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A novel cathepsin B active site motif is shared by helminth bloodfeeders
Experimental Parasitology 101 (2002) 83–89 www.academicpress.com
A novel cathepsin B active site motif is shared by helminth bloodfeeders 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, USA b 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 hemoglobin degradation by these cathepsin B-like enzymes. This motif should be useful in identifying additional helminth hemoglobinases 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 Ó 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-complex lifecycles. Helminth parasites employ these enzymes during skin penetration, and to evade host immune responses through digestion of host immune effector molecules (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 *
Corresponding author. Fax: 1-706-542-0059. E-mail address: [email protected] (D.S. Peterson).
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 converting 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 (Cheng and Ngo, 1997; Roberts et al., 1990). Due to the importance of protease activity at crucial stages of a parasiteÕs lifecycle, it has been proposed that protease inhibitors may prove to be highly effective in the treatment of parasitic diseases (McKerrow, 1989). Proteases are been being investigated as targets for chemotherapeutic intervention for parasitic diseases, and several studies have now demonstrated the effectiveness of inhibitor treatment in vivo. Mice infected with murine malaria can be cured by administration of cysteine protease inhibitors, thought to target falcipain, a
0014-4894/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 4 - 4 8 9 4 ( 0 2 ) 0 0 1 0 5 - 4
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protease involved in hemoglobin degradation (Olson et al., 1999). Experiments with murine models of ChagasÕ 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 demonstrated anti-helminth activity in in vitro and in vivo studies (Rhoads and Fetterer, 1996; Wasilewski et al., 1996). In addition, recent studies have demonstrated the potential for clearance of T. crassiceps from infected mice through administration of parasite specific protease 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 between the proteases of the host and the infectious agent. In the present study, we have discovered and correlated specific primary sequence motifs of helminth cathepsin B-like cysteine proteases with the biochemical characteristics of those enzymes. These motifs are not present in non-parasitic worms, nor in the cathepsin Blike 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 hemoglobin. This effort identified sequences from Ostertagia ostertagi (A48454), Schistosoma japonicum SJ31 (S31907), Schistosoma mansoni ‘‘SM31 prec’’ (P25792), Ancylostoma caninum ACCP-1 (AAC46877), Haemonchus 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 well as from other non-helminth species, papain (CAB42883) Rattus norvegicus (CAA57792), Aedes aegypti (AAA 79004), and Leishmania major (AAB48119). These sequences were aligned using the MACAW program which can be used to search for short highly conserved sequence blocks (Schuler et al., 1991). From this alignment emerged an amino acid motif from the active site region that was present only in the bloodfeeding helminths. 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-SW-X–X–D-W-G-E). Also the BLAST search tool was used to search GenBank with the individual permutations of the Asn active site motif present in different bloodfeeding helminths. These searches resulted in the
discovery of an additional eight gene sequences, all cathepsin 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 minimize insertions and deletions. For all gene sequences the GenBank literature reference, and related references, were reviewed to determine stage specificity of expression, pH optimum, requirements for a reducing environment, and other data relevant to potential proteolytic activity against hemoglobin. 2.2. Phylogenetic analysis Neighbor joining, maximum likelihood, and maximum parsimony were constructed with MEGA 1.0 (Kumar et al., 1993), MOLPHY 2.3 (Adachi and Hasegawa, 1994), and Phylip 3.5 (Felsenstein, 1989), respectively. Amino acid sequence data were analyzed with the JTT-F model for maximum likelihood and a c correction for neighbor joining. The c distribution parameter a was assessed from the amino acid sequence data 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). Statistical confidence estimates of topologies and branches were obtained with the RELL bootstrap method for maximum likelihood and the standard bootstrap method (10,000 bootstrap resamplings) for maximum parsimony and neighbor-joining. Trees were constructed using Treeview 1.5 (Page, 1998) and Tree Explorer (Tamura, 1997). Trees generated by maximum likelihood, parsimony, and by the distance methods neighbor joining and Fitch were compared using the userÕs tree option of protml to confirm that protml had derived the optimal tree. Alignments are available from the corresponding author.
3. Results and discussion 3.1. Identification of hemoglobinase specific motifs To identify motifs specific to a particular class of cysteine protease, we obtained the primary sequences of a number of cathepsin B-like cysteine proteases from both bloodfeeding helminth parasites, and other organism found in GenBank (listed in Fig. 2) and employed the MACAW (Schuler et al., 1991) alignment program to identify conserved domains in cathepsin B subsets. MACAW was chosen since it employs an
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interactive approach to constructing an alignment based upon 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 other classes 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 observations. First, a PROSITE (Bucher and Bairoch, 1994) search of the TrEMBL and SwissProt databases using 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 helminth, C. elegans, which represents the entire complement 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. japonicum (Merckelbach et al., 1994) and S. mansoni (reviewed 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 degradation based upon such criteria as expression in bloodfeeding 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 secreted (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
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expressed in adult stages, localized in the hookworm esophogeal, 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 asparagine active site region. Here hydrogen bonding plays a key role in catalysis. Notably, in the hemoglobinase motif containing enzymes, this region is characterized by hydrogen-bond-donating residues that replace non-hydrogen-bond-donating residues found in other cathepsin Bs. For example, Asp, a hydrogen-bond-donating amino acid, is present in the hemoglobinase motif at position 11 (Fig. 1), while Tyr at position 1 has the ability to function as a weak acid. Interestingly, four out of five of the C. elegans cathepsin B-like proteases are characterized 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 residue, Phe (Fig. 2). By virtue of changes in hydrogen bonding character within the motif region, such cathepsin 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 difference between the specificity of a cathepsin B for a general substrate or a hemoglobin substrate may occur through the mechanism of changes in the nature of hydrogen 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.
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region can cause significant modifications in the proteolytic 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 alteration is especially notable because only 29% identity exists between papain and cathepsin B enzymes at the primary structure level. Substrate specificity alterations have also been accomplished through protein engineering 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
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 nonconserved 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 mexicana (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 americanus (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 ‘‘AC5’’ (B48435), H. contortus ‘‘GCP7’’ (AAC05262); O. ostertagi ‘‘cath-B like CP’’ (A48454); H. contortus ‘‘HCCP6’’ (CAB03627); H. contortus ‘‘HMCP4’’ (CAA93278); H. contortus ‘‘HMCP3’’ (CAA93277).
To assess whether the motif represents selection for catalytic function as opposed to inheritance of an ancestral helminth pattern, we analyzed two alignments, both constructed with CLUSTAL W (Higgins et al., 1996) with one manually edited to improve the alignment. We developed phylogenetic trees of the aligned cysteine protease sequences based upon the criteria of maximum likelihood (ML), maximum parsimony, c corrected Neighbor Joining, and Fitch–Margoliash, as well as non-c corrected Kitsch. We employed bootstrapping methods (Felsenstein, 1985) to assess the accuracy of the phylogeny and limited our analysis to complete cathepsin B sequences in GenBank. All phylogenetic methods using either of the two constructed alignments produced substantially similar trees with preservation of inner branches in all cases. Although we show only the results obtained from ccorrected 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 hemoglobinase from Ascaris is most closely related to two C. elegans cathepsin B sequences that lack the motif. The motif 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 molecular level. Moreover, in all cases, our analysis demonstrates that it is most parsimonious for the motif to have emerged evolutionarily in a minimum of three separate 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 enzymes are encoded by a multiple gene family (e.g., 5 in
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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.
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 suggested 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 hemoglobins revealed that they are 81–93% identical in their a and b chains. This suggests that conserved hemoglobin sequences could have provided a uniform selective pressure for the emergence of a single hemoglobinase motif in the active sites of helminth cathepsin B 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 (Brinkworth et al., 2000). Is it reasonable to propose therefore that the relatively high sequence similarity of mammalian hemoglobin could have provided a uniform selective force for the emergence of a hemoglobinase motif?
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While there clearly is high overall similarity, some regions 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 selected for the emergence of hemoglobinases with a shared motif include the similar gastrodermal environment 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), developmental 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 from red 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 proteases 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 mammalian host 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 worms could satisfy that need. It is also possible that the Ascaris protease is expressed during larval liver/lung migration where it is likely that red cells are ingested. Unfortunately no expression data are available to establish 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 degrade hemoglobin, including mammals during recycling of senescent erythrocytes, bloodfeeding insects, and intra-erythrocytic parasites such as Plasmodium falciparum. Our studies have not detected the helminth bloodfeeder motif in any of these organisms. One explanation is that the hemoglobin degrading cysteine proteases of other organisms may have arisen from different paralogous gene copies in this large gene family, 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
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P. falciparum and Schistosomes that a number of different 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 degradation is provided by the two cathepsin B enzymes of S. japonicum (GenBank Accession Nos. S31907 and S31909). 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 observation suggests that the protease lacking the motif may be generalized for ‘‘housekeeping’’ functions, and the SJ31 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 of potential hemoglobinase activity in other parasites. Because cathepsin B enzymes of humans and other pertinent 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 for critically reading the manuscript.
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Krohn, A., et al., 1990. Rational design of peptide-based HIV proteinase inhibitors. Science 248 (4953), 358–361. Rullmann, J.A., Bellido, M.N., van Duijnen, P.T., 1989. The active site of papain. All-atom study of interactions with protein matrix and solvent. J. Mol. Biol. 206 (1), 101–118. Schuler, G.D., Altschul, S.F., Lipman, D.J., 1991. A workbench for multiple alignment construction and analysis. Proteins 9 (3), 180– 190. Shompole, S., Jasmer, D.P., 2001. Cathepsin B-like cysteine proteases confer intestinal cysteine protease activity in Haemonchus contortus. J. Biol. Chem. 276 (4), 2928–2934. Smith, M.H., Lee, D.L., 1963. Metabolsim of haemoglobin and haematin compounds in Ascaris lumbricoides. Proc. Roy. Soc. London 157, 234–257. Strimmer, K., von Haeseler, A., 1996. Quartet puzzling: a quartet maximun-likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13 (7), 964–969. Tamura, K., 1997. TreeExplorer. Tort, J., Brindley, P.J., Knox, D., Wolfe, K.H., Dalton, J.P., 1999. Proteinases and associated genes of parasitic helminths. Adv. Parasitol. 43, 161–266. Wang, J., Xiang, Y.F., Lim, C., 1994. The double catalytic triad, Cys25–His159–Asp158 and Cys25–His159–Asn175, in papain catalysis: role of Asp158 and Asn175. Protein Eng. 7 (1), 75– 82. Wasilewski, M.M., Lim, K.C., Phillips, J., McKerrow, J.H., 1996. Cysteine protease inhibitors block schistosome hemoglobin degradation in vitro and decrease worm burden and egg production in vivo. Mol. Biochem. Parasitol. 81 (2), 179–189. Zerda, K.S., Dresden, M.H., Chappell, C.L., 1988. Schistosoma mansoni: expression and role of cysteine proteinases in developing schistosomula. Exp. Parasitol. 67 (2), 238–246.
A novel cathepsin B active site motif is shared by helminth bloodfeeders 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, USA b 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 hemoglobin degradation by these cathepsin B-like enzymes. This motif should be useful in identifying additional helminth hemoglobinases 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 Ó 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-complex lifecycles. Helminth parasites employ these enzymes during skin penetration, and to evade host immune responses through digestion of host immune effector molecules (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 *
Corresponding author. Fax: 1-706-542-0059. E-mail address: [email protected] (D.S. Peterson).
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 converting 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 (Cheng and Ngo, 1997; Roberts et al., 1990). Due to the importance of protease activity at crucial stages of a parasiteÕs lifecycle, it has been proposed that protease inhibitors may prove to be highly effective in the treatment of parasitic diseases (McKerrow, 1989). Proteases are been being investigated as targets for chemotherapeutic intervention for parasitic diseases, and several studies have now demonstrated the effectiveness of inhibitor treatment in vivo. Mice infected with murine malaria can be cured by administration of cysteine protease inhibitors, thought to target falcipain, a
0014-4894/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 4 - 4 8 9 4 ( 0 2 ) 0 0 1 0 5 - 4
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protease involved in hemoglobin degradation (Olson et al., 1999). Experiments with murine models of ChagasÕ 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 demonstrated anti-helminth activity in in vitro and in vivo studies (Rhoads and Fetterer, 1996; Wasilewski et al., 1996). In addition, recent studies have demonstrated the potential for clearance of T. crassiceps from infected mice through administration of parasite specific protease 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 between the proteases of the host and the infectious agent. In the present study, we have discovered and correlated specific primary sequence motifs of helminth cathepsin B-like cysteine proteases with the biochemical characteristics of those enzymes. These motifs are not present in non-parasitic worms, nor in the cathepsin Blike 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 hemoglobin. This effort identified sequences from Ostertagia ostertagi (A48454), Schistosoma japonicum SJ31 (S31907), Schistosoma mansoni ‘‘SM31 prec’’ (P25792), Ancylostoma caninum ACCP-1 (AAC46877), Haemonchus 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 well as from other non-helminth species, papain (CAB42883) Rattus norvegicus (CAA57792), Aedes aegypti (AAA 79004), and Leishmania major (AAB48119). These sequences were aligned using the MACAW program which can be used to search for short highly conserved sequence blocks (Schuler et al., 1991). From this alignment emerged an amino acid motif from the active site region that was present only in the bloodfeeding helminths. 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-SW-X–X–D-W-G-E). Also the BLAST search tool was used to search GenBank with the individual permutations of the Asn active site motif present in different bloodfeeding helminths. These searches resulted in the
discovery of an additional eight gene sequences, all cathepsin 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 minimize insertions and deletions. For all gene sequences the GenBank literature reference, and related references, were reviewed to determine stage specificity of expression, pH optimum, requirements for a reducing environment, and other data relevant to potential proteolytic activity against hemoglobin. 2.2. Phylogenetic analysis Neighbor joining, maximum likelihood, and maximum parsimony were constructed with MEGA 1.0 (Kumar et al., 1993), MOLPHY 2.3 (Adachi and Hasegawa, 1994), and Phylip 3.5 (Felsenstein, 1989), respectively. Amino acid sequence data were analyzed with the JTT-F model for maximum likelihood and a c correction for neighbor joining. The c distribution parameter a was assessed from the amino acid sequence data 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). Statistical confidence estimates of topologies and branches were obtained with the RELL bootstrap method for maximum likelihood and the standard bootstrap method (10,000 bootstrap resamplings) for maximum parsimony and neighbor-joining. Trees were constructed using Treeview 1.5 (Page, 1998) and Tree Explorer (Tamura, 1997). Trees generated by maximum likelihood, parsimony, and by the distance methods neighbor joining and Fitch were compared using the userÕs tree option of protml to confirm that protml had derived the optimal tree. Alignments are available from the corresponding author.
3. Results and discussion 3.1. Identification of hemoglobinase specific motifs To identify motifs specific to a particular class of cysteine protease, we obtained the primary sequences of a number of cathepsin B-like cysteine proteases from both bloodfeeding helminth parasites, and other organism found in GenBank (listed in Fig. 2) and employed the MACAW (Schuler et al., 1991) alignment program to identify conserved domains in cathepsin B subsets. MACAW was chosen since it employs an
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interactive approach to constructing an alignment based upon 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 other classes 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 observations. First, a PROSITE (Bucher and Bairoch, 1994) search of the TrEMBL and SwissProt databases using 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 helminth, C. elegans, which represents the entire complement 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. japonicum (Merckelbach et al., 1994) and S. mansoni (reviewed 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 degradation based upon such criteria as expression in bloodfeeding 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 secreted (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
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expressed in adult stages, localized in the hookworm esophogeal, 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 asparagine active site region. Here hydrogen bonding plays a key role in catalysis. Notably, in the hemoglobinase motif containing enzymes, this region is characterized by hydrogen-bond-donating residues that replace non-hydrogen-bond-donating residues found in other cathepsin Bs. For example, Asp, a hydrogen-bond-donating amino acid, is present in the hemoglobinase motif at position 11 (Fig. 1), while Tyr at position 1 has the ability to function as a weak acid. Interestingly, four out of five of the C. elegans cathepsin B-like proteases are characterized 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 residue, Phe (Fig. 2). By virtue of changes in hydrogen bonding character within the motif region, such cathepsin 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 difference between the specificity of a cathepsin B for a general substrate or a hemoglobin substrate may occur through the mechanism of changes in the nature of hydrogen 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.
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region can cause significant modifications in the proteolytic 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 alteration is especially notable because only 29% identity exists between papain and cathepsin B enzymes at the primary structure level. Substrate specificity alterations have also been accomplished through protein engineering 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
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 nonconserved 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 mexicana (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 americanus (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 ‘‘AC5’’ (B48435), H. contortus ‘‘GCP7’’ (AAC05262); O. ostertagi ‘‘cath-B like CP’’ (A48454); H. contortus ‘‘HCCP6’’ (CAB03627); H. contortus ‘‘HMCP4’’ (CAA93278); H. contortus ‘‘HMCP3’’ (CAA93277).
To assess whether the motif represents selection for catalytic function as opposed to inheritance of an ancestral helminth pattern, we analyzed two alignments, both constructed with CLUSTAL W (Higgins et al., 1996) with one manually edited to improve the alignment. We developed phylogenetic trees of the aligned cysteine protease sequences based upon the criteria of maximum likelihood (ML), maximum parsimony, c corrected Neighbor Joining, and Fitch–Margoliash, as well as non-c corrected Kitsch. We employed bootstrapping methods (Felsenstein, 1985) to assess the accuracy of the phylogeny and limited our analysis to complete cathepsin B sequences in GenBank. All phylogenetic methods using either of the two constructed alignments produced substantially similar trees with preservation of inner branches in all cases. Although we show only the results obtained from ccorrected 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 hemoglobinase from Ascaris is most closely related to two C. elegans cathepsin B sequences that lack the motif. The motif 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 molecular level. Moreover, in all cases, our analysis demonstrates that it is most parsimonious for the motif to have emerged evolutionarily in a minimum of three separate 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 enzymes are encoded by a multiple gene family (e.g., 5 in
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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.
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 suggested 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 hemoglobins revealed that they are 81–93% identical in their a and b chains. This suggests that conserved hemoglobin sequences could have provided a uniform selective pressure for the emergence of a single hemoglobinase motif in the active sites of helminth cathepsin B 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 (Brinkworth et al., 2000). Is it reasonable to propose therefore that the relatively high sequence similarity of mammalian hemoglobin could have provided a uniform selective force for the emergence of a hemoglobinase motif?
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While there clearly is high overall similarity, some regions 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 selected for the emergence of hemoglobinases with a shared motif include the similar gastrodermal environment 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), developmental 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 from red 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 proteases 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 mammalian host 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 worms could satisfy that need. It is also possible that the Ascaris protease is expressed during larval liver/lung migration where it is likely that red cells are ingested. Unfortunately no expression data are available to establish 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 degrade hemoglobin, including mammals during recycling of senescent erythrocytes, bloodfeeding insects, and intra-erythrocytic parasites such as Plasmodium falciparum. Our studies have not detected the helminth bloodfeeder motif in any of these organisms. One explanation is that the hemoglobin degrading cysteine proteases of other organisms may have arisen from different paralogous gene copies in this large gene family, 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
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P. falciparum and Schistosomes that a number of different 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 degradation is provided by the two cathepsin B enzymes of S. japonicum (GenBank Accession Nos. S31907 and S31909). 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 observation suggests that the protease lacking the motif may be generalized for ‘‘housekeeping’’ functions, and the SJ31 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 of potential hemoglobinase activity in other parasites. Because cathepsin B enzymes of humans and other pertinent 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 for critically reading the manuscript.
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