270 Parasitology Today, vol. 6, no. 8, 1990

Cysteine Proteinases of Parasitic Protozoa

M.J. North, J.C. Mottram and G.H. Coombs

Proteinases are involved with many pro- cesses in living organisms. In recent years, there has been increasing interest in eluci- dating the functions the enzymes perform in parasites. These studies have revealed that one class of proteinases, the cysteine proteinases, predominates in many para- sitic protozoa. In this article Mick North, ]eremy Mottram and Graham Coombs review what is known about the cysteine proteinases of parasitic protozoa and dis- cuss the approaches being pursued in attempts to design antiparasite drugs based on inhibitors or substrates of these enzymes.

The proteinases of parasites have attrac- ted considerable attention during the last five years. It is now recognized that, in addition to their role in general protein turnover, proteinases (Box I) are cru- cially involved in many aspects of host- parasite interactions. The advent of new types of inhibitors that show high spe- cificity for particular proteinases has raised hopes that such compounds could prove to be useful antiparasitic agents. The possibilities seem clear, but which approaches are likely to bring success most rapidly? In this article, we review the current status of research into a group of proteinases that appears to be the most abundant in parasitic protozoa and that seem to be particularly suscep- tible to chemotherapeutic exploitation - the cysteine proteinases.

O c c u r r e n c e

Cysteine proteinases have been detected in most groups of parasitic pro- tozoa (Box 2). The enzymes typically have high activity in at least one stage of the life cycle - often a stage present in the mammalian host. Cysteine pro- teinases are the only type of proteinase that has been detected in some proto- zoa so far, but in the majority of species investigated other proteinases are also present: a well characterized example is the surface-located metalloproteinase of Leishmania species 47. Many of the proto- zoan cysteine proteinases are compar- able in size to plant and animal enzymes (see Box 3), but some species contain cysteine proteinases that are apparently considerably larger. In most instances, the enzymes are lysosomal.

Recent A d v a n c e s and Studies in Progress

Many studies have been concerned with identifying the types of proteinases present in parasites at different stages of their life cycle. Compounds considered to act specifically against the proteinases of different groups (see Table I) are used widely as tools for identification. They have been extremely useful for this purpose, but it is essential that the results are interpreted with caution, as it is clear that some of the inhibitors are not totally specific. These investigations have revealed some interesting differences between the proteinase content of para- sites and mammals. Most mammalian tissues contain only three major cysteine proteinases (cathepsins B, L and H) whereas some parasitic protozoa, no- tably trichomonads, trypanosomes and L. mexicana, contain relatively large num- bers of distinct enzymes.

Many of the protozoan proteinases appear to be unusually large (Box 2). The molecular mass data in many cases have been derived simply from the relative mobility of enzymes in substrate gels, but for Trypanosoma cruzi and Trichomonas vaginalis confirmatory evidence for the large size of the enzymes has come from SDS-PAGE and gel filtration. Another

feature of many protozoan cysteine pro- teinases is their relative stability to alkali, as compared with mammalian enzymes, most of which are rapidly inactivated. Further analysis is required to find expla- nations for these differences between parasite and mammalian enzymes, but whatever the cause, it seems likely that they will be reflected in the enzymes' activities.

The substrate specificity of individual proteinases in cell lysates can be deter- mined by separating them on gels and determining their activity in situ towards a range of fluorogenic substrates s'9'26. Combination of this method with gelatin SDS-PAGE has allowed comparisons to be made of the activities of individual enzymes towards proteins and peptide substrates. Studies of this type can provide extensive data on peptide sequences that can be hydrolysed by individual enzymes. Such information is central to the design of specific inhibi- tors, especially since many of the most promising compounds are peptide de- rivatives whose amino acid composition can be changed to increase specificity (see Table I). However, the natural sub- strates of the enzymes are most likely to be proteins with complicated tertiary structures that affect their susceptibility to hydrolytic attack, and identifying these physiological substrates and hence the

Box I. Proteinases The classes of enzymes that are grouped together under the general name proteinase (or endopeptidase) share the ability to catalyse the hydrolysis of peptide bonds within proteins. Four main classes of proteinase have been detected: aspartic, serine, metallo- and cysteine proteinases. The names were coined in recognition of key active site residues of the enzymes. Some proteinases are very specific, enabling them to perform highly specialized roles within the cell; for example, activation of a proprotein. Other proteinases are active towards a wide range of proteins. These enzymes are involved in more general proteolytic events such as those that occur within lysosomes. For the majority of enzymes, however, very little is known about substrate specificity. Conse- quently, the parts played by the enzymes in the cells can only be guessed, taking into account such factors as subcellular distribution, pH optimum and occurrence during cell differentiation. The specificity of proteinases is determined in part by the amino acid sequence of the target protein. The accepted nomenclature for the different amino acid sites is:

peptide bond hydrolysed

N terminus-.. P3- P2- P i - P'l - P~- P'3'" C terminus

The binding site on the proteinase for each of these amino acids is known as the corresponding S pocket. For instance, the S 2 pocket is where the P2 amino acid binds.

Some of the features of proteinases that are crucial for activity have been described, mainly as a result of extensive investigations of a few 'model' enzymes, for example, papain (see Fig. I and Box 3).

© 1990, Elsevier Science Publishers Ltd, (UK) 01694707/90/$0200

Parasitology Today, vol. 6, no. 8, 1990 271

Box 2. Occurrence of Cysteine Proteinases in Parasitic Protozoa

Leishmania mexicana I-s

Apparent sizes. Multiple (at least seven enzymes), 18-31 kDa~ Occurrence. Amastigotes., low activities detectable in stationary- phase promastigotes (putative metacyclic stages). Purification. Several enzymes of L. m. mexicana. Sequence. Complete sequence derived from cDNA (J.C. Mottram et al., unpublished). Typical proregion but unusual sequence at N terminus of central region. Short C-terminal extension. Comments. Stage-specific. Associated with unusual lysosomes (megasomes) present in amastigotes only. Include three distinct groups of enzymes on the basis of substrate specificity. Responsible for activation of leish- manicidal amino acid e,~ters. Probably required for parasite survival in macrophages. Similar enzymes have not been detected in other species of Leishmania, although a cysteine proteinase gene fragment has been amplified from/_ major (J.H. McKerrow, J.A. Sakanari and J. Bouvier, pets. commun.).

Trypanosoma b. brucei 6-9

Apparent sizes. Multiple (at least four enzymes), 25-45 kDa, major enzyme 27-28 kDa. Occurrence. Major enzyme detected at all stages investigated, higher levels reported in short stumpy bloodstream forms. Purification. None with T. b. bruceL A 31 kDa proteinase has been purified from bloodstream forms of T. congolense I°. Sequence. Complete sequence derived from cDNA, includes a typical proregion and central region but has an unusual C-terminal extension. Comments. Preference for sub- strates with bulky, hydrophobic residues at the P2 position (cathepsin L-like). Present in the lysosomal fraction.

Trypanosoma cruz/I 1-20

Apparent sizes. Four enzymes (50->200 kDa) detected by electrophoresis. Occurrence. 200 kDa enzyme isolated from epimastigotes. 60 kDa enzyme (cruzipain) present at 10-fold higher levels in epimastigotes than in amastigotes and trypomas- tigotes. Purification. 60 and 200 kDa enzymes. Sequence. Those of the N terminus and a short segment near the C terminus have been derived for the 60 kDa enzyme. A gene fragment has been amplified by PCR and sequenced: it overlaps with the 60 kDa proteinase amino acid sequence. No unusual features yet appar- ent. Comments (60 kDa enzyme). Preference for Arg-Arg and Phe-Arg derivatives as substrates (cathepsin B-like). Sequence closer to cathepsin L. Explanation for the unusually large size not yet apparent from the sequence data. Glycosylation accounts for 16 kD~ Located in lysosomes but also detected on the cell surface. Involved in penetration into host cell?

Trichomonas vaginalis z I-z6

Apparent sizes. Multiple enzymes (between II and 23 distinct activities detected), 18- I I 0 kDa. Larger enzymes more active. Occurrence. Many at hig!h activity intracellularly in lysosomes. Enzymes also released in large quantities. Purification. Partial purification of two intracellular enzymes, 18 and 64 kDa, and two extracellular enzymes, 23 and 60 kDa. Sequence. PCR-derived gene fragments now avaiilable for sequencing. Comments. Quali- tative differences between intracellular and extracellular enzymes. Enzymes of at least three groups according to substrate specificities. 43 kDa proteinase reported to reside on parasite surface. Enzymes may be involved in cytoadherance and cytotox- icity.

Tritrichomonas foetus 24-27

Apparent sizes. Multiple enzymes (at least ten enzymes detected), the most active have molecular weights between 18 and 34 kDa. Occurrence. Lysosomal, but also released in large quantities. Purification. 18-20 kDa enzyme. Sequence. PCR-derived gene fragments now available for sequencing. Comments. Qualitative differences between some intracellular and extracellular enzymes. Differences in specificity between individual enzymes demonstrated.

Giardia lamblia 28-3°

Apparent sizes. Two major enzymes, 38-40 and 105 kDa. Occur- rence. Enzyme detected in trophozoites and cysts of G. muris. Purification. None. Comments. Lysosomal. Hydrolyse human immunoglobulin.

Entamoeba h istolytica 314o

Apparent sizes. Various sizes reported, 16, 26-29, 40, 56 kDa, but similarity of properties suggests that the enzymes are closely related and, in some cases, the same. Occurrence. Trophozoites, the 56 kDa enzyme is secreted. Purification. A number of enzymes have been purified to apparent homogeneity. Sequence. N-terminal sequence of a 26-29 kDa enzyme (histolysin) deter- mined. Gene fragment amplified by PCR and sequenced. Com- ments. High degree of specificity towards peptides containing Arg at the Pz position. Sequence closer to cathepsin L than cathepsin B. Active towards physiologically significant substrates (cartilage proteoglycan, collagen, laminin, fibronectin, comple- ment C3b). Probably involved in tissue invasion. Cytotoxic factor identified as 16 kDa proteinase. Histolysin detaches tissue cul- ture cells. Evidence available that secreted enzyme (56 kDa) responsible for cytopathic effect, but secretion not observed in all cases. The 56 kDa enzyme may be a precursor form of the intracellular 26-29 kDa enzyme (J.H. McKerrow, pets. commun.).

Plasmodium falciparum 41-43

Apparent size. 28 and 68 kDa. Occurrence. Erythrocytic forms: trophozoites (28 kDa); schizonts and merozoites (68 kDa). Purification. 68 kDa proteinase only. Sequence. A fragment of the 28 kDa proteinase gene has been amplified and sequenced (P.J. Rosenthal, pets. commun.). Comments. 28 kDa enzyme stage- specific, located in acidic food vacuole and involved in haemoglo- bin digestion. Inhibitors prevent parasite multiplication. 68 kDa enzyme of narrow specificity with a role in reinvasion of erythro- cytes. Additional note. The malarial I I I-K major antigen contains a region homologous to the mature section of cysteine pro- teinases but lacks the active site cysteine 44-46. It is not known whether this protein is a proteinase.

Other species

Little information is available, although preliminary studies in our laboratory indicate that none of the following possess high activities of cysteine proteinases: Crithidia fasciculata, Herpeto- monas spp, Toxoplasma gondii, Cryptosporidium spp.

roles of the proteinases is a difficult task. The malarial enzyme responsible for the digestion of host cell haemoglobin is one of the few well characterized examples 42,43.

The only approach for many studies of individual enzymes is to purify them

first. Unfortunately, this is still relatively time-consuming, and only very few cys- teine proteinases of parasitic protozoa have been purified to homogeneity. The N-terminal amino acid sequences of cys- teine proteinases of Trypanosoma cruzi 14 and Entamoeba histolytica 34 have been

determined, and both show greater similarities with cathepsin L than cathep- sin B. This approach is being pursued with other parasite proteinases, notably the multiple enzymes that characteris- tically occur in abundance in the amasti- gotes of L. mexicana, although data are

272 Parasitology Today, vol. 6, no. 8, 1990

Box 3. Cysteine Proteinases of Plants and Vertebrates Mature enzymes. All the well characterized cysteine proteinases belong to the papain superfamily and, with the exception of calpains (see below), have closely related primary structures (see Fig. I). There are two branches of the superfamily, one includes mammalian cathepsins L and H and the plant proteinases, the other contains mammalian cathepsin B and helminth cysteine proteinases. The enzymes range in size from 212 amino acid residues (papain, 23.4 kDa) to 252 amino acid residues (cathepsin B, 29.0 kDa). The N-terminal amino acids of the vast majority of mature cysteine proteinases can be aligned exactly with one another, the second amino acid being proline. The position of the C terminus can vary by up to three residues.

Preproregions. All of the cysteine proteinases appear to be synthesized as preproproteins. The preregions represent typical hydrophobic signal peptides. The proregions are N-terminal extensions of between 62 residues (cathepsin B) and 123 residues (aleurain, a barley proteinase) in length and are relatively hydrophilic in character. Some sections are highly conserved between different proproteinases.

C-terminal extensions. The C-terminal residue of most mature cysteine proteinases for which there is information corresponds to that predicted from the complete cDNA sequence. In a few cases, the cDNA sequence predicts a longer protein that includes an extension at the C terminus of the mature enzyme, ranging in size from six residues (cathepsin B) to 109 residues (tomato putative proteinase).

Post-translational changes. In addition to the removal of N-terminal and C-terminal extensions, some cysteine proteinases are subject to further proteolysis. For example, most mammalian cysteine proteinases can exist as two chain forms produced by cleavage of the mature enzyme. All vertebrate cysteine proteinases are N-glycosylated, but bromelain is the only plant enzyme known to be so modified. Several plant cysteine proteinases are not glycosylated.

Three-dimensional structure. This has been solved for the plant enzymes papain, actinidin and calotropin. Essentially, the enzymes consist of two domains that flank the active site. The most highly conserved portions of the polypeptide chain are in the vicinity of the active site. Six cysteine residues involved in three disulphide bridges in papain and actinidin are highly conserved among cysteine proteinases. This, together with the conservation of hydrophobic core residues, suggests that the three-dimensional struc- tures of all the mature enzymes are likely to be similar.

Mechanism. Cysteine proteinases contain a thiol-imidazolyl system within the active site. Catalysis proceeds via a thiol ester formed transiently between the substrate and Cys25 (papain number). Ester formation is facilitated by side chains of adjacent residues, including His159.

Activity. Cysteine proteinases are usually most active at slightly acid or neutral pH. For maximum activity, an agent such as dithiothreitol is required to maintain the active site cysteine in its reduced form. Cysteine proteinases tend to have broad specificity with respect to protein and peptide sequences, but often show a preference for arginine at the PI position (see Box I).

Inhibitors. See Table I.

Cellular location. Mature mammalian cysteine proteinases are all lysosomal. They are synthesized on membrane-bound ribosomes of the endoplasmic reticulum as glycosyl- ated precursors. The preregion is removed immediately. Precursors containing the proregion are transferred to the Golgi. Conversion to the mature enzyme occurs rapidly on arrival at the lysosomes and could be due to the lysosomal aspartic proteinase, cathepsin D or to autoactivation. Any further proteolytic processing occurs within the lysosomes. In some instances, for example with certain transformed cell lines and turnout cells, precursors containing the proregion are secreted. These precursors are either inactive or have a restricted specificity, but can be activated in vitro. Interference with glycosylation or with other aspects of Golgi function also leads to precursor secretion. Plant cysteine proteinases are frequently isolated from extracellular material, but their location and targeting within cells have not been studied in detail.

Calpains. Animal cells contain cytosolic calcium-dependent cysteine proteinases called calpains. These are distantly related to other members of the papain superfamily: domain II of the large subunit of calpain has some homology with the mature region of the other cysteine proteinases. No equivalent protozoan enzymes have been reported.

yet to be published. A joint biochemical and molecular approach is vital, so that the products of individual genes can be identified and correlations made be- tween the gene and protein structure and the functional features of the mature enzyme, such as substrate specificity, susceptibility to inhibitors and subcellular location.

Several cysteine proteinase genes have been identified using degenerate oligonucleotides, either as primers for the polymerase chain reaction (PCR) 16'49 or directly as probes 8. The oligonucleotides have been based upon two highly conserved regions that form part of the active site of the enzymes (see Fig. 2). The spacing of these con- served sequences in cysteine proteinase genes makes them particularly suitable for amplification by PCR and the tech- nique should, in theory, allow equivalent fragments to be amplified from any parasite. PCR has been applied very successfully by Jim McKerrow and his colleagues to clone serine and cysteine proteinase gene fragments from a num- ber of parasites, including E. histolytica, T. cruzi and T. brucei t6'49. Cysteine pro- teinase fragments have also been iso- lated in this way from Plasmodium falciparum (P.J. Rosenthal, unpublished), Trichomonas vaginalis and Tritnchomonas foetus (M.J. North, unpublished) and Theileria annulata (R. Hall and J.C. Mot- tram, unpublished). Sequencing of the fragments amplified in this way has con- firmed that they do indeed have good homology with the sequences of mam- malian and plant enzymes 16. These frag- ments have been used to isolate the full-length genes from genomic or cDNA libraries, and so the complete sequence of cysteine proteinase-encoding genes in a range of parasitic protozoa will soon be known.

To date, however, only one complete sequence of a protozoan cysteine pro- teinase gene has been published 8. The protein structure predicted from this Trypanosoma brucei gene sequence dif- fers significantly from those known for other cysteine proteinases. Although the pre-, pro- and central regions of the predicted enzyme have considerable homology to the mammalian enzyme cathepsin L, the trypanosome gene encodes an unusually long C-terminal extension. On the basis of size esti- mations using gelatin SDS-PAGE of the cysteine proteinases detectable in extracts of 7-. brucei, it seems likely that a significant part of the C-terminal exten- sion remains in the mature protein 8'9. The presence of this region may there- fore be reflected in the enzyme's properties, including its catalytic activity,

Parasitology Today, vol. 6, no. 8, 1990 273

and so offers promise for the design of specific inhibitors. The gene appears to be present in more than 20 copies ar- ranged in a tandem array in the trypano- some genome. No information is yet available on the transcription of this array, nevertheless it is a surprisingly high number of repeats for an enzyme that is not present at a particularly high activity in any of the stages of the parasite that have been examined 7'9.

The gene sequence of the T. brucei enzyme reveals that all of the main amino acids that comprise the $2 pocket (the binding site of the P2 amino acid, see Box I) in cathepsin L are also present in the trypanosome enzyme, suggesting that the enzymes have similar speci- flcities for amino acids at this site. The biochemical studies so far reported sup- port this conclusion. Indeed, with the exception of E. histolytica enzymes (see Box 2), the cysteine pro!:einases of all parasitic protozoa that have been bio- chemically characterized are similar to cathepsin L in showing preference for bulky amino acids at the. P2 position. However, some enzymes, for instance cruzipain of T. cruzi, differ from cathep- sin L in that they will also accommodate basic P2 residues. Nevertheless, it seems likely that if there are major differences in substrate specificity between proto- zoan and mammalian cysteine pro- teinases, they must be at positions other than P2 (the P3 and P' sites, for instance) but this has been little studied.

All mammalian lysosomal cysteine proteinases are glycosylated, with the carbohydrate moiety possibly playing a role in targeting the enzymes to lyso- somes. The gene sequence of the T. brucei enzyme shows two possible glycosylation sites. Neither, however, is in the central region, one being located in the proregion and the other in the C-terminal extension 8. In contrast, the part of the T. cruzi gene sequence that has been determined shows potential glycosylation sites on the central region of the protein ~6. Indeed, the enzyme purified from this parasite has been shown to be a glycoprotein 17, as have some of the enzymes of L. rnexicana s, but for most enzymes it is simply not known.

Many protozoan cysteine proteinases are located in lysosomes, but there is scant information on how they, or other enzymes, are targeted to the organelles. Some parasitic protozoa have been shown to release o/steine pro- teinases 23'2s'33'5°. Attempts have been' made to explain the invas.ive behaviour or other pathological effects of parasites in terms of this release, using the ob- served toxicity of the enzymes towards

Table I. Inhibitors of proteinases a (a) Inhibitors of cysteine proteinases

Name Concentration b Reversible (..)

Iodoacetate I O- 100 No

Tosyl I 0- 100 No lysylchloromethane (TLCK)

Leupeptin I 0-I 00 Yes

E-64 I-I 0 No

Cystatins 0. I - I Yes

Peptidyl I - I 0 No diazomethanes

Peptidyl I - I 0 No fluoromethanes

Mechanism and specificity Alkylates active site cysteine -SH. Not specific for proteinases.

Active-site-directed lysine derivative. Alkylates active site cysteine -SH. Also inhibits trypsin-like serine proteinases.

Peptide containing arginine aldehyde. Binds to active site. Also inhibits trypsin-like serine proteinases.

Peptide epoxide which probably alkylates the active site cysteine. Highly specific. Excellent active site titrant.

Superfamily of protein inhibitors. Bind tightly to the active site. Highly specific.

Alkylate active site cysteine -SH, probably via a thiohemiketal derivative. Peptide determines selectivity. With one well characterized exception (plasma kallikrein), specific for cysteine proteinases.

Alkylate active site cysteine -SH. Peptide determines selectivity. Many are inhibitors ofserine proteinases, although some appear to be specific for cysteine proteinases.

(b) Commonly used inhibitors of other types of proteinase

N a m e Pepstatin Phenylmethane sulphonyl fluoride (PMSF) Diisopropyl fluorophosphate (DipF) 3,4-Dichloroisocoumarin (3,4-DCI) EDTA I, I 0-phenanthroline

Type of proteinase inhibited Aspartic Serine Serine Serine Metallo- Metallo-

"See Ref. 48 for further details. b Recommended concentration range for inhibition of enzymes in parasite lysates.

mammalian cell lines in vitro as evi- dence 33'34. The finding that cysteine proteinase inhibitors abrogate the pathogenicity of some parasites sup- ports this idea s~'s2. Proteinase release is also widespread among helminths, although the enzymes involved in most cases are serine or metallo-protein- ases 53, and cysteine proteinases have also been implicated with the allergens of mites s4, so this is an aspect of current interest. The mechanisms by which para- sitic protozoa release proteinases are not known, but the equivalent of the proregion (see Box 3) is still present on

cysteine proteinases released from some cancer cells. It will also be interest- ing to discover whether enzymes se- creted by protozoa are processed differently from those cysteine pro- teinases that remain within the cells.

A p p r o a c h e s to D r u g Design

The advent of families of compounds that have good and relatively specific activity against cysteine proteinases (see Table t) has resulted in a rush to test them against both parasite enzymes and

274 Parasitology Today, vol. 6, no. 8, 1990

Region Pre Pro Central/mature C-terminal

Length ~20 62-123 212-252 0-109 (amino acids)

N-glycosylation ~ "k ~ = sites 22-63 153-200

lie S-S-

s,,,, ..... "m [__$_$/56_9s CYS 25

[~S- S---I • ~///////.~//////Z//////////~

I "V. . . . . s i t e 2

ASN, SER, TRP 175-177

HIS 159

Fig. I. The primary structure of a cysteine proteinase. The diagram gives an outline of the structure of a member of the papain superfamily, showing some of the key conserved residues involved in the active site (numbers refer to papain residues). Note that Cys153, in the right-hand disulphide bridge, is absent from cathepsin B and the Sin31 proteinase 0fSchistosoma mansoni, and that Asp 175 and Ser176 are absent from bromelain. N-Glycosylation sites: < >, region in mature enzyme containing known sites; *, putative sites in some proregions. Site I and Site 2 refer to conserved sequences used to design oligonucleotides (see Fig. 2).

also the parasites themselves. Many in- hibit a variety of parasitic protozoa pro- teinases and some have been reported to have antiprotozoal activity in v i t ro 9'26'42'55-57. As yet, however, there is only one report of a proteinase in- hibitor showing antiprotozoal activity in vivo s2. Nevertheless, this is an area of current research and it is to be hoped that some compounds will eventually become useful drugs.

There are different views on the best approach to follow to develop such drugs. The compounds currently avail- able have not been designed with the peculiarities of protozoan proteinases in mind; rather they have been used in parasitology after they have been shown to be potent against mammalian enzymes. It is hoped that compounds especially active against parasite pro- teinases will be developed, but for this to be achieved most efficiently extensive data on the substrate specificity of the parasite enzymes and how this differs

from that of the mammalian enzymes are essential. This rational approach, however, entails a considerable amount of preliminary work and it remains to be seen if simply producing compounds randomly may achieve the goals more quickly and cheaply.

All the unusual features of the proto- zoan enzymes are theoretically open to selective chemotherapeutic attack. For instance, proteinases located on the cell surface or released are potentially vul- nerable to inhibitors that do not per- meate into cells and so have no toxicity towards cells possessing only intracellu- lar cysteine proteinases. Most current efforts, however, are aimed at pro- ducing active-site directed inhibitors. A major problem is frequently the difficulty of obtaining sufficient pure enzyme from the relatively small amounts of parasite material available. The recent advances in molecular technology mean that it is now possible to express cloned parasite genes in heterologous systems. This

a 20 25

Dictyostelium CP1 Actinidin Papain Human Cathepsin L

consensus

Q G Q C G S C W Q G E C G G C W Q G S C G S C W Q G Q C G S C W

Q G Q C G S C W

170 175

Y W I V K N S W Y W I V K N S W Y I L I K N S W Y W L V K N S W

Y W I V K N S W

b 5' primer: EcoR1 Q G Q C G S C W

5' CCGAATTC CAR GGI CAR TGY GGI XNI TGY TGG 3'

3' primer: 5' CCAAGCTT CCA INX RTT YTT IAC PAT CCA RTA 3' Hind111 W S N K V I W Y

Fig. 2. (a) Two highly conserved regions of cysteine proteinases and (b) the oligonucleotides used for PCR. The oligonucleotides contained all possible nucleotide combinations that could encode the nucleic acid sequences, with inosine being used to maximize base-pairing in positions where all four bases were specified for a codon. A great advantage of PCR is that the use of degenerate oligonucleotides accommodates differences in the codon bias and protein sequence between organisms. X = T/A, N = G/C, Y = T/C, R = A/G, I = inosine, P = A/G/T.

should allow the proteins to be charac- terized in much greater detail, including both crystallographic and modelling studies, which will provide the detailed information necessary for the design of specific inhibitors.

Another approach being pursued in the search for new drugs is the design of prodrugs that would be specifically acti- vated by the parasite cysteine pro- teinases 58's9. Again, knowledge of the substrate specificity of the parasite enzymes is an essential prerequisite, although some selectivity towards the parasite may be possible because of dif- ferences in enzyme distribution or abun- dance between parasite and host. Parasite-activated prodrugs are attract- ive possibilities, for the toxicity to the host should be minimal. However, their design will not be easy, for not only should molecules be toxic to the parasite only upon hydrolysis, but they must also possess pharmacokinetic properties that allow them to reach and enter the para- site. Such compounds have yet to be reported, but their potential advantages make the idea worth pursuing.

Acknowledgements We thank J.H. McKerrow, P.J. Rosenthal and J.J. Cazzulo for providing us with unpublished information and the Wellcome Trust for financial support.

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31 McLaughlin, J. and Faubert, G. (I 977) Can. ]. Microbial. 23,420-425

32 Lushbaugh, W.B., Hofbauer, A.F. and Pittman, F.E. (1985) EXp. Parasitol. 59, 328-336

33 Keene, W.E. et al. (1986)J. Exp. Med. 163,

Insecticide Resistance

As you point out in This Month in the May issue of Parasitology Today I , agricultural pesticides have been clearly shown to be responsible for insecticide resistance in Anopheles albimanus, the principal malaria vector in Central America. You also mention that the same accusation has been made about malathion resistance in the malaria vectors of Sudan and Sri Lanka.

Since agricultural chemicals are so often accused in this way, it seems important to set the record straight about the few cases where there is adequate evidence one way or the other. In both Sudanese An. arabiensis and Sri Lankan An. culicifacies, the resistance is specific to malathion, does not give cross- resistance to the chemicals used in agriculture, and cannot have been selected for bythem. In Sudan, resistance inAn. arabiensis increased wherever anti-malaria spraying with malathion was continued, Where fenitrothion was used instead, resistance remained constant or declined, in spite of continued aerial spraying of insecticide cocktails throughout the area.

These facts (for more details, see Ref. 2) clearly incriminate anti-malaria house- spraying, rather than agricultural chemicals, as the source of selection for resis:ance in these cases.

J. Lines Department of Medical Parasitology London School of Hygiene and Tropical Medicine Keppel Street London WC I E 7HT, UK

536-549 34 Luaces, A.L. and Barrett, A.J. (1988) Biochem.].

250, 903-909 35 Scholze, H. and Werries, E. (1986) Mol. Bio-

chem. Parasitol. 18, 103- I 12 36 Reed, S.L., Keene, W.E. and McKerrow, J.H.

( 1989)]. Clin. Microbial. 27, 2772-2777 37 Reed, S.L. et oL (1989) ]. Immunol. 143,

189-195 38 Otte, J. and Werries, E. (I 989) Mol. Biochem.

Parasitol. 33,257-264 39 Scholze, H. and Schulte, W. (1988) Blamed.

Biochim. Acta47, 115 123 40 Perez-Montfort, R. et al. (1987) Mol. Biochem.

Parasitol. 26, 87-98 41 Grellier, P. et al. (1989) Parasitol. Res. 75,

455-460 42 Rosenthal, P.J. et al. (1989) Mol. Biochem. Para-

sitoL 35, 177-184 43 Rosenthal, P.J. et al. (1988)J. Clin. Invest. 82,

1560-1566 44 Higgins, D.G., McConnell, D.J. and Sharp, P.M.

(1989) Nature 340, 604 45 Eakin, A.E. et al. (1989)Nature 342, 132 46 Mottram, J.C., Coombs, G.H. and North, M.J.

(1989)Nature 342, f 32 47 Bouvier, J. et al. (1989) Mol. Biochem. Parasitol.

37, 235-246 48 Beynon, R.J. and Bond, J.S. (1989) Proteolytic

Enzymes: A Practical Approach, IRL Press 49 Sakanari, J.A. et al. (1989) Proc. Natl Acad. Sci.

USA 86, 48634867 50 Lockwood, B.C., North, M.J. and Coombs,

References I Ash, C. Parasitology Today 6, 14 I 2 Lines, J. Parasitology Today (1988) 4 (Suppl.),

S 17-$20

Schistosome Surface Protein Anchors

In response to J. Havercroft's review of the GPI-anchoring of schistosome proteins ~ , we can now provide some additional information.

of our article further Since the publication 2 studies have been carried out to characterize the release of GPI-anchored antigens from adult worms in viva. Significant amounts of the 200 kDa protein are released from live adult worms following treatment with PIPLC from Bacillus thuringiensis, as assessed by anti-CRD crossreactivity. This release was readily seen when the supematants, rather than the total worm extracts, were analysed by SDS- PAGE. Under the same experimental conditions, the 22 kDa protein was not released from adult worms in viva, possibly due to the presence of an additional palmitate molecule attached to the inositol moiety of the GPI-anchored portion, blocking the release of the protein, as had been previously described for human erythrocyte acetylcholinesterase 3.

With respect to the expression of these proteins in the early schistosomular stage, we have observed that the 22 kDa protein can

G.H. (1988) Mol. Biochem. Parasitol. 30, 135-[42

51 Keene, W.E. etal. Exp. Parasital. (in press) 52 Bremner, A.F., Coombs, G.H. and North, MJ.

(1986)IRCSMed ScL 14, 555-556 53 McKerrow, J.H. (1989) Exp. Parasitol. 68,

111-115 54 Chua, K.Y. et al. (1988)]. Exp. Meal 167,

175-182 55 Rockett, K.A. et al. (1990) FEBS Lett. 259,

257-259 56 Coombs, G.H., Hart, D.T. and Capaldo, J.

(I 982) Trans. Roy. Sac. Trap. Med. Hyg. 76, 660-663

57 Coombs, G.H. and Baxter, J. (I 984) Ann. Trap. Meal Parasitol. 78, 21-24

58 Coombs, G.H. (I 989) in Leishmaniasis: The Current Status and New Strategies for Control (Hart, D.T., ed.), pp 851-858, Plenum Press

59 Coombs, G.H. (I 986)]. Roy. Army Meal. Corps 132, 147-148

Michael North is at the Department of Biologi- ca/and Molecular Sciences, University of Stir- ling, Stirling FK9 4LA, UK, Jeremy Mot t ram is at the Wellcome Unit o f Molecular Parasitology, Institute of Genetics, University of Glasgow, Church Street, Glasgow G I I 5JS, UK and Graham Coombs is at the Laboratory for Bio- chemical Parasitology, Department of Zoology, University of Glasgow, Glasgow G 12 8QQ, UK.

be metabolically labelled with [3SS]- methionine and 3H-fatty acids as early as 3 h after transformation, whereas it required more than 4 days before the 200 kDa protein was expressed.

S.Y. Sauma and M. Strand Department of Pharmacology and Molecular Sciences Johns Hopkins School of Medicine Baltimore, MD 21205, USA

References I Havercroft, J,C, (1990) Parasitology Today 6,

142 2 Sauma, S.Y. and Strand, M. (1990) Mol. Bio-

chem. Parasitol. 38, 199-210 3 Roberts, W.L. (1988)J. Biol. Chem. 263,

18766-18775

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