(Received for publication, April 11, 1995; and in revised form, June 27, 1995)
From the
A cDNA encoding a Schistosoma japonicum aspartic
proteinase was cloned, sequenced, and found to encode a zymogen of 380
amino acid residues, and its gene was shown to be present as a single
copy in the S. japonicum genome. Identity comparisons showed
that the enzyme (Sjpasp) was most closely related to the cathepsin Ds.
The deduced amino acid sequence has four potential glycosylation sites,
two of which are in identical positions to the two glycosylation sites
of human kidney lysosomal cathepsin D. Furthermore, all four disulfide
bonds found in mammalian cathepsin D sequences are present in Sjpasp,
although the -hairpin (loop 3), which is cleaved during maturation
of vertebrate cathepsin Ds to yield light and heavy chain subunits, is
absent from Sjpasp. While most residues involved in substrate
specificity and catalysis of aspartic proteinases are preserved in
Sjpasp, several residues in these regions exhibit changes that may
result in a novel substrate specificity. Aspartic proteinase activity
is present in extracts of adult S. japonicum and Schistosoma mansoni and in culture media in which schistosomes
were maintained and was capable of digesting hemoglobin. The
schistosome aspartic proteinase may play a pivotal role in the
catabolism of hemoglobin obtained from host erythrocytes.
Blood flukes of the genus Schistosoma are the cause of schistosomiasis, which afflicts more than 250 million people in the tropics. Schistosomes feed on red blood cells, and it has been estimated that male and female adult Schistosoma mansoni ingest 39,000 and 330,000 red blood cells/h, respectively(1) . Accordingly, catabolism of host hemoglobin is likely to be integrally involved in schistosome nutrition. It was suggested in the 1950s that acid proteinases were secreted by schistosomes to digest hemoglobin extracellularly(2) . The precise enzyme activities involved in the catabolism of hemoglobin into readily absorbable peptides have still not been determined, although both cysteine and aspartyl proteinases have been implicated(3) .
We have already described the
characterization of a cathepsin L proteinase secreted by adult
schistosomes, which we suggested was involved in the degradation of
host hemoglobin (Hb), and we also reported the gene sequence for
schistosome cathepsin L(4) . Now we describe the isolation of a
cDNA encoding the Schistosoma japonicum aspartic proteinase.
Gene fragments were initially isolated from a cDNA library using a
primer that we designed based on the consensus sequence adjacent to the
active site Asp-32 of 16 eukaryotic aspartic proteinases. The larger
fragment was used as a probe to screen a cDNA library to isolate a
full-length clone. In addition, aspartic proteinase activity was
detected using the synthetic substrate
Phe-Ala-Ala-Phe(NO)-Phe-Val-Leu-OM4P (5) in
extracts of adult schistosomes and in culture medium in which
schistosomes had been maintained, and we demonstrate that this type of
activity can degrade human Hb. Since Southern hybridization indicated
that the gene encoding the aspartic proteinase was present as a single
copy in the parasite genome, it is likely that the cDNA we characterize
here encodes these proteinase activities. Therefore, adult schistosomes
secrete both aspartic proteinase and cathepsin L cysteine proteinases,
which may all be involved in the degradation of Hb in
vivo(3) .
Analysis of nucleotide and deduced amino acid sequences was performed using the FASTA program of the GCG Package software, version 7.3.1 (1993, Genetics Computer Group, Madison, WI) and the EGCG software (EMBL, Heidelberg, Germany) and using the GenBank (release 87) and SwissProt (release 31) data bases. A dendrogram to predict the relationship between members of the subclasses of aspartic proteinases was constructed by pairwise alignment using the algorithm of Feng and Doolittle (9) and identity scores based on FASTA comparisons of amino acid sequences of full preproenzyme sequences of 37 aspartic proteinases (see Fig. 1).
Figure 1: Dendrogram of multiple pairwise alignments of deduced amino acid sequences of aspartic proteinases from Schistosoma japonicum and other species producing clusters of similar sequences (GenBank accession numbers from the top: X75787, L10740, P10977, P07267, P20142, P03955, P04073, P25796, P14091, P03954, P11489, P27677, P00790, P28712, P27821, P07822, P00792, P00793, P18276, P16476, P27823, P06281, P00796, P08424, P00797, P18242, P24268, P07339, P00795, Q05744, Q03168, L41346 (Sjpasp), P28871, P22929, P06026, P10602, P17946). The dendrogram predicts evolutionary relationships of Sjpasp to other members of the aspartic proteinase class(9) . Similarities (%) to Sjpasp were obtained using the FASTA program. Boldface denotes the group of cathepsin Ds that includes Sjpasp.
The UNI Zap-XR cDNA library was
screened by nucleic acid hybridization using the P-labeled
insert of the cloned 1.3-kb PCR product as the probe, using Hybond-N
(Amersham Corp.) nylon membranes. Insert DNA (15 ng ml
hybridization solution) radiolabeled
[
-
P]dCTP (DuPont NEN) by random oligomer
priming and Klenow polymerase (AMRAD-Pharmacia, North Ryde, Australia)
was hybridized to membranes at 65 °C overnight in 1 mM EDTA, 0.5 M Na
HPO
, 7% SDS, pH
7.2, and washed at 65 °C for 2 h in 1 mM EDTA, 40 mM Na
HPO
, 5% SDS, pH 7.2, and then for 2 h in
1 mM EDTA, 40 mM Na
HPO
, 1%
SDS, pH 7.2, as described previously(10) . Autoradiography was
performed at -70 °C using Kodak X-AR film and intensifying
screens. pBluescript SK(-) phagemids in positive
clones
were excised using R408 interference resistant helper phage and E.
coli strain XL1-Blue (Stratagene).
The open reading frame encodes an entire preproenzyme of 380 amino acid residues. Since Tang and Wong (6) consider that all aspartic proteinases have evolved from a common, primordial enzyme, we constructed a dendrogram of aspartic proteinase sequences in order to predict the phylogenetic relatedness of the S. japonicum aspartic proteinase (Sjpasp) to 36 other aspartic proteinases (Fig. 1). Closest identities for Sjpasp were with mammalian cathepsin Ds (52.5-55.1%) and to the aspartic proteinase of the mosquito Aedes aegypti (53.1%)(13) , and accordingly we consider the schistosome aspartic proteinase can be characterized as cathepsin D-like (EC3.4.23). By contrast, Sjpasp showed less identity to renins, pepsinogens, cathepsins E, or to several other aspartic proteinases including two putative hemoglobinases (pfaasphem and pfaaspprot) of the malaria parasite Plasmodium falciparum(14, 15) .
Figure 2:
Alignment of deduced amino acid sequences
of cathepsin D aspartic proteinases from S. japonicum (catD
pSjpasp, accession number L41346), mouse (catd_mouse, P18242),
rat (tDcatd_rat, P24268), human (catd_human, P07339), chicken
(catd_chick, Q05744), and the mosquito A. aegypti (aspp_aedae)
(Q03168). The numbering system is based on the sequence of human kidney
cathepsin D (catd_human); the presumed NH-terminal residue
of the mature enzyme is Gly-1. The residues of the mature enzymes are
counted in the positive from this residue, while the pre- and proenzyme
regions are numbered in the negative from Gly-1. Blocks denote
conserved residues, and gaps have been introduced to maximize
alignment. Symbols on the schistosome sequence pSjpasp designate the
aspartic proteinase catalytic Asp residues (solidtriangles), S
subsite residues (solidcircles), S
subsite residues (opencircles), potential Asn glycosylation sites (diamonds), phosphorylation determinants (dottedlines), cleavage of signal peptide (solidarrow), beginning of mature proteinase (openarrow), and cysteine residues (opentriangles). The eight loops characteristic of aspartic
proteinases (15) are indicated by numberedbars.
A major structural difference
within cathepsin Ds is the sequence involved in the processing of the
single chain form to the two-chain form(6) . Processing of
vertebrate cathepsin Ds involves the proteolytic cleavage of a
-hairpin (loop 3), which in human cathepsin D is formed by
residues 94-107. This
-hairpin loops outside the molecular
surface, where it is readily hydrolyzed to transform the enzyme from a
single chain into a dimeric polypeptide. This proteolytic cleavage also
stabilizes the tertiary structure of the cathepsin D(17) .
However, the schistosome aspartic acid, like those of chicken and A. aegypti, does not contain this kind of
-hairpin.
Aspartic proteinases that lack the
-hairpin are processed in other
ways; the aspartic proteinase of barley and that of A. aegypti are cleaved at other sites to generate hetero- and homodimeric
enzymes, respectively(12) . Whether the native schistosome
aspartic proteinase exists as one of these kinds of bilobed enzymes
requires further investigation.
The mature Sjpasp enzyme has four potential Asn-linked glycosylation sites at positions Asn-70, Asn-172, Asn-199, and Asn-212, although only sites at Asn-70 and Asn-199 align with conserved, glycosylated asparagine residues of mammalian cathepsin Ds. Lys-203, a determinant for phosphorylation of high-mannose oligosaccharides(13) , is conserved in cathepsin Ds and in Sjpasp. However, Arg-202 and Ala-204, conserved residues on each side of Lys-203 in mammalian, avian, and mosquito cathepsin Ds, are exchanged for Glu-202 and Ser-204 in the schistosome enzyme. A second phosphorylation determinant spans 28 residues from Cys-265 to Leu-292(13) . 19 of these determinant residues are conserved in mammalian cathepsin Ds, whereas only 13 of these 19 are conserved in Sjpasp. Elucidation of the extent of glycosylation/phosphorylation of the schistosome aspartic proteinase will provide insight into the mechanism by which adult schistosomes process this enzyme in the gastrodermal cells and secrete it into the cecal lumen (see below). Three disulfide linkages present in other cathepsin Ds are preserved in the schistosome enzyme, including Cys-46-Cys-53 (within loop 1), Cys-222-Cys-226 (within loop 5), and Cys-265-Cys-302(6, 7, 15) .
Figure 3:
Southern hybridization of a P-labeled, 1.4-kb BamHI-HindI11 fragment
containing the full coding sequence of the Schistosoma japonicum aspartic proteinase to genomic DNAs from S. japonicum after EcoRI (lane 1), BsaW 1 (lane
2) and PstI (lane 3) digestion, and from S.
mansoni after PstI digestion (lane 4). Size
standards in kb are shown at the left.
Figure 4:
Panel A, inhibition profile for
activity in S. japonicum extracts capable of cleaving
Phe-Ala-Ala-Phe(NO)-Phe-Val-Leu-OM4P. The following
inhibitors were employed; pepstatin (2 mM),
Z-Phe-Ala-CHN
(5 µM), AEBSF (2 mM),
and EDTA (5 mM). PanelB, pH optima of
proteinase activities in Schistosoma japonicum extracts.
Aspartic proteinase activity was measured at 310 nm using the specific
peptide substrate Phe-Ala-Ala-Phe(NO
)-Phe-Val-Leu-OM4P.
Cathepsin L activity was assayed with the fluorogenic peptide substrate
Z-Phe-Arg-NHMec. Mean values from triplicate assays are shown in panelsA and B.
We have previously observed cathepsin L activity in these schistosome extracts(4) . Using Z-Phe-Arg-NHMec as substrate, we now show that the schistosome cathepsin L is active in the pH range 3.5-6.0, with an optimum pH of 5.5 (Fig. 4B)(4) . Therefore, we conclude that the aspartic proteinase and the cathepsin L have activity through overlapping ranges of pH. Similar aspartic proteinase and cathepsin L activities were detected in extracts of S. mansoni, and in ES from both S. japonicum and S. mansoni (not shown).
Figure 5:
Hemoglobin-degrading activity in S.
japonicum tissue extracts and ES. PanelA,
inhibition of activity in extracts by pepstatin and partial inhibition
by Z-Phe-Ala-CHN; lane1, S.
japonicum extract alone; lane2, Hb alone; lane3, extract and Hb; lane4, Hb,
extract, and pepstatin (2 mM); lane5,
extract, Hb, and Z-Phe-Ala-CHN
(5 µM); lane6, extract, Hb, and AEBSF (2 mM); lane7, extract, Hb, and EDTA (5 mM). Largearrows indicate position of the whole molecule
(64 kDa), dimer (32 kDa), and monomer of Hb (16 kDa); the smallarrow indicates position of a
10-kDa product of
digestion that appears only when the inhibitor Z-Phe-Ala-CHN
is added. PanelB, pH optimum of
Hb-degradation. Reactions were carried out in sodium acetate, pH 3.5 (lanes1-3), sodium acetate, pH 4.5 (lanes4 and 5), and sodium phosphate, pH 5.5. Lane1, extract alone; lanes2, 4,
and 6, Hb alone; lanes3, 5, and 7, Hb and extract. PanelC, degradation of
Hb by S. japonicum ES. Lanes1 and 4, Hb and ES; lane2, Hb, ES, and pepstatin
(0.2 mM); lane3, Hb, ES, and pepstatin (2
mM); lane5, Hb, ES, and Z-Phe-Ala-CHN
(0.5 µM); lane6, Hb, ES, and
Z-Phe-Ala-CHN
(5 µm).
Bogitsh et al. (19) reported hemoglobinase activity
in extracts of S. japonicum that was inhibited by pepstatin
but was unaffected by leupeptin, an inhibitor of cysteine proteinases.
In contrast, Chappell and Dresden (20) attributed the
hemoglobinase activity in schistosome ES to a cysteine proteinase since
it was inhibited by leupeptin. It is now apparent, based on our present
results, that soluble extracts of S. japonicum contain both
aspartic and cysteine proteinase activities, each of which can degrade
Hb. In our inhibition analysis, the cathepsin L inhibitor
Z-Phe-Ala-CHN partially blocked the digestion of Hb by
activities in the crude parasite extract (as evidenced by the
appearance of a 10-kDa fragment) and completely blocked Hb digestion by
activities in the parasite ES. Our results are therefore consistent
with the reports of both Bogitsh et al. (19) and
Chappell and Dresden (20) and indicate that the cathepsin L-like proteinase may be comparatively more active or abundant
in ES relative to crude extracts, suggesting that it functions in the
schistosome gut.
In contrast to the cathepsin L-like activity, the aspartic proteinase may be more pervasive in schistosome tissue(18) . Using anti-bovine cathepsin D sera, Bogitsh and Kirschner (21) localized a cathepsin D-like enzyme not only to the gastrodermis and cecal lumen but also to the dorsal tegument and tubercles of male schistosomes. Presumably, the aspartic proteinase activity that we have identified in ES is secreted from the gastrodermis or cecum. However, the distribution of aspartic proteinases in tissues distant from the gut (21) suggests than the aspartic proteinase may be expressed in diverse tissues, given that our Southern hybridization indicated that only a single copy of its gene was present in the schistosome genome. Thus, it is likely that the hemoglobinolytic activity we report here is encoded by the Sjpasp transcript.
The aspartic proteinase described here, along with the cathepsin L-like activity that we reported previously(4) , may comprise the acid proteinase activities originally described nearly four decades ago by Timms and Bueding (2) as being responsible for the digestion of Hb. Expressed recombinant active enzymes of both the aspartic proteinase and the cathepsin L can now be employed to define the precise mechanism of Hb digestion by schistosomes. In addition, these molecules may provide novel targets for drug and/or vaccine development.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L41346[GenBank].