A single exon-encoded Theileria parva strain Muguga cysteine protease (ThpCP): Molecular modelling and characterisation.

The tick-transmitted apicomplexan Theileria parva causes East Coast fever, a bovine disease of great economic and veterinary importance in Africa. Papain-like cysteine proteases play important roles in protozoan parasite host cell entry and egress, nutrition and host immune evasion. This study reports the identification and characterisation of a T. parva strain Muguga cathepsin L-like (C1A subfamily) cysteine protease (ThpCP). Molecular modelling confirmed the papain-like fold of ThpCP, hydrophobic character of the S2 substrate binding pocket and non-covalent interaction between the pro- and catalytic domains preceding low pH autoactivation. ThpCP was recombinantly expressed in a protease deficient E. coli (Rosetta (DE3)pLysS strain) expression host as a 46 kDa proenzyme. Following Ni-chelate affinity chromatography and acidification, the 27 kDa mature ThpCP was purified by cation-exchange chromatography. Purified ThpCP hydrolysed typical cathepsin L substrates N-α-benzyloxycarbonyl (Z)-Phe-Arg-7-amino-4-methyl-coumarin (AMC) (kcat/Km = 4.49 × 105 s-1M-1) and Z-Leu-Arg-AMC (kcat/Km = 4.20 × 105 s-1M-1), but showed no activity against the cathepsin B-selective substrate Z-Arg-Arg-AMC. Recombinant ThpCP was active over a broad pH range from pH 4.5 to 7.5, thereby showing potential activity in the acidic parasite food vacuole and close to neutral pH of the host lymphocyte cytoplasm. Recombinant ThpCP was inhibited by the cysteine protease inhibitors E64, iodoacetate, leupeptin, chymostatin, Z-Phe-Ala-diazomethylketone (DMK) and Z-Phe-Phe-DMK and hydrolysed bovine proteins: haemoglobin, immunoglobulin G, serum albumin and fibrinogen as well as goat IgG at pH 6 and 7. Functional expression and characterisation of Theileria cysteine proteases should enable high throughput screening of cysteine protease inhibitor libraries against these proteases.

Low Dose Gamma Irradiation of Trypanosoma evansi Parasites Identifies Molecular Changes That Occur to Repair Radiation Damage and Gene Transcripts That May Be Involved in Establishing Disease in Mice Post-Irradiation.

The protozoan parasite Trypanosoma evansi is responsible for causing surra in a variety of mammalian hosts and is spread by many vectors over a wide geographical area making it an ideal target for irradiation as a tool to study the initial events that occur during infection. Parasites irradiated at the representative doses 100Gy, 140Gy, and 200Gy were used to inoculate BALB/c mice revealing that parasites irradiated at 200Gy were unable to establish disease in all mice. Cytokine analysis of mice inoculated with 200Gy of irradiated parasites showed significantly lower levels of interleukins when compared to mice inoculated with non-irradiated and 100Gy irradiated parasites. Irradiation also differentially affected the abundance of gene transcripts in a dose-dependent trend measured at 6- and 20-hours post-irradiation with 234, 325, and 484 gene transcripts affected 6 hours post-irradiation for 100Gy-, 140Gy- and 200Gy-irradiated parasites, respectively. At 20 hours post-irradiation, 422, 381, and 457 gene transcripts were affected by irradiation at 100Gy, 140Gy, and 200Gy, respectively. A gene ontology (GO) term analysis was carried out for the three representative doses at 6 hours and 20 hours post-irradiation revealing different processes occurring at 20 hours when compared to 6 hours for 100Gy irradiation. The top ten most significant processes had a negative Z score. These processes fall in significance at 140Gy and even further at 200Gy, revealing that they were least likely to occur at 200Gy, and thus may have been responsible for infection in mice by 100Gy and 140Gy irradiated parasites. When looking at 100Gy irradiated parasites 20 hours post-irradiation processes with a positive Z score, we identified genes that were involved in multiple processes and compared their fold change values at 6 hours and 20 hours. We present these genes as possibly necessary for repair from irradiation damage at 6 hours and suggestive of being involved in the establishment of disease in mice at 20 hours post-irradiation. A potential strategy using this information to develop a whole parasite vaccine is also postulated.

Homology modelling of Trypanosoma brucei major surface proteases and molecular docking of variant surface glycoproteins and inhibitor ligands for drug design.

Trypanosomes, which cause animal African trypanosomiasis, escape host immune responses by renewing their variable surface glycoprotein (VSG) coat. Chemotherapy is currently the only form of external intervention available. However, the efficacy of current trypanocides is poor due to overuse leading to an increase in drug resistance. Major surface proteases (MSPs) of trypanosomes, which are zinc-dependent metalloproteases, are possible drug targets. A Trypanosoma brucei MSP-B (TbMSP-B) mediates parasite antigenic variation via cleavage of 60% of VSG molecules. Whilst TbMSP-A has no apparent role in VSG cleavage; it is not known if TbMSP-C is involved in VSG cleavage. In this study, three-dimensional structures of TbMSP-A, TbMSP-B and TbMSP-C were modelled. By comparing the docking poses of the C-terminal domains of VSG substrates into the models, TbMSP-C showed an affinity for similar VSG substrate sites as TbMSP-B, but these sites differed from those recognised by TbMSP-A. This observation suggests that TbMSP-C may be involved in VSG cleavage during antigenic variation. Furthermore, by docking small inhibitor ligands into the TbMSP-B and TbMSP-C homology models, followed by molecular dynamics simulations, ligands with potential anti-trypanosomal activity were identified. Docking studies also revealed the depth of the S1' pockets of TbMSP-B and TbMSP-C, which is influential in ligand and substrate binding, thereby identifying the protease subsite pocket that should be targeted in drug design.

Trypanosoma brucei gambiense-iELISA: A Promising New Test for the Post-Elimination Monitoring of Human African Trypanosomiasis.

BACKGROUND: The World Health Organization targeted Trypanosoma brucei gambiense human African trypanosomiasis (gHAT) for elimination as a public health problem and for elimination of transmission. To measure gHAT elimination success with prevalences close to zero, highly specific diagnostics are necessary. Such a test exists in the form of an antibody-mediated complement lysis test, the trypanolysis test, but biosafety issues and technological requirements prevent its large-scale use. We developed an inhibition ELISA with high specificity and sensitivity that is applicable in regional laboratories in gHAT endemic countries. METHODS: The T. b. gambiense inhibition ELISA (g-iELISA) is based on the principle that binding of monoclonal antibodies to specific epitopes of T. b. gambiense surface glycoproteins can be inhibited by circulating antibodies of gHAT patients directed against the same epitopes. Using trypanolysis as reference test, the diagnostic accuracy of the g-iELISA was evaluated on plasma samples from 739 gHAT patients and 619 endemic controls and on dried blood spots prepared with plasma of 95 gHAT and 37 endemic controls. RESULTS: Overall sensitivity and specificity on plasma were, respectively, 98.0% (95% CI 96.7-98.9) and 99.5% (95% CI 98.6-99.9). With dried blood spots, sensitivity was 92.6% (95% CI 85.4-97.0), and specificity was 100% (95% CI 90.5-100.0). The g-iELISA is stable for at least 8 months when stored at 2-8°C. CONCLUSION: The g-iELISA might largely replace trypanolysis for monitoring gHAT elimination and for postelimination surveillance. The g-iELISA kit is available for evaluation in reference laboratories in endemic countries.

Isolation of Nile crocodile (Crocodylus niloticus) serum immunoglobulin M and Y (IgM and IgY).

Crocodile immunity has not been fully characterised with more studies on crocodile innate immunity than cell-mediated or humoral immunity. Crocodile immunoglobulin genes have been described but immunoglobulin proteins have not been isolated or studied biochemically. Two large proteins proposed to be crocodile IgM and IgY were isolated and purified from Crocodylus niloticus sera using two different protocols. A 50% (w/v) ammonium sulfate and a 15% (w/v) polyethylene glycol precipitation step was followed by Cibacron blue F3GA affinity- and Sephacryl-S300 gel filtration chromatography. An alternate purification protocol, with only two steps, involved thiophilic affinity- and Sephacryl-S300 gel filtration chromatography. The purified crocodile IgM resolved on reducing SDS-PAGE with an apparent mass of 180 kDa. Purified crocodile IgY resolved at 180 kDa alongside chicken IgY on a non-reducing SDS-PAGE gel, and is deduced to consist of two 66 kDa heavy and two 23 kDa light chains under reducing conditions. The thiophilic/gel filtration two-step protocol gave three-fold higher yields of isolated protein than the four-step precipitation/chromatography protocol. Antibodies against the isolated crocodile IgM and IgY were raised in chickens and affinity purified. The chicken antibodies differentiated between crocodile IgM and IgY and have the potential for use in the diagnosis of crocodile infections. The purified crocodile antibodies can be biochemically characterised and compared to mammalian and avian antibodies to give a better understanding of crocodile humoral immunity.

Expression and copper binding properties of the N-terminal domain of copper P-type ATPases of African trypanosomes.

Copper is an essential component of cuproproteins but can be toxic to cells, therefore copper metabolism is very carefully regulated within cells. To gain insight into trypanosome copper metabolism, Trypanosoma spp. genomic databases were screened for the presence of copper-containing and -transporting proteins. Among other genes encoding copper-binding proteins, a copper-transporting P-type ATPase (CuATPase) gene was identified. Sequence and phylogenetic analyses suggest that the gene codes for a Cu+ transporter belonging to the P1B-1 ATPase subfamily that has an N-terminal domain with copper binding motifs. The N-terminal cytosolic domains of the proteins from Trypanosoma congolense and Trypanosoma brucei brucei were recombinantly expressed in Escherichia coli as maltose binding protein (MBP) fusion proteins. These N-terminal domains bound copper in vitro and within E. coli cells, more than the control MBP fusion partner alone. The copper binding properties of the recombinant proteins were further confirmed when they inhibited copper catalysed ascorbate oxidation. Native CuATPases were detected in a western blot of lysates of T. congolense IL3000 and T. b. brucei ILTat1.1 bloodstream form parasites using affinity purified IgY antibodies against N-terminal domain peptides. The CuATPase was also detected by immunofluorescence in T. b. brucei bloodstream form parasites where it was associated with subcellular vesicles. In conclusion, Trypanosoma species express a copper-transporting P1B-1-type ATPase and together with other copper-binding proteins identified in the genomes of kinetoplastid parasites may constitute potential targets for anti-trypanosomal drug discovery.

Expression, purification and characterisation of Trypanosoma congolense metacaspase 5 (TcoMCA5) - a potential drug target for animal African trypanosomiasis.

The metacaspases (MCAs) are attractive drug targets for the treatment of African trypanosomiasis as they are not found in the metazoan kingdom and their action has been implicated in cell cycle and cell death pathways in kinetoplastid parasites. Here we report the biochemical characterisation of MCA5 from T. congolense. Upon recombinant expression in E. coli, autoprocessing is evident, and MCA5 further autoprocesses when purified using nickel affinity chromatography, which we term nickel-induced over autoprocessing. When both the catalytic His and Cys residues were mutated (TcoMCA5H147A/C202G), no nickel-induced over autoprocessing was observed and was enzymatically active, suggesting the existence of a secondary catalytic Cys residue, Cys81. Immunoaffinity purification of native TcoMCA5 from the total parasite proteins was achieved using chicken anti-TcoMCA5 IgY antibodies. The full length native TcoMCA5 and the autoprocessed products of recombinant TcoMCA5H147A/C202G were shown to possess gelatinolytic activity, the first report for that of a MCA. Both the native and recombinant enzyme were calcium independent, had a preference for Arg over Lys at the P1 site and were active over a pH range between 6.5 and 9. Partial inhibition (23%) of enzymatic activity was only achieved with leupeptin and antipain. These findings are the first step in the biochemical characterisation of the single copy MCAs from animal infective trypanosomes towards the design of novel trypanocides.

Recombinant and native TviCATL from Trypanosoma vivax: Enzymatic characterisation and evaluation as a diagnostic target for animal African trypanosomosis.

African animal trypanosomosis (nagana) is caused by tsetse-transmitted protozoan parasites. Their cysteine proteases are potential chemotherapeutic and diagnostic targets. The N-glycosylated catalytic domain of Trypanosoma vivax cathepsin L-like cysteine protease, rTviCATLcat, was recombinantly expressed and purified from culture supernatants while native TviCATL was purified from T. vivax Y486 parasite lysates. Typical of Clan CA, family C1 proteases, TviCATL activity is sensitive to E-64 and cystatin and substrate specificity is defined by the S2 pocket. Leucine was preferred in P2 and basic and non-bulky, hydrophobic residues accepted in P1 and P3 respectively. Reversible aldehyde inhibitors, antipain, chymostatin and leupeptin, with Arg in P1 and irreversible peptidyl chloromethylketone inhibitors with hydrophobic residues in P2 inhibited TviCATL activity. TviCATL digested host proteins: bovine haemoglobin, serum albumin, fibrinogen and denatured collagen (gelatine) over a wide pH range, including neutral to slightly acidic pH. The recombinant catalytic domain of TviCATL showed promise as a diagnostic target for detecting T. vivax infection in cattle in an indirect antibody detection ELISA.

Plasmodium glyceraldehyde-3-phosphate dehydrogenase: A potential malaria diagnostic target.

Malaria rapid diagnostic tests (RDTs) are immunochromatographic tests detecting Plasmodial histidine-rich protein 2 (HRP2), lactate dehydrogenase (LDH) and aldolase. HRP2 is only expressed by Plasmodium falciparum parasites and the protein is not expressed in several geographic isolates. LDH-based tests lack sensitivity compared to HRP2 tests. This study explored the potential of the Plasmodial glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a new malaria diagnostic biomarker. The P. falciparum and P. yoelii proteins were recombinantly expressed in BL21(DE3) Escherischia coli host cells and affinity purified. Two epitopes (CADGFLLIGEKKVSVFA and CAEKDPSQIPWGKCQV) specific to P. falciparum GAPDH and one common to all mammalian malaria species (CKDDTPIYVMGINH) were identified. Antibodies were raised in chickens against the two recombinant proteins and the three epitopes and affinity purified. The antibodies detected the native protein in parasite lysates as a 38 kDa protein and immunofluorescence verified a parasite cytosolic localization for the native protein. The antibodies suggested a 4-6 fold higher concentration of native PfGAPDH compared to PfLDH in immunoprecipitation and ELISA formats, consistent with published proteomic data. PfGAPDH shows interesting potential as a malaria diagnostic biomarker.

A systematic review of pentacyclic triterpenes and their derivatives as chemotherapeutic agents against tropical parasitic diseases.

Parasitic infections are among the leading global public health problems with very high economic and mortality burdens. Unfortunately, the available treatment drugs are beset with side effects and continuous parasite drug resistance is being reported. However, new findings reveal more promising compounds especially of plant origin. Among the promising leads are the pentacyclic triterpenes (PTs) made up of the oleanane, ursane, taraxastane, lupane and hopane types. This paper reviews the literature published from 1985 to date on the in vitro and in vivo anti-parasitic potency of this class of phytochemicals. Of the 191 natural and synthetic PT reported, 85 have shown high anti-parasitic activity against various species belonging to the genera of Plasmodium, Leishmania, Trypanosoma, as well as various genera of Nematoda. Moreover, structural modification especially at carbon 3 (C3) and C27 of the parent backbone of PT has led to improved anti-parasitic activity in some cases and loss of activity in others. The potential of this group of compounds as future alternatives in the treatment of parasitic diseases is discussed. It is hoped that the information presented herein will contribute to the full exploration of this promising group of compounds as possible drugs for parasitic diseases.

The Drosophila retinoblastoma binding protein 6 family member has two isoforms and is potentially involved in embryonic patterning.

The human retinoblastoma binding protein 6 (RBBP6) is implicated in esophageal, lung, hepatocellular and colon cancers. Furthermore, RBBP6 was identified as a strong marker for colon cancer prognosis and as a predisposing factor in familial myeloproliferative neoplasms. Functionally, the mammalian protein interacts with p53 and enhances the activity of Mdm2, the prototypical negative regulator of p53. However, since RBBP6 (known as PACT in mice) exists in multiple isoforms and pact-/- mice exhibit a more severe phenotype than mdm2-/- mutants, it must possess some Mdm2-independent functions. The function of the invertebrate homologue is poorly understood. This is complicated by the absence of the Mdm2 gene in both Drosophila and Caenorhabditis elegans. We have experimentally identified the promoter region of Snama, the Drosophila homologue, analyzed potential transcription factor binding sites and confirmed the existence of an additional isoform. Using band shift and co-immunoprecipitation assays combined with mass spectrometry, we found evidence that this gene may be regulated by, amongst others, DREF, which regulates hundreds of genes related to cell proliferation. The potential transcription factors for Snama fall into distinct functional groups, including anteroposterior embryonic patterning and nucleic acid metabolism. Significantly, previous work in mice shows that pact-/- induces an anteroposterior phenotype in embryos when rescued by simultaneous deletion of p53. Taken together, these observations indicate the significance of RBBP6 proteins in carcinogenesis and in developmental defects.

Phylogenetic analysis of the polyprotein coding region of an infectious South African bursal disease virus (IBDV) strain.

Infectious bursal disease virus (IBDV) causes Gumboro disease, which is highly contagious and immunosuppressive in young chickens. A virulent form of IBDV reached South Africa in 1989 and to date there has been little molecular information available for this strain. In this study, the polyprotein coding region of the South African strain SA-KZN95 was sequenced and analysed along with 52 representative sequences of other serotype I and II strains. We explored the relative impact of recombination on phylogenetic reconstruction using a multidimensional scaling approach. Phylogenetic analyses consistently placed the South African isolate within the very virulent IBDV clade. Selection analyses were also conducted to identify evolutionarily relevant amino acid residues. Previously, 19 residues in the polyprotein were shown to be potentially diagnostic for the different IBDV pathotypes. This study identified an additional two unique residues in the polyprotein which may be used as genetic signatures in future viral identifications. Better strain identification would aid in the development and application of vaccines.

Recombinant expression and biochemical characterisation of two alanyl aminopeptidases of Trypanosoma congolense.

Trypanosoma congolense is a haemoprotozoan parasite that causes African animal trypanosomosis, a wasting disease of cattle and small ruminants. Current control methods are unsatisfactory and no conventional vaccine exists due to antigenic variation. An anti-disease vaccine approach to control T. congolense has been proposed requiring the identification of parasitic factors that cause disease. Immunoprecipitation of T. congolense antigens using sera from infected trypanotolerant cattle allowed the identification of several immunogenic antigens including two M1 type aminopeptidases (APs). The two APs were cloned and expressed in Escherichia coli. As the APs were expressed as insoluble inclusion bodies it was necessary to develop a method for solubilisation and subsequent refolding to restore conformation and activity. The refolded APs both showed a distinct substrate preference for H-Ala-AMC, an optimum pH of 8.0, puromycin-sensitivity, inhibition by bestatin and amastatin, and cytoplasmic localisation. The two APs are expressed 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.

Trypanosoma brucei brucei oligopeptidase B null mutants display increased prolyl oligopeptidase-like activity.

African trypanosomosis is a parasitic disease in man and animals caused by protozoan parasites of the genus Trypanosoma. Nagana, the cattle form of the disease, is caused by Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei brucei. 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. One such pathogenic factor is oligopeptidase B (TbOPB), a trypanosome peptidase that hydrolyses Arg/Lys containing peptides smaller than 30 amino acid residues and is suspected to be involved in the hormonal deregulation associated with the disease. To better understand the role TbOPB plays in parasite physiology and host pathogenesis, oligopeptidase B null mutant parasites (Δopb) were generated in the T. b. brucei Lister 427 strain. Δopb Trypanosoma brucei parasites grew at a significantly faster rate 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 TbOPB is not involved in assisting T. brucei parasites to cross microvascular endothelial cells. Gelatine gel analysis of Δopb null mutants showed an increase in discrete cysteine peptidase activities when compared to wild type strains. Enzymatic activity assays were carried out to identify how closely related oligopeptidases are affected by TbOPB gene deletion. A significant increase of T. brucei prolyl oligopeptidase (TbPOP) activity was observed, but no concomitant increase in TbPOP protein levels, suggesting that a POP-like enzyme might compensate for a loss in OPB activity in Δopb null mutants.

Production of congopain, the major cysteine protease of Trypanosoma (Nannomonas) congolense, in Pichia pastoris reveals unexpected dimerisation at physiological pH.

African animal trypanosomosis (nagana) is arguably the most important parasitic disease affecting livestock in sub-Saharan Africa. Since none of the existing control measures are entirely satisfactory, vaccine development is being actively pursued. However, due to antigenic variation, the quest for a conventional vaccine has proven elusive. As a result, we have sought an alternative 'anti-disease vaccine approach', based on congopain, a cysteine protease of Trypanosoma congolense, which was shown to have pathogenic effects in vivo. Congopain was initially expressed as a recombinant protein in bacterial and baculovirus expression systems, but both the folding and yield obtained proved inadequate. Hence alternative expression systems were investigated, amongst which Pichia pastoris proved to be the most suitable. We report here the expression of full length, and C-terminal domain-truncated congopain in the methylotrophic yeast P. pastoris. Differences in yield were observed between full length and truncated proteins, the full length producing 2-4 mg of protein per litre of culture, while the truncated form produced 20-30 mg/l. The protease was produced as a proenzyme, but underwent spontaneous activation when acidified (pH <5). To investigate whether this activation was due to autolysis, we produced an inactive mutant (active site Cys→Ala) by site-directed mutagenesis. The mutant form was produced at a much higher rate, up to 100mg/l culture, as a proenzyme. It did not undergo spontaneous cleavage of the propeptide when subjected to acidic pH suggesting an autocatalytic process of activation for congopain. These recombinant proteins displayed a very unusual feature for cathepsin L-like proteinases, i.e. complete dimerisation at pH >6, and by reversibly monomerising at acidic pH <5. This attribute is of utmost importance in the context of an anti-disease vaccine, given that the epitopes recognised by the sera of trypanosome-infected trypanotolerant cattle appear dimer-specific.

Expression, purification and characterisation of two variant cysteine peptidases from Trypanosoma congolense with active site substitutions.

Congopain, the major cysteine peptidase of Trypanosoma congolense is an attractive candidate for an anti-disease vaccine and target for the design of specific inhibitors. A complicating factor for the inclusion of congopain in a vaccine is that multiple variants of congopain are present in the genome of the parasite. In order to determine whether the variant congopain-like genes code for peptidases with enzymatic activities different to those of congopain, two variants were cloned and expressed. Two truncated catalytic domain variants were recombinantly expressed in Pichia pastoris. The two expressed catalytic domain variants differed slightly from one another in substrate preferences and also from that of C2 (the recombinant truncated form of congopain). Surprisingly, a variant with the catalytic triad Ser(25), His(159) and Asn(175) was shown to be active against classical cysteine peptidase substrates and inhibited by E-64, a class-specific cysteine protease inhibitor. Both catalytic domain clones and C2 had pH optima of either 6.0 or 6.5 implying that these congopain-like proteases are likely to be expressed and active in the bloodstream of the host animal.

Anti-peptide antibodies differentiate between plasmodial lactate dehydrogenases.

Malaria lactate dehydrogenase, a glycolytic enzyme, is a malaria diagnostic target in lateral flow immunochromatographic rapid diagnostic tests. Recombinant Plasmodium yoelii LDH was cloned into the pET-28a vector, expressed and the expressed protein purified from a Ni-NTA affinity matrix. A pan-malarial LDH antibody directed against a common malaria LDH peptide (APGKSDKEWNRDDLL) and two anti-peptide antibodies, each targeting a unique Plasmodium falciparum (LISDAELEAIFDC) and Plasmodium vivax (KITDEEVEGIFDC) LDH peptide were raised in chickens. The antibodies were affinity purified with the appropriate peptide affinity matrix. The affinity purified anti-peptide antibodies detected recombinant P. falciparum, P. vivax and P. yoelii LDH and native P. falciparum and P. yoelii LDH in western blots and immunofluorescence studies. The pan-malarial antibody detected LDH from the three malaria species in western blots. The species-specific anti-peptide antibodies differentiated between P. falciparum and P. vivax LDH. Affinity purified chicken antibodies against recombinant PfLDH, PvLDH and PyLDH proteins each detected the parent and orthologous proteins with similar titers in an ELISA. The study supports an anti-peptide antibody approach to the development of diagnostic reagents.

Oligopeptidase B: a processing peptidase involved in pathogenesis.

Oligopeptidase B is a "processing peptidase" from the prolyl oligopeptidase family of serine peptidases present in Gram negative bacteria, protozoa and plants. Unlike the prototype prolyl oligopeptidase, oligopeptidase B hydrolyses peptides on the carboxyl side of pairs of basic amino acid residues. Molecular modelling and mutation studies have identified carboxyl dyads in the C-terminal catalytic domain that mediate substrate and inhibitor binding. The peptidase is efficiently inhibited by non-peptide irreversible serine peptidase inhibitors, peptidyl-chloromethylketones, -phosphonate alpha-aminoalkyl diphenyl esters with basic residues at P1, and tripeptide aldehydes, but not by proteinaceous host plasma inhibitors such as alpha2-macroglobulin and serpins. Access of these large molecular mass inhibitors and substrates larger than approximately 30 amino acid residues to the catalytic cleft is restricted by the N-terminal beta-propeller domain. The physiological role of oligopeptidase B from various sources has not yet been elucidated. However, the peptidase has been identified as an important virulence factor and therapeutic agent in animal trypanosomosis. This review highlights the structure-function properties of oligopeptidase B in context with its physiological and/or pathological roles which make the enzyme a promising drug target.

Raising antibodies in chickens against primaquine, pyrimethamine, dapsone, tetracycline, and doxycycline.

Antibodies against primaquine, pyrimethamine, dapsone, tetracycline, and doxycycline were raised in chickens inoculated with each drug conjugated to a rabbit albumin carrier. Antibody titres against drug and carrier were highest during week 6 postinoculation. Affinity purified anti-primaquine antibodies did not recognise other drugs, but affinity purified anti-doxycycline and anti-tetracycline antibodies recognised both tetracycline and doxycycline in addition to primaquine. Primaquine was detected in urine from 6 to 12 hours after ingestion of therapeutic doses of the drug by anti-primaquine antibodies in a competitive ELISA. Affinity purified anti-primaquine antibodies detected primaquine in the cytoplasm and localised in organelles in monocytes that had been incubated with therapeutic concentrations of the drug.

Evidence that serpin architecture intrinsically supports papain-like cysteine protease inhibition: engineering alpha(1)-antitrypsin to inhibit cathepsin proteases.

The closely related serpins squamous cell carcinoma antigen-1 and -2 (SCCA-1 and -2, respectively) are capable of inhibiting cysteine proteases of the papain superfamily. To ascertain whether the ability to inhibit cysteine proteases is an intrinsic property of serpins in general, the reactive center loop (RCL) of the archetypal serine protease inhibitor alpha(1)-antitrypsin was replaced with that of SCCA-1. It was found that this simple substitution could convert alpha(1)-antitrypsin into a cysteine protease inhibitor, albeit an inefficient one. The RCL of SCCA-1 is three residues longer than that of alpha(1)-antitrypsin, and therefore, the effect of loop length on the cysteine protease inhibitory activity was investigated. Mutants in which the RCL was shortened by one, two, or three residues were effective inhibitors with second-order rate constants of 10(5)-10(7) M(-)(1) s(-)(1). In addition to loop length, the identity of the cysteine protease was of considerable importance, since the chimeric molecules inhibited cathepsins L, V, and K efficiently, but not papain or cathepsin B. By testing complexes between an RCL-mimicking peptide and the mutants, it was found that the formation of a stable serpin-cysteine protease complex and the inhibition of a cysteine protease were both critically dependent on RCL insertion. The results strongly indicate that the serpin body is intrinsically capable of supporting cysteine protease inhibition, and that the complex with a papain-like cysteine protease would be expected to be analogous to that seen with serine proteases.