresa Helen Taillefer.; Lonsdale-Eccles, John David.
Bloodstream forms of African trypanosomes, which endocytose macromolecules exclusively through their flagellar pockets, contain an acid phosphatase (AcP) activity in this organelle. In the present thesis, AcP activity was demonstrated cytochemically in some intracellular vesicles and on the surface of Trypanosoma congolense as well as in the flagellar pocket. Unlike other trypanosomatids such as Leishmania spp. and Trichomonas spp., these trypanosomes, while viable, did not release this enzyme into the surrounding medium. In contrast to mammalian cells, the AcP in T. congolense was shown by cell fractionation to be a non-lysosomal enzyme. The enzyme was mostly recovered in the microsomal and cytosolic fractions which had 52.7% and 44.4% of the total activity, respectively. Further separation of the microsomal fraction showed an association of AcP activity with vesicles derived from the plasma membrane, Golgi apparatus and endoplasmic reticulum. After ammonium sulfate precipitation and chromatography on a succession of columns containing Sephacryl S-300, DEAE-cellulose and Sephadex G-75, two acid phosphatases (AcPi and ACP2) were produced from the cytosolic fraction. A membrane-bound acid phosphatase (ACP3) was isolated from the microsomal pellets extracted with Triton X-l 14 and subjected to the above chromatographic procedures. The molecular mass of AcP 1 was higher than 700 kDa. It had an isoelectric point of 4.7. AcP2 (pi 5.3) and AcP3 (pi 6.5) had molecular masses of 33 and 320 kDa, respectively. AcPi and ACP3 were strongly inhibited by vanadate while ACP2 was strongly inhibited by p-chloromercuribenzoate. None of the enzymes was inhibited by tartrate but all were inhibited by NaF. The Km values for each of the various substrates differed widely between the three AcPs indicating that the binding site of each enzyme was distinct. The best of all the substrates tested was para-nitrophenyl phosphate. On non-denaturing gels the enzymes exhibited very high molecular masses but on denaturing SDS-PAGE, two similar bands of activity, localised at 62 and 65 kDa, were observed in all three AcP preparations. Thus the three isolated enzymes may be derived from the same base 62 and 65 kDa units. Differences between enzymes may be derived from differential processing of the isoenzymes for different functions at different locations.
A study of the African horse sickness virus using High Resolution Melt, multivariate and phylogenetic analyses for a potential serotyping assay.
(2014) Groenink, Shaun Reinder.; Coetzer, Theresa Helen Taillefer.; Young, Marion Belinda.; Watson, Gregory M. F.
African horse sickness (AHS) is a viral disease that afflicts all equine species and has a 90% mortality rate in unvaccinated horses. The disease has a devastating effect on the national herd of South Africa each year and affects both the sport and racehorse industries, including the export of prized bloodstock, as well as the rural and subsistence economies that depend on animal traction. Transmitted by the Culicoides spp. of biting midge, the virus belongs to the Orbivirus genus of the Reoviridae family with nine known serotypes and ten genome segments. Segment 2 (which encodes VP2) is responsible for serotype determination while segment 10 (which encodes NS3) is merely serotype-divergent. Knowledge of the seroprevalence of the virus is poor. The increasing reluctance of horse owners to use the registered vaccine due to perceived inefficacy is of concern. As a means to increase knowledge output in this regard, and potentially provide a service to horse owners, a rapid serotyping assay is sought based on High Resolution Melt (HRM) analysis. HRM analysis is a powerful tool that is based on the release of a DNA intercalating dye from polymerase chain reaction products through gradual and controlled heating. The dye is released at a specific point that is dependent on the unique sequence of the amplicon. It can thus be used to distinguish, very sensitively, differences in divergent amplicons. Using a range of freely available bioinformatics software, such as Clustal X2, Primaclade, Treeview and BLAST analysis, primers were designed based on segment 2 that sought to differentiate the individual serotype from previously defined clades based on a pair of segment 10 primers. Reference and field isolates of the AHS virus were obtained from the National Institute of Communicable Disease and the Onderstepoort Veterinary Institute, South Africa, and were propagated on Vero cell monolayers. Total RNA was extracted using guanidine-thiocyanate and verified as containing AHSV genomic material using primers recognised by the World Organisation for Animal Health that target the genome segment encoding VP7. Variable amounts of total RNA did not influence the downstream analysis as individual serotypes were easily distinguished using HRM despite wide ranging template concentrations. Through testing the primers designed in the present study, various serotype anomalies were discovered with regard to the isolates obtained from the Onderstepoort Veterinary Institute. Serotype-specific primers and the segment 10 primers were used to interpret the serotype anomalies through High Resolution Melt analysis. Sequencing confirmed the anomalies: serotype 2 isolates were serotype 6 isolates and a serotype 5 isolate was serotype 8. A proposed protocol for a rapid serotyping assay was investigated. This involved an initial PCR to determine into which clade of segment 10 the sample fits. Following this, the serotype was elucidated for each clade using segment 2 clade-specific primers. These reactions were performed in the Corbett Rotor-Gene™ 6000 and its in-built software was used. However, limitations of the software soon became apparent, as it was not able to completely distinguish the serotypes. Alternate methods were sought and included ScreenClust HRM® Software, principal component analysis (PCA) and discriminant analysis (DA). The use of normalised HRM fluorescence curves for PCA and DA was effective in standardising the template concentrations. These methods were successful in determining the serotype and rendered results with greater statistical confidence. Finally, a phylogenetic analysis was performed of all the available AHS virus sequences to determine the degree of possible genetic drift in the virus that may give rise to a new serotype. Minimal to no genetic drift could be found comparing sequences from the 1960s to sequences from the 2000s. This study presents the ability of HRM analysis to recognise and define AHS virus serotype anomalies, provide a new protocol for the serotyping of the virus with an extensive statistical analysis, the first for an orbivirus. Furthermore, the protocols described can be extrapolated to other orbiviruses.
Trypanopain : a possible target for anti-trypanosomal agents?
(1997) Troeberg, Linda.; Coetzer, Theresa Helen Taillefer.; Lonsdale-Eccles, John David.
The protozoan parasite Trypanosoma brucei brucei causes nagana in cattle and is a widely used model for human sleeping sickness. The major lysosomal cysteine proteinases (trypanopains) of African trypanosomes may contribute to pathogenesis by degrading proteins in the mammalian bloodstream and also appear to be essential for the viability of T. cruzi and T. congolense. This study describes the first purification to electrophoretic homogeneity of trypanopain-Tb from T. b. brucei and the first reported characterisation of its enzymatic properties. Trypanopain-Tb was purified from bloodstream forms of T. b. brucei by a combination of three phase partitioning (between ammonium sulfate and tertiary butanol), and chromatography on quaternary amine or pepstatin A-Sepharose resins. Trypanopain-Tb was found to be a typical cysteine proteinase, in that it is inhibited by typical cysteine proteinase inhibitors and requires reducing agents for full activity. Trypanopain has cathepsin L-like specificity for synthetic substrates and readily degrades various proteins. In vitro analysis of the kinetics of trypanopain interaction with cystatins suggested that these are likely to inhibit any trypanopain released into the mammalian bloodstream. Furthermore, no trypanopain-like activity was detectable in the blood of infected hosts, so it appears that trypanopain is unlikely to contribute directly to pathogenesis by degrading bloodstream host proteins. Antibodies against a peptide corresponding to a region of the trypanopain active site were produced in rabbits and chickens. Both enzyme activity-enhancing and enzyme activity inhibiting antibodies were produced and these effects varied with the substrate tested. Thus, the in vivo effects of anti-trypanopain antibodies will only become clearly understood once the physiological substrates of trypanopain have been identified. Various cysteine proteinase inhibitors, including peptidyl diazomethylketones, killed cultured bloodstream forms of T. b. brucei. Use of biotinylated derivatives of peptidyl diazomethylketone and fluoromethylketone inhibitors suggested that trypanopain is the likely intracellular target of these inhibitors, indicating that the enzyme is essential for parasite viability. Furthermore, chalcones (a class of reversible cysteine proteinase inhibitors) killed in vitro cultured parasites and also prolonged the life of T. b. brucei-infected mice. Thus, trypanopain-Tb seems to be a possible target for new anti-trypanosomal drugs.
Type IV collagenase and cathepsins L and H : proteinases involved in tumour invasion.
(1992) Coetzer, Theresa Helen Taillefer.; Dennison, Clive.
The collagenolytic proteinases, type IV collagenase and cathepsins Land H, have been implicated in tumour invasion and metastasis, by virtue of their degradative action on the extracellular matrix barriers traversed by migrating tumour cells. Type IV collagenase was isolated from human leucocytes using anti-peptide antibody immunoaffinity chromatography. The highly specific targeting of both native and denatured forms of human type IV collagenase by these anti-peptide antibodies holds much promise for immunolocalisation studies in human tumour tissue. Cathepsin L was purified in both a free; single-chain form from sheep liver, and as complexes with the endogenous cysteine proteinase inhibitor, stefin B. These complexes comprised mixtures of the usual tight-binding non-covalent, inhibitory complexes, and novel, proteolytically active, covalent cathepsin L/stefin B complexes. The latter form spontaneously in a pH-dependent manner in vitro from purified, active constituents. The primary structures of these complexing moieties from sheep liver are reported here for the first time, and showed a high degree of sequence homology with their human counterparts. Single-chain cathepsin L, both in the free, and novel, covalently complexed forms, manifested stability and increased activity at neutral pH, thus suggesting a role in extracellular tissue destruction. This potential involvement in tumour invasion was strengthened by demonstrating that the single-chain form of the enzyme, and similar covalent complexes, active under physiological conditions, could be isolated from liver tissue homogenates of higher primates, baboon (Papio ursinus) and man. A battery of versatile polyclonal anti-sheep cathepsin L and anti-human cathepsins L and H peptide antibodies were raised in chickens and rabbits. The chicken egg yolk antibodies were often of a higher titre than the corresponding rabbit serum antibodies, and additionally manifested unique immunoinhibitory properties. In the case of the polyclonal chicken anti-sheep cathepsin L antibodies, this was derived from their ability to target a peptide located in the active site of cathepsin L. The chicken anti-human cathepsins L and H peptide antibodies constitute the immunological probes of choice for immunolocalisation and in vitro tumour invasion studies to elucidate the relative contributions of these collagenolytic cathepsins to tumour invasion, and could ultimately find application in tumour immunotherapy.

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Browsing Doctoral Degrees (Biochemistry) by Author "Coetzer, Theresa Helen Taillefer."