Oviduct-specific expression of two therapeutic proteins in transgenic hens.

Recent advances in avian transgenesis have led to the possibility of utilizing the laying hen as a production platform for the large-scale synthesis of pharmaceutical proteins. Ovalbumin constitutes more than half of the protein in the white of a laid egg, and expression of the ovalbumin gene is restricted to the tubular gland cells of the oviduct. Here we describe the use of lentiviral vectors to deliver transgene constructs comprising regulatory sequences from the ovalbumin gene designed to direct synthesis of associated therapeutic proteins to the oviduct. We report the generation of transgenic hens that synthesize functional recombinant pharmaceutical protein in a tightly regulated tissue-specific manner, without any evidence of transgene silencing after germ-line transmission.

The Leishmania mexicana proteasome.

As a start to understanding the importance of intracellular proteolysis in the protozoon Leishmania mexicana, the parasite proteasome has been purified and characterised. The L. mexicana proteasome is similar to proteasomes from other eukaryotes. It is soluble, and the 20S form has a mass of around 670 kDa, composed of at least 10 distinct subunits in the 22 to 32 kDa size range. The molecular mass of the L. mexicana proteasome increases to 1200 kDa in the presence of adenosine-5'-triphosphate, consistent with there being a 26S proteasome in the parasite. The purified 20S proteasome has activity towards substrates with hydrophobic, basic and acidic P, residues, and is sensitive to a range of peptide aldehyde inhibitors, as well as the proteasome-specific inhibitor lactacystin. The peptide aldehydes are able to arrest parasite growth in vitro with the same relative effectiveness as against the purified proteasome activity. The parasite population arrests with an increased 4N DNA content, indicating that, in part, the essential nature of the proteasome for L. mexicana proliferation is due to a role in the parasite cell cycle. Surprisingly, lactacystin is a relatively inefficient inhibitor of L. mexicana growth in vitro.

Identification and isolation of three proteasome subunits and their encoding genes from Trypanosoma brucei.

We have determined peptide sequences of three Trypanosoma brucei proteasome subunit proteins by mass spectrometry of tryptic digests of the proteins purified by two-dimensional (2-D) polyacrylamide gel electrophoresis. Three genes identified by the sequence of their cDNA encode the peptides identified in these three proteins. The three proteins predicted from the gene sequences have significant similarity to other known proteasome subunits and represent an alpha6 type subunit (TbPSA6), and two beta-type subunits belonging to the beta1-type (TbPSB1) and beta2 type (TbPSB2). The sequences of both beta-subunits predict formation of catalytically active subunits through proteolytic processing. The prediction is supported by the presence in each of the two beta-subunits of a tryptic peptide that has the correctly processed N-terminus that creates the threonine nucleophile of the mature protein. This peptide cannot be generated by trypsin because of the required cleavage of a glycine-threonine bond. It is thus likely that there are at least two catalytically active beta-subunits, TbPSB1 and TbPSB2, present in the mature 20S proteasome from T. brucei.

Analysis of the cysteine proteinases of Leishmania mexicana and Trypanosoma cruzi using specific antisera.

Antisera raised against papain and cysteine proteinases (CPs) purified from Leishmania mexicana and Trypanosoma cruzi have been used to study the proteins in the two parasites. The antisera against the major CP of T. cruzi (cruzipain) not only cross-reacted with known CPs of L. mexicana but also detected stage-specific molecules that may represent previously unrecognised CPs. The binding of the same antisera to extracts of different life cycle stages of T. cruzi suggested that the stages possess different isoforms of cruzipain. The lack of cross-reactivity of anti-papain antiserum against cruzipain suggests that the major immunogenic epitopes of these CPs are different, whereas the detection of the major CPs of L. mexicana with both heterologous antisera shows that the parasite's enzymes share epitopes with the other CPs.

Expression of cysteine proteinases by metacyclic promastigotes of Leishmania mexicana.

The expression of cysteine proteinases by metacyclic promastigotes of Leishmania mexicana was investigated using gelatin polyacrylamide gel electrophoresis. Two prominent bands were detected which distinguished metacyclics from multiplicative promastigotes, lacking detectable cysteine proteinase activity, and amastigotes, with a distinct banding pattern composed of multiple enzymes. A correlation between relative activity of the metacyclic-specific bands and the prevalence of metacyclics was found both during the growth cycle in vitro as metacyclogenesis occurred, and by comparison of stationary phase populations from consecutive subpassages in vitro. Irreversible inhibition of the metacyclic activities using N-benzyloxycarbonyl-phenylalanyl-alanyl diazomethane did not inhibit metacyclic to amastigote transformation in vitro. These activities provide a useful biochemical marker for the metacyclic promastigotes of L. mexicana.

Multiple high activity cysteine proteases of Leishmania mexicana are encoded by the Imcpb gene array.

The interrelationship of the multiple cysteine proteases (CPs) found characteristically at high activity in Leishmania mexicana amastigotes has been investigated. The mature forms of the five enzymes of groups B and C, which have subtly different substrate preferences, are the same size. Enzymically deglycosylated group A CP proteins also have the same molecular mass. Proteases of all three groups are specifically recognized by antisera raised against the group B or group C CPs. In addition, CPs of groups A, B and C have highly similar N-terminal amino acid sequences. The consensus sequence matches that predicted from the sequenced Imcpb gene, which occurs in a tandem array of over ten similar genes. Thus, the results are consistent with the groups A, B and C CPs being products of different Imcpb genes within the array, the different genes encoding CPs with identical N-termini, but with limited amino acid substitutions within the mature enzyme accounting for the different properties of the CPs. Evidence is also presented to indicate membrane-association of proteolytically active but less processed forms of Imcpb products.

Cathepsin B-like cysteine proteases of Leishmania mexicana.

A group of Leishmania mexicana cysteine proteases that differ from those previously found in this protozoon are described. The enzymes characteristically have a preference for peptidyl substrates with a phenylalanyl-valyl-arginyl moiety, do not hydrolyse gelatin in substrate-sodium dodecyl sulphate polyacrylamide gels, are stimulated by thiol-reducing agents and are sensitive to inhibitors specific for cysteine proteases. They have unusual solubility properties that indicate that the enzymes are amphiphilic proteins. Two of the cysteine proteases have been purified from L. mexicana amastigotes and shown to have molecular masses of 31 and 33 kDa. Their N-terminal amino acid sequences are very similar and show high homology to the mammalian cysteine protease, cathepsin B.

Fasciola hepatica: a secreted cathepsin L-like proteinase cleaves host immunoglobulin.

Adult Fasciola hepatica secrete a cysteine proteinase capable of cleaving host IgG close to the papain cleaving site. The proteinase was separated by size permeation chromatography. Gelatin-substrate polyacrylamide gel electrophoresis analysis revealed that the proteinase migrates as 6 proteolytic bands in the apparent molecular size range 60-90 kDa. Based on pH profiles of activity, inhibition studies using diethylpyrocarbonate and the diazomethylketone Z-phe-ala-CHN2, and characterising the substrate specificity of the enzymes using fluorogenic peptide substrates we have shown that the 60-90-kDa proteinases are cathepsin L-like proteinases.

Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms.

A new method is described which has made possible the long-term axenic cultivation of Leishmania mexicana amastigote-like forms in Schneider's Drosophila medium supplemented with 20% (v/v) foetal calf serum. Unlike previous methods, it utilizes direct culture of parasites obtained from the lesions of infected animals rather than adaptation of promastigotes in vitro. Ultrastructural (possession of megasomes), biochemical (cysteine proteinase activity and gelatin SDS-PAGE banding pattern) and infectivity (in vivo) data are presented which show the close similarity of the cultured forms to lesion amastigotes. The axenically cultured forms grew optimally at a temperature of 32-33 degrees C, providing further evidence for their amastigote nature. It was found that adjustment of the pH of the growth medium to 5.4 was required in order to retain the amastigote morphology of the cultured parasites. This supports the notion that leishmanial amastigotes are acidophiles.

Stage-specific proteinases of Leishmania mexicana mexicana promastigotes.

Four distinct bands of cysteine proteinase activity were detected when stationary-phase populations of Leishmania mexicana mexicana were subjected to gelatin-SDS-PAGE. The highest mobility band contained at least three isoforms separable by mono Q anion exchange chromatography. These high mobility activities were distinct from all the major amastigote enzymes. Stationary-phase promastigote populations also contained two acid-activable precursor forms of the promastigote-specific band. It is suggested that these promastigote-specific activities occur in the infective metacyclic stage of the parasite and may have a role in parasite survival upon inoculation into a mammal.

A developmentally regulated cysteine proteinase gene of Leishmania mexicana.

We have isolated a gene encoding a previously unreported class of trypanosomatid cysteine proteinase (CP) from the protozoan parasite Leishmania mexicana. The single-copy gene (lmcpa) [corrected]. has several unusual features that distinguish it from CP genes cloned from the related species Trypanosoma brucei and Trypanosoma cruzi. These include a shorter C-terminal extension of only 10 amino acids and a three-amino-acid insertion, GlyValMet, close to the predicted N-terminus of the mature protein. Northern blot analysis showed that the gene is expressed in all life-cycle stages but at higher levels in the amastigote stage in the mammal and in stationary phase promastigote cultures which contain the infective metacyclic form of the parasite. A precursor protein of 38 kDa was detected in amastigotes and stationary phase promastigotes with antisera specific to the LmCPa pro-region, but was barely detectable in early log-phase promastigotes. Anti-central domain antisera recognized the 38 kDa precursor and 24 and 27 kDa proteins. The major CPs of L. mexicana amastigotes, previously designated types A, B and C, were not detected with the antisera, suggesting that the gene codes for a previously uncharacterized CP in L. mexicana. The 24 kDa protein detected by the antiserum has no activity towards gelatin but apparently hydrolyses the peptide substrate BzPheValArgAMC. The relative levels of the 24 and 27 kDa proteins vary between the different life-cycle stages. The results indicate that expression of this CP is regulated at both the RNA and protein level.

Characterisation of three groups of cysteine proteinases in the amastigotes of Leishmania mexicana mexicana.

The multiple cysteine proteinases characteristic of the amastigote forms of Leishmania mexicana mexicana have been shown to be of three types. The groups of enzymes are distinguished by their substrate specificities and physical properties and have been purified free from other proteinases and most other proteins. One group (A) comprises at least four enzymes that bind to Con A. The cysteine proteinases comprising the other two groups (B and C) were separated by ion exchange chromatography. These last two groups of enzymes show different specificities towards a range of peptidyl aminomethylcoumarins. Notably, the two group C proteinases are more active towards compounds with a basic amino acid in the P1 position, whereas the three enzymes of group B are as active towards substrates with tyrosine in this position. All the cysteine proteinases show preference for compounds with bulky amino acids at positions P2 and P3 and all are equally susceptible to a range of inhibitors characteristically active against cysteine proteinases. Other proteinases present in amastigotes of L. m. mexicana were shown to differ significantly from the cysteine proteinases with respect to their substrate preferences and susceptibility to inhibitors.

Analysis of the proteinases of Trypanosoma brucei.

A method comprising enzyme separation by SDS-PAGE and subsequent use of peptidyl aminomethylcoumarins as substrates has been used to study proteinases of the protozoan parasite Trypanosoma brucei. The application of this method has allowed investigation of the substrate specificities of individual proteinases in cell lysates without the need for enzyme purification. The results show that T. brucei contains a group of cysteine proteinases, probably four in number, with substrate and inhibitor specificities similar to those of cathepsin L. A second group of proteinases, larger enzymes with significantly different substrate specificities and sensitivity to inhibitors, was also detected. Peptidyl diazomethanes inhibited the cysteine proteinases and also parasite growth, offering promise that peculiarities in the substrate specificity of trypanosomal cysteine proteinases could be exploited by compounds of this type.

The specificity of trichomonad cysteine proteinases analysed using fluorogenic substrates and specific inhibitors.

The multiple cysteine proteinases of Trichomonas vaginalis and Tritrichomonas foetus, both those retained intracellularly and those released, were separated using gelatin-SDS-PAGE, and their activity towards a range of 15 fluorogenic peptidyl aminomethylcoumarins determined together with their relative sensitivity to inhibitors. Three types of enzyme were apparent in T. vaginalis: (i) an 86-kDa enzyme active only on Z-Arg-Arg-NHMec; (ii) a 54-kDa proteinase which was most active on Z-Phe-Arg-NHMec but also able to hydrolyse N-t-Boc-Val-Leu-Lys-NHMec, Suc-Ala-Phe-Lys-NHMec, H-Pro-Phe-Arg-NHMec and Z-Arg-Arg-NHMec; and (iii) a group of six enzymes which preferentially hydrolysed substrates with bulky residues at the P2 and P3 positions. N-t-Boc-Val-Leu-Lys-NHMec and H-Leu-Val-Tyr-NHMec were the best substrates for the latter group. The 86-kDa proteinase was inactivated by E-64, but only at high concentrations, and was relatively insensitive to the peptidyl diazomethanes. The other proteinases were inhibited by low concentrations of E-64 and by Z-Phe-Ala-CHN2, and to a lesser extent by Z-Phe-Phe-CHN2. Differences between the proteinases of T. foetus were also demonstrated. All of them were active on Z-Arg-Arg-NHMec, but their activity towards other substrates varied. Three predominantly extracellular proteinases (25, 27 and 34 kDa), hydrolysed Z-Arg-Arg-NHMec specifically. Other proteinases (apparent Mr of 20,000 and 32,000) hydrolysed a number of other substrates, with the 32-kDa enzyme having greater activity towards N-t-Boc-Val-Leu-Lys-NHMec and H-Leu-Val-Tyr-NHMec than towards Z-Arg-Arg-NHMec. At a high concentration (270 microM), E-64 inhibited all of the T. foetus enzymes, but lower concentrations were less effective, with the 18-kDa proteinase being particularly insensitive. Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2 were relatively poor inhibitors. The results demonstrate that the proteinases of both species are a heterogeneous group with respect to specificity, and have highlighted significant differences between the enzymes of T. vaginalis and T. foetus. The information on the specificities will be useful for assessing the features required in proteinase inhibitors if they are to be of potential value as antitrichomonal agents.