Molecular & Biochemical Parasitology 120 (2002) 1 – 21

www.parasitology-online.com.

Review

Cysteine proteases of parasitic organisms

M. Sajid *, J.H. McKerrow
Tropical Disease Research Unit, Uni6ersity of California, San Francisco, CA 94122, USA

Received 24 November 2000; accepted in revised form 7 November 2001

Abstract

Cysteine proteases play numerous indispensable roles in the biology of parasitic organisms. Aside from previously known
general catabolic functions and protein processing, cysteine proteases may be key to parasite immunoevasion, excystment/encyst-
ment, exsheathing and cell and tissue invasion. Parasite cysteine proteases are unusually immunogenic and have been exploited as
serodiagnostic markers and vaccine targets. Although host homologues exist, parasite cysteine proteases have distinct structural
and biochemical properties including, pH optima and stability, the alteration in peptide loops or domain extensions, diverse
substrate specificity and cellular location. The disparate nature of parasite cysteine protease compared to the host orthologous
proteins has opened opportunities for chemotherapy. This review will highlight recent research on the ‘papain-like’ class of
cysteine proteases, the most abundant family, and the newly discovered class of asparaginyl-endopeptidases. Cysteine protease
classification will be re-examined in light of the diversity uncovered within parasitic organisms. © 2002 Published by Elsevier
Science B.V.

Keywords: Cysteine protease; Papain-family; Legumain-like; GPI:protein transamidase; Classification; Protease trafficking; Evolution; Nutrition;
Biological function; S2 pocket; Occluding loop; Chemotherapy; pH; Immunoevasion; Tissue and cell invasion; Protein processing; Excystment and
serodiagnostics

1. Introduction biological systems from viruses to vertebrates. As

Peptide hydrolases (proteases) catalyse the cleavage
of amide linkages in macromolecular proteins and
oligomeric peptides. Proteases have been identified in
Abbre6iations: AMC, amidomethyl coumarin; CA074, N(L-trans-
propylcaramoyloxirane-2-carbonyl)-L-isoleucyl-proline; Der, Derma-
tophagus allergen; E64, L-trans-epoxysuccinyl-leucyl-amido
(4-guanidino)butane; EMBL, european molecular biology laboratory;
ER, endoplasmic reticulum; Fp2, Plasmodium falciparum cysteine
protease 2; GlCP, Giardia lamblia cathepsin B-like protease; GPI,
glycosylphosphatidylinositol; K11777, N-methyl piperazine-urea-
phenylalanyl-homophenylalanyl-vinylsulfone-benzene; MSP-1, mero-
zoite surface protein 1; RDG, Arg–Asp–Gly; SERA, serine repeat
antigen; SmCB1, Schistosoma mansoni cathepsin B-like protease 1;
Z –FR– AMC, Z – Phe– Arg–AMC; Z–RR–AMC, Z–Arg– Arg–
AMC.

Supplementary material to this article can be found at http://
www.elsevier.com/PII/S016668510104388.
* Corresponding author.
E-mail address: sajid@socrates.ucsf.edu (M. Sajid).
genome-sequencing projects are completed it has be-
come clear that proteases comprise approximately 2%
of all expressed genes with little variance between or-
ganisms (A. Barrett, personal communication). It is
estimated that without proteases as biological catalysts
it would take hundreds of years to hydrolyse a peptide
bond; in comparison a protease can degrade as many as
one million peptide bonds per second. Proteases range
from monomers of 10 kDa to multimeric complexes of
several hundred kDa. Catalysis can be initiated either
within a polypeptide chain (endoprotease activity) or
from amino or carboxyl ends (exopeptidase activity).
Proteases have been divided into groups on the basis of
the catalytic mechanism used during the hydrolytic
process. The main catalytic types are serine, threonine,
aspartate, metallo and cysteine proteases, other ‘un-
defined’ or cryptic proteases may also exist. There are a
number of excellent reviews covering general parasite
proteinases [1–6]. This review will concentrate on the

0166-6851/02/$ - see front matter © 2002 Published by Elsevier Science B.V.

PII: S 0 1 6 6 - 6 8 5 1 ( 0 1 ) 0 0 4 3 8 - 8
2 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21
cysteine class referred to as thiol or sulfhydryl
proteinases [7] and review more recent novel areas of
research. The enhanced interest in cysteine proteases is
reflected in the literature. Five years ago there were a
few hundred citations on cysteine proteases per year,
whereas over 1200 per year are published now.
It is also now established that many parasite
proteases are promising chemotherapeutic or vaccine
targets [4,8,9]. Interest in cysteine proteases as targets
derives from the recognition that they are critical to the
life cycle or pathogenicity of many parasites. This func-
tional diversity is in turn derived from their unique
nucleophilicity, adaptability to different substrates, and
stability in different biological environments. Parasite
derived cysteine proteases play key roles in immunoeva-
sion, enzyme activation, virulence, tissue and cellular
invasion as well as excystment, hatching and moulting.
2. Background
Prior to hydrolysis of peptide bonds, a protease must
bind the protein or peptide substrate in its active site.
The binding efficiency is a function of both the respec-
tive chemical environments that the protease subsites
create, and the chemical nature of the peptide that
interacts directly with the active site groove. Important
factors affecting interactions include; size, polarity,
charge, hydrophobicity and accessibility. Although a
single peptide bond is cleaved during catalysis, a num-
ber of amino acids either side of the site of cleavage are
critical to fix the register and specificity of an enzyme
(Fig. 1A). Sequences that directly flank the active site
cysteine and histidine are also highly conserved to
maintain catalytic register. These conserved regions
have been used to classify proteases and to clone or-
thologous genes in a reverse genetic approach. Sub-
strate specificity may also be exploited for identification
of selective anti-protease inhibitors, and in some cases
to predict natural substrates. Cysteine proteases have
mechanistic similarities with serine proteases but are
better nucleophiles due to the extra shell of electrons
present in the sulphur of the thiol group.
Cysteine proteases require an essential cysteine
residue in the active site for hydrolysis. The thiol-group
is enhanced as a nucleophile due to the close proximity
of an active site histidine which acts as a proton
donor/general base. The sulfhydryl or ‘SH’ group of
cysteine side chain, and the imidazole of histidine give
rise to a thiolate–imidazolium charge relay diad (Fig.
1B). This frequently, but not always, is stabilised by a
highly conserved asparagine. A highly conserved glu-
tamine forms the oxyanion hole, a crucial element in
forming an electrophilic centre to stabilise the tetrahe-
dral intermediate during hydrolysis. The two ionisable
groups of the thiolate– imidazolium diad allow a broad
pH range of enzymatic activity, consistent with a pKa of
cysteine of around 4.0 and pKa of histidine ionisation of
around 8.5. The charge relay system is further stabilised
by the chemical environment of the active site. It is
thought that the main interactions of papain-like cys-
teine proteases with their substrate occur at S2, S1 and
S1% [10]. The mechanism of hydrolysis has been eluci-
dated and well documented, the enzyme is transiently
bound to the substrate and forms unstable tetrahedral
intermediates prior to returning to an active enzyme
state.
Many cysteine proteases are synthesised as precur-
sors that contain a pro-domain and a mature (catalytic)
domain. In some cases like the proteases from Leishma-
nia, Trypanosoma and several plants, a carboxy termi-
nal extension may also be present. The pro-region has
evolved to provide a number of independent functions,
including: acting as an intramolecular chaperone to
assist in protein folding; an endogenous inhibitor to
regulate protease activity (Ki of 0.4 nM for human
cathepsin B); and in some cases a signal that targets the
protease to its intracellular destination. Cysteine
proteases that are targeted to an intracellular compart-
ment or those that are secreted also include a hydro-
phobic amino terminal sequence of around 15–22
amino acid residues, termed the signal or leader
sequence.
3. Families, structure and phylogeny
3.1. Classification and e6olution
Cysteine proteases of parasitic organisms are divided
into two main groups referred to as clans, CA and CD
[7,11]. In 1879 the first cysteine protease was purified
and characterised from Carica papaya, the papaya fruit,
and was thus named papain. Papain was also the first
cysteine protease structure to be solved. Since its dis-
covery numerous proteases that have sequences in com-
mon with papain have been loosely called ‘papain-like’.
Papain-like, or Clan CA proteases, are further divided
into families. Important parasite proteases are located
to family C1 (cathepsin B and cathepsin L-like) and
family C2 (calpain-like). Other clans and relevant
families of pathogenic organisms include clan CB and
CC (viral proteases) and clan CD (family C13; legu-
main-like) (Fig. 2). Conventionally, proteases are as-
signed to clans and families depending on a number of
characteristics including sequence similarity, possession
of inserted peptide loops, and biochemical specificity to
small peptide substrates. Robust classification relies on
sequence homology directly spanning the catalytic cys-
teine and histidine, and where known, the catalytic
asparagine and the glutamine of the oxyanion hole (see
below). It is clear from work on parasite proteases that
 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21 3

the previous dogma of biological classification based on
mammalian cysteine proteases and papain, requires re-
vision. This will be further discussed in the section on
S2 specificity and the occluding loop.
The order of the catalytic Cys/His (as in clan CA) or
His/Cys (as in clan CD) in the linear peptide sequence
of parasite cysteine proteases suggests that the cysteine
proteases arose from a number of independent evolu-
tionary events. Within each superfamily of proteases it
is thought that the progenitor cysteine protease under-
went rapid initial gene duplication generating
paralogous proteins. This event predates prokaryote/
eukaryote divergence [12]. Gene duplication is also
apparent in that some mammalian cysteine proteases
are located on the same chromosome [13]. The evolu-
tion and specificity seen in present organisms was
driven by cellular location and biochemical function.
Simultaneous diversification occurred under positive

Fig. 1. Diagramatic representation of peptide substrate interaction with the active site pockets of a cysteine protease. Amino acid residues from
the peptide are denoted by ‘P’ and the sub-sites that the peptide interact with is given the letter ‘S’. The active site cysteine sulfhydryl nucleophile
is represented as SH, and the corresponding peptide bond that is attacked prior to hydrolysis (scissile bond) is labelled. The carboxyl side of the
peptide and corresponding subsites are conventionally referred to as the prime side and are termed P1% , P2% , Pn% and S1% , S2% and Sn% , respectively. The
amino side of the peptide and corresponding subsites are assigned the non-prime side and are designated P1, P2, Pn and S1, S2 and Sn, respectively.
1B, Schematic of the close spacial proximity in the active site showing: (1) the sulfhydryl of cysteine to the imidazole group of histidine, and the
equilibrium with (2) the thiolate –imidazolium charge relay diad. The shaded area represents a delocalised electron dense cloud.
4 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21

Fig. 2. Schematic diagram of the cysteine protease superfamily highlighting how cysteine proteases of parasitic organisms are related. Subfamilies
were determined by sequence homology around the active site amino acid residues.

natural selection as mutations arose more rapidly than
would be expected by random. There is little evidence
of lateral transfer of cysteine protease genes between
organisms. One exception may be the bacterial
bleomycin hydrolase (EMBL, Bh – Lac), which to date
has no other prokaryotic orthologues [14]. This review
will concentrate on the legumain-like family and also
the papain-like family of proteases which are the most
abundant family of proteases.
3.2. Legumain-like family
3.2.1. Asparaginyl endopeptidases
Asparaginyl endopeptidases exclusively hydrolyse
peptides and proteins on the carboxyl side of as-
paragine residues. These enzymes are often referred to
as ‘legumain-like’ as the template protease was first
identified and characterised from the plant legume,
Cana6alia ensiformis, the jack bean. Legumain-like
proteases have been identified in many plants and
mammals including, human, mouse, rat and pig. The
hepatic flukes, S. mansoni and F. hepatica both contain
these proteases [15,16]. Legumains are thought to lo-
calise to lysosome like compartments where they may
function to trans-process other proteins. In S. mansoni
the asparaginyl-endopeptidase is detected in the gut
gastrodermis and the parasite caecal-lumen. This fluke
enzyme is expressed in two forms derived from distinct
gene products in approximately equal amounts (C.R.
Caffrey and A.M. Gaffney, unpublished data). The
 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21 5

native active protein has been biochemically charac-
terised and contains the active site Cys197 (S. mansoni
asparaginyl-endopeptidase numbering). In addition to
this protease there is a distinct transcript which has
an asparagine residue in place of the active site cys-
teine. This is referred to as the ‘inactive’ form (Fig.
3cysteine were compiled using ClustalW at A; [15]).
Substitution of the cysteine in place of the asparagine
in the inactive form of the schistosome asparaginyl
endopeptidase (Asn197 “Cys197) restores enzymatic ac-
tivity and exhibits a similar substrate profiles to the
wildtype enzyme when using combinatorial peptide li-
braries (M.A. Mathieu and C.R. Caffrey, personal
communication). Why the parasite maintains a prote-
olytically inactive enzyme with apparently intact sub-
sites is unclear.
One of the characteristics of the legumain-like class
of cysteine proteases is their insensitivity to generic
cysteine protease inhibitors such as E64 and leupeptin.
Proteolytic activity is inhibited by the general thiol-
blocking reagents iodoacetamide and iodoacetic acid as
well as the broad specificity macromolecular protease
inhibitors, a2-macroglobulin and chicken ovamucoid
cystatin. This lack of selective inhibition has hampered
detailed biochemical characterisation. Recently a vinyl
sulfone combinatorial inhibitor library with a fixed
asparagine at the P1 position (synthesised by D. Green-
baum and M. Bogyo) was used to screen human and S.
mansoni asparaginyl endopeptidases (H. Chapman; M.
Mathieu; unpublished data). Results clearly indicate
that vinyl sulfones can inhibit asparaginyl endopepti-
dase activity and more importantly, that there is specifi-

Fig. 3. Comparison of the legumain-like superfamily showing (A) sequence alignment spanning the active site histidine and cysteine were compiled
using ClustalW at www.sacs.ucsf.edu. Active site residues are labelled with arrows. (B) Phylogenetic tree using the sequences shown in the
alignment was generated using the ClustalW method. Sequences were obtained either from GeneBank or EMBL, SmAP(N) was from [15] and the
T. brucei sequence was from Simon Lillico. Abbreviations: AP, asparaginyl endopeptidase; Ce, C. elegans; hum, human; Dros, Drosophila; Sm,
S. mansoni; Sj, S, japonicum; Sacc, Sacchromyces; Pf, P. falciparum; L. mex, L. mexicana; Tryp, T. brucei; all alignments are available on request.
6 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21
city at the P2 and P3 positions. Targeting the S. mansoni
asparaginyl endopeptidase for chemotherapeutic inter-
vention is particularly attractive as it is likely that to
affect a number of downstream pathways where
protease trans-processing and activation is inhibited.
3.2.2. GPI:protein transamidases
DNA sequences have been identified from C. elegans,
Drosophila melanogaster, T. brucei and Plasmodium fal-
ciparum (and a fragment from Cryptosporidium) that
show striking similarities at the amino acid level to
legumain (Fig. 3A). However, these proteins are as-
signed to a sub-family within the legumain-like proteins
called glycosylphosphatidylinositol (GPI): protein
transamidases. In human, Sacchromyces and L. mexi-
cana [17,18] these proteins have been shown to play a
role in the attachment of pre-formed GPI anchors to
precursor proteins in the ER (J.C. Mottram, personal
communication). The two distinct clades of the legu-
main-like family, the transamidase-like clade and the
asparaginyl-endopeptidase like clade, represent the dif-
ferent functions of the two classes of C13 proteases
within the phylogenetic relationship seen in Fig. 3B. It
is likely that the D. melanogaster, C. elegans and P.
falciparum proteases may function in GPI:protein
transamidation. There is significant sequence similarity
within the asparaginyl endopeptidase-clade around the
active site His/Cys; whereas, there is little sequence
homology around the His/Cys within the transamidase
clade suggesting an extremely early evolutionary
divergence.
3.3. Papain-like proteases
The majority of parasite cysteine proteases belong to
the family C1 within clan CA. Whether this number
reflects the true protease profile within an organism or
if the application of degenerate PCR to isolate novel
genes has biased the known proteases to those that
have related sequences is still open to question. In
addition, a number of parasitic organisms may have
further skewed our knowledge as they are of veterinary
or medical importance and more extensively studied.
Nonetheless, as genome sequencing projects are com-
pleted, a clearer picture is developing of molecular
evolution of this protease family.
Members of the CA clan are either targeted to intra-
cellular vesicle compartments (these include cathepsins
B, C, H, K, L and S [13]), or are secreted, and thus
possess a leader peptide. Clan CA proteases are also
characterised by their sensitivity to the general cysteine
protease inhibitor, E64 (L-trans-epoxysuccinyl-leucyl-
amido (4-guanidino) butane) and by having substrate
specificity defined by the S2 pocket. Initial detailed
studies classified lysosomal cathepsins using vertebrate
systems. Hydrolysis of small peptides that contain
arginine at P2 has often been used to discriminate
cathepsin B activity over cathepsin L activity (see be-
low). Specificity at the S2 pocket has been ascribed to
the chemical properties of the residue corresponding to
position 205 (papain numbering); in human cathepsin B
and cathepsin L this residue is glutamate and alanine,
respectively. In addition, there are structural and se-
quence variances between cathepsin B and cathepsin L.
Cathepsin Ls (including the closely related cathepsin S
and cathepsin K) have a conserved inter-spaced motif
in the pro-region, Glu, X3, Arg, X2, (Ile/Val), Phe, X2,
Asn, X3, Ile, X3, Asn (‘ERFNIN’; named after the
single letter code for amino acids; X is any amino acid
[19]). Cathepsin Bs lack the ERFNIN motif but do
have an inserted peptide loop in the catalytic domain,
referred to as the ‘occluding loop’. In addition to
endopeptidase activity (like cathepsin L), cathepsin Bs
have a dipeptidyl carboxypeptidase activity. This has
been attributed to the occluding loop (see below; [20]).
The cathepsin B and L subfamily can be further delin-
eated by the length and sequence similarity within
respective pro-regions as well as the number and order
of cysteine residues involved in disulfide bond forma-
tion. Indeed the pro-region can be used to selectively
purify respective mature domain of cysteine proteases
including those from Trypanosomes [21]. Calpains, the
Ca2 + dependent cytosolic cysteine proteases, are ‘pa-
pain-like’ but lack a signal sequence and have a calcium
binding regulatory domain.
If parasite cathepsin B-like and cathepsin L-like se-
quences are aligned using sequences immediately span-
ning the active site cysteine and including the glutamine
of the oxyanion hole (Fig. 4, cysteine, histidine and
asparagine can be viewed at http::/XXXXXXXX/
cathepsin B; Fig. 5, cysteine, histidine and asparagine
can be viewed at http::/XXXXXXXX/cathepsin L), the
cathepsin B-like subfamily is quite homologous. This
may suggest a more recent evolutionary divergence. All
known parasite cathepsin Bs have highly conserved
active site residues. The phylogenetic tree of the cathep-
sin B subfamily confirms the Giardia cathepsin B as the
earliest lineage in keeping with its primitive cell evolu-
tion denoted by other gene sequences (Fig. 4B, [22]).
One cathepsin B clade is exclusively nematode while the
other is divided into trypanosomatids, digenean and
nematodes. Other digenean and human sequences show
little sequence divergence. The cathepsin C-like se-
quences differ notably from the cathepsin Bs, and in
addition have a small peptide insert. For example the
tripeptide PQY of O. 6ol6ulus is located between the
oxyanion glutamine and the active site cysteine.
The currently characterised cathepsin L-like parasite
proteases share relatively less similarity compared with
the cathepsin Bs (Fig. 5A, B). There is great sequence
 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21 7

Fig. 4. Comparison of the cathepsin B-like protease alignment comparing amino acid sequences spanning the active site cysteine which is labelled
with a white arrow the predicted glutamine of the oxyanion hole which is marked with a filled arrow. The alignment was produced as in Fig. 3.
The full comparison of the active site cysteine, histidine and http::/XXXXXXXX/cathepsinB. 4B, Phylogenetic tree of the parasite cathepsin B-like
proteases using the sequences shown in the above alignment including the active site histidine and asparagine generated using the ClustalW
method. Abbreviations: Lmaj, L. major; L. mex, L. mexicana; Tc, T. cruzi; hum, human; Sm, S. mansoni; Sj, S, japonicum; Fh, F. hepatica; Ce,
C. elegans; Hc, H. contortus; Oo, O. ostertagi; Ancyl, Ancylostoma; Gl, G. lamblia.

discordance within the cathepsin L subfamily suggest-
ing that cathepsin Ls either evolved early in evolution
or that they have faster mutation rates to adapt to their
divergent cellular functions. Cathepsin L-proteases in
parasites duplicated and evolved following speciation
the exception being Plasmodium cathepsin Ls which can
be found in more that one class. Cryptosporidia is the
earliest known lineage in the cathepsin L-like family.
The remainder of the proteases are less clearly grouped
and a number of clades are evident. One branch is
8 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21

Fig. 5. Comparison of the cathepsin L-like protease alignment comparing amino acid sequences spanning the active site cysteine which is labelled
with a white arrow the predicted glutamine of the oxyanion hole which is marked with a filled arrow. For ease of comparison, large peptide inserts
within the active site Cys –His and His– Asn of Plasmodium spp. were omitted. The alignment was produced as in Fig. 3. The full comparison of
the active site http::/XXXXXXXX/cathepsinL. Abbreviations; EH, E. histolytica. 5B, Phylogenetic tree of the parasite cathepsin L-like proteases
using the sequences shown in the above alignment including the active site histidine and asparagine generated using the ClustalW method. For
ease of sequence alignment, relatively large and unique inserts between the catalytic cysteine and histidine, and the histidine and the asparagine
in Plasmodium spp were omitted. Abbreviations: Tc, T. cruzi; Trang, T. rangeli; Tcong, T. congolense; Tbruc, T. brucei; Lmaj, L. major; Pcyn,
P. cynomolgi; pviv, P. 6i6ax; Pf, P. falciparum; Dermat, Dermatophagus;Toxo, Toxoplasma; Fh, F. hepatica; Sarcoph, Sarcophaga; Booph,
Boophilus; Dicty, Dictyostelium; Eh, E. histolytica; Crypto, Cryptosporidia.
 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21
exclusively the Entamoeba proteases. Other closely re-
lated sequences are from the digeneans and mammalian
proteins. The remainder are more difficult to define and
sequences from parasitic arthropods and Dictyostelium
are interspersed suggesting early sequence divergence.
The serine-repeat antigen (SERA) from Plasmodium
diverged prior to all parasite cathepsin L-like sequences
in this analysis and in addition, the SERA-C have
particular sequence disparity around the active site
residues compared with other parasite cathepsin Ls
(data not shown).
The mature (catalytic) domain of mammalian cathep-
sin B, cathepsin H and cathepsin L is itself a two
domain structure. Following removal of respective
propeptides the mature domain of cathepsin B under-
goes further limited proteolysis, which may be in cis- or
trans, and which involves the removal of small
oligopeptides. It is unclear what biological significance
this has as the secondary proteolysis has no effect on
enzymatic function (D. Turk, personal communica-
tion). All parasite proteases that have been biochemi-
cally studied to date, like mammalian cathepsin S and
plant papain, are single chain polypeptides. The evolu-
tionary consequence of this also remains unclear but
may be involved in the control of proteolytic activity at
higher pH.
3.4. The S2 pocket of cathepsins
The three dimensional structure of vertebrate cathep-
sin L and cathepsin B revealed the important amino
acid residues that contribute to substrate specificity.
These cathepsins have primary substrate preference at
the S2 subsite. Previously, cathepsin B-like and cathep-
sin L-like proteases have been distinguished by their
ability to degrade small peptide substrates that vary at
the P2 position. Peptide substrates that have been used
are Z–Phe –Arg – AMC (Z – FR – AMC) and Z –Arg –
Arg –AMC (Z –RR – AMC); where Z, is an N-terminal
blocking group, AMC is a fluorescent leaving group
after hydrolysis. Mammalian cathepsin B can hydrolyse
both substrates (Km values; Z– FR – AMC 364 mM,
Z–RR – AMC 160 mM), whereas cathepsin L is limited
to Z–FR –AMC only (Km 2 mM). The chemical nature
of the protease S2 pocket, and in particular the amino
acid residue 205 (papain numbering), is thought to be
critical for this substrate preference. Vertebrate cathep-
sin B-like proteases have an acidic group at this posi-
tion and can accommodate and stabilise the polar
guanadino group of arginine. Conversely, mammalian
cathepsin L proteases have alanine at this position
which cannot contribute to arginine binding. Protein
engineering of this single amino acid can alter enzyme
activity versus small substrates.
There are a number of parasite cathepsin L-like and
B-like proteases that conform to the predicted struc-
9
ture-function features of mammalian orthologues with
the equivalent residue at the bottom of the S2 pocket
(Fig. 6). However, many parasite proteases do not
possess ‘mammalian’ residues at this position. Indeed,
the difference in the nature of the residue is consistent
with observed proteolytic activity. The cathepsin L
from T. cruzi, referred to as ‘cruzain’ or ‘cruzipain’,
contains Glu330 (cruzain numbering) and is able to
degrade Z–RR –AMC. In the G. lamblia cathepsin Bs
there are two variants at this position, either glutamic
acid (for GlCP1) or glutamine (in GlCP2, GlCP3).
Consistent with this observation is the occurrence of
two discrete proteolytic activities in G. lamblia lysate.
One activity that degrades only Z–FR –AMC (GlCP2,
GlCP3) and another that is active on both Z–FR –
AMC and Z –RR –AMC (GlCP1; M. Sajid, work in
progress).
Further confirmation of the importance of this single
amino acid has come from L. major cathepsin B-like
enzyme [23]. Although, clearly a cathepsin B by se-
quence and predicted structure this protease is unable
to hydrolyse the ‘classic’ cathepsin B substrate Z– RR –
AMC. This inability is attributed to Gly234 and replac-
ing this with glutamic acid by mutagenesis restored
Z–RR –AMC activity. The pitfall of trying to classify
cysteine proteases as B or L-like by substrate preference
is further underscored by assays with the Ancylostoma
cathepsin B. This enzyme like, the G. lamblia GlCP2
and GlCP3, has a glutamine at the bottom of the
predicted S2 pocket. Once again this cathepsin B
(defined by sequence and structural homology) has
‘cathepsin L-like’ substrate specificity.
3.5. The occluding loop of cathepsin B
Cathepsin B-like proteases possess an exopeptidase
activity in addition to their endopeptidase function.
This has been attributed to a 20 amino acid peptide
loop insert unique to this family of enzymes [24]. The
‘occluding loop’ is so called because its position par-
tially occludes the active site cleft. Large protein sub-
strates are still able to gain access to the active site,
although macromolecular inhibitors are less effective
compared with their activity versus cathepsin L. The
occluding loop was used previously to identify cathep-
sin Bs, and it is also responsible for inhibition by the
cathepsin B specific inhibitor, CA074. The additional
dipeptidyl carboxypeptidase (exopeptidase) activity is
accounted for by two histidine residues, His110 and
His111 (human cathepsin B numbering [20]). The histidi-
nes serve to anchor the carboxylate at the P%2 of the
substrate and direct the C-terminal dipeptide into the
active site for hydrolysis. In addition, the histidines in
the occluding loop have also been shown to govern the
pH dependency of cathepsin B by its propeptide [25].
Removal of the occluding loop abolishes exopeptidase
10 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21

activity and binding by CA074, but increases endopep-
tidase activity as well as the ability for macromolecular
inhibitors such as cystatin to bind the active site [26].
Comparison of sequences spanning the predicted oc-
cluding loop of parasite cathepsin Bs versus vertebrate
cathepsin Bs has been carried out (Fig. 7). The Giardia
cathepsin B proteases do not contain the occluding
loop suggesting that either the loop was lost or the
insert evolved later in evolution; this is consistent with
diplomonads being the earliest evolutionary lineage of
eukaryotes [22]. Vertebrate cathepsin B proteases have
all maintained an intact occluding loop suggesting that
it provides an important biological activity. Many pro-
tozoan and helminth parasite proteases also have con-
served the general occluding loop sequence and
presumably function as exopeptidases. However, a
number of organisms have modified the loop sequence,
either with amino acid or small peptide inserts as in
Toxoplasma, or large deletions as in the C. elegans
cathepsin B3, and the Aedes cathepsin B. Conversely,
the Trichuris cathepsin B occluding loop has an addi-
tional nine amino acid peptide insert within the loop.
The essential His–His moiety in the loop is also main-
tained in many parasitic cathepsin Bs, particularly the
apicomplexa and trypanosomatids. However, some par-
asite or invertebrate enzymes have either lost one his-
tidine, as in the cathepsin Bs from F. hepatica, H.
contortus (B7, B5), Ancylostoma (B2), Necator, O. os-
tertagi (B3) and Trichuris; or mutated and substituted
both such as the C. elegans cathepsins B4 and B5.
While the consequences or selective advantage, of these
alterations is unknown, the latter enzymes would not be
predicted to possess exopeptidase activity. However,
human cathepsin C also has a loop with only one

Fig. 6. Sequence comparison around the residue that is predicted to determine the S2 pocket specificity of (A) cathepsin L-like and (B) cathepsin
B-like protease. The critical residue is marked with an arrow. Abbreviations; EH, E. histolytica.
 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21 11

Fig. 7. Sequence alignment around the predicted ‘occluding loop’ of vertebrate and parasite cathepsin B-like proteases. The loop region is shown
with a black box, the essential His110 and His111 (human cathepsin B numbering) are arrowed and sequences with predicted N-glycosylation site
(N-X-S/T) within the loop are marked with X and underlined.

histidine that functions to stabilise the substrate and act
as an exopeptidase. Therefore, parasite cathepsin B
proteases that only have a single histidine within the
loop may maintain exopeptidase activity akin to
cathepsin C (B. Turk, personal communication).
Vertebrate cathepsins are N-glycosylated midway in
the occluding loop. Mutants that lack this motif are not
catalytically impaired and the function of glycosylation
is unclear. In cathepsin L, glycosylation confers protein
stability and correct intracellular targeting [27]. Many
parasite cathepsin Bs have maintained a potential gly-
cosylation site, whilst others have mutated the essential
asparagine. Detailed biochemical analysis will be re-
quired to understand the effects of these modifications.
3.6. Protease trafficking
The manose-6-phosphate receptor is required by
proteases, like cathepsin B, to correctly traffic to the
lysosomal compartment. Mutation of the essential N-
glycosylation site of proteases or overexpression (as
seen with cathepsin L in transformed cells) enhances
extracellular secretion [28]. However, proteases such as
cathepsin H are lysosome resident but do not possess
glycosylation sites [29]. The manose-6-phosphate inde-
pendent pathway is, therefore, considered to be the
‘primitive state’. Indeed, the trypanosomatids, Leishma-
nia and T. cruzi maintain a manose-6-phosphate inde-
pendent trafficking pathway which may involve a nine
amino acid peptide loop in the pro-domain [30,31].
Mutations within this motif result in inappropriate
delivery to extra-lysosomal compartments. Comparison
of the pro-domain of cruzain with the conserved pep-
tide motif of the Giardia cathepsin Bs, suggest that the
manose-6-phosphate independent pathway evolved
early in the eukaryotic evolution and was concomitant
with the organisation of the secretory system (Fig. 8).
Work is currently underway to confirm the function of
12 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21
this conserved motif in Giardia (M. Abodeely; personal
communication) and to identify and characterise its
receptor in T. cruzi (J. A. Huete-Perez; personal
communication).
Other receptors that may aid correct cellular target-
ing and function are integrins. In higher eukaryotes
these transmembrane heterodimers bind many matrix
proteins, all of which have a tripeptide consensus se-
quence in common, namely Arg– Gly – Asp (RGD).
This may serve to anchor proteins that have no
transmembrane spanning domain or do not possess a
GPI moiety. Many parasite cysteine proteases contain
the RGD binding motif. These include the parasite; G.
lamblia B3, S. mansoni L1, S. japonicum L1, SERA-S,
E. histolytica CP5, E. histolytica CP112 and Parago-
nimus L as well as the mammalian and Drosophila
legumain-like proteases. It is as yet undetermined if the
RGD motifs identified in parasite cysteine proteases are
accessible and functional.
4. Localisation and function
4.1. pH dependence
Parasite cysteine proteases function in a broader
chemical environment than the homologous host en-
zymes. Mammalian lysosomal cysteine proteases are
relatively unstable at neutral pH when compared with
parasite orthologues, the exception being mammalian
cathepsin S. The stability of mammalian cathepsins
may be a regulation mechanism to minimise unwanted
proteolysis in inappropriate cellular or tissue compart-
ments. The lysosomal pH has been estimated to be as
low as pH 4.0 and low pH, can activate lysosomal
enzymes as well as denature protein substrates [32].
While parasite cathepsins are also active at low pH, in
marked contrast to the vertebrate proteases the parasite
proteins are more active and remain stable at neutral
pH (Fig. 9). This broad pH profile of the parasite
cysteine protease is consistent with the numerous extra-
lysosomal functions that have been characterised.
Fig. 8. Cysteine protease prodomain sequence alignments showing the conserved motif implicated in the manose-6-phosphate independent protein
trafficking. The conserved nine amino acid peptide loop is shown boxed. Abbreviations; Gl, Giardia lamblia.
4.2. Nutrition
Molecular phylogeny suggests that cysteine
proteinases arose early in evolution to degrade proteins
both intra- and extracellularly. Parasitic organisms are
a ‘window’ on early eukaryotic evolution because they
represent the earliest lineages of eukaryotic cells present
today. There is a wealth of examples where exogenous
proteins are degraded in parasitic protozoa [3],
helminths [5] and arthropods. Some examples include
cysteine proteases from; Trypanosoma brucei, Tri-
chomonas 6aginalis, P. falciparum, E. histolytica, Schis-
tosoma mansoni, S. japonicum, Fasciola hepatica, P.
westermani, Haplometra cyclindracea, Dictyocaulus
6i6iparus, Gymnophalloides seoi, Taenia solium, Ascaris
suum, Dirofilaria immitis, N. americanus, Strongylus
6ulgaris, Trichenella spirallis, Neodiplostomum seoulense,
Haemonchus contortus, Teladorsargia circumcincta, T.
muris, Globodera pallida and Caenorhabditis elegans. In
addition, a multitude of additional parasite cysteine
proteases have been either partially characterised or
identified by gene sequence.
Two of the best characterised parasite protease sys-
tems that catalyse the degradation of host proteins are
the haemoglobin degrading activity of the S. mansoni
cathepsin-B1 (SmCB1 [33]) and falcipain 2 of P. falci-
parum, (Fp2 [34]; see Fig. 10 for comparison). SmCB1
has been immunolocalised to the gastrodermis and the
intestine. Isolated regurgitant from adult worm gut
degrades haemoglobin and the majority of the activity
was found to be sensitive to the cathepsin-B specific
inhibitor CA074. Cathepsin-L1, asparaginyl endopepti-
dase, cathepsin-C and an aspartyl-protease may also
play a role in haemoglobin hydrolysis via a protease
cascade. Cysteine protease inhibitors have been shown
to reduce worm size and egg number in S. mansoni,
confirming the importance of this class of protease to
the parasite [35]. SmCB1 is a particularly attractive
drug target due to its location in the gut lumen and
accessibility to inhibitors; the orthologous host protease
being found intracellularly in lysosomes. Work is cur-
rently underway to obtain structural information to aid
in drug selection (M. Sajid, work in progress). It is
 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21
Fig. 9. pH comparison of cysteine protease activity from plant and humans (filled symbols) compared with parasitic organisms (empty symbols)
using small peptide substrates. Where pH curves are not available pH optima are marked ([7,11] S. mansoni B1 and G. lamblia B1 from M. Sajid,
unpublished data).
likely that an analogous haemoglobinolytic protease
system also exists in F. hepatica, where a number of
cysteine proteases including cathepsin B-like, cathepsin
L-like, dipeptidylpeptidase I and asparaginyl-endopep-
tidase activities have been identified.
During its asexual erythrocytic cycle the human
malaria parasite degrade grams of haemoglobin even in
a moderate infection. There is controversy as to which
class of protease (cysteine or aspartate) is responsible
for the initial hydrolysis of haemoglobin, although both
classes of enzyme appear to contribute to the degrada-
tion of haemoglobin to small peptides. Nevertheless,
thiol-protease inhibitors block haemoglobin degrada-
tion in vitro and confer protection in murine models
[36]. Three P. falciparum cathepsin-L like proteases
genes have been cloned (falcipain 1, 2 and 3) and
heterologously expressed [34,37]. The main cysteine
protease activity in the food vacuole appears to be
falcipain 2. It is a promising target for chemotherapeu-
tic intervention [38]. Recently, falcipain 3 has been
expressed and is also very active against haemoglobin
(P.J. Rosenthal, personal communication). A number
of lead inhibitors of the falcipains have already been
identified and have striking effects in vitro. These in-
clude fluoromethyl ketones [39], phenothiazines [40],
vinyl sulfones [41], bis-hydrazides and acyl- and cyclic-
diamino ketones.
4.3. Tissue/cell in6asion
During migration within or between hosts, parasites
use proteases to aid tissue and cellular invasion as well
as to facilitate tissue migration. This process has many
parallels with tumour cell invasion. There are many
examples of serine and metallo-proteases that are in-
volved in tissue/cell invasion in parasitic organisms; in
13
contrast relatively few cysteine proteases have been
identified as being directly involved.
Several Plasmodium spp. have been shown to possess
cysteine proteases and those from P. falciparum, P.
chabaudi and P. berghei have been partially character-
ised from the merozoite and/or schizont stage and have
pH optima around pH 7.0. A number of merozoite
surface proteins are degraded prior to or during the
invasion process [42] and there is evidence for the
involvement of a serine protease [43,44]. However, in-
hibitors of cysteine as well as serine proteases have an
inhibitory effect on the invasion process. Peptidyl cys-
teine protease inhibitors [45], leupeptin [46], chymo-
statin [47] and calpain-specific inhibitors [48] had
marked affects on reducing P. falciparum merozoite
invasion of erythrocytes. Cysteine proteases identified
in P. falciparum [49], P. chabaudi [50] and P. berghei
[51] were immunolocalised to the anterior, apical end of
the merozoite, and may be released during invasion.
While the contribution of a thiol-protease to the
invasion process is still unclear, there is far more com-
pelling evidence for the involvement of cysteine
proteases in late schizont mediated rupturing of red
blood cells [52]. Cysteine protease inhibitors reduce
protein processing prior to erythrocyte lysis as well as
cause an accumulation of P. falciparum late schizonts
or non-released merozoites in P. knowlesi [53]. A P.
falciparum cysteine protease activity has been identified
in synchronised mature schizonts [54] and has also been
shown to destablise the ankyrin-band 3 cystoskeleton of
erythrocytes and facilitate parasite release [55]. In addi-
tion, the cysteine protease dependent release of mero-
zoites of P. falciparum involves an initial exit from the
host cell followed by proteolytic rupturing of the para-
sitophorous vacuolar membrane [56].
14 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21
An intriguing gene family of Plasmodium is that of
the cysteine protease-like SERA genes that contain a
serine rich domain. Curiously, a serine residue has been
substituted for the active site cysteine in ESTs ho-
mologous with cysteine protease catalytic domains.
Other family members, SERP Hs, maintain the active
site thiol; nevertheless, none of this family has been
shown to be catalytically active. Whether the SERA/
SERP H family are involved in protein processing or
erythrocyte rupture is still unclear, but, vaccination
with recombinant SERA conferred immunity against P.
falciparum in aotus [57] and squirrel monkeys [58], and
anti-SERA antibodies raised in mice inhibit in vitro
erythrocyte invasion by P. falciparum [59]. Cysteine
proteases have also been implicated in the invasion of
hepatocytes in vitro by sporozoites of P. berghei [60].
In T. cruzi, cysteine proteases have been implicated
as being involved in a number of cellular processes and
hypothesised as having a role in cell invasion. Peptidyl
diazomethane inhibitors were reported to reduce para-
site invasion and subsequent development in vitro [61].
Leishmania parasites that lack the multicopy cathepsin
L-like genes in the CPB array (Dcpb) are considerably
less effective at invading macrophages in vitro [62].
However, there is far more compelling evidence for the
involvement of the serine protease, prolyl endopepti-
dase in the actual invasion process of T. cruzi [63].
Cysteine proteases in E. histolytica have been impli-
cated in tissue invasion or destruction and are impor-
tant virulence factors [64,65]. In patients that have
invasive amoebiasis (approximately 10%) there is a
correlation with levels of extracellular cysteine protease,
Fig. 10. Schematic flow diagram comparing haemoglobin degradation by the intracellular protozoan parasite, P. falciparum and the hepatic liver
fluke, S. mansoni.
epithelial damage and production of anti-cysteine
protease antibodies [66]. It is thought that tissue dam-
age is not an adaptive consequence of the cysteine
protease expression and release. Rather the increased
protease activity of the pathogenic species aids in the
degradation of host proteins or luminal bacteria for
nutrition and unintentionally leads to tissue destruc-
tion. There are five cysteine protease genes expressed in
E. histolytica to date (S. Reed, personal communica-
tion). The gene for CP5 is found both in E. histolytica
and in the non-pathogenic E. dispar. However, func-
tional CP5 is only expressed in pathogenic E. histolyt-
ica. The related Naegleria fowleri amoeba, is the
causative agent for amoebic meningoencephalitis, a fa-
tal disease of humans initiated by invasion via the nasal
mucosa [67]. In N. fowleri secreted cysteine protease
may be involved in tissue destruction and invasion. The
non-pathogenic N. gruberi, also possesses a similar
cysteine protease, however unlike N. fowleri, it is unable
to grow at temperatures above 30 °C.
T. 6aginalis resides in the urogenital system and
causes trichomosis. In order to colonise T. 6aginalis has
to overcome the primary defense properties of the
mucin rich mucous layer to bind to the vaginal epithe-
lium. It is believed that trichomonads traverse the
mucin layer and attach to the host epithelium cells by
producing a number of cysteine proteases, a hypothesis
supported by in vitro studies [68]. The protease is active
in the reducing environment of the vagina and allows
cellular attachment.
Cysteine protease inhibitors block invasion of Eime-
ria sporocysts into epithelial cells [69], Theileria par6a
 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21
sporozoites into bovine lymphocytes [70] and a cysteine
protease has been localised to the rhoptries of Toxo-
plasma gondii and is thought to also play a role in cell
invasion (S. Reed, personal communication).
Animal and plant parasitic nematodes express a
number of metallo- and serine proteases that are essen-
tial for penetration and tissue migration. Cysteine
proteases have been implicated in larval invasion by
the, L3 stage of Strongyloides stercoralis, the L2 stage
of A. suum, the L3/L4 stages of D. immitis, adult H.
contortus and the L3 stage N. americanus.
Cestodes reside in the gut of their host but tissue
invasion involves larval stages. Tissue invasion proba-
bly requires proteases; however, these activities are
poorly characterised. Metallo proteases are most likely
the enzymes facilitating tissue invasion, although a
cysteine protease activity has been associated with tis-
sue penetration of T. saginata [71] and the plerocercoid
Gymnorhynchus gigas [72]. The pseudophyllidean tape-
worm, Spirometra mansonoidese, expresses a cathepsin
B-like protease in the tegument of the plerocercoid.
This cysteine protease has a number of putative roles
including, invasion, evasion, immunomodulation and
potentiating effects on growth [73].
Parasitic trematodes migrate through tissue in one or
more stages of their life cycle, however, there are few
examples of cysteine proteases that are involved in
tissue migration. The cathepsin L-like proteases
secreted into the gut and regurgitated from F. hepatica
degraded lamanin, collagen and other matrix proteins
[74] and a cysteine protease located in the cercarial
penetration glands of Diplostomum pseudospathaceum is
thought to be involved skin penetration of aquatic birds
[75].
4.4. Excystment/encystment, exsheathment and
hatching
Proteases are required for the emergence of protozoa,
helminths and arthropods from protective cysts, cuticles
or eggs. G. lamblia is the causative agent for gas-
trointestinal giardiasis in humans. Initiation of an infec-
tion by cyst rupture as well as cyst formation requires
cysteine proteases [22]. In P. westermani a cysteine
protease is involved in excystment of the metacercaria
in the host intestinal lumen [76]. In the filarial ne-
matodes, irreversible cysteine protease inhibitors arrest
molting of the L3 larval stages of Onchercerca 6ol6ulus
[77], D. immitis [78] and Brugia pahangi [79]. In the
aforementioned nematodes, the L3 larvae were found
to be viable, but were unable to exsheath. Labeled
cysteine protease inhibitors or antibodies located the
cathepsin L-like protease activity to the fluid-filled
space between the nematode tegument and the
exsheathing cuticle. A cysteine protease may also be
involved in the exsheathment of the L3 of N. ameri-
15
canus [80]. Cysteine proteases have been identified in
eggs of S. mansoni and were proposed to be involved in
hatching or host tissue invasion. These are localised to
the miracidial penetration glands of schistosome eggs
[81].
4.5. Protein processing and acti6ation
Some proteases have relatively indiscriminate sub-
strate preference and probably function in general
catabolism. Other proteases have a very limited sub-
strate preference and facilitate specific cell processes as
typified by the furin/PACE or the caspase family. In the
human malaria, P. falciparum, merozoites invade ery-
throcytes to asexually replicate culminating in the re-
lease of an expanded number of merozoites. Prior to, or
during rupturing of the erythrocytes, a large and abun-
dant merozoite surface protein, MSP-1, undergoes an
unusual processing event where the processed polypep-
tides do not dissociate from each other, instead of
forming a multimeric polyprotein complex. Evidence
suggest that this processing event is a pre-requisite for
cellular release. The identity of this protease is un-
known except that inhibitors of cysteine proteases abol-
ish MSP-1 primary processing and merozoite release
(A.A. Holder, personal communication). Another api-
complexan, T. gondii, releases a surface protein, MIC2,
which is thought to participate in the cellular invasion
process. MIC2 is proteolyticaly processed by at least
two independent activities, one of which is in part
sensitive to cysteine protease inhibitors [82]. C. par6um
is an intracellular protozoan parasite that activates the
host’s caspases and induces apoptosis. In this instance
the parasite activates a host cysteine protease cascade
to aid exit from intestinal cells [83].
G. lamblia has three cathepsin B-like proteases, which
may function in excystation or encystation. G. lamblia
is also the host for a double stranded RNA giardiavirus
which synthesises a large capsid protein precursor. Un-
like many viral precursors which are proteolyticaly
processed by a viral derived protease, the giardiavirus
employs one of the host’s cysteine proteases to specifi-
cally process the 100 kDa capsid protein [84]. Current
data suggest that the processing activity is due to either
the G. lamblia GlCP2 or GlCP3 (A.L. Wang, personal
communication).
S. mansoni secretes a cathepsin B-like protease (re-
ferred to as, SmCB1) into the caecal lumen of adult
worms. This protease is the major cysteine protease
activity in the gut [85] and is thought to be fundamental
in the process of haemoglobin degradation (see above).
SmCB1 is translated as a larger precursor protein which
is known to be processed prior to activation. Analysis
of SmCB1 produced in the yeast, Pichia pastoris, sug-
gested that, unlike many cysteine proteases, SmCB1
could not be activated by autocatalysis. Instead recom-
16 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21
binant SmCB1 was found to rely on trans-processing
by the S. mansoni asparaginyl endopeptidase at
HNDWN86-¡-VEIPS (predicted by Dalton et al. [16]
SmCB1 numbering). Moreover, when the polypeptide
chain of SmCB1 is modelled using human cathepsin B
as a template, the scissile bond that is hydrolysed by the
S. mansoni asparaginyl endopeptidase is located in a
solvent accessible loop (C.R. Caffrey, personal commu-
nication). There are a number of precedents of an
asparaginyl endopeptidase processing proteins or acti-
vating enzymes in mammalian [86] and plant systems
[87]. Indeed, one could speculate that a similar activa-
tion system exists in F. hepatica, a close relative of S.
mansoni, and in which the orthologous cathepsin B and
asparaginyl endopeptidase-like proteins have been
identified.
4.6. Immunoe6asion
Proteases have long been hypothesised as aiding par-
asites in evading the immune response of the host by
degrading immune effectors or modulating the cellular
immune response. There are a number of examples
where cysteine proteases cleave immunoglobulins in
vitro. However, this is by no means evidence that they
play such a role in vivo. Degradation of antibodies by
parasite cysteine proteases has been documented in T.
6aginalis, T. foetus, E. histolytica, G. muris, G. lamblia,
T. cruzi, G. gigas, H. contortus, F. hepatica, F. gigan-
tica, T. solium, T. crassiceps and Spirometra. To
confirm such a role in vivo would require analysis of
immune clearance of parasites in a living host following
chemical or genetic knockout of the protease. This is
still beyond the scope of current technology for most of
these systems.
Secretory leukocyte protease inhibitor is a general
inhibitor found in body fluids such as saliva, blood,
tears and vaginal secretions. Patients infected with T.
6aginalis had lower levels of this protective inhibitor;
the reduction was due to proteolytic degradation and
inactivation by the secreted cathepsin-L like proteases
[88]. T. 6aginalis cysteine proteases have been impli-
cated in the avoidance of immunological damage. Selec-
tive cysteine protease inhibitors blocked degradation of
the C3 protein component of the host’s alternative
complement pathway, increasing subsequent cell lysis of
parasites [89].
L. mexicana has a number of cysteine proteases
belonging to the cathepsin L-like and cathepsin B-like
protease family [1,90]. Targeted gene disruption of both
the gene loci encoding the L. mexicana cathepsin L-like
proteases, Dcpa and Dcpb, resulted in marked reduction
in virulence and a shift in the immune response from a
predominantly Th-2 (wildtype) to a Th-1 response [91].
In T. cruzi the major cysteine protease, cruzain, has
been linked to plasma leakage in post-capillary venules
and may recruit macrophages for invasion [92]. The
fluke, P. westermani secretes a number of cysteine
proteases in the gut and is known to degrade collagen
and haemoglobin. It has been speculated that cysteine
proteases from Paragonimus may suppress the immune
system and induce immune tolerance, hindering para-
site elimination. Other examples where parasites may
use proteases to evade the immune system are still
preliminary and include Ancylostoma, F. hepatica,
Boophilus microplus and the kininogenase activity of
cruzain from T. cruzi.
4.7. Serodiagnostics, allergies and 6accines
It has long been known that many cysteine proteases
are unusually immunogenic. This characteristic of cys-
teine proteases has been exploited in their use as conve-
nient immunodominant markers for infectious diseases;
in a number of cases the serodiagnostic marker’s iden-
tity as a cysteine protease was confirmed only after
extensive use.
Examples include infections of F. hepatica, F. gigan-
ticum, T. canis, P. westermani, E. histolytica, T. 6agi-
nalis, L. major, T. cruzi, P. falciparum, P. 6inckei, S.
mansoni and Dermatophagus pteronyssinus. Cysteine
proteases may also have an affect on the specific nature
of the humoral response. N. brasiliensis evokes a pre-
dominantly IgE/IgG1 antibody response whilst
Spirometra and D. pteronyssinus induce a strong IgE
specific response.
Cysteine proteases are also responsible for allergic
responses in humans and animals. Allergy to plant
pollens, detergents and dust mites is due to hypersensi-
tisation to proteases. Among the best characterised
allergens are the cysteine proteases found in faeces of
the Dermatophagus dust mite [93], like Der f1 and Der
p1. This cysteine protease, Der p1, can disrupt tight
junctions in the lung and nasal mucosa and facilitate its
own passage across blood vessels, possibly promoting
the allergy and associated asthma, rhinitis and atopic
dermatitis. Recent work on Der p1 has compared the
sequence of the mite antigen to homologues in parasites
and animals. The results of this study suggest that a
potentially accessible and generic IgE epitope exists in
Der p1 and the other cysteine proteases. In the case of
the mite allergen the sequence is LDAFRRHYDGR-
TIIQ [94]. Alternatively, other investigators have hy-
pothesised that the proteolytic activity of papain family
cysteine proteases contributes to their allergic potential.
Allergic hypersensitivity responses to cysteine proteases
have also been documented for filarial Setaria digitata
in cattle, S. mansoni in mice, T. cruzi in mice and
enzymatically active papain in mice.
Antibodies directed against cysteine proteases can
have an inhibitory affect on their proteolytic activity as
seen with the anti-F. hepatica cathepsin L antibodies
 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21
[95], anti-D. 6i6iparus cysteine protease and the anti-D.
pteronyssinus Der p1 antibodies [96]. Immunological
protection using cysteine proteases has also been ob-
served against P. falciparum SERA/SERP antigens in
aotus monkeys and mice, the L. major cysteine protease
in mice, E. histolytica cysteine protease antigens in vitro
and in animal models and S. mansoni anti-9B antibod-
ies. An antibody mediated protection was reported
against T. cruzi and L. mexicana. The S. mansoni
calpain is implicated as a target of protective immunity
and has been used as a vaccine in murine models; later
studies identified the minimum protective oligopeptide
epitope sequence of calpain as EWKGAWCDGS.
CD4+ T lymphocytes that used this calpain epitope as
a target antigen could be passively transferred and
continued to protect against subsequent challenges of
S. mansoni [97].
The most encouraging work to verify the application
of an anti-cysteine protease vaccine against parasitic
organisms is that with F. hepatica and H. contortus
[98,99]. Significant protection using cysteine protease
vaccine antigens against challenge of either parasite in
sheep (and in the case of F. hepatica, in cattle too) has
been documented, with reduced infection, reduced fae-
cal egg number and reduced egg viability. Similar
strategies may be effective against many parasitic or-
ganisms, particularly blood feeding parasites such as
gastrointestinal nematodes, other hepatic fluke and the
larvae and adult stages of arthropods such as ticks,
mosquitoes and mites.
5. Chemotherapy
Due to the ubiquitous presence of cysteine proteases
in both protozoan and helminth parasites, they repre-
sent intriguing targets for antiparasitic drug develop-
ment. Most of the work to date has focused on the
papain family proteases (cathepsin L and B-like)
proteases, and this review will, therefore, summarise
that body of data.
The potential advantages of targeting this family are
several-fold. First, because homologous family mem-
bers occur in nearly every major parasitic organism,
and in light of the fact that the catalytic mechanism is
identical in each of these homologues, it is reasonable
to assume that a relatively large inhibitor library would
contain potential leads for a number of different para-
sitic diseases. This would provide a very cost-effective
approach to new antiparasitic chemotherapy, a neces-
sity when dealing with diseases which are endemic in
economically poor regions of the world. Secondly,
much is known about structure/function relationships
and the catalytic mechanism in this enzyme family,
providing a foundation for synthetic and computational
drug efforts. Thirdly, many of the parasite enzymes
17
have homologues in mammalian systems that are cur-
rently being targeted by large pharmaceutical compa-
nies for their role in human diseases with larger market
value. This has meant that a considerable amount of
effort has already gone into evaluating efficacy, phar-
macokinetics and toxicology for a group of inhibitors
that include suitable candidates for antiparasitic leads.
Protease inhibitors targeting this family of enzymes,
derived from a number of different chemical scaffolds
(both peptide-like and nonpeptide), have already been
evaluated against specific enzyme targets [100,101].
Before the promise of targeting this family of en-
zymes could be realised, a number of key questions
needed to be answered. First and foremost was whether
compounds could be developed that are selective for
the parasite targets. While this enzyme family includes
potential targets for many parasitic diseases, there are
also homologues in humans. Proteases like cathepsin B,
cathepsin L, and cathepsin S provide important cellular
immunologic functions for the human host. The ‘proof
of concept’ that such an approach is possible has,
however, now been largely substantiated. Rosenthal
and colleagues showed that fluoromethyl ketone-deriva-
tised pseudopeptides could cure a malarial infection in
mice [36,39]. This same class of compounds was shown
to arrest infections with T. cruzi, and decreased worm
burden and egg burden in murine schistosome infec-
tions [35]. These initial studies indicated that the para-
site targets could be selectively hit, even in vivo.
However, the fluoromethyl ketone moiety produced
toxic effects at the higher doses necessary to cure,
rather than just arrest infections. This led to the screen-
ing of other types of inhibitors including vinyl sulfones.
Vinyl sulfone-derivitised pseudopeptides eliminated the
toxicity associated with the fluoromethyl ketone homo-
logues and were used to demonstrate the cure infection
by T. cruzi in the mouse model [102]. More impor-
tantly, this class of compounds has undergone thor-
ough rodent and dog toxicology tests and
pharmacokinetic analysis (SRI International project c
1382-377). It is likely that at least one member of this
class (N-met pip–urea–Phe –homoPhe –VSO – benzene
(K11777; Fig. 11)) will enter human clinical trials for
Chagas’ disease within the next two years [103]. The
development of K11777 as a selective cruzain inhibitor
is a significant step and has important implications for
the entire strategy of targeting this class of enzymes. It
indicates that cysteine protease inhibitors can be viable
drug candidates, both in terms of efficacy (cure), safety,
pharmacokinetics and oral availability. The concern
that a cryptic host cysteine protease might be inadver-
tently targeted by the antiparasitic compound has been
answered in this case but must be assessed for every
new class of compound. In fact, the lack of toxicity
from even less selective inhibitors such as the vinyl
sulfone series was at first surprising. It is now clear that
18 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21
Fig. 11. Chemical structure of the anti-Chaga’s drug, K11777 (N-methylpiperazine-urea-phenylalanyl-homophenylalanyl-vinylsulfone-benzene)
currently undergoing pre-clinical trials [107].
the blood levels achieved with these compounds, while
sufficient to target the parasite enzyme, do not reach
intracellular compartments where host enzymes are res-
ident in sufficient quantity to inhibit vital cellular func-
tions (SRI International Reports B031-99 and
M158-01, communicated by C.Litterst, NIAID,
Bethesda, MD). Thus, a ‘bioselectivity’ is achieved even
in the absence of further structural modifications that
could potentially make the inhibitors even more selec-
tive for the parasite target.
Several other chemical scaffolds are also being evalu-
ated in parallel with the vinyl sulfone series. A program
at Glaxo SmithKline targeting cathepsin K yielded
several potential leads for targeting falcipain from
malaria, and cruzain and rhodesain from try-
panosomes. The ketone scaffold developed at Glaxo
SmithKline for their cathepsin K program is mecha-
nism-based but reversible [104]. Successful interruption
of both the malarial and trypanosome life cycle in vitro
with these compounds indicates that nonpeptide in-
hibitors are also viable candidates as antiparasitics.
Concurrent work on synthesis of other cysteine
protease inhibitors using a variety of chemical scaffolds
is now focused on economy of synthesis, the last impor-
tant criteria for an ideal antiparasitic drug.
6. Conclusion
Cysteine proteases have numerous functions in para-
sites, many that are very different from homologous
enzymes of the host. There are fundamental differences
in substrate specificity, domain extensions and stability
over a wide pH range. It is clear that in many parasites,
cysteine proteases have a number of functions that are
extra-lysosomal. In addition, parasite cysteine
proteases, in particular the cathepsins, do not conform
to the previously outlined classifications described for
vertebrate protease systems. Notable differences are
seen with the S2 subsite of parasite cathepsins and the
presence or absence of a functional occluding loop in
parasite cathepsin B-like proteases. Further, the natural
in vivo substrates that parasite proteases target are
often inferred and not well characterised. Work using
combinatorial positionally scanning substrate libraries
in conjunction with phage display technologies has
provided insights to substrate preference and the nature
of natural substrates [105]. Combinatorial technology
has also been employed in generating inhibitor libraries
that can guide to the identification of lead compounds.
Work with labelled inhibitors has made it possible to
profile the cysteine proteases of an organism [106].
Proteases that have cysteine protease homology but
lack the active site sulfhydryl have been identified and
partially characterised. Two notable examples are the
‘inactive’ form of S. mansoni asparaginyl endopeptidase
and the SERA family from malaria. The functions of
these ‘inactive’ proteases are unknown and may suggest
additional non-proteolytic functions such as im-
munomodulation. It is becoming apparent that cysteine
proteases often are part proteolytic cascades and this
theme may be more evident as parasite systems are
thoroughly analysed.
Acknowledgements
Thanks to Conor Caffrey, Phil Rosenthal, Sharon
Reed, Jeremy Mottram, Joey Hansell and Judy Saka-
nari for helpful and encouraging discussions. The au-
 M. Sajid, J.H. McKerrow / Molecular & Biochemical Parasitology 120 (2002) 1–21
thors would also like to acknowledge Jeremy Mottram,
David Roos, Phil Rosenthal, Alan Barrett, Boris Turk,
Dusan Turk, Tony Holder, Mary Mathieu, Alan
Gaffney, Harold Chapman, Marla Abodeely, Matt Bo-
gyo, Doron Greenbaum, Jorge Huete-Perez, Sharon
Reed, Phil Rosenthal and Alice Wang for use of un-
published work and helpful suggestions. Thanks to
Conor Caffrey for supplying the sequence for the S.
mansoni cathepsin B2 and Simon Lillico for the T.
brucei GPI:protein transamidase sequence. We also ap-
preciate Chris Franklin’s assistance with the figures. M.
Sajid is an Advanced Wellcome Fellow in Tropical
Medicine and J.H. McKerrow is a Burroughs-Well-
come Scholar in Molecular Parasitology.
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