In this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid
Trang 1Differences in substrate specificities between cysteine protease CPB
changes
Maria A Juliano1, Darren R Brooks2, Paul M Selzer3, Hector L Pandolfo1, Wagner A S Judice1,
Luiz Juliano1, Morten Meldal4, Sanya J Sanderson5, Jeremy C Mottram2and Graham H Coombs5
1
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de Sa˜o Paulo, Brazil;2Wellcome Centre for Molecular Parasitology, The Anderson College, University of Glasgow, UK;3Akzo Nobel, Intervet Innovation GmbH, BioChemInformatics, Schwabenheim, Germany;4Center for Solid-Phase Organic Combinatorial Chemistry, Department of Chemistry, Carlsberg Laboratory, Valby, Denmark; 5 Division of Infection and Immunity, Institute of Biomedical and Life Sciences,
Joseph Black Building, University of Glasgow, UK
The CPB genes of the protozoan parasite Leishmania
mex-icanaencode stage-regulated cathepsin L-like cysteine
pro-teases that are important virulence factors and are in a
tandem array of 19 genes In this study, we have compared
the substrate preferences of two CPB isoforms, CPB2.8 and
CPB3, and a H84Y mutant of the latter enzyme, to analyse
the roles played by the few amino acid differences between
the isoenzymes in determining substrate specificity CPB3
differs from CPB2.8 at just three residues (N60D, D61N and
D64S) in the mature domain The H84Y mutation mimics
an additional change present in another isoenzyme, CPB18
The active recombinant CPB isoenzymes and mutant were
produced using Escherichia coli and the S1-S3 and S1¢-S3¢
subsite specificities determined using a series of fluorogenic
peptide derivatives in which substitutions were made on
positions P3to P3¢ by natural amino acids Carboxydipep-tidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK(Dnp)-OH and some of its ana-logues The kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S3to S3¢ subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84 Particularly noteworthy was the large preference for Pro in the P2¢ position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the
L mexicanaCPBs
Keywords: carboxydipeptidase; cysteine protease; fluoro-genic peptides; Leishmania; parasite
Cysteine proteases (CPs) are present in almost all organisms
and are associated with numerous physiological and
pathological conditions [1,2] Cysteine proteases of the
papain superfamily, designated Clan CA, family C1 [3], are
synthesized as zymogens that are activated by cleavage of
the pro-domain to generate mature enzymes located
predominantly within lysosomes The mature protease folds
into an ellipsoid conformation with the active site cleft
located between two structural domains One domain
consists predominantly of b-barrel folds, while a prominent central helix of the second domain is adjacent to, and helps define, the opposite side of the active site cleft [4]
Leishmania mexicanapossesses three CPs of the papain superfamily, designated CPA and CPB, both of which are cathepsin L-like, and CPC, which is cathepsin B-like [5] The CPB proteases exist as multiple isoenzymes, which are encoded by a tandem array of 19 similar CPB genes located
in a single locus [6,7] L mexicana CPB isoenzymes are expressed as inactive zymogens comprising an 18 amino acid pre-region that is thought to be rapidly removed by a signal peptidase upon transfer into the endoplasmic reticu-lum, a 106 amino acid pro-region, a 218 amino acid mature domain that includes the active site, and a C-terminal domain of either 16 or 100 amino acids [7] The first two genes of the array, CPB1 and CPB2, are atypical because they encode enzymes with a C-terminal domain of just 16 amino acids [7] Furthermore, CPB1 and CPB2 are expressed almost exclusively in the infective metacyclic stage, whereas the remaining isogenes, namely CPB3– CPB17(which include CPB2.8 and CPB3) and CPB18 are expressed predominantly in amastigotes, whereas CPB19 is
a pseudogene [8] The role of the C-terminal domain remains uncertain Roles in intracellular targeting to the megasomes, immune evasion and modulation of the
Correspondence to G H Coombs, Division of Infection & Immunity,
Institute of Biomedical and Life Sciences, Joseph Black Building,
University of Glasgow, Glasgow, G12 8QQ, UK.
Fax: +44 141 330 3516, Tel.: +44 141 330 4777,
E-mail: G.Coombs@bio.gla.ac.uk
Abbreviations: Abz, ortho-amino-benzoyl; AMC,
7-amino-4-methyl-coumarin; CTE, C-terminal extension; DMF, dimethylformamide;
EDDnp, N-[2,4-dinitrophenyl]-ethylenediamine; K(Dnp), (2,4
di-nitrophenyl)-e-NH 2 -lysine; MCA, 4-methylcoumarin-7-amide;
rCPB2.8, recombinant Leishmania mexicana cysteine protease CPB2.8
lacking the C-terminal extension, originally designated CPB2.8DCTE;
Suc-LY-MCA, N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin;
t-Boc, tert-butyloxycarbonyl.
(Received 25 May 2004, revised 12 July 2004, accepted 28 July 2004)
Trang 2enzyme’s activity have all been postulated, although
defin-itive data are lacking [5]
Information about the functions and importance of the
Leishmaniaenzymes in host–parasite interactions has been
obtained by the generation of mutants deficient in the
multicopy CPB gene array (Dcpb) L mexicana Dcpb
mutants have reduced virulence with poor lesion growth
in BALB/c mice and induce a protective Th1 response [6,9]
Reinsertion of the amastigote-specific CPB2.8 or
meta-cyclic-specific CPB2 into Dcpb mutants failed to restore
either a Th2 response or sustained virulence, and only the
re-expression of multiple CPB genes from a cosmid
significantly restored virulence [10]
A recombinant form of the enzyme encoded by CPB2.8
but lacking the C-terminal extension, originally designated
CPB2.8DCTE but herein named rCPB2.8 (Table 1 lists
nomenclature of all proteins analysed), was expressed [11],
and its substrate specificity has been studied extensively
[12–15] and several peptide inhibitors have also been
reported for it [16–18] The CPB3 gene, originally
designa-ted cDNA CPB as it was isoladesigna-ted from a cDNA library [19],
is another CPB gene from the central region of the array [7]
The corresponding protein, CPB3, when expressed in Dcpb
mutants was devoid of the gelatinase activity in
nondenat-urating gel electrophoresis that was observed for CPB2.8 [7]
These two CPB isoforms differ from each other in the
mature enzyme domain in only three positions, CPB2.8 has
Asn60, Asp61 and Asp64 whereas CPB3 has Asp60, Asn61
and Ser64 Interestingly, CPB18, which also has Asp60,
Asn61 and Ser64 but also Tyr84 and Asn18 instead of the
His84 and Asp18 in CPB2.8, is active towards gelatin but
differs from CPB2.8 in its activity towards some short
peptidyl-7-amido-4-methylcoumarin substrates [7] Thus the
substrate preferences of some CPB isoenzymes seem to be
determined by just a few amino acids
In order to explore the effects on substrate utilization of
the restricted local amino acid variations of the CPB
isoen-zymes of L mexicana, the recombinant CPB3 (rCPB3), and
a recombinant H84Y mutant of CPB3 that was generated,
were expressed in Escherichia coli and their S1-S3and S1¢-S3¢
subsite (based on the Schechter & Berger nomenclature [20])
specificities investigated in a systematic way using
intramole-cularly quenched fluorescence substrates derived from
Abz-KLRFSKQ-EDDnp (where Abz is ortho-amino-benzoyl
and EDDnp is N-[2,4-dinitrophenyl]-ethylenediamine),
which were previously used to study the specificity of
rCPB2.8 [13] Moreover, the locations of the varying residues
were analyzed via molecular modeling to gain insight into how the changes may impinge upon enzyme activity The recombinant cysteine protease cruzain from Trypan-osoma cruziand rCPB2.8 of L mexicana are both cathepsin L-like and characteristically endopeptidases However, we have shown that these enzymes have carboxydipeptidase activities and have compared them with those of human recombinant cathepsin B and cathepsin L [21] Therefore we also comparatively analyzed the carboxydipeptidase activit-ies of rCPB3 and rH84Y using the internally quenched fluorescent peptide Abz-FRFK(Dnp)-OH and some of its analogues, where K(Dnp) is (2,4 dinitrophenyl)-e-NH2 -lysine, in order to characterize further the importance of the varying amino acids
Materials and methods Parasites
Leishmania mexicana(MNYC/BZ/62/M379) promastigotes were grown in modified Eagle’s medium (designated com-plete HOMEM medium when supplemented with 10% (v/v) heat-inactivated fetal bovine serum, pH 7.5) at 25C as described previously [7] The required antibiotics were added
as follows: hygromycin B (Sigma) at 50 lgÆmL)1, phleomy-cin (Cayla
1 , Toulouse, France) at 10 lgÆmL)1, and neomycin (G418, Geneticin, Life Technologies Inc.) at 25 lgÆmL)1 Molecular modeling
A homology-based protein model of a Leishmania major cath-epsin L-like cysteine protease (GenBank locus U43706, PDB identification code 1bmj) was built usingINSIGHTII[22] soft-ware (Accelrys Inc, San Diego, CA, USA) and the crystal structures of papain [23] and cruzain [24] as reference proteins The L mexicana CPB isoforms were then modeled by super-imposition usingMIDASPLUS(Computer Graphics Laboratory, University of California San Francisco, CA, USA) [25,26]
Mutagenesis, constructs, transfections and production
of recombinant enzymes Mutations were incorporated into pGL27, a pBluescript SK– plasmid containing the SWB1a CPB cDNA gene [19] now designated CPB3, using the QuikChange Site-Directed mutagenesis kit (Stratagene) and the following reverse-phase-purified oligonucleotides (only the sense strand
Table 1 Plasmids, L mexicana cell lines, and proteins used in this study CTE, C-terminal extension; r, recombinant.
Plasmid
Cell lines (plasmids
expressed in Dcpb) Abbreviation
Expressed cysteine protease Abbreviation Comments pGL37 Dcpb [pXCPB3] DcpbCPB3 CPB3 CPB3 native CPB3
pGL43 Dcpb [pXCPB3 D18N ] DcpbCPB3D18N CPB3(D18N) D18N CPB3 mutated D18N pGL44 Dcpb [pXCPB3H84Y] DcpbCPB3H84Y CPB3(H84Y) H84Y CPB3 mutated H84Y pGL45 Dcpb [pXCPB3D60N, N61D, S64D] DcpbCPB3M3 CPB3(D60N,
N61D, S64D)
M3 CPB mutated D60N,
N61D, S64D pGL46 Dcpb [pXCPB2.8] DcpbCPB2.8 CPB2.8 CPB2.8 native CPB2.8
pGL180 not applicable CPB2.8DCTE rCPB2.8 rCPB2.8 lacking CTE [11]
6
pGL400 not applicable CPB3DCTE rCPB3 rCPB3 lacking CTE pGL401 not applicable CPB3(H84Y)DCTE rH84Y rCPB3(H84Y) lacking CTE
Trang 3primers are shown and the mutated sites are given in
bold): OL416 to generate pGL40: 5¢-GACGCCGGTGA
AGAATCAGGGTGCGTG-3¢, OL418 to generate pGL41:
5¢-CGAACGGGCACCTGTACACGGAGGACAGC-3¢
and OL420 to generate pGL42: 5¢-GCTGCGATGACA
TGAACGATGGTTGCGACGGCGGGCTGATGC-3¢
These mutant constructs were verified by sequence
analysis using an ABI 373 automated DNA sequencer
(PerkinElmer) The native and mutant CPB3 genes were
excised from pBluescript SK– with XbaI–XhoI Blunt ends
were created using Klenow fragment (NEB) and these were
ligated to the SmaI site of the pX episomal shuttle vector
[27] to generate the pGL plasmids detailed in Table 1 The
CPB2.8gene [6] was excised from pBluescript SK– (pGL28)
as a 2.0 kb EcoRV fragment and ligated to the SmaI site of
pX to generate pGL46 (Table 1) All pX-based constructs
were then used to transfect Dcpb [6]
Transfection of L mexicana promastigotes was as
des-cribed previously [6] Briefly, pX-based constructs were
prepared using Qiagen Tip100 columns as outlined by the
manufacturer Transfection utilized 10 lg of DNA and
4· 107 late-log phase Dcpb promastigotes Following
electroporation, cells were allowed to recover in 10 mL
complete HOMEM medium for 24 h at 25C and then
transfectants were selected in complete HOMEM medium
containing 25 lg G418ÆmL)1
To generate recombinant L mexicana CPBs, the 203 bp
KpnI–SacI fragment of pGL180 (pQE-30 CPB2.8DCTE)
[11] was replaced with the corresponding fragments from
pGL27 and pGL41 to give expression constructs pGL400
(pQE-30 CPB3) and pGL401 (pQE-30 H84Y) (Table 1)
These encode proteins comprising the pro- and mature
domains of the enzymes, and which lack the pre- and
C-terminal domains The recombinant enzymes were
pro-duced without the C-terminal extension (CTE) to aid
refolding from the insoluble inclusion body phase The
production of active, mature recombinant enzyme using
E coliwas essentially as described previously for isoenzyme
CPB2.8 [11] The concentration of the enzyme stock
solutions ( 11 lM) were determined by active site titration
with human cystatin C, which was a generous gift from
M Abrahamson (University of Lund, Sweden), using
Z-FR-7-amido-4-methylcoumarin (Sigma) as the substrate
Activity analyses of cysteine proteases using gelatin
SDS/PAGE
Parasite cysteine protease activities were analysed using
substrate SDS/PAGE as described previously [7,28]
Parasite cell lysates (107 cells) were subjected to
electro-phoresis under nonreducing conditions using 12% (w/v)
acrylamide gels containing 0.2% (w/v) gelatin Following
electrophoresis, the gel was washed for 1 h with 2.5%
(v/v) Triton X-100 and then incubated for 2 h in 0.1M
sodium acetate, pH 5.5, containing 1 mM dithiothreitol
Gelatin hydrolysis was detected by staining with
Coomas-sie Blue R-250 (0.25% w/v) When analysing activities
towards N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin
(Suc-LY-MCA;
2 Sigma), the peptidyl
amidomethylcou-marin fluorogenic substrate was added to 0.01 mM and,
following a 10 min incubation, fluorescence was detected
by exposure of the gel to low intensity UV light [28]
Western blotting Western blotting utilized polyclonal anti-CPB serum (1 : 2500) raised against rCPB2.8 expressed in and purified from E coli [11]
Synthesis of Abz-peptidyl-Q-EDDnp All the intramolecularly quenched fluorogenic peptides contain N-[2,4-dinitrophenyl]-ethylenediamine (EDDnp) attached to glutamine This is a necessary result of the solid-phase peptide synthesis strategy employed, the details
of which were provided elsewhere [29] An automated bench-top simultaneous multiple solid-phase peptide syn-thesizer (PSSM 8 system; Shimadzu, Tokyo, Japan) was used for the solid-phase synthesis of all the peptides by the Fmoc-procedure The final de-protected peptides were purified by semipreparative HPLC using an Econosil C-18 column (10 lm, 22.5· 250 mm) and a two-solvent system: (A) trifluoroacetic acid/H2O (1 : 1000) and (B) trifluoro-acetic acid/acetonitrile/H2O (1 : 900 : 100) The column was eluted at a flow rate of 5 mLÆmin)1with a 10 (or 30))50 (or 60)% gradient of solvent B over 30 or 45 min Analytical HPLC was performed using a binary HPLC system from Shimadzu with a SPD-10AV Shimadzu UV-vis detector and a Shimadzu RF-535 fluorescence detector, coupled to
an Ultrasphere C-18 column (5 lm, 4.6· 150 mm) which was eluted with solvent systems A1 (H3PO4/H2O, 1 : 1000) and B1 (acetonitrile/H2O/H3PO4, 900 : 100 : 1) at a flow rate of 1.7 mLÆmin)1and a 10–80% gradient of B1 over
15 min The HPLC column eluates were monitored by their absorbance at 220 nm and by fluorescence emission at
420 nm following excitation at 320 nm The molecular mass and purity of synthesized peptides were checked by MALDI-TOF mass spectrometry (TofSpec-E, Micromass, Manchester, UK)
3 and or peptide sequencing using a protein sequencer PPSQ-23 (Shimadzu) The concentrations of the solutions of the substrates were determined by colorimetric determination of 2,4-dinitrophenyl group (extinction coef-ficient at 365 nm is 17 300ÆM )1Æcm)1)
Enzymatic hydrolysis of fluorescent quenched substrates Hydrolysis of the fluorogenic peptide substrates by rCPB2.8, rCPB3 and rH84Y were carried out in 0.1M sodium acetate, 2 mM EDTA, 200 mM NaCl, pH 5.5, at
37C All kinetic analyses were carried out at 37 C with
5 min enzyme preincubation in 2.5 mMdithiothreitol and
by measuring the fluorescence at 420 nm, following excita-tion at 320 nm, using a Hitachi F-2500 spectrofluorometer
to follow the Abz-peptidyl-Q-EDDnp substrate hydrolysis The kinetic parameters were calculated according Wilkinson [30] as well as by using Eadie–Hofstee plots The standard derivations of Kmand kcatdeterminations were in no case higher than 5% of the obtained value
HPLC analysis of the enzymatic hydrolysis products
of the synthetic fluorogenic substrates The cleaved peptide bonds in each substrate were identified
by isolation of the fragments by HPLC reverse-phase chromatography on a C18 column equilibrated in 10%
Trang 4solvent B (90% acetonitrile, 0.1% trifluoroacetic acid, v/v).
The column was eluted at a flow rate of 1 mLÆmin)1with 10–
80% gradient of solvent B over 28 min The elution profile
was monitored by absorbance at 220 nm and by fluorescence
at 420 nm after excitation at 320 nm The Abz-containing
fragments were compared with authentic synthetic sequences
and/or by amino acid sequencing and molecular mass
determination by MALDI-TOF mass spectrometry
Results
Correlation between structure and activity of CPB
isoenzymes using substrate SDS/PAGE
The CPB locus of L mexicana consists of 19 genes in a
tandem array [7] The proteins encoded by three of these
genes differ in just a few amino acids The present study was
based on the finding that CPB2.8 and CPB18 are both
highly active towards gelatin as a substrate when assessed by
in situsubstrate SDS/PAGE, whereas CPB3 was inactive
[6,7] CPB3 differs from CPB2.8 in just three residues (60, 61
and 64) whereas the only difference between CPB3 and
CPB18 are residues 18 and 84 Thus it was reasoned that
one or more of these changes must play a key role in
modulating the enzyme activity There appeared two
possible explanations for the activity differences observed:
that the amino acid substitutions modulated enzyme activity
directly, or they had an indirect effect by influencing the
enzyme’s folding and stability To investigate the role of the
different amino acid residues we generated two mutants of
CPB3 in which residues 18 and 84 were changed to those
present in CPB18 We also mutated residues 60, 61 and 64
of CPB3 to those present in CBP2.8 – as a positive control
for the procedure
Incorporating mutations into the CPB3 gene and
intro-ducing the mutated genes into Dcpb by transfection allowed
an investigation into the functional roles of these amino
acids The activity of the enzyme expressed in the parasite
was then analyzed towards gelatin and a small fluorogenic
peptidyl substrate, in both cases using in situ substrate SDS/
PAGE Conversion of three variant residues in the mature
domain of the CPB3 isoenzyme (Asp60, Asn61 and Ser64)
to those present in CPB2.8 (Asn60, Glu61 and Glu64) to
give a protein designated M3, restored gelatinase activity to
the protease as expected (Fig 1A, lane 6) This
demon-strates that one or more of these residues play an important
role either in modulating the activity towards gelatin or in
enabling the enzyme to reactivate after the electrophoresis
procedure used Mutation of His84 to Tyr84 (to give H84Y)
also restored gelatinase activity to the CPB3 isoenzyme
(Fig 1A, lane 5), whereas mutation of Asp18 to Asn18 (to
give D18N) did not (Fig 1A, lane 4) The level of
re-expressed CPBs was to the same order in all of the cell
lines, as assessed by Western blotting (Fig 1B, lanes 3–7),
although there were differences in expression levels that may
account in part for the differences in proteolytic activities
apparent (for example between lanes 5–7 of Fig 1A)
Interestingly, the H84Y activity appeared to be somewhat
greater than that of M3 However, such in situ gel assays,
although they are useful in providing qualitative results,
need to be interpreted with caution with respect to
quantitative data
The mutated forms of CPB3 were also assessed for activity towards a small fluorogenic peptide using the gel-based assay, which also is useful for assessing activity but is not very quantitative (Fig 2) As observed for gelatin, the mutants expressing the CPB3 or D18N were inactive towards Suc-LY-MCA (Fig 2A, lanes 1 and 2) In contrast, the H84Y isoenzyme hydrolyzed Suc-LY-MCA well (Fig 2A, lane 3) The M3 enzyme was also active towards this fluorogenic compound (Fig 2A, lane 4) This was to be expected, and so served as a positive control, as the mutant has the same mature domain as CPB2.8 (Fig 2A, lane 5), which is active towards this substrate [7] The higher molecular mass activities towards Suc-LY-MCA (approxi-mately 35 kDa) correspond to activated precursor forms of the isoenzymes [11] To confirm that similar amounts of the re-expressed proteases had been applied to these activity gels, a Western blot was performed on duplicate samples with the CPB-specific antiserum (Fig 2B)
These results indicated that residues 60, 61, 64 and 84 influenced the activity of CPB, thus we produced as recombinant enzymes CPB3 and also H84Y in order to carry out a fuller analysis of their substrate specificities and
1 2 3 4 5 6 7
1 2 3 4 5 6 7
A
B
30
22
42
30
22 -kDa
kDa
Fig 1 Gelatin SDS/PAGE and Western blot analyses of L mexicana CPB isoenzymes expressed in Dcpb (A) Extracts from 10 7 stationary phase promastigotes were used for gelatin SDS/PAGE Wild type parasites (lane 1), Dcpb (lane 2), DcpbCPB3 (lane 3), DcpbCPB3D18N (lane 4), DcpbCPB3H84Y (lane 5), DcpbCPB3M3 (lane 6) and DcpbCPB2.8 (lane 7) The higher molecular mass activities (approxi-mately 35 kDa) evident with some samples resulted from the activation
in situ of precursor forms of the isoenzymes [11] (B) Extracts from
5 · 10 6 stationary phase promastigotes were used for Western blotting with anti-CPB serum (1 : 2500) Samples were applied to lanes 1–7 as denoted for (A) Molecular mass markers are shown in kDa The anti-CPB serum recognized anti-CPB isoenzymes that migrated as a major band with a molecular mass of 27 kDa and a minor band of molecular mass
26 kDa in cell lysates of stationary phase promastigotes of wild type
L mexicana (lane 1) The specificity of the antiserum was confirmed by absence of detected proteins in Dcpb (lane 2).
Trang 5compare them with that of CPB2.8 [13] All enzymes were produced without the C-terminal extension as previously [11]
S1subsite specificity characterization of CPB3 and H84Y
A series derived from the peptide Abz-KLRFSKQ-EDDnp were synthesized with systematic variation of Arg at P1 using all natural amino acids as previously reported for the subsite specificity studies of rCPB2.8 [13] Table 2 shows the kinetic parameters for the hydrolysis of this series of peptides by CPB3 and H84Y, and, for comparison, also the
kcat/Kmand Kivalues of rCPB2.8
The substrate inhibition with the peptides containing hydrophobic and non-charged amino acids that occurred with rCPB2.8 was not observed with rCPB3 and rH84Y, and some hydrolysis occurred with all substrates However, the specificity constants (kcat/Km) values for these proteases were considerably lower than those obtained with rCPB2.8 The higher kcat/Kmvalues for rCPB2.8 were due to both lower Kms and higher kcats For example, with X¼ R, the rCPB2.8 values were 0.04 lM (Km) and 2.60 s)1 (kcat) compared with the values of 0.3 lM(Km) and 0.48 s)1(kcat) for rCPB3 (Table 2)
rCPB3 hydrolyzed with highest kcat/Km values the peptides containing Ser and Thr, followed by those with Phe, Arg, Lys and Tyr These higher catalytic efficiencies are mainly due to the low Kmvalue rather than the catalytic component Similar hydrolytic behaviour was observed with rH84Y and the best substrates were those containing Met, Ser and Thr
30 -
22 -
42 -
A
B
kDa
2
2
30
22
42
-kDa
Fig 2 Fluorogenic SDS/PAGE and Western blot analyses of
L mexicana CPBs expressed in Dcpb (A) Extracts from 107stationary
phase promastigotes of Dcpb re-expressing the following proteases
were analysed for activity towards Suc-LY-MCA
12 DcpbCPB3 (lane 1),
DcpbCPB3D18N (lane 2), DcpbCPB3H84Y (lane 3), DcpbCPB3M3
(lane 4) and DcpbCPB2.8 (lane 5) (B) Extracts from 5 · 10 6 stationary
phase promastigotes were analysed with anti-CPB serum to show that
roughly equivalent protein loadings were applied for the fluorogenic
SDS/PAGE analyses: samples were applied to lanes 1–5 as denoted for
(A) Molecular mass markers are shown in kDa The mature CPBs are
arrowed.
Table 2 Kinetic parameters for hydrolysis, by CPB, of the peptides derived from Abz-KLXFSKQ-EDDnp with modifications in X (P 1 ) Conditions of hydrolysis: 100 m M NaOAc, 200 m M NaCl, 2 m M EDTA, pH 5.5 and 37 C The enzymes were preactivated by 2.5 m M dithiothreitol
The cleavage site is indicated by fl, and the numbers following give
8 the percentage of hydrolysis at each peptide bond All the other hydrolyses were
at the X–F bond rCPB2.8 data from reference [13] The units for K i values are n M , and k cat /K m values are in (m M Æs))1 X indicates the residue varied.
Modification
k cat /K m
(m M Æs))1
K m
(l M )
k cat
(s)1)
k cat /K m
(m M Æs))1
K m
(l M )
k cat
(s)1)
k cat /K m
(m M Æs))1
L K i ¼ 15 k cat /K m ¼ 600 (XflF ¼ 55, FflS ¼ 45) k cat /K m ¼ 700 (XflF ¼ 50, FflS ¼ 50)
I K i ¼ 9 k cat /K m ¼ 350 (XflF ¼ 15, FflS ¼ 85) k cat /K m ¼ 100 (XflF ¼ 19, FflS ¼ 81)
V K i ¼ 29 k cat /K m ¼ 250 (XflF ¼ 24, FflS ¼ 76) k cat /K m ¼ 300 (XflF ¼ 28, FflS ¼ 72)
N K i ¼ 32 k cat /K m ¼ 300 (XflF ¼ 67, FflS ¼ 33) k cat /K m ¼ 133 (XflF ¼ 67, FflS ¼ 33)
Trang 6Most of the peptides were hydrolyzed by rCPB3 and
rH84Y at the X–F peptide bond, where X represents all the
substitutions of Arg A second cleavage, at the F–S bond,
was observed for the peptides with X¼ Leu, Ile, Val and
Asn Like cathepsin L, the S2–P2interaction is determinant
in defining the cleavage point and these cysteine proteases
prefer hydrophobic amino acids at P2position [31,32] Thus
the cleavage of the F–S bond with Asn at the S2subsite by
rCPB3 and rH84Y is surprising, although this cleavage
corresponded to only 33% of the total (Table 2)
S2and S3subsite specificity characterization of rCPB3
and rH84Y
The kinetic parameters for hydrolysis of the peptides
modified at positions P2and P3 are shown in Table 3 S2
specificity is considered critical for the activity of clan CA
5cysteine proteases [31] The rCPB3 preferred Leu at P2
position of the substrate, while Pro is the worst amino acid
of those tested for activity On the other hand, rH84Y
hydrolyzed the peptide with Arg at P2with higher kcat/Km;
this was mainly due to kcatcontribution because the Kmwas
relatively high The extended binding site of rCPB3 and
rH84Y also included the S3subsite, as the modifications at
P3position of the substrates resulted in significant variations
on the kcat/Km values (Table 3) rCPB3 hydrolyzed with
better efficiency the peptides with Lys and Leu at P3,
whereas rH84Y was most efficient with the peptides
containing Leu and Ala
S1¢ to S3¢ subsite specificity characterization of rCPB3
and rH84Y
The kinetic parameters for hydrolysis of the peptides
modified at positions P1¢ to P3¢ are shown in Table 4 The
trend of the kcat/Kmvalues obtained with the substrates with
modifications at P1¢ position were similar between rCPB3 and rCPB2.8, however, the latter enzyme hydrolyzed all the peptides of this series with kcat/Kmvalues at least one order
of magnitude higher In contrast to rCPB3 and rCPB2.8, the mutant enzyme rH84Y hydrolyzed the peptide with Phe only poorly This enzyme hydrolyzed best the peptide containing Ala at P1¢, followed by those with Leu and Arg The peptide with Pro was almost resistant to all three proteases, although a low rate of hydrolysis was observed at the P–S bond
The modification at the P2¢ position of the substrates revealed the very significant effect of Pro, resulting in considerably higher kcat/Km values for the hydrolysis by rCPB3 and rH84Y – even above those with rCPB2.8 These high values mainly reflected a marked decrease in the Km value The kcat/Km values for the hydrolysis of Abz-KLRFPKQ-EDDnp by these two proteases were the highest found Such preference for Pro in the S2¢ subsite is
a peculiarity of cruzain (of T cruzi) and Leishmania CPB These enzymes accept Pro in this position in synthetic substrates very well [13,15,33,34] and also in the auto-processing of their pro-enzymes to active enzymes – Pro is the second amino acid in the mature form of the proteases [11,35] The importance of S2¢–P2¢ interaction was further evidenced by the variations in the kcat/Kmvalues for the hydrolysis of various substrates with modifications at P2¢ position by rH84Y – changing the Ser favoured by rCPB2.8
in each case resulted in increased activity
Table 3 Kinetic parameters for hydrolysis, by CPB, of the peptides
derived from Abz-KLRFSKQ-EDDnp, containing modifications at the
Leu (P 2 ) and Lys (P 3 ) residues Conditions of hydrolysis: 100 m M
NaOAc, 200 m M NaCl, 2 m M EDTA, pH 5.5 and 37 C The enzymes
were preactivated by 2.5 m M dithiothreitol for 5 min rCPB2.8 data
from reference [13] X indicates the residue varied The cleavage site is
indicated by fl.
9
Substrate
rCPB2.8 rCPB3 rH84Y
k cat /K m
(m M Æs))1
K m
(l M )
k cat
(s)1)
k cat /K m
(m M Æs))1
K m
(l M )
k cat
(s)1)
k cat /K m
(m M Æs))1 Abz-KXRflFSKQ-EDDnp
L 65000 0.3 0.48 1600 1.6 0.22 138
F 15517 0.9 0.13 144 0.8 0.17 212
A 2204 0.2 0.02 100 0.4 0.04 100
R 3295 0.3 0.04 133 1.4 0.66 471
P 471 0.5 0.01 20 4.8 0.39 81
Abz-XLRflFSKQ-EDDnp
K 65000 0.3 0.48 1600 1.6 0.22 138
L 6167 0.05 0.07 1400 0.05 0.13 2600
A 16769 0.1 0.08 800 0.1 0.25 2500
H 6320 0.7 0.28 400 2.2 0.46 209
R 10421 0.4 0.12 300 0.5 0.29 630
Table 4 Kinetic parameters for hydrolysis, by CPB, of the peptides derived from Abz-KLRFSKQ-EDDnp, containing modifications at the Phe (P 1 ¢), Ser (P 2 ¢) and Lys (P 3 ¢) residues Conditions of hydrolysis:
100 m M NaOAc, 200 m M NaCl, 2 m M EDTA, pH 5.5 and 37 C The enzymes were preactivated by 2.5 m M dithiothreitol for 5 min rCPB2.8 data from reference [13] The units for K i values are n M
X indicates the residue varied.
10 The cleavage site is indicated by ‘fl’.
11
Substrates
rCPB2.8 rCPB3 rH84Y
k cat /K m
(m M Æs))1
K m
(l M )
k cat
(s)1)
k cat /K m
(m M Æs))1
K m
(l M )
k cat
(s)1)
k cat /K m
(m M Æs))1 Abz-KLRflXSKQ-EDDnp
F 65000 0.3 0.48 1600 1.6 0.22 138
L 14117 0.7 0.28 400 0.06 0.03 500
A 14882 0.2 0.09 450 0.3 0.28 933
R 5438 0.4 0.11 275 0.4 0.17 453
P a K i ¼ 490 0.5 0.01 20 0.3 0.03 93 Abz-KLRflFXKQ-EDDnp
S 65000 0.3 0.48 1600 1.6 0.22 138
F 27778 0.2 0.18 977 0.3 0.19 633
A 8909 0.1 0.10 774 0.06 0.11 1833
R 15833 0.3 0.32 944 0.3 0.50 1667
P 11875 0.01 0.18 18000 0.01 0.16 16000 Abz-KLRflFSXQ-EDDnp
K 65000 0.3 0.48 1600 1.6 0.22 138
F K i ¼ 40 0.5 0.04 80 0.1 0.23 2300
A 12727 0.6 0.20 333 0.6 0.12 200
R 24286 0.4 0.15 375 0.2 0.31 1550
P 11000 0.2 0.02 100 0.6 0.27 450
a Cleavage at P–S bond.
Trang 7The S3¢ subsite also influenced the binding of rCPB3 and
rH84Y Significant variations in the kcat/Km values were
observed with the amino acid substitutions at P3¢ position of
the substrates (Table 4) The peptide with Lys was
hydro-lyzed best by rCPB3 (it is also favoured by rCPB2.8),
whereas rH84Y was more efficient on the peptides
contain-ing Phe and Arg
Carboxydipeptidase activity of rCPB3 and rH84Y
The kinetic parameters for the carboxydipeptidase
activit-ies of rCPB3 and rH84Y on the internally quenched
fluorescent peptide Abz-FRFK(Dnp)-OH and some of its
analogues are shown in Table 5 The kcat/Km values for
human recombinant cathepsin L and rCPB2.8 [21] are also
shown for comparison The carboxydipeptidase activities
of rCPB3, and rH84Y were lower than those of cathepsin
L and rCPB2.8, although relative activities towards the
different substrates were rather similar and in each case
Abz-FRAK(Dnp)-NH2 was the best substrate The
sub-strates with free C-terminal carboxyl group were
hydro-lyzed with Kmvalues that are an order of magnitude higher
than those presented in Tables 2–4 The unfavorable
effects of the C-terminal negatively charged carboxyl
group on the protease activity of rCPB3 and rH84Y is
demonstrated by the pH-profiles of the carboxydipeptidase
activities of these enzymes on the peptides
Abz-FRAK(Dnp)-OH and Abz-FRAK(Dnp)-NH2 (Fig 3)
The pH optima of the carboxydipeptidase activities
towards Abz-FRAK(Dnp)-OH of rCPB3 and rH84Y are
displaced to 4.0–4.5 and the activity decreases greatly by
pH 5.5 This pH range corresponds to the pK of
carboxylate group formation from the substrate, indicating
that the protonated carboxyl group fits better to the
enzymes than its carboxylate form This is confirmed by
the pH-profiles for the hydrolysis of
Abz-FRAK(Dnp)-NH2 In this case, the pH optimum is in the range 6–8 It is
noteworthy that the pH-profile of carboxydipeptidase
activity of rCPB2.8 on Abz-FRAK(Dnp)-OH contrasts
greatly with those of rCPB3 and rH84Y It has optimal
activity at pH 6–8, showing that rCPB2.8 accommodates
much better the negatively charged carboxylate group
Interestingly, the pH-profile of hydrolysis of
Abz-FRAK(Dnp)-OH by rH84Y presents a second small but
significant peak of activity around pH 7.5 It is difficult to
assign the group responsible for this effect because there
are several groups that change their ionization status around this pH
Analysis of amino acid locations via modelling The location of the amino acids that differ between the CPB isoenzymes at positions 18, 60, 61, 64 and 84 was determined by constructing a model of the L mexicana CPB isoenzymes by superimposing upon that obtained for the enzymes’ homologue in L major [22] (Fig 4) The crystal structure of papain [23] and of a recombinant cysteine protease of T cruzi, cruzain [24], which has a high degree of sequence identity to CPB, was used as a template for the leishmanial CPB model The mature regions of the L mexicana and L major enzymes used for the modelling have an overall 80% amino acid sequence identity, which reaches even higher values within the structurally conserved regions and especially within the active site (Table 6) Therefore, their protein structures appear to be very similar as determined by secondary structure alignments The polypeptide backbone of these cysteine proteases folds into a series of a-helices and b-sheets and the active site cleft is located between two structural domains The C-terminal extension is not shown
on the model, as the structure of this domain has not been solved
The protease activity of all papain-like cysteine proteases
is associated with the catalytic triad (L mexicana residues Cys25, His163 and Asn183; Fig 4) but the substrate specificity is defined by the binding affinities of the subsites The catalytic residues and the S1and S1¢ subsites are highly conserved between the three parasite species, and key residues at the three major subsites are completely conserved between the L mexicana CPB isoenzymes and the L major homologue (Table 6) Consequently, differences in activities between the L mexicana CPB isoenzymes must be associ-ated with amino acid variations in more peripheral positions
of the molecule
The model revealed the location of the amino acids that differ between the CPB isoenzymes under study and so mediate the observed activity changes (the residues are highlighted in Fig 4B) Residues 60, 61 and 64 are located above the a-helix that forms a wall of the active site cleft Amino acid 18 is relatively close to the active site cleft and also near to one disulphide bridge (Cys22–Cys63), whereas residue 84 is sited on a surface loop of one domain of the
Table 5 Kinetic constant parameters for carboxydipeptidase activities Conditions of hydrolysis: 100 m M NaOAc, 200 m M NaCl, 2 m M EDTA,
pH 5.5 and 37 C The enzymes were preactivated by 2.5 m M dithiothreitol for 5 min Cath L and rCPB2.8 data are from reference [21].
Substrates
Cath L rCPB2.8 rCPB3 rH84Y
k cat /K m
(m M Æs))1
k cat /K m
(m M Æs))1
K m
(m M )
k cat
(s)1)
k cat /K m
(m M Æs))1
K m
(m M )
k cat
(s)1)
k cat /K m
(m M Æs))1 Abz-FRFK(Dnp)-OH 256 306 3.3 0.23 70 3.9 0.51 130 Abz-RRFK(Dnp)-OH 2.2 4.5 K i ¼ 4.3 l M Resistant
Abz-ARFK(Dnp)-OH 0.5 1.1 K i ¼ 2.3 l M 4.7 0.004 0.85 Abz-FRK(Dnp)W-OH 477 625 0.7 0.14 210 1.9 0.24 121 Abz-FRAK(Dnp)-OH 667 389 2.9 0.35 118 2.2 0.46 203 Abz-FRAK(Dnp)-NH 2 5739 4909 1.2 0.90 780 0.85 1.14 1340
Trang 8enzyme but adjacent to another disulphide bridge (Cys56–
Cys101)
Discussion
The CPB cysteine proteases of L mexicana have been
shown to be virulence factors [6], therefore it is
important to understand the relative contributions that
different isoenzymes play in the pathogenicity of the
parasite Those isoenzymes that have been characterized
so far are highly conserved (98% identical) and yet some activity differences were apparent [7] The aim of this study was to determine the extent to which these amino acid variations generated activity differences The CPB sequences obtained to date all possess identical residues aligning the substrate subsites so it seemed that other residues outside of the active site cleft must affect activity
Abz-FRAK(Dnp)-OH
pH
3 4 5 6 7 8 0
400 800 1200 1600 2000 2400 2800
10 9
pH
3 4 5 6 7 8 9 1 0
200 400 600 800 1000 1200 1400 1600 1800 2000
0
pH
3 4 5 6 7 8 9 10 0
200
400
600
800
1000
pH
3 4 5 6 7 8 9 10 0
20
40
60
80
100
120
140
160
180
pH
3 4 5 6 7 8 9 10 0
40
80
120
160
200
240
pH
3 4 5 6 7 8 9 10
-1 s -1 )
-1 s -1 )
-1 s -1 )
-1 s
-1 )
-1 s
-1 )
-1 s
-1 )
0 2000 4000 6000 8000 10000 12000 14000 16000
Fig 3 pH-profile activity (k cat /K m ) for the hydrolysis of Abz-FRAK(Dnp)-OH and Abz-FRAK(Dnp)-NH 2 by rCPB2.8, rCPB3 and rH84Y The reactions were carried out in standard buffer containing 25 m M acetic acid, 25 m M Mes, 75 m M Tris base, 25 m M glycine, and 2 m M EDTA The
pH range was 3.5–10 and adjusted with 2 M NaOH or HCl The enzymes were preactivated by 2.5 m M dithiothreitol for 5 min at 37 C.
Trang 9The activity results presented show that the few amino
acid variations known to exist between some isoenzymes of
CPB of L mexicana are indeed important in modifying the
substrate specificities of the CPB isoenzymes Both rCPB3
and rH84Y have lower activity towards some substrates
than does rCPB2.8, but they are able to accommodate a
wider range of amino acids at P1 (Table 2) The kinetic
parameters of hydrolysis by rCPB3, rH84Y and rCPB2.8 of
the substrates with variations at P1position indicated that
the specificity of S1 subsite is greatly influenced by the
modifications at 60, 61, 64 and 84 However, the enzymes
have an extended binding site that goes at least from S3to
S3¢ and importantly each CPB isoform also shows
signifi-cant differences in the specificity of each subsite (Tables 3
and 4) Thus, the overall conclusion is that the different
isoenzymes do indeed have different hydrolytic capabilities
and presumably this is important for the parasite’s survival
This view is supported by the recent observation that the
expression of multiple CPB genes encoding cysteine
proteases, rather than just one, is required for L mexicana
virulence in vivo [10]
A particularly noteworthy finding was the effect of Pro in
the P2¢ position in decreasing considerably the Km value
with rCPB3 and rH84Y Clearly these isoenzymes greatly
favour Pro at this site This may reflect an important role for
the enzymes in the activation of CPB in the parasite, as Pro
is the second amino acid of the mature domain in all of the CPB isoenzymes for which the structure is known The variation of amino acid residues 60, 61 and 64 between the CPB isoenzymes examined in this work involve amino acids with charged side chains and this necessarily results in significant modifications on the electrostatic potential on the surface of the enzymes The modeling analysis (Fig 4) shows clearly that residues 60, 61 and 64 are located near the catalytic groove of the enzyme and so it
is likely that the localized charge variations resulting from the introduction or removal of residues with charged side groups to these positions could be the basis for the differences observed with respect to the utilization of substrates The pH-profile differences for the carboxydi-peptidase activities of rCPB3 and rCPB2.8 clearly suggest that the latter isoform accommodates the substrate carb-oxylate group better than the former isoform, perhaps indicating that residue 60 (Asn in CPB2.8 but Asp in CPB3)
is particularly important for this binding These findings agree well with a study of the pH-activity profile of cruzain,
a related cathepsin L-like cysteine protease of T cruzi, which highlighted the importance of several ionizable groups and suggested that Asn60 is potentially involved in substrate recognition [36] Thus the data obtained provide further evidence for the role of electrostatic potential in defining the substrate specificity of the CPB isoforms
84
60 61 64
18
SS 63
22
25 163
183
SS 204
156
SS 101 56
Fig 4 Homology-based protein model of the mature domain of L mexicana CPB The protein is shown as a ribbon structure (spi-rals, a-helices; arrows, b-sheets) (A) High-lighted in yellow are the location of the active site triad (Cys25, His163 and Asn183) and disulphide bonds (Cys22–Cys63, Cys56– Cys101 and Cys156–Cys204) (B) Highlighted
in red are the residues that differ between CPB2.8, CPB3 and CPB18.
Table 6 Key active site residues of cathepsin L -like cysteine proteases The major variations between the active sites of parasite CPs and papain occur between the S subsites Additional S and S subsites are not listed because they are so far not identified by the cocrystallization of a peptide substrate and the proteases of the parasites.
Protease S2 subsite S1 subsite Catalytic triad S1¢ subsite Papain W69, S205, F207 Y67, P68, V133, A160 C25, H159, N175 Q19, W177 Cruzain N69, E208, S210 L67, M68, A138, G163 C25, H162, N182 Q19, W177
L mexicana CPB L69, Y209, V211 L67, M68, A139, G164 C25, H163, N183 Q19, W185
L major CPB L69, Y209, V211 L67, M68, A139, G164 C25, H163, N183 Q19, W185
Trang 10Residue 84 is located near the surface of the mature
domain structure and so it is less easy to understand how it
plays a role in affecting the binding of substrates and their
hydrolysis However, it is positioned near to a disulphide
bridge (Cys56–Cys101) and it is conceivable that the
replacement of His with Tyr may impinge upon this
structure and so change the active site is some way Clearly
the findings on the activity of this mutant compared with
rCPB3 suggest that mutation had some effects, although
more minor than those resulting from the three changes
(N60D, D61N and D64S) between CPB2.8 and the other
isoenzymes
The results show that rCPB3 has activity towards peptide
substrates and yet the enzyme showed no activity in
substrate SDS/PAGE analysis, whereas both CPB2.8 and
CPB18 were both highly active in similar analyses (Figs 1
and 2) This suggests not only that one or more of amino
acid changes H60D, D61N and D64S plays a key role in
modulating the enzyme activity directly but may also be
able to do so indirectly by affecting the enzyme’s refolding
and/or stability under the conditions employed for the
gelatin SDS/PAGE Moreover, H84Y but not D18N can
counteract this effect It is too early to be able to interpret
the way is which this is achieved, but the results show the
complexity of the interactions that occur both within the
mature protein and in the acquisition of its tertiary
structure
In conclusion, the data reported suggest that the set of
CPB isoenzymes with only a few sequence modifications
have the modifications at strategic positions such that the
enzyme’s substrate specificity is changed and that these
variations between isoenzymes provide the parasite with an
array of hydrolytic activity that is needed for its interaction
with the mammalian host, and ensure its survival and
success as a parasite
Acknowledgements
This work was supported by Fundac¸a˜o de Amparo Pesquisa do Estado
de Sa˜o Paulo (FAPESP), Conselho Nacional de Desenvolvimento
Cientı´fico e Tecnolo´gico (CNPq) and Human Frontiers for Science
Progress (RG 00043/2000-M) J.C.M and G.H.C are supported by the
Medical Research Council.
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