Analysis of Leishmania proteinases reveals developmental changes in species-specific forms and a common 68-kDa activity

Abstract The proteinases of three species of Leishmania have been analysed by electrophoresis. Amastigotes of L. mexicana mexicana have several high-activity, low- M _r cysteine proteinases which are absent from log-phase promastigotes of L. m. mexicana and from all developmental stages of the other species analysed ( L. donovani and L. major ). Low-activity, low- M _r proteinases were present in populations of stationary-phase promastigotes of L. m. mexicana . All three species of Leishmania had higher M _r proteinases, a number of which showed developmental regulation, some of them being stage-specific. Significantly, at all stages of the life cycle in all three species a 68-kDa proteinase was apparent. In its size, sensitivity to inhibitors and ability to bind concanavalin A-agarose, this resembles the major surface protein thought to be present in all Leishmania species and which has recently been reported to possess proteinase activity in L. major promastigotes.     

Leishmania amazonensis: involvement of cysteine proteinases in the killing of isolated amastigotes by L-leucine methyl ester

L-leucine-methyl ester (Leu-OMe) kills Leishmania mexicana amazonensis amastigotes by a mechanism which requires proteolytic cleavage of the ester. N-Benzyloxycarbonyl-phenylalanyl-alanyl diazomethane (Z-Phe-AlaCHN2), a specific and irreversible inhibitor of cysteine proteinases, was used to characterize the enzymes involved in parasite destruction. It was shown that (1) amastigotes preincubated with micromolar concentrations of Z-Phe-AlaCHN2 survived challenge with Leu-OMe concentrations lethal to control parasites; (2) the proteolytic activity of 25- to 33-kDa cysteine proteinases in parasite lysates subjected to electrophoresis in gelatin-containing acrylamide gels was selectively inhibited in parasites pretreated with Z-Phe-AlaCHN2 and chased in inhibitor-free medium; and (3) cysteine proteinase activity was also inhibited in gels incubated with amino acid and dipeptide esters, possibly because the compounds were acting either as substrates (e.g., Leu-Leu-OMe) or as inhibitors (e.g., Ile-OMe) of the enzyme. The results support the involvement of low molecular weight cysteine proteinases in the destruction of amastigotes by Leu-OMe. Characterization of the structure and substrate specificity of the enzymes may permit the rational development of more selectively leishmanicidal amino acid derivatives.     

Purification of cysteine proteinases from trichomonads using bacitracin-sepharose

Abstract Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus . It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteines from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 μg of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10⁹ cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusuall features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.     

Inhibition of cysteine proteinases by a protein inhibitor from potato

The inhibitory specificity of a protein from potato tubers that inhibits cysteine proteinases (potato cysteine proteinase inhibitor, PCPI) has been compared with that of chicken egg-white cystatin. Most proteinases that are inhibited by cystatin were also inhibited by PCPI, but the potato inhibitor inhibited stem bromelain and fruit bromelain, which are not inhibited by cystatin, and for which no protein inhibitor of comparable potency has previously been described. In contrast, papaya proteinase IV was unaffected by PCPI as it is by the cystatins, and the exopeptidase, dipeptidyl peptidase I, is inhibited by cystatins, but was unaffected by PCPI. The differences in inhibitory specificity between these proteins may well reflect differences between superfamilies of cysteine proteinase inhibitors.     

Characterization of a multi-copy gene for a major stage-specific cysteine proteinase of Leishmania mexicana.

lmcpb, a gene from Leishmania mexicana that encodes a major cysteine proteinase in the parasite, has been cloned and sequenced. LmCPb is related more to cysteine proteinases from Trypanosoma brucei and Trypanosoma cruzi than to a previously characterized cysteine proteinase, LmCPa, of L. mexicana. It contains a long C-terminal extension characteristic of similar enzymes of T. brucei and T. cruzi. The gene is multi-copy and tandemly arranged. lmcpb RNA levels are developmentally regulated with steady state levels being high in amastigotes, low in metacyclic promastigotes and undetectable in multiplicative promastigotes. This variation correlates with and may account for the stage-specific expression of LmCPb enzyme activity.     

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.     

Proteinase activity in rumen ciliate protozoa

Azocasein-degrading proteinase activity was detected in all rumen ciliate protozoa that were examined from four entodiniomorphid and two holotrich genera. All of the activities were optimal in the range pH 4.0-5.0 and were inhibited by cysteine proteinase inhibitors, notably leupeptin. The inhibition profiles and extent of inhibition observed with the different groups of inhibitors were organism-specific. Gelatin-SDS-polyacrylamide gel electrophoresis of protozoal lysates revealed multiple forms of the proteinases in the species examined. The number of enzymes detected, their molecular masses, the level of activity and inhibitor susceptibility was genus-dependent. The proteinase profiles of the two holotrich species differed and inter-species differences were also apparent among species of the genus Entodinium. The characteristics and molecular size distribution of rumen bacterial proteinases were different to the protozoal proteinases. Low levels of proteinase activity, of apparently bacterial origin, were detected by gelatin-SDS-PAGE analysis of cell-free rumen liquor.     

Leishmania mexicana: amastigote hydrolases in unusual lysosomes

Leishmania mexicana mexicana (M379) amastigotes were found to contain much higher activities than cultured promastigotes of five putative lysosomal enzymes: cysteine proteinase; arylsulfatase (EC 3.1.6.1); beta-glucuronidase (EC 3.2.1.31); DNase (EC 3.1.22.1), and RNase (EC 3.1.27.1). The release profiles of the first three of these enzymes from digitonin-permeabilized amastigotes suggests that they are located within organelles. Cytochemical staining for cysteine proteinase, using gold labeled antibodies and arylsulfatase, showed that both were present in large organelles previously named "megasomes." Comparative studies with L. mexicana amazonensis (LV78), L. donovani donovani (LV9), and L. major (LV39) revealed that L. mexicana amazonensis was similar to L. mexicana mexicana in possessing both high amastigote cysteine proteinase activity and large numbers of megasome organelles in amastigotes, whereas the other two species lacked both these features. The results suggest that the presence of numerous lysosome-like organelles in the amastigote is a characteristic of the L. mexicana group of parasites.     

Cloning and sequencing of cDNA encoding a rat salivary cysteine proteinase inhibitor inducible by beta-adrenergic agonists

The beta-adrenergic agonist isoproterenol induces a unique secretory protein (LM) in the salivary glands of developing and adult rats. In order to study the regulation of growth and gene expression by catecholamines, we have isolated and sequenced several cDNA clones encoding the LM protein. Each of the LM cDNA clones described identifies, by Northern blot analyses, a single mRNA species of approximately 900 bases in size. The mRNA encoding this secreted protein was not detected in submandibular glands or brains of untreated adult rats. Sequence analyses of the LM cDNA clones revealed a striking similarity to the family 2 of cysteine proteinase inhibitors. Furthermore, when purified LM protein was used to assay for inhibition of cysteine proteinases, the data demonstrated that it is indeed a type of cysteine proteinase inhibitor. This inhibitor, termed rat cystatin S, provides the first example of cysteine proteinase inhibitors that can be induced by beta-adrenergic agonists.     

Electrophoretic analysis of the polypeptides of Leishmania amastigotes and promastigotes

The polypeptides of Leishmania mexicana mexicana (M379), L. m. amazonensis (LV78), L. major (LV39) and L. d. donovani (LV39) amastigotes and cultured promastigotes have been analysed by SDS-polyacrylamide gel electrophoresis. The polypeptide banding patterns of the promastigotes of the four species were quite similar, but distinct differences were detected between those of amastigotes. The results suggest that the various species of Leishmania are adapted differently for survival and growth in the mammalian host. The polypeptides of L. m. mexicana amastigotes were very rapidly hydrolysed unless protected by the cysteine proteinase inhibitor leupeptin.     

Purification and characterization of proteolytic enzymes of Leishmania mexicana mexicana amastigotes and promastigotes

Leishmania mexicana mexicana amastigote and promastigote soluble proteinases were purified using gel filtration and ion exchange chromatography. For the amastigotes, two main proteinase activity peaks were separated with both methods. These accounted for approximately 10% and 90% of the total activity. Characterization of the two activities for substrate specificity and sensitivity to inhibitors indicated that the major peak from both column methods contained enzymes with the characteristics of cysteine proteinases. SDS-polyacrylamide gel electrophoresis of the enzyme from the major peak purified by gel filtration revealed one polypeptide with a molecular weight in the region of 31 000. In contrast, the activity of the minor peak eluted from the columns was of higher molecular weight (67 000) and was similar to metalloproteinases. Purification of the soluble proteinases in the promastigote of L. m. mexicana produced only one activity peak from both column techniques. This activity (mol. wt 67 000) corresponded to the high molecular weight proteinase of the amastigote. The purified proteinases were active on 4-nitroanilide and 7-amino-4-methylcoumarin derivatives of various small peptides. The high molecular weight proteinases of both amastigotes and promastigotes were similarly active against most of the peptides, suggesting a low specificity of the enzymes. In contrast, the low molecular weight amastigote proteinases were particularly active against two of the substrates, namely BZ-Pro-Phe-Arg-Nan and Z-Phe-Arg-MCA. These results indicate that a highly active, substrate-specific, soluble proteinase, with characteristics of a cysteine proteinase, is produced upon transformation of the L. m. mexicana promastigote to amastigote. The discovery and characterization of this enzyme offers opportunities for the development of new antileishmanial agents.     

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.     

Expression of Cysteine Proteinases by Metacyclic Promastigotes of Leishmania mexicana

ABSTRACT. 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 .     

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.     

Hydrolysis of L-leucine methyl ester by Leishmania mexicana mexicana amastigote cysteine proteinases.

The main cysteine proteinases of the amastigote form of Leishmania mexicana mexicana were partially purified by gel filtration and ion exchange chromatography. The latter procedure resulted in the separation of some individual cysteine proteinases, as demonstrated by gelatin-sodium dodecyl sulphatepolyacrylamide gel electrophoresis. Fractions containing the partially purified proteinases rapidly hydrolysed L-leucine methyl ester to leucine. The activity towards this compound co-eluted with and resembled the parasite's cysteine proteinase activity. The results suggest that amastigotes of L.m.mexicana are susceptible to L-leucine methyl ester because this compound is rapidly hydrolysed by cysteine proteinases that occur in abundance in the megasomes of this stage.     

Cystatin S: a cysteine proteinase inhibitor of human saliva

An acidic protein of human saliva, which we named SAP-1 previously, is now shown to be an inhibitor of several cysteine proteinases. The protein inhibited papain and ficin strongly, and stem bromelain and bovine cathepsin C partially. However, it did not inhibit either porcine cathepsin B or clostripain. The mode of the inhibition of papain was found to be non-competitive. The name cystatin S has been proposed for this salivary protein in view of the similarities in activity and structure to other cysteine proteinase inhibitors such as chicken egg-white cystatin and human cystatins A, B, and C. The cystatin S antigen was detected immunohistochemically in the serous cells of human parotid and submaxillary glands.     

Proteolysis of enteric cell villin by Entamoeba histolytica cysteine proteinases

Invasive microorganisms efface enteric microvilli to establish intimate contact with the apical surface of enterocytes. To understand the molecular basis of this effacement in amebic colitis, we seeded Entamoeba histolytica trophozoites on top of differentiated human Caco-2 cell layers. Western blots of detergent lysates from such cocultures showed proteolysis of the actin-bundling protein villin within 1 min of direct contact of living trophozoites with enterocytes. Mixtures of separately prepared lysates excluded detergent colysis as the cause of villin proteolysis. Caspases were not responsible as evidenced by the lack of degradation of specific substrates and the failure of a specific caspase inhibitor to prevent villin proteolysis. A crucial role for amebic cysteine proteinases was shown by prevention of villin proteolysis and associated microvillar alterations through the treatment of trophozoites before coculture with synthetic inhibitors that completely blocked amebic cysteine proteinase activity on zymograms. Moreover, trophozoites of amebic strains pSA8 and SAW760 with strongly reduced cysteine proteinase activity showed a reduced proteolysis of villin in coculture with enteric cells. Salmonella typhimurium and enteropathogenic Escherichia coli disturb microvilli without villin proteolysis, indicating that the latter is not a consequence of the disturbance of microvilli. In conclusion, villin proteolysis is an early event in the molecular cross-talk between enterocytes and amebic trophozoites, causing a disturbance of microvilli.     

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.     

Assay of alpha-cysteine proteinase inhibitor in serum or plasma

A new proteinase inhibitor has recently been found in human serum or plasma which specifically inhibits cysteine proteinases such as ficin, papain, bromelain and cathepsin B. However, serum contains alpha 2-macroglobulin which also inhibits these cysteine proteinases and, consequently, interferes with the assay of the new alpha-cysteine proteinase inhibitor. Therefore, assay of the inhibitor in serum has not been established previously. In the present method, the alpha 2-macroglobulin is inactivated by preincubating the serum in methylamine solution at 55 degrees C, while the alpha-cysteine proteinase inhibitor retains its activity. The inhibitory power against cysteine proteinases is found to be due mainly to this protein in human serum. This inhibitor is also found in mammals such as cows, pigs and rats. Vitamin E deficient rats show a very high inhibitor level. Therefore, the present method will enable us to investigate the relation between diseases and the activity of the alpha-cysteine proteinase inhibitor. Also, this method is simple and inexpensive. The necessary amount of serum is only 10 microliter.