(Received for publication, December 30, 1996, and in revised form, March 6, 1997)
From the The cpb genes of
Leishmania mexicana encode stage-regulated, cathepsin
L-like cysteine proteinases that are leishmanial virulence factors.
Field inversion gel electrophoresis and genomic mapping indicate that
there are 19 cpb genes arranged in a tandem array. Five
genes from the array have been sequenced and their expression analyzed.
The first two genes, cpb1 and cpb2, differ
significantly from the remaining 17 copies
(cpb3-cpb19) in that: 1) they are expressed
predominantly in metacyclic promastigotes (the form in the insect
vector which is infective to mammalian macrophages) rather than
amastigotes (the form that parasitizes mammals); 2) they encode enzymes
with a truncation in the COOH-terminal extension, an unusual feature of
these cysteine proteinases of trypanosomatids. Transfection of
cpb1 into a cpb null mutant resulted in
expression of an active enzyme that was shown by immunogold labeling
with anti-CPB antibodies to be targeted to large lysosomes. This
demonstrates that the 100-amino acid COOH-terminal extension is not
essential for the activation or activity of the enzyme or for its
correct intracellular trafficking. Transfection into the
cpb null mutant of different copies of cpb and
analysis of the phenotype of the lines showed that individual
isoenzymes differ in their substrate preferences and ability to restore
the loss of virulence associated with the null mutant. Comparison of
the predicted amino acid sequences of the isoenzymes implicates five
residues located in the mature domain (Asn18,
Asp60, Asn61, Ser64, and
Tyr84) with differences in the activities of the encoded
isoenzymes. The results suggest that the individual isoenzymes have
distinct roles in the parasite's interaction with its host. This
complexity reflects the adaptation of cathepsin L-like cysteine
proteinases to diverse functions in parasitic protozoa.
Cathepsin L is a mammalian member of the papain superfamily, which
comprises the largest family of cysteine proteinases identified to date
(1). Cathepsin L is generally a lysosomal protein involved in protein
degradation (2), although secreted enzyme has been implicated in a
number of physiological processes including bone resorption (3),
tumor invasion (4), and arthritis (5). The protein is synthesized as an
inactive precursor that is processed (by removal of the
NH2-terminal pro-peptide extension in the pre-lysosomal compartment) to produce a single chain mature enzyme. The pro-region is
a potent inhibitor of the enzyme's activity (6) through its binding to
the active site (7), thus maintaining the proteinase in an inactive
form during trafficking to the lysosome. Cathepsin L-like enzymes have
been characterized from a wide range of eukaryotes, including some
protists that are thought to form the earliest branches of the
eukaryotic tree (1, 8). There are many cathepsin L-like cysteine
proteinases in parasitic protozoa; frequently they are highly abundant
and stage-regulated, and in some instances they occur in unusual
cellular locations, such as on the surface, or are secreted (9-12).
Thus these enzymes are considered to be good targets for exploitation
with anti-parasite agents (10, 11, 13).
Trypanosomatid flagellated protozoa of the genus Leishmania
are responsible for a number of human diseases in the tropics and
subtropics (14). The diseases, the leishmaniases, range in severity
from a visceral infection that is frequently fatal unless treated to a
cutaneous lesion that self-cures. The parasite has two principal
morphological forms: a motile extracellular flagellated form, the
promastigote, which resides in the sandfly vector; and a nonmotile
form, the amastigote, which resides in a phagolysosomal vacuole in a
mammalian macrophage. Two distinct developmental types of promastigote
live in the sandfly: the multiplicative promastigote in the mid-gut,
and the nondividing metacyclic promastigote in the mouthparts. The
metacyclic promastigote is infective to mammals and has distinct
biochemical properties that distinguish it from the multiplicative
promastigote.
The major cysteine proteinase activities detected in Leishmania
mexicana are cathepsin L-like in their substrate specificity (15),
are encoded by the multicopy cpb genes (16), found in an
array of 2.8-kb1 repeat units (17), and
have been classified as belonging to the papain superfamily (1). They
show in general a marked stage-regulated expression with there being a
very low level of mRNA detected in multiplicative promastigotes,
higher levels in metacyclic promastigotes, and very high mRNA
levels in amastigotes (17). The encoded cysteine proteinases similarly
show stage regulation in that most of the multiple enzymes are at the
highest activity in amastigotes (15, 16). There are, however, two CPB
activities that appear to be predominantly expressed in metacyclic
promastigotes (18). The amastigote's CPB activity has been categorized
into four groups (A, B, C, and E) on the basis of their physical
characteristics and substrate preferences (15, 16). Group A cysteine
proteinases are glycosylated, as assessed by their ability to bind
concanavalin A, whereas groups B and C do not bind this lectin. Groups
B and C enzymes differ in their elution from anion exchange resin
(reflecting charge differences) and have different substrate
preferences; group C enzymes are less efficient at cleaving substrates
with a bulky amino acid in the substrate P1 position (15). Group E
enzymes differ from the other amastigote cysteine proteinases in having
slower mobilities in gelatin-SDS-polyacrylamide gels and apparently
being membrane-associated (16). NH2-terminal sequence
analysis confirmed that cysteine proteinases of groups A, B, and C are
encoded by cpb genes, leading to the hypothesis that
different copies of the repeat encode cysteine proteinase isoenzymes
with distinct characteristics (16).
The cpb genes, together with homologs from other
Leishmania species (19, 20), Trypanosoma brucei
(21, 22) and Trypanosoma cruzi (23, 24), encode enzymes of
the type I class of trypanosomatid cysteine proteinases (12). Type I
cysteine proteinases are cathepsin L-like in the mature domain but are
characterized by the presence of an unusual, long COOH-terminal
extension (CTE). CTEs have also been found on cathepsin L-like cysteine
proteinases of rice and other plants, although they have no sequence
homology with the CTEs of the trypanosomatid enzymes (25, 26); none has
been observed with vertebrates' cysteine proteinases. The two copies of cpb which have been described previously encode a
100-amino acid CTE, some residues of which are apparently processed in
the production of the mature enzyme (17, 27). The function of the CTE
is unknown, although several roles have been postulated, including
trafficking of the enzyme to the lysosome, modulating the substrate
specificity of the enzyme, inhibiting the activity of the enzyme until
correctly processed and targeted, and diverting the host's immune
system (12).
We have described the generation, by targeted gene disruption, of
L. mexicana mutants null for the cpb gene array,
which showed that the CPBs are virulence factors (27). Null mutants are
less able than wild type L. mexicana to survive in
macrophages in vitro and to infect mice. Re-expression of
one internal copy of the tandem array in the null mutant gave an active
cysteine proteinase that restored the in vitro virulence
phenotype (27). This approach of expressing individual copies of the
cpb gene array in the null mutant provides a powerful method
for determining the activity of, and functional differences between,
individual isoenzymes and so allows analysis of the relationship
between structure and function. We have now cloned and sequenced three
more cpb genes from the array, re-expressed them
individually in the null mutant, and investigated their substrate
specificities and their ability to restore the virulence phenotype.
L. mexicana (MNYC/BZ/62/M379)
promastigotes were grown in HOMEM medium with 10% (v/v)
heat-inactivated fetal calf serum, pH 7.5, at 25 °C as described
elsewhere (28). Mid-log phase (multiplicative) promastigotes were
harvested from cultures at a density of 5 × 106 ml 15-23-kb fragments of L. mexicana
DNA partially digested with Sau3AI were purified on a 1-3
M NaCl gradient and ligated into cpb genes were cloned directionally
into the pTEX vector (33). cpb1 was subcloned on a
EcoRI/HindIII fragment from pNE2 into the
EcoRI/HindIII of pTEX to give pTEXNE2,
cpb18 on a EcoRI/HindIII fragment from
pNX2 into the EcoRI/HindIII of pTEX to give
pTEXNX2, and cpb19 on an HindIII/XhoI
fragment from NX3 into the HindIII/XhoI site of
pTEX to give pTEXNX3. The cloning of pTEXG2.8 and pTEXSWB1a has been
described (27). Transfection of L. mexicana has been described previously (27, 34).
Wild type L. mexicana DNA was isolated using a Nucleon kit (Scotlab,
Coatbridge). RNA was isolated from different life cycle stages of
L. mexicana (mid-log phase promastigotes, stationary phase
promastigotes, and amastigotes) using a Qiagen total RNA kit. Southern
and Northern blots were carried out as described previously (28).
223-bp DNA probes corresponding to sequence in the CTE of
cpb2.8 and the equivalent position in cpb1 were generated by PCR using, respectively, primers DB1 and DB2 (on plasmid
template pNE2) and primers DB3 and DB4 (on plasmid template pTEXG2.8;
Ref 27). A 1.4-kb EcoRI/XhoI fragment from
plasmid pSWB1a was used as a cpb cDNA probe (17).
DB1: ACTGATATGTACTGCACTCAG
DB2: TATAGGGAGGTGGTCTTCCTC
DB3: TTCGATAAGAACTGCACTCAG
DB4: TAGTGACAGGTGTTCATGATC
DNA fragments were labeled by random priming (Stratagene) and
hybridized as described (29).
FIGE was carried out at
10 °C using a Hoefer Switchback Pulse Controller. 2.5 µg of
L. mexicana wild type genomic DNA was digested to completion
with XhoI and run at 150 volts (at 5.2 V/cm) on a 0.9% GTG
agarose gel in 0.5 × TBE buffer. The run-in time was 10 min, run
time was 28 h, pulse time was 0.6 to 2.0 s in reverse mode
with a F/R ratio of 3:1. The gel was blotted onto Hybond N (Amersham
Corp.) and probed with a 1.4-kb EcoRI/XhoI fragment from cpb cDNA clone pSWB1a as described
(17).
Proteinase
activity was analyzed using substrate-SDS-PAGE. Gelatin-SDS-PAGE
involved electrophoresis under reducing conditions on 12% (w/v)
acrylamide gels incorporating 0.2% gelatin and staining with Coomassie
Blue R-250 to detect hydrolysis of gelatin as described (15, 27).
Substrate specificity of the proteinases was also assessed following
separation on nonreducing SDS-PAGE (12% w/v acrylamide) gels by
incubation with 10 µM peptidyl amidomethylcoumarin fluorogenic substrates (BzFVR-NHMec and SucLY-NHMec, from Sigma) as
described previously (18). Western blotting with anti-CPB antiserum
(16) was performed as described (28).
Axenic amastigotes
were centrifuged at 1,000 × g for 5 min at 20 °C
and the pellet fixed in freshly prepared 2% (w/v)
p-formaldehyde, 0.1% glutaraldehyde in phosphate-buffered
saline, pH 7.4, at 4 °C for 30 min. Pellets were washed twice for 5 min in cold phosphate-buffered saline and cold processed (modified from
Ref. 35) through an increasing series of alcohol concentrations: 50%
ethanol, 4 °C for 10 min; then 70%, 95%, and 100% ethanol steps
at Proteinases encoded by
the cpb gene array are polymorphic (16), but it was not
known whether this is because of differences in gene sequence between
copies of the array or because of post-translational modifications, or
a combination of both. We have characterized in detail the
cpb locus (Fig. 1A) using genomic
mapping, FIGE (Fig. 1B), analysis of
A library was constructed in
Details of the protein characteristics predicted from the sequences
determined for the five cpb genes are shown in Table
I. cpb1 has 98% nucleotide identity with
cpb2.8 in the sequence that encodes the pre-pro-region
(nucleotides 170-544; see cpb2.8, accession no. Z49962[GenBank]) and
99% identity for the mature domain (nucleotides 545-1197), but only
70% identity for the CTE (nucleotides 1198-1497). This divergence in
the sequence encoding the CTE of cpb1 is also found with the
second copy of the array, cpb2 (data not shown). This makes
the first two copies of the array significantly different in this
domain from the internal genes, exemplified by cpb2.8 (see
Fig. 3; cpb2.8 is used as the benchmark for
comparisons as it encodes an active cysteine proteinase that is able to
restore virulence toward macrophages in vitro, Ref. 27).
cpb1 predicts an open reading frame of 38.7 kDa. This is
less than the 47.7 kDa of CPB2.8 and is due to the truncation of the
CTE to 15 amino acids. This truncation is due to a single base pair
insertion (relative to cpb2.8) in the cpb1
sequence which causes a frameshift and the introduction of a stop
codon. This single base insertion is also present in cpb2
producing a correspondingly truncated protein (data not shown). In
contrast, cpb2.8 and the cDNA encode proteins with 100 amino acid CTEs (Fig. 3).
Table I.
Characteristics of predicted proteins encoded by cpb genes
The nucleotide sequence of cpb19, its predicted open reading
frame, and the unique 3 cpb18 encodes a 47.9-kDa protein that also has a high degree
of sequence homology with CPB2.8. CPB18 has five amino acid differences in the mature domain compared with CPB2.8 (Fig. 3). Three of these changes (N60D, D61N and D64S)2 are also
found in the cDNA and CPB19, one (H84Y) is present in CPB1 and
CPB19, whereas D18N is conserved only in CPB19. CPB18 encodes a
full-length CTE of 100 amino acids, which has eight amino acid
differences from CPB2.8. Of these changes, six are the same as those
that differ between the cDNA and CPB2.8. 10 cysteine residues are
present in all three gene products containing the full-length CTEs
(Fig. 3). Cysteine residues are thought to be involved in the tertiary
structure of the CTE of T. cruzi (40, 41). One of the
differences in the CTEs of CPB18 and the cDNA (T248R) abolishes a
potential N-linked glycosylation site (NCT) which is found
in the COOH-terminal extension of CPB2.8. This may have relevance to
the glycosylation status of different CPB isoenzymes as the single
N-linked glycosylation site in the mature domain (at
position Asn103) is conserved in all cpb genes
sequenced.
We reported
previously on the marked stage-regulated expression of the
cpb genes with high levels of mRNA being detected in amastigotes, lower levels in stationary phase (metacyclic)
promastigotes, and very low levels in multiplicative promastigotes
(17). To determine at which stages during the life cycle
cpb1 and cpb2 are expressed, Northern blots were
performed with a PCR-derived probe specific to these genes (Fig.
4). The probe was derived from 223 bp of sequence just
downstream of the stop codon of cpb1. On a Southern blot of
L. mexicana DNA digested with XhoI, the probe
detected 2.8- and 4.0-kb fragments (Fig. 4A, lane
1) corresponding to fragments containing the cpb2 and
cpb1 genes, respectively (see Fig. 1, A and
B). No hybridization was detected with the remaining copies
of the array located on the 47-kb fragment, confirming that the probe
is specific for cpb1 and cpb2. In contrast, a
PCR-derived probe generated to the corresponding region of
cpb2.8 detected the 47-kb fragment but not the
2.8- or 4.0-kb fragments (Fig. 4A, lane 2). This
probe would hybridize to cpb2.8,
cpb18, the genomic copy encoding the cDNA, and possibly
all the other genes located between cpb3 and
cpb18 inclusive.
Total RNA was prepared from the three life cycle stages of L. mexicana: mid-log phase (multiplicative) promastigotes, stationary phase (metacyclic) promastigotes, and amastigotes isolated from a mouse
lesion. Relative hybridization intensity between life cycle stages on
the Northern blots were quantified on a PhosphorImager and normalized
to an We had
previously generated, via targeted gene disruption, an L. mexicana cell line null for cpb (designated
One of the criteria used previously to assess the phenotype of the
cpb mutants was their ability to survive in macrophages in vitro (27). Peritoneal exudate cells were removed from
BALB/c mice, exposed to stationary phase promastigotes, and assessed for parasite survival after 7 days in culture (Table
II). As reported previously (27), the null mutant
Table II.
Infectivity of transfected lines to peritoneal exudate cells
Wellcome Unit of Molecular Parasitology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Parasites
1 and stationary phase (metacyclic)
promastigotes at a density of approximately 2 × 107
ml
1, as described previously (29). Amastigotes were
isolated from infected BALB/c mice (30). Amastigote-like forms (axenic
amastigotes) were grown in vitro in Schneider's
Drosophila medium (Life Technologies, Inc.) with 20% (v/v)
fetal calf serum at pH 5.5 and 32 °C (31). Antibiotics appropriate
for the selection of transfectants were added as follows: hygromycin B
(Sigma) at 50 µg ml
1; phleomycin (Cayla, France) at 10 µg ml
1; neomycin (G418, Geneticin, Life Technologies
Inc.) at 25 µg ml
1. The infectivity of parasites to
peritoneal exudate cells was determined as described previously
(27).
Dash II (Stratagene).
A library of 3 × 105 independent clones was
generated. The library was screened with 32P-labeled
cpb cDNA clone pSWB1a (17) and positive clones isolated by plaque purification according to standard methods (32).
DNA was
isolated using Wizard bacteriophage minipreparations (Promega).
clones were mapped using restriction enzyme digestion and hybridization with probes able to distinguish the 5
- and 3
-flanking sequences. cpb genes were subcloned into pBluescript SKII plasmid (pBS)
for sequence analysis. A 2.8-kb SalI fragment from
13,
containing cpb1 (the first gene of the array), was cloned
into the SalI site of pBS(SK
) to give plasmid clone pNE2.
A 2.8-kb SalI fragment from
20, containing
cpb18 (the penultimate gene in the array), was cloned into
the SalI site of pBS(SK
) to give plasmid pNX2. A
3.0-kb SalI fragment from
20, containing cpb19
(the last gene of the array), was cloned into the SalI site
of pBS(SK
) to give plasmid pNX3. The nucleotide sequence covering the
cpb open reading frame for each gene was determined in both
orientations using a series of overlapping oligonucleotide primers
about 300 bp apart. Sequencing was carried out on an ABI 373 DNA
sequencer (Perkin-Elmer), and analysis was performed using the
Wisconsin GCG package.
20 °C for 20 min each, with agitation at 10 min. The samples
were subsequently treated with a 50:50 mixture of LR White Medium resin
and absolute ethanol at
20 °C for 30 min, then with a 75:25
resin/ethanol mixture at
20 °C for 30 min, then with 100% resin
for 8 h (during which time the specimens were brought up to
20 °C) and subsequently allowed to infiltrate fully overnight with
mild agitation. Parasite pellets were transferred to gelatin capsules
in fresh LR White Medium resin and polymerized at 20 °C under 365-nm
indirect UV light for 2 days. Ultrathin sections were collected onto
300 mesh nickel grids. On-grid immunostaining was performed essentially as described previously (36). Blocking and conditioning steps using 0.2 M glycine and 1% bovine serum albumin in
phosphate-buffered saline for 30 min each were followed by incubation
for 30 min in anti-CPB antibody diluted 200-fold in phosphate-buffered
saline, 1% acetylated bovine serum albumin (Aurion) and then a 30-min incubation in goat anti-rabbit antibody, 10 nm of gold conjugate (Aurion) diluted 20-fold in the same buffer. Sections were briefly counterstained in 0.5% uranyl acetate and viewed with a Zeiss 902 EFTEM at 0 eV to improve contrast.
Polymorphism within the cpb Gene Array
clones containing
sections of the array, and sequence analysis. Southern blot analysis of
genomic XhoI fragments from wild type L. mexicana
DNA separated by FIGE and probed with a cpb-specific probe
revealed three hybridizing fragments of 47, 4.0, and 2.8 kb (Fig.
1B). XhoI sites are located in the unique 5
- and
3
-flanking sequences of the array and also just downstream of the
cpb open reading frame for the first two copies of the
array, but they are not present in any other of the cpb repeat units (Fig. 1A and Ref. 27). The 4.0-kb
XhoI fragment contains cpb1, the first copy of
the array, the 2.8-kb fragment the second copy, cpb2, and
the 47-kb fragment the remaining copies. As all the copies occur on
2.8-kb SalI fragments (17), and taking into account the
position of the XhoI site downstream of the last copy of the
array (Fig. 1A), it was calculated that there are 17 gene
copies present on the 47-kb XhoI fragment and hence a total
of 19 copies in the whole array. The single band at 47 kb indicates
that the two allelic arrays contain the same number of copies of
cpb. This is in contrast to the type I cysteine proteinase genes of the X10.6 strain of T. cruzi which have 14 and 23 copies in allelic clusters (37).
Fig. 1.
Panel A, map of the cpb
locus. 19 copies of the cpb gene (black boxes)
are arranged in a tandem array of 2.8-kb repeat units (numbered
cpb1-cpb19). The unique 5- and 3
-flanking
sequences are shown (gray boxes).
clones that span part
of the array are drawn above the map. The position of the
cpb2.8 gene has not been determined but lies somewhere
between cpb2 and cpb18. Restriction sites are
indicated as follows: S, SalI; X,
XhoI; Sc, SacI; H, HindIII. Expanded maps of cpb1,
cpb2.8, and cpb19 are shown; and the relative
sizes of the pro-region (black), central domain
(white), and CTE (gray) are shown, as are
restriction sites. Panel B, FIGE analysis of the array.
L. mexicana genomic DNA was restricted with XhoI
and the DNA fragments separated by FIGE. The gel was transferred to
nylon membrane and probed with a cpb cDNA probe (17).
The markers shown to the left of the Southern blot are a
Bio-Rad 8-48-kb ladder. Estimated sizes of the three XhoI
fragments are shown to the right.
[View Larger Version of this Image (13K GIF file)]
by partial digestion of L. mexicana genomic DNA with Sau3AI. The library was
screened with the cpb cDNA, and several
clones were
isolated. Southern blot analysis of
clones digested with diagnostic
restriction enzymes and hybridized with 5
and 3
cpb
cDNA probes identified fragments that contained either unique 5
-
or 3
-flanking sequence with varying amounts of the cpb
repeat (Fig. 1A) and
clones that contained
cpb repeat units only.
14 was found to contain the complete cpb1 gene and part of cpb2, whereas
13 contained cpb1, cpb2, and cpb3.
20 contained the last two copies in the array (cpb18 and
cpb19) and the 3
-flanking sequence. Several of the cpb genes were subcloned into pBluescript for sequence
analysis. cpb1 was cloned on a 2.8-kb SalI
fragment from
14 (to give pNE2), cpb18 on a 2.8-kb
SalI fragment from
20 (to give pNX2), and
cpb19 on a 3.0-kb SalI fragment from
20 (to
give pNX3). The sequences of the cpb cDNA and internal
gene cpb2.8 have been reported previously (17, 27). The
nucleic acid sequence of the 2.1-kb HindIII/XhoI fragment containing cpb1 is shown in Fig.
2A. The first 453 bp do not occur within the
intergenic sequence of the cpb array; and a probe specific
to this region, generated by PCR with primers to the sequences
indicated (underlined in Fig. 2A), recognized a
single band in Southern blots (Ref. 27 and data not shown), indicating
that this sequence is unique. Sequence downstream of the divergence
point (uppercase in Fig. 2A) is present within the 2.8-kb cpb repeat unit. The predicted ATG start codon is
located at position 804. Protein coding genes in Leishmania
and other trypanosomatids are transcribed polycistronically and then
processed by trans-splicing and polyadenylation (38, 39).
Sequence analysis of the cDNA clone pSWB1a (17) revealed the
position of spliced leader addition for the copy of the cpb
repeat from which the cDNA was derived. An AG dinucleotide splice
site is located at the same position upstream of the cpb1
start codon (Fig. 2A, position 661, shown in
bold). Other possible AG dinucleotides are located between
the one used for the cpb cDNA and the ATG start codon, although the one indicated is a good candidate for the cpb1
splice site as upstream of it are a number of polypyrimidine stretches (Fig. 2A, double underlined) which have been
shown to be important for accurate splice site selection (39). No
significant open reading frames were located in the sequence
immediately 5
to the first cpb gene of the array.
Fig. 2.
Nucleic acid sequence and predicted protein
sequence for cpb1 (A) and cpb19
(B). Underlined nucleotides show the positions of oligonucleotides used to make DNA probes specific for the 5 and 3
flanks (27). The lowercase sequence is unique to the 5
(panel A) or 3
(panel B) flank. The sequence in
uppercase is found in the cpb gene array. The AG
dinucleotide splice site used for spliced leader addition in the
cDNA (17) is shown in bold, a polypyrimidine stretch
upstream of the splice site which may have a role in splice site
selection is double underlined, and the SalI site
used for cloning is dot underlined.
[View Larger Version of this Image (77K GIF file)]
Proteinase
Residues ORF
Mass,
ORF
I.P., ORF
Residues, M
Mass, M
I.P., M
Residues, M + C
Mass, M + C
I.P., M + C
kDa
kDa
kDa
CPB1
359
38.7
5.43
218
23.4
4.39
234
24.9
4.34
CPB2.8
443
47.7
6.19
218
23.3
4.34
318
34.0
4.83
cDNA
443
47.8
6.59
218
23.3
4.40
318
34.1
5.38
CPB18
443
47.9
6.73
218
23.4
4.38
318
34.1
5.40
CPB19
366
39.5
5.79
218
23.4
4.38
241
25.7
4.37
Fig. 3.
Amino acid sequence alignment for predicted
cpb gene products. The sequence of CPB2.8 (27) is
shown in full and compared with predicted open reading frame from the
cpb cDNA (17), CPB1, CPB18, and CPB19. Dashes
represent sequence identity. In the numbering system used, the
NH2 terminus of the mature CPB2.8 enzyme is designated as
residue 1, and the pro-region is assigned negative numbers decreasing
toward the NH2 terminus. The arrow shows the
last residue of the mature domain. The two potential
N-linked glycosylation sites are shown in bold.
Stars show the conserved cysteine residues in the CTE.
[View Larger Version of this Image (37K GIF file)]
-flanking sequence downstream of the gene are
shown in Fig. 2B. The underlined sequences show
the DNA to which oligonucleotide primers were designed for the PCR of a
590-bp fragment for use as a 3
-flanking probe (27). cpb19 encodes a protein of 39.5 kDa with 98% nucleotide sequence identity with cpb2.8 over the pre-pro-region and the mature domain.
However, the sequences diverge totally near the start of the sequence
encoding the CTE. The point of divergence is coincident with the
beginning of the 3
- flanking sequence (lowercase, Fig.
2B), which introduces a stop codon that truncates the CTE of
cpb19 to 22 amino acids (Fig. 3). No significant open
reading frames were located in the sequence immediately 3
of the
cpb array.
Fig. 4.
Stage-regulated expression of cpb
genes. Panel A, Southern blot of XhoI-digested
L. mexicana DNA (2.5 µg) probed with a PCR fragment
specific for cpb1/cpb2 (lane 1) or
cpb3-18 inclusive (lane 2). Sizes of the
hybridizing fragments are indicated. Panels B-E, Northern
blots. 5 µg of L. mexicana total RNA was separated by
formaldehyde-agarose gel electrophoresis, blotted onto nylon membrane,
and hybridized with the cpb cDNA (panel B) or
PCR fragments specific for cpb1/cpb2 (panel
C), cpb3-18 inclusive (panel D), or
-tubulin (panel E). L. mexicana multiplicative
promastigotes (P), stationary phase (metacyclic)
promastigotes (M), and amastigotes isolated from mice
(A).
[View Larger Version of this Image (30K GIF file)]
-tubulin control (Fig. 4E). The cpb
cDNA probe, which should hybridize to all the cpb genes,
detected very low levels of cpb mRNA in multiplicative
promastigotes, higher levels in stationary phase (metacyclic)
promastigotes, and the highest level in amastigotes (in the ratio of
1:10:25, Fig. 4B). In contrast, the
cpb1/cpb2-specific probe detected a significantly different stage expression with low multiplicative promastigote expression, predominant metacyclic promastigote expression, and very
low amastigote expression (the ratio was 1:6.5:0.2, Fig. 4C). The probe designed to the genes cpb3-cpb18
detected 2.2-kb mRNA predominantly present in the amastigote (ratio
of 1:6:33, multiplicative promastigote:metacyclic
promastigote:amastigote, Fig. 4D). The expression of
cpb19 was assessed using a PCR-derived probe generated to
sequence immediately downstream of the stop codon. This contains a
3
-flanking sequence that would be predicted to be part of a
3
-untranslated region for the cpb19 gene. No hybridization,
however, was detected with this probe in any of the three life cycle
stages (not shown). Furthermore, when cpb19 was expressed
from the pTEX vector in the cpb null mutant (see below) it
was not possible to detect either cysteine proteinase activity by
gelatin gel electrophoresis or protein by Western blotting. Our
conclusion is that cpb19 is a pseudogene created through
recombination events that produced the tandem array and that the
3
-flanking sequence immediately downstream of the gene does not
contain the sequence elements required for the correct processing of a
functional cpb19 mRNA. A similar truncation in the CTE
and lack of expression were observed in the last copy of the type I
cysteine proteinase gene array of T. cruzi (37).
cpb) (27) and also cell lines that re-expressed the
cpb cDNA or cpb2.8 in the
cpb null mutant background (27). To compare the cysteine proteinase activities encoded by different copies of cpb and
to assess their biological roles in the parasite, the genes were cloned
from Bluescript into the pTEX vector and transfected into
cpb to generate cell lines expressing either CPB1
(
cpb[pTEXNE2]), CPB18
(
cpb[pTEXNX2]) or CPB19
(
cpb[pTEXNX3]). Cell lines were grown at a high
concentration (500 µg ml
1) of G418 to increase
expression levels. Extracts were prepared from stationary phase
promastigotes of each cell line and analyzed for the presence of CPB
protein by Western blotting and for cysteine proteinase activity by
substrate-SDS-PAGE using gelatin and fluorogenic substrates (Figs.
5 and 6). Two main bands of cysteine
proteinase activity are characteristically detected on gelatin gels
with wild type L. mexicana stationary phase promastigote
extracts (Fig. 5A, lane 1). These are
stage-regulated and occur predominantly in this parasite form (18, 42).
When CPB1 was re-expressed in the null mutant (lane 2) it
had a single cysteine proteinase activity that co-migrated with the
slower band detected in wild type extracts, whereas the CPB2.8
re-expressor (lane 3) had activities with mobilities between
those in the wild type extracts, similar to the situation with wild
type amastigotes (15, 18). The activity of the CPB18 re-expressor
(lane 4) had a mobility very similar to that of the slower
band detected in wild type extracts, but analysis of several gels using
variable amounts of material and incubation times suggested that in
fact it has a slightly faster mobility. The CPB2.8 re-expressor
(lane 3) and to a lesser extent the CPB18 re-expressor
(lane 4) also had significant activities with much slower
mobilities (apparently 30-40 kDa; but it should be noted that proteins
do not migrate strictly according to molecular mass in
gelatin-SDS-PAGE). Equivalent activities were not readily detected with
wild type or in cell extracts prepared from the CPB1 re-expressor,
although activity was detected if sufficient material was used. Western
blotting of wild type stationary phase promastigote extracts with
anti-CPB antiserum detected two major proteins (25 and 29 kDa, Fig.
5B, lane 1). It should be noted that the sample
preparation for the activity gels and Western blots differ, the former
samples are not denatured by boiling, which accounts for the different
mobilities observed for the same proteins with the two procedures. The
CPB1 was also 25 kDa (lane 2), whereas the main
cpb products in the CPB2.8 re-expressor (lane 3)
and CPB18 re-expressor (lane 4) migrated with a molecular
mass of 28 kDa. The lines expressing CPB2.8 or CPB18 (lanes
3 and 4) also contained larger molecular mass proteins
(about 38 kDa) detected by the antiserum. The cell lines were also
analyzed for their ability to hydrolyze two fluorogenic peptide
substrates (Fig. 6). Both cysteine proteinase activities detected in
wild type extracts (lane 1) hydrolyzed each of the
substrates, although the slower mobility activity showed greater
activity toward SucLY-NHMec whereas the reverse was true for the higher
mobility activity. The cysteine proteinase in each of the three cell
lines expressing CPB1 (lane 2), CPB2.8 (lane 3),
or CPB18 (lane 4) were almost equally proficient at
hydrolyzing BzFVR-NHMec (Fig. 6A), whereas CPB18 had a
significantly higher activity toward SucLY-NHMec than either CPB1 or
CPB2.8 (Fig. 6B). The slower mobility activities toward
gelatin (Fig. 5) detected for CPB2.8 (lane 3) or CPB18 (lane 4) did not hydrolyze either of the fluorogenic
substrates to any appreciable degree (Fig. 6).
Fig. 5.
Analysis of CPB isoenzymes expressed in the
cpb null mutant. Extracts from 1 × 107 (panel A) or 5 × 106
(panel B) stationary phase promastigotes were used for
gelatin-SDS-PAGE (panel A) and Western blotting using
anti-CPB antibody (panel B). Wild type L. mexicana (lane 1); cpb re-expressing CPB1
(lane 2), CPB2.8 (lane 3), or CPB18 (lane
4). Molecular mass markers are shown in kDa.
[View Larger Version of this Image (36K GIF file)]
Fig. 6.
Substrate specificity differences between CPB
isoenzymes. Activity of CPB isoenzymes expressed in the
cpb null mutant toward peptidyl amidomethylcoumarin
fluorogenic substrates BzFVR-NHMec (panel A) and SucLY-NHMec
(panel B). Wild type L. mexicana (lane 1); cpb re-expressing CPB1 (lane 2),
CPB2.8 (lane 3), or CPB18 (lane 4). Molecular
mass markers are shown in kDa.
[View Larger Version of this Image (68K GIF file)]
cpb survived in just a few macrophages, whereas
expressing CPB2.8 in the null mutant restored levels of infectivity
almost back to wild type levels. In contrast, expression of CPB1 or
CPB18 in the null mutant increased infectivity only a minor extent.
Cell
line
CPB expressed
% infected PECs
Amastigotes/infected cell
Wild
type
CPB1-CPB18
38
± 9.0
5.2 ± 0.9
cpb
None
1.5
± 0.2
1.7 ± 0.2
cpb[pTEX2.8]
CPB2.8
29 ± 9.2
3.7
± 0.4
cpb[pTEXNE2]
CPB1
3.5
± 2.8
1.8 ± 0.9
cpb[pTEXNX2]
CPB18
4.6 ± 1.4
2.6
± 0.4
Immunogold
labeling of wild type L. mexicana axenic amastigotes with
anti-CPB antiserum (Fig. 7A) resulted in
strong labeling of the large lysosomes (termed megasomes) as reported
previously (43, 44). No labeling was detected in the cpb
null mutant axenic amastigotes (Fig. 7B) or in a negative
control with no antibody (not shown but similar to Fig. 7B),
confirming the specificity of the antiserum used to detect the CPB
enzymes. The megasomes were also labeled in axenic amastigotes of
null mutants expressing CPB2.8 and CPB1, indicating that both these
isoenzymes were targeted to megasomes (Fig. 7, C and
D). With each of the three cell lines expressing CPB, no
significant labeling was detected outside megasomes, indicating that
the trafficking of the proteinase was efficient in these parasites, and
there was no accumulation of precursors in other compartments of the
cell such as the Golgi. Some of the gold-labeled megasomes were
adjacent to the flagellar pocket (see Fig. 7D), which could
reflect some interaction between the two (either discharging of the
megasomes into the flagellar pocket or uptake into megasomes). The
lack of gold labeling in the pocket does not support the former
possibility, although it is not yet possible to rule this out.
This study has shown that the cathepsin L-like cpb cysteine proteinase genes of L. mexicana are found in allelic tandem arrays of 19 copies. We postulated previously (16) that the diversity in biochemical characteristics of the parasite's numerous stage-regulated cysteine proteinases detected using gelatin gels (15, 16) was the result of small differences between the genes in the cpb array, although post-translational modifications could also be involved. Support for this working hypothesis has now been provided through the sequence analysis of five independent copies of cpb. The data generated show that although the genes have a very high level of overall nucleotide sequence identity (97-99%), there are a number of differences that alter the amino acid sequence of the encoded proteins or introduce premature stop codons. The significance of these amino acid substitutions became apparent when the individual isoenzymes were expressed in a cpb null mutant. Three of the re-expressed gene products (CPB1, CPB2.8, and CPB18) showed activity toward gelatin and fluorogenic substrates, but the enzymes differed both in their relative activities toward the different substrates and also their physical characteristics as displayed by the mobilities on gels. These differences correlated with evidence from biochemical studies which showed that isoenzymes have distinct properties (15, 16). CPB1 has a lower molecular mass than either CPB2.8 or CPB18, as predicted from the gene sequences. The predicted size of the cpb1 gene product for the mature proteinase, including the 15-amino acid CTE, is 24.9 kDa (Table I), which correlates well with the Western blot data (Fig. 5B). The predicted size of the cpb2.8 and cpb18 gene products for the mature proteinase, including the 100-amino acid CTE, is 34 kDa, considerably larger than the 28-kDa protein detected by Western blotting (Fig. 5B). It is likely, therefore, that more than half of the CTE is processed to give the mature CPB2.8 and CPB18 cysteine proteinases. The finding that the antibody detected essentially a single mature cysteine proteinase (see below for discussion on the larger molecular mass forms) suggests that the CTE is processed at a single site to give a homogeneous isoenzyme encoded by each individual gene. Therefore, the cysteine proteinase isoenzymes that occur in wild type L. mexicana are unlikely to be caused by multiple processing of CTEs from one gene product but probably reflect the polymorphisms in the DNA sequence which introduce amino acid changes.
CPB1 co-migrates on gelatin gels with one of the two major activities characteristically present in stationary phase (metacyclic) promastigotes of wild type cells (Fig. 5A). The protein also co-migrates on Western blots with a protein in the stationary phase wild type promastigotes (Fig. 5B). These results correlate with the Northern blot data demonstrating that levels of mRNA transcribed from cpb1 and/or cpb2 are elevated in stationary phase promastigotes relative to multiplicative promastigotes or amastigotes (Fig. 4C). Taken together, these results suggest that some of the proteinase activity in wild type metacyclic promastigotes is encoded by cpb1. The differences observed between the relative activities of wild type parasites and CPB1 expressed in the null mutant toward the two fluorogenic substrates (Fig. 6, lanes 1 and 2) suggest, however, that the slower mobility activity in wild type cells may comprise more than one gene product. It has been shown previously that some activity bands detected using gelatin gels do indeed represent several isoenzymes (15). CPB2 is likely to account for some of this activity, although the detection of cpb mRNA in metacyclic promastigotes using the cpb3-cpb18-specific probe (Fig. 4D) indicates that there is also a low level of expression of one or more of these genes in this life cycle stage.
Overall, the data show that expression of the first two genes of the array, which are characterized by the lack of sequence encoding a full-length CTE, is stage-regulated and occurs primarily in the infective metacyclic promastigote. The stage-specific expression of cpb1 and cpb2 suggests that they play some important role at this stage of the life cycle. This could involve the interaction of the metacyclic promastigote with its sandfly vector or the process of differentiation itself, but it is attractive to consider that it may be important for the early events associated with invasion of a macrophage following inoculation into a mammal. The finding that the null mutant cell line expressing CPB1 was less able than the wild type parasites to survive in macrophages shows that CPB1 is not able to restore the infectivity lost due to the deletion of the cpb array under these circumstances, and in this way it differs from CPB2.8. This inability to restore infectivity is not due to the lack of a CTE as CPB18, although apparently expressed in amastigotes naturally, also is unable to restore infectivity to the null mutant. This indicates that the individual genes of the array indeed perform different functions, a suggestion supported by the observed differences in substrate specificities. The precise timing of expression of the different isoenzymes may be an important factor for parasite invasion and survival in the macrophage.
The expression of the msp genes of L. chagasi has
some similarities with the expression of cpb genes of
L. mexicana. The msp genes encode a
metalloproteinase known as gp63. It is the major surface proteinase of
Leishmania and in L. chagasi is encoded by 18 msp genes arranged in a tandem array. These genes have been grouped into three classes based on their differential gene expression (45, 46). RNAs from the multiple mspL genes are present
predominantly in multiplicative promastigotes, whereas RNAs from the
five mspS genes are present predominantly in stationary
phase promastigotes (46). The regulation of the msp genes
occurs at a post-transcriptional level, possibly via sequences located
in the 3-untranslated region of the mRNAs. This may also be the
case for the cpb genes since there are differences in the
noncoding DNA sequence between the cpb1 and
cpb2.8 genes3 which
may be important for the developmentally regulated gene expression
observed. Heterogeneity observed for the msp gene products on gels has been attributed to amino acid variation between different copies of the array (47), but these have not been correlated with
differences in enzymatic activity of the metalloproteinase or function
in different life cycle stages. The finding that cpb isogenes encode enzymes differing both in amino acid sequences and
substrate specificity suggests that this may also be the case for the
msp genes.
The null mutants re-expressing CPB2.8 or CPB18 exhibit a number of lower mobility bands in gelatin gels (30-40 kDa) and also larger molecular mass species in Western blots (about 38 kDa) (Fig. 5). By contrast, similar larger molecular mass proteins were not readily seen with the null mutant line re-expressing CPB1, which exhibited just the fully processed protein, or with wild type parasites unless a much larger amount of lysate was applied. These slower mobility activities in the mutants re-expressing CPB2.8 or CPB18 may be precursors of the mature cysteine proteinase which are activated in situ following gel electrophoresis. Noncovalent complexes between lysosomal cathepsin B and its pro-peptide which are formed during autocatalytic maturation of the precursor have been described (48, 49). These complexes are believed to keep the proteinase in an inactive state until it is secreted, when it is activated by local acidification. It is possible that either the CPB pro-region or CTE (or both) could be acting as an inhibitory peptide in a similar fashion, and the processing of the gel subsequent to electrophoresis provides conditions in which the inhibitory peptide is removed. The finding that CPB1 lacks a full CTE and does not appear to produce these putative precursor forms that are activated in situ provides some evidence that it may be this domain that is important with regard to any such processing events. Indeed, processing of the CTE may be a prerequisite for correct and efficient processing of the pro-domain.
Previous speculations on the function of the CTE have included a role in the targeting of the protein to lysosomes, a theory strengthened by the finding that a trypanosome cysteine proteinase apparently lacks mannose 6-phosphate, which therefore cannot play a role in the enzyme's trafficking (50). The finding using immunoelectron microscopy that the mature CPB1 enzyme, which has a truncated CTE, is located in lysosomes that are typical of the amastigotes of L. mexicana shows that a full-length CTE is not essential for successful intracellular trafficking. It is clearly also not essential for activity of these cysteine proteinases, as CPB1 is highly active toward gelatin (Fig. 5A) and other substrates (Fig. 6). This is consistent with previous reports that demonstrated that recombinant cysteine proteinases from T. cruzi and T. brucei lacking the CTE can be expressed as active enzymes (51, 52). All of the CTEs, irrespective of size, have a serine/threonine-rich region bounded by proline residues (Fig. 3). It is not known whether this region has any role in targeting or processing of the enzyme, but this deserves study.
When the cpb cDNA was expressed in the null mutant cell
line cpb it produced a protein of the predicted molecular
size, indicating correct processing, but it was inactive toward gelatin
under the standard conditions tested (27). This contrasts with the
results reported here for the cpb1,
cpb2.8, and cpb18 genes, which are expressed in the null mutant to produce active cysteine proteinases. The cpb cDNA encodes a protein that differs within the
mature domain from CPB2.8 in only three amino acids (N60D; D61N, and D64S; see Fig. 3). A further six amino acid differences are present in
the CTE. As more than half of the CTE would appear to be cleaved off to
produce the mature protein, it is difficult to predict the effect that
these amino acid variations in the CTE could have on the enzyme
activity. The differences in the mature regions between CPB1, CPB2.8,
and CPB18 must also explain the differences in relative activity toward
the fluorogenic substrates observed in this study. More detailed
studies on the substrate specificities of the individual isoenzymes are
required, but it is already clear that the ability to express
individual cpb genes in the null mutant (and also genes
modified via site-directed mutagenesis) and subsequently analyze the
expressed cysteine proteinase biochemically and functionally will
provide a powerful approach for investigating the function of these
proteinases.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Z49963[GenBank] (cpb1), Z49965[GenBank] (cpb19), and Y09958[GenBank] (cpb18).
We thank Phil McCready, Margaret Mullin, and David Laughland for technical assistance.