A novel cathepsin B active site motif is shared by helminthbloodfeeders

Salman Baig,a Raymond T. Damian,a,b and David S. Petersonb,c,*

a Department of Cellular Biology and ZymeX Pharmaceuticals, Inc., University of Georgia, Athens, GA 30602, USAb Center for Tropical and Emerging Global Diseases, University of Georgia, USA

c Department of Medical Microbiology and Parasitology, University of Georgia, Athens, GA 30602, USA

Received 30 July 2001; received in revised form 3 June 2002; accepted 22 August 2002

Abstract

This study compared specific protein sequence motifs present within cathepsin B-like cysteine proteases from a number of

helminth parasites. We have focused our efforts on cathepsin B-like proteases of Haemonchus contortus, Caenorhabditis elegans,

Schistosoma mansoni, Schistosoma japonicum, Ostertagia ostertagi, and Ancylostoma caninum. The goal of this work is to correlate

specific features, or proposed roles, of the cathepsin B-like proteases with primary sequence motifs discovered within the proteins.

We report here a general motif for the identification of cathepsin B enzymes, and more significantly, a motif within this pattern that

is found, with one exception, only in cathepsin B-like proteases of helminth bloodfeeders. We suggest that the ‘‘hemoglobinase’’

motif arose evolutionarily in a minimum of three independent events as a specialized response to increase the efficiency of hemo-

globin degradation by these cathepsin B-like enzymes. This motif should be useful in identifying additional helminth hemoglo-

binases and may provide a specific target for drug design efforts.

Index Descriptors and Abbreviations: Haemonchus contortus, Caenorhabditis elegans, Schistosoma mansoni, Schistosoma japonicum,

Ostertagia ostertagi, Ancylosotoma caninum, Ascaris suum, hemoglobinase; helminth; motif; phylogenetic analysis

� 2002 Elsevier Science (USA). All rights reserved.

1. Introduction

Parasitic worms remain important causes of disease

in both animals and humans. Proteases are utilized by

helminth parasites at several stages of their often-com-

plex lifecycles. Helminth parasites employ these enzymes

during skin penetration, and to evade host immune re-

sponses through digestion of host immune effectormolecules (Tort et al., 1999). Also, many helminths are

bloodfeeders and rely upon hemoglobin obtained from

the vertebrate host as a significant source of nutrition. A

number of proteases have been proposed to play a role

in the digestion of hemoglobin, including cathepsin L-,

D-, C-, and B-like cysteine proteases. The role of the

cathepsin B-like cysteine proteases as hemoglobinases in

bloodfeeding parasites has been well established in some

helminths, and suggested in others (Brindley et al., 1997;

Harrop et al., 1995; Pratt et al., 1990; Shompole and

Jasmer, 2001).

The use of protease inhibitors for the treatment of

disease is increasing. Inhibitors of angiotensin convert-

ing enzyme are widely used to treat hypertension, while

the HIV aspartyl protease is the target of one of the first

such inhibitors produced by rational drug design (Chengand Ngo, 1997; Roberts et al., 1990). Due to the im-

portance of protease activity at crucial stages of a par-

asite�s lifecycle, it has been proposed that protease

inhibitors may prove to be highly effective in the treat-

ment of parasitic diseases (McKerrow, 1989). Proteases

are been being investigated as targets for chemothera-

peutic intervention for parasitic diseases, and several

studies have now demonstrated the effectiveness of in-hibitor treatment in vivo. Mice infected with murine

malaria can be cured by administration of cysteine

protease inhibitors, thought to target falcipain, a

Experimental Parasitology 101 (2002) 83–89

www.academicpress.com

*Corresponding author. Fax: 1-706-542-0059.

E-mail address: peterson@vet.uga.edu (D.S. Peterson).

0014-4894/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.

PII: S0014 -4894 (02 )00105-4

protease involved in hemoglobin degradation (Olsonet al., 1999). Experiments with murine models of

Chagas� disease have demonstrated that mice infected

with a lethal dose of trypanosomes can be rescued by

treatment with cysteine protease inhibitors (Engel et al.,

1998). Cysteine protease inhibitors have also demon-

strated anti-helminth activity in in vitro and in vivo

studies (Rhoads and Fetterer, 1996; Wasilewski et al.,

1996). In addition, recent studies have demonstrated thepotential for clearance of T. crassiceps from infected

mice through administration of parasite specific prote-

ase inhibitors (Baig and Damian, submitted). Some

studies have found toxic effects due to the treatment,

indicating the need for inhibitors highly specific for

parasite proteases. The development of such drugs relies

on the discovery of clear biochemical differences be-

tween the proteases of the host and the infectious agent.In the present study, we have discovered and corre-

lated specific primary sequence motifs of helminth ca-

thepsin B-like cysteine proteases with the biochemical

characteristics of those enzymes. These motifs are not

present in non-parasitic worms, nor in the cathepsin B-

like proteases of their vertebrate hosts.

2. Materials and methods

2.1. Cathepsin B sequences and motif searches

This study was initiated by a literature search to

identify cysteine proteases of blood feeding helminths

reported to play a role in the degradation of host he-

moglobin. This effort identified sequences from Ost-

ertagia ostertagi (A48454), Schistosoma japonicum SJ31

(S31907), Schistosoma mansoni ‘‘SM31 prec’’ (P25792),

Ancylostoma caninum ACCP-1 (AAC46877), Haemon-

chus contortus ‘‘AC-3’’ (D48435), H. contortus ‘‘AC-4’’

(C48435), and H. contortus ‘‘AC-5’’ (B48435). For

comparison we also included several cysteine proteases

of the non-parasitic helminth Caenorhabditis elegans,

‘‘cpr3’’ (AAA98789), and ‘‘cpr4’’ (AAA98785), as wellas from other non-helminth species, papain (CAB42883)

Rattus norvegicus (CAA57792), Aedes aegypti (AAA

79004), and Leishmania major (AAB48119). These se-

quences were aligned using the MACAW program

which can be used to search for short highly conserved

sequence blocks (Schuler et al., 1991). From this align-

ment emerged an amino acid motif from the active site

region that was present only in the bloodfeeding helm-inths. To find additional species harboring these motifs

we utilized the pattern search tool Prosite with the motif

from the Asn active site region (Y-W-[IL]-[IV]–A-N-S-

W-X–X–D-W-G-E). Also the BLAST search tool was

used to search GenBank with the individual permuta-

tions of the Asn active site motif present in different

bloodfeeding helminths. These searches resulted in the

discovery of an additional eight gene sequences, allcathepsin B-like cysteine proteases containing the

helminth bloodfeeder motif. All of these additional

sequences were, with the exception of Ascaris suum,

from helminths described as bloodfeeders. Alignments

for the phylogenetic studies utilized cathepsin B genes

for which complete sequence data was available, and

were made with ClustalW and visually refined to mini-

mize insertions and deletions. For all gene sequencesthe GenBank literature reference, and related references,

were reviewed to determine stage specificity of expres-

sion, pH optimum, requirements for a reducing

environment, and other data relevant to potential pro-

teolytic activity against hemoglobin.

2.2. Phylogenetic analysis

Neighbor joining, maximum likelihood, and maxi-

mum parsimony were constructed with MEGA 1.0

(Kumar et al., 1993), MOLPHY 2.3 (Adachi and Ha-

segawa, 1994), and Phylip 3.5 (Felsenstein, 1989), re-

spectively. Amino acid sequence data were analyzed

with the JTT-F model for maximum likelihood and a ccorrection for neighbor joining. The c distribution pa-

rameter a was assessed from the amino acid sequencedata set to be 0.88 (S.E. 0.13) using the Quartet Puzzle

algorithm (Strimmer and von Haeseler, 1996) with the

Dayhoff model of substitution (Dayhoff et al., 1978).

Tree topology did not change significantly for values of

the a parameter within 1 SE (from 0.75 to 1.01). Sta-

tistical confidence estimates of topologies and branches

were obtained with the RELL bootstrap method for

maximum likelihood and the standard bootstrap meth-od (10,000 bootstrap resamplings) for maximum parsi-

mony and neighbor-joining. Trees were constructed

using Treeview 1.5 (Page, 1998) and Tree Explorer

(Tamura, 1997). Trees generated by maximum likeli-

hood, parsimony, and by the distance methods neighbor

joining and Fitch were compared using the user�s tree

option of protml to confirm that protml had derived the

optimal tree. Alignments are available from the corre-sponding author.

3. Results and discussion

3.1. Identification of hemoglobinase specific motifs

To identify motifs specific to a particular class ofcysteine protease, we obtained the primary sequences of

a number of cathepsin B-like cysteine proteases from

both bloodfeeding helminth parasites, and other or-

ganism found in GenBank (listed in Fig. 2) and em-

ployed the MACAW (Schuler et al., 1991) alignment

program to identify conserved domains in cathepsin B

subsets. MACAW was chosen since it employs an

84 S. Baig et al. / Experimental Parasitology 101 (2002) 83–89

interactive approach to constructing an alignment basedupon short segments of sequence and is thus ideal for

finding novel motifs present in a subset of sequences.

The identification of cysteine proteases is currently

based upon motifs that encompass the catalytic cysteine,

asparagine, and histidine active site regions (Hofmann

et al., 1999). Using MACAW, we detected a pattern

located in the histidine and asparagine active site regions

that distinguishes cathepsin B-like enzymes from otherclasses of cysteine proteases (Fig. 1). A second motif

within this region was found only in cathepsin B-like

cysteine proteases of helminth bloodfeeders (Fig. 1).

The hypothesis that this second motif identifies

cathepsin B-like cysteine proteases with a substrate

specificity for hemoglobin is supported by several ob-

servations. First, a PROSITE (Bucher and Bairoch,

1994) search of the TrEMBL and SwissProt databasesusing the motif identifies only cathepsin B-like cysteine

proteases from known helminth bloodfeeders. Second, it

is notable that the hemoglobinase motif is absent in all

seven cathepsin B sequences of the non-parasitic hel-

minth, C. elegans, which represents the entire comple-

ment of such enzymes within this organism. Lastly, the

cathepsin B enzymes which contain this motif have been

suggested to be hemoglobinases in the parasites S. ja-

ponicum (Merckelbach et al., 1994) and S. mansoni (re-

viewed in Brindley et al. (1997)), H. contortus (Pratt

et al., 1990; Shompole and Jasmer, 2001), O. ostertagi

(Pratt et al., 1992), and A. caninum (Harrop et al., 1995).

While not providing conclusive proof that these motif

containing proteases digest hemoglobin, these reports

propose a role for these proteases in hemoglobin deg-

radation based upon such criteria as expression inbloodfeeding stages of the worms life cycle, localization

of protease activity to sites of hemoglobin degradation,

and the fact that the proteases are reported to be se-

creted (reviewed in Tort et al. (1999)). For example, the

H. contortus cathepsin B-like proteases have been shown

to be developmentally expressed primarily in adult

worm blood-feeding stages (Cox et al., 1990). In A.

caninum, the described cathepsin B-like enzymes are

expressed in adult stages, localized in the hookwormesophogeal, emphidial, and in excretory glands from

where they would be released into the gut (Harrop et al.,

1995). In O. ostertagi, the cathepsin B-like proteases are

also expressed in the adult stages, secreted, and localized

in the gut area, (Pratt et al., 1992) and may have acidic

pH optima similar to many hemoglobinases (De Cock

et al., 1993).

The hemoglobinase motif is located in the asparagineactive site region. Here hydrogen bonding plays a key

role in catalysis. Notably, in the hemoglobinase motif

containing enzymes, this region is characterized by hy-

drogen-bond-donating residues that replace non-hy-

drogen-bond-donating residues found in other cathepsin

Bs. For example, Asp, a hydrogen-bond-donating ami-

no acid, is present in the hemoglobinase motif at posi-

tion 11 (Fig. 1), while Tyr at position 1 has the ability tofunction as a weak acid. Interestingly, four out of five of

the C. elegans cathepsin B-like proteases are character-

ized by non-hydrogen-bond-donating residues in place

of Asp at position 11 in the motif (shaded in Fig. 2).

Moreover, two cathepsin B-like cysteine proteases (AC1

and AC2) of the bloodfeeder H. contortus lack the motif

by virtue of the replacement of the weak acid Tyr at

position 1 with the non-hydrogen-bond-donating resi-due, Phe (Fig. 2). By virtue of changes in hydrogen

bonding character within the motif region, such ca-

thepsin B proteases may be generalized to perform

housekeeping functions including endogenous lysosomal

protein turnover as opposed to being specialized for

hemoglobin degradation. The key point is that the dif-

ference between the specificity of a cathepsin B for a

general substrate or a hemoglobin substrate may occurthrough the mechanism of changes in the nature of hy-

drogen bonding ability in this motif region. Indeed, it

has been well established that hydrogen bonding is of

paramount importance in cysteine protease catalysis

(Kamphuis et al., 1984; Menard et al., 1991; Rullmann

et al., 1989; Wang et al., 1994).

One issue that must be considered is the feasibility

that single- or double-point mutations within the motif

Fig. 1. The histidine and asparagine active site signature regions for cysteine proteases, cathepsin B enzymes, and proteases with the hemoglobinase

motif. The cysteine protease motif pattern is displayed in PROSITE format. Catalytic residues are in bold face. (a) Active site histidine signature

region. Brackets designate residue possibilities for that position. Individual residues are separated by dashes. (b) Active site asparagine region.

PROSITE pattern for the asparagine active site region has been truncated here to illustrate the differences in the cathepsin B and hemoglobinase

subsets.

S. Baig et al. / Experimental Parasitology 101 (2002) 83–89 85

region can cause significant modifications in the prote-olytic character of cysteine proteases to the extent that

substrate specificity is altered (Khouri et al., 1991; Wang

et al., 1994). However, various observations suggest that

this is possible. For example, the substrate preference of

the cathepsin L-like protease papain was altered to a

cathepsin B-like specificity by mutation of Val133 into

Ala and Ser205 into Glu (Khouri et al., 1991). This al-

teration is especially notable because only 29% identityexists between papain and cathepsin B enzymes at the

primary structure level. Substrate specificity alterations

have also been accomplished through protein engineer-

ing for a spectrum of other enzymes including subtilisin

(Estell et al., 1985) and trypsin (Graf et al., 1987).

3.2. Phylogenetic analysis and evolution of cathepsin

B-like hemoglobinases

To assess whether the motif represents selection for

catalytic function as opposed to inheritance of an an-

cestral helminth pattern, we analyzed two alignments,

both constructed with CLUSTAL W (Higgins et al.,

1996) with one manually edited to improve the align-

ment. We developed phylogenetic trees of the aligned

cysteine protease sequences based upon the criteria ofmaximum likelihood (ML), maximum parsimony, ccorrected Neighbor Joining, and Fitch–Margoliash, as

well as non-c corrected Kitsch. We employed boot-

strapping methods (Felsenstein, 1985) to assess the ac-

curacy of the phylogeny and limited our analysis to

complete cathepsin B sequences in GenBank. All phy-

logenetic methods using either of the two constructed

alignments produced substantially similar trees withpreservation of inner branches in all cases.

Although we show only the results obtained from c-corrected Neighbor Joining here (Fig. 3), all resulting

phylogenetic trees clustered the cathepsin B sequences

into six clades with most of the parasitic nematodes

clustered on one branch. However, the putative hemo-

globinase from Ascaris is most closely related to two C.

elegans cathepsin B sequences that lack the motif. Themotif does not cluster solely based upon the phylogeny,

as it is found in both nematode and trematode groups.

Therefore, the possession of the hemoglobinase motif in

phylogenetically diverse helminths may constitute an

interesting example of convergent evolution at the mo-

lecular level. Moreover, in all cases, our analysis dem-

onstrates that it is most parsimonious for the motif to

have emerged evolutionarily in a minimum of three sep-arate events. This conclusion was reached by examining

two initial conditions: (1) that an ancestral cathepsin B

contained the motif, or (2) lacked the motif. We then

accounted for the current distribution of the motif with

the fewest events that would result in its gain or loss.

In all metazoans examined to date, cathepsin B en-

zymes are encoded by a multiple gene family (e.g., 5 in

Fig. 2. Asparagine active site region of the papain family including

cathepsin B and hemoglobinase motif containing proteases. The motif

in cathepsin B enzymes of bloodfeeders is boldfaced. At top, the active

site asparagine residue are indicated by a ‘‘*.’’ Dashes represent non-

conserved residues. Protease accession numbers from GenBank follow.

Cysteine proteases: Papain (CAB42883); Caricain (JN0633); Aleurain

prec (P05167); human cathepsin H (NP_004381); human cathepsin L

(NP_001903). Cathepsin B proteases: Mus musculus (CAA38713); R.

norvegicus (CAA57792); Thaliana aestevium (CAA46811); Arabidopsis

thaliana (AAC24376); C. elegans ‘‘gut specific cp’’ (P25807), C. elegans

‘‘cpr3’’ (AAA98789), C. elegans ‘‘cpr4’’ (AAA98785), C. elegans

‘‘cpr5’’ (P43509), C. elegans ‘‘CPR6’’ (AAC70871); Leishmania mexi-

cana (CAA88490), L. major (AAB48119); Sarcophaga peregrina

(S38939); Gallus gallus (P43233); Nicotinica rustica (S60479); Bos

taurus (AAA80198), A. aegypti (AAA79004); Trypanasoma cruzi

(AAD03404); Human (NP_001899), Hemoglobinases: Necator amer-

icanus (CAB53367); S. japonicum ‘‘cathepsin-B like cp’’ (S31909), S.

japonicum SJ31 (S31907), S. mansoni ‘‘SM31 prec’’ (P25792), A. suum

(AAB40605); A. caninum ACCP-1 (AAC46877), A. caninum ACCP-2

(AAC46878), Ancylostoma ceylanicum (AAD17287); H. contortus

‘‘AC-1’’ (AAA29175), H. contortus ‘‘AC-2’’ (AAA29171), H. contortus

‘‘AC-3’’ (D48435), H. contortus ‘‘AC-4’’ (C48435), H. contortus ‘‘AC-

5’’ (B48435), H. contortus ‘‘GCP7’’ (AAC05262); O. ostertagi ‘‘cath-B

like CP’’ (A48454); H. contortus ‘‘HCCP6’’ (CAB03627); H. contortus

‘‘HMCP4’’ (CAA93278); H. contortus ‘‘HMCP3’’ (CAA93277).

86 S. Baig et al. / Experimental Parasitology 101 (2002) 83–89

C. elegans and a minimum of 7 in H. contortus). Thus

they may be specialized to perform different functions,

which suggests that our phylogenetic analysis is more

likely a representation of the relatedness of paralogous

sequences rather than a true helminth phylogeny

(Brooks, 1992). An exception may be C. elegans CPR6

and the protease from Ascaris, which have been sug-

gested to be orthologs (Rehman and Jasmer, 1999).Independent emergence of this motif in separate

helminth lineages implies the action of a similar selective

pressure. An alignment of human, sheep, and cow he-

moglobins revealed that they are 81–93% identical in

their a and b chains. This suggests that conserved he-

moglobin sequences could have provided a uniform

selective pressure for the emergence of a single hemo-

globinase motif in the active sites of helminth cathepsinB enzymes. If true, it is unlikely that the motif is older

than mammalian adult hemoglobin, which emerged

approximately 100 million years ago (Czelusniak et al.,

1982). Recently it has been proposed that sequence

differences in host hemoglobin may play a role in the

host range of bloodfeeding helminth parasites (Brink-

worth et al., 2000). Is it reasonable to propose therefore

that the relatively high sequence similarity of mamma-lian hemoglobin could have provided a uniform selective

force for the emergence of a hemoglobinase motif?

While there clearly is high overall similarity, some re-gions in mammalian hemoglobin are far more conserved

than others. Therefore it is possible that hemoglobin of

different mammalian hosts could have contributed to

both host specificity and selection for a conserved active

site motif depending upon whether the cleavage site of

the relevant protease falls in a conserved or variable

region. Other evolutionary pressures that may have se-

lected for the emergence of hemoglobinases with ashared motif include the similar gastrodermal environ-

ment where many of these proteases are localized and

secreted (Chappell and Dresden, 1986; Dowd et al.,

1994; Harrop et al., 1995; Karanu et al., 1993), devel-

opmental variables (Dowd et al., 1994; Harrop et al.,

1995; Pratt et al., 1990; Zerda et al., 1988) (because most

of the cathepsin B proteases are reported to be expressed

in similar stages), and the release of glutathione fromred blood cells (Chappell et al., 1987), which would

provide for a similar reducing environment that is

physiologically required for proteolytic activation of

cysteine proteases.

As defined, the motif identifies cathepsin B-like pro-

teases expressed by helminth bloodfeeders, however the

motif is also present in a cathepsin B-like protease of A.

suum. Adult Ascaris in the intestine of the mammalianhost may feed on blood, but are generally thought to

survive by ingestion of the liquid contents of the lumen.

Therefore the inclusion of Ascaris in this group requires

comment. Smith and Lee (1963) discussed the need of

Ascaris for host hemoglobin to supply haematin for

synthesis of its own hemoglobin. They suggested that

internal bleeding from ulceration and abrasion of the

intestinal wall by the movements of these large wormscould satisfy that need. It is also possible that the As-

caris protease is expressed during larval liver/lung mi-

gration where it is likely that red cells are ingested.

Unfortunately no expression data are available to es-

tablish the stage at which the Ascaris motif containing

protease is expressed. However it must be acknowledged

that Ascaris stands apart from the other helminths with

the motif, that clearly are bloodfeeders.Should we expect to find this motif in hemoglobin

degrading cysteine proteases of non-helminth species?

There are many examples of organisms that must de-

grade hemoglobin, including mammals during recycling

of senescent erythrocytes, bloodfeeding insects, and in-

tra-erythrocytic parasites such as Plasmodium falcipa-

rum. Our studies have not detected the helminth

bloodfeeder motif in any of these organisms. One ex-planation is that the hemoglobin degrading cysteine

proteases of other organisms may have arisen from

different paralogous gene copies in this large gene fam-

ily, and therefore natural selection has acted upon a

different initial gene sequence in different organisms.

Also, these various organisms may degrade hemoglobin

by different routes; it is certainly clear from studies in

Fig. 3. Phylogenetic analysis of cathepsin B like cysteine proteases.

Confidence bootstrap values are separated by slash marks and based

upon the following three methods: c corrected neighbor-joining

(a ¼ 0:68, 10,000 replicates), maximum parsimony (1000 resamplings),

and maximum likelihood, respectively. Dashes represent cases where

the phylogenetic grouping was not consistent for the given method.

Aedes was designated as the outgroup in all cases. Motif containing

proteases are in boldface.

S. Baig et al. / Experimental Parasitology 101 (2002) 83–89 87

P. falciparum and Schistosomes that a number of dif-ferent proteases play a role in hemoglobin degradation

(Brindley et al., 1997; Francis et al., 1997).

We predict that this motif will be found in other

bloodfeeding helminths but not in parasitic cestodes,

which do not digest hemoglobin. A potential means of

testing the relevance of this motif to hemoglobin deg-

radation is provided by the two cathepsin B enzymes of

S. japonicum (GenBank Accession Nos. S31907 andS31909). Although 77.2% identical at the amino acid

level, one protease (Accession No. S31907) contains the

motif. Moreover there is experimental evidence that

this cathepsin B enzyme, ‘‘SJ31,’’ is a hemoglobinase,

whereas the second enzyme lacking the motif is not

known to be (Caffrey and Ruppel, 1997). This obser-

vation suggests that the protease lacking the motif may

be generalized for ‘‘housekeeping’’ functions, and theSJ31 hemoglobinase for hemoglobin breakdown.

Therefore, we would predict that the protease lacking

the motif would not degrade hemoglobin as readily as

the other, motif-containing protease. Furthermore, in

vitro mutagenesis to either include or remove the motif

should alter the relative activity against hemoglobin

shown by these proteases.

This motif may provide clues to the identification ofpotential hemoglobinase activity in other parasites. Be-

cause cathepsin B enzymes of humans and other perti-

nent hosts lack this pattern, future experimental

directions may include a focus on this region for the

development of potential chemotherapeutic inhibitors

and/or immunization strategies against helminth

bloodfeeders as a group.

Acknowledgments

The authors thank R. Kaplan and E. Kipreos forcritically reading the manuscript.

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