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Process Biochemistry
j o u r n a l h o m e p a g e :
w w w . e l s e v i e r . c o m / l o c a t e / p r o c b i o
Biochemical characterization of a cysteine proteinase from
Bauhinia forficata
leaves and its kininogenase activity
Sheila S. Andrade
a
, Rosemeire A. Silva-Lucca
c
, Lucimeire A. Santana
a
, Iuri E. Gouvea
b
, Maria A. Juliano
b
,
Adriana K. Carmona
b
, Mariana S. Araújo
a
, Misako U. Sampaio
a
, Maria Luiza V. Oliva
a
,
∗
aDepartment of Biochemistry, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil bDepartment of Biophysics, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil
cCentro de Engenharias e Ciências Exatas, Universidade Estadual do Oeste do Paraná, 85903-000 Toledo, PR, Brazil
a r t i c l e
i n f o
Article history: Received 20 July 2010
Received in revised form 7 October 2010 Accepted 20 October 2010
Keywords: Circular dichroism Cysteine proteinase Papain
Plant enzyme Protein purification Substrate specificity
a b s t r a c t
In this work, the proteinase activity detect in the acetone precipitate (80%, v/v) ofB. forficataleaves, trivially known as cow paw, and popularly used in folk medicine for treatment of diabetes mellitus, was purified by chromatography on Sephadex G-25, Canecystatin-Sepharose, and on Con A-Sepharose. The molecular weight 30 kDa was estimated by SDS-PAGE and zymography, and the N-terminal sequence and CD spectra indicated a relationship with the papain family of cysteine proteinases. Denominated baupain, the enzyme was activated by dithiotreitol and inhibited by E-64 and iodoacetamide, but not by benzamidine, TLCK, TPCK and EDTA. The S2 and S1 substrate specificity of baupain, assayed with two series of fluorescence resonance energy transfer (FRET) peptide substrates derived from Abz-KLRSSK-Q-EDDnp, indicates a preference for Phe and Tyr at P2 position over Leu found in papain. Baupain releases bradykinin from HMWK (human high molecular weight kininogen) though its proteolytic activity is blocked by the sequence motif QVVA of kininogen (Kiapp= 1.9×10−8M). Canecystatin, from sugar cane, which also lodges the QVVA sequence, inhibits baupain (Kiapp= 0.18×10−9M).
© 2010 Elsevier Ltd.
1. Introduction
Within the members of cysteine proteinases expressed in
viruses, bacteria, animals and plants
[1]
, papain is the archetypical
member of these endopeptidyl hydrolases (EC 3.4.22). This group
also comprise the mammalian lysosomal cathepsins, the
cytoso-lic calpains (i.e., calcium activated cysteine proteinases) as well as
several parasitic proteinases
[2,3]
.
The action of these enzymes can be controlled by members
of a family known as cystatins super family, which comprises
three families, on the basis of sub-cellular localization,
molecu-lar weight, disulfide bonds and sequence simimolecu-larity including the
QXVXG motif. Specifically from plant, this group is known as plant
cystatins or phytocystatins (PhyCys) whose primary sequences
Abbreviations: FRET, fluorescence resonance energy transfer; Q-EDD, npglutaminyl-[N-(2,4-dinitrophenyl)-ethylenediamine]; Abz, ortho-aminobenzoic acid; HPLC, high performance liquid chromatography; LC/MS, liquid chromatogra-phy/mass spectrometry.
∗Corresponding author at: Universidade Federal de São Paulo, Departamento de Bioquímica, Rua Três de Maio 100, 04044-020 São Paulo, SP, Brazil.
Tel.: +55 11 55764444; fax: +55 11 55723006. E-mail address:olivaml.bioq@epm.br(M.L.V. Oliva).
share a high degree of similarity within the cystatin family,
includ-ing the sequence motif QXVXG
[4,5]
.
In plants, cysteine proteinases are widely distributed in various
tissues, and are involved in physiological events such as
germina-tion, senescence and environmental stress responses. Papain-like
cysteine proteinases are often found in senescing organs
partic-ularly leaves
[6–8]
, flowers
[9]
, legume nodules
[10]
as well as
in germinating seeds
[10–13]
. Cysteine proteinases have been
intensively studied with various expression patterns, reported for
different stages of plant development
[14–16]
. Equally studied is
cysteine proteinases role in processing and degradation of seed
storage proteins
[17,18]
, in legume nodule development
[19]
, in
response to stresses such as wounding, cold, and drought
[20]
as
well as in programmed cell death
[21,22]
.
In the present work, the purification and the functional
charac-terization of a new cysteine proteinase from
B. forficata
leaves are
described.
B. forficata
is a Leguminosae known as cow paw, due to
the characteristic bilobed aspect of its leaves from which the
home-made extract is prepared and used in popular therapy for diabetes
mellitus. The studies reported that the beneficial of leaf extracts
(aqueous and alcoholic) in the prevention of diabetes
complica-tions is associated with oxidative stress
since B. forficata
and other
plant extracts have significant antioxidant activity
[23,24]
.
1359-5113 © 2010 Elsevier Ltd. doi:10.1016/j.procbio.2010.10.008
Open access under the Elsevier OA license.
Although this plant has been the subject of several studies
[24–26]
, but a characterization of the enzyme activity is not yet
described.
The substrate specificity of the enzyme, designated as baupain
was assayed using peptides derived from the leading sequence
Abz-KLRSSKQEDDnp
[27–31]
and the series Abz-KXRSSKQ-EDDnp and
Abz-KLXSSKQ-EDDnp (X = different amino acids) used for mapping,
respectively, the S2 and S1 substrate specificity
[29–31]
. To
fur-ther compare the hydrolytic properties of baupain with papain and
human cathepsin L, we explored in vitro the ability of baupain to
release kinin from HMWK.
2. Materials and methods
2.1. Enzyme purification
Leaves (50 g) collected from wildBauhinia forficatatrees were homogenized in a blender with 0.15 M NaCl (700 ml). The proteins in the crude extract were pre-cipitated by 80% (v/v) acetone at 4◦C. The sediment separated by centrifugation
was dried at room temperature, and dissolved in 0.1 M sodium phosphate buffer, pH 6.3. The enzyme fraction (1.5 ml) was applied on a size exclusion chromatogra-phy (Sephadex G-25) equilibrated with 0.1 M sodium phosphate buffer, pH 6.3. The enzyme activity was followed using Z–Phe–Arg–MCA as substrate. The fractions containing enzyme activity were pooled and subsequently chromatographed on a canecystatin-Sepharose, equilibrated with the same phosphate buffer. The active material, eluted by 2.0 mMl-cysteine, was chromatographed on Con A Sepharose,
equilibrated with 0.1 M sodium phosphate buffer, pH 6.3, used for removal of pig-ments. The whole purification procedure was carried out at 4◦C. The unbound
fractions containing enzyme activity were separated and subsequently purified by HPLC on a-Bondapak C18reverse phase column. The separation was achieved by an acetonitrile gradient (0–100%) in 0.1% TFA (v/v) during 75 min, at 1 ml/min flow rate and room temperature. Proteins were estimated spectrophotometrically (A280) as well as by Bradford[32]assay using bovine serum albumin as the standard.
2.2. N-terminal amino acid sequence
Purified baupain was denatured and reduced by addition of 200l 50 mM Tris/HCl buffer, pH 8.5, containing 6.0 M guadinium HCl, 1.0 mM EDTA, and 5.0 mM dithiothreitol for 3 h, at 37◦C.S-pyridylethylation of cysteines was achieved by
addi-tion of (5l) 4-vinylpyrimidine for 3 h at 37◦C in the dark and nitrogen atmosphere.
The excess reagents were removed by reversed-phase HPLC on aC18column using the same gradient conditions already described. N-terminal amino acid sequences were determined by Edman degradation using a PPSQ-23 Model Protein Sequencer (Shimadzu, Tokyo, Japan). Phenylthiohydantoin derivates of amino acids were iden-tified.
2.3. Electrophoresis and gelatin zymography
The homogeneity and the molecular weight of baupain were assessed by SDS-PAGE under reducing and non-reducing conditions according to Laemmli[33], using 12% acrylamide gel. HMWK (10g) limited proteolysis cleavage by baupain (0.23M) was demonstrated by SDS-polyacrylamide gel electrophoresis[34]. Bau-pain and HMWK were incubated in 0.1 M sodium phosphate buffer, pH 6.3 for 10 and 60 min at 37◦C. The proteins were stained with Coomassie brilliant blue R-250.
A broad range of molecular weight protein markers, from 25 to 175 kDa and 20 to 94 kDa New England BioLabs Inc. (Ipswich, MA, USA), was used.
The zymography experiment was performance under non-reducing conditions according to Becker et al.[35]. Baupain activity was detected using 10% (w/v) acry-lamide (Gelatin-PAGE), with 0.04% (w/v) copolymerized gelatin included in the gel as substrate for the proteinase. After electrophoresis, the gels were incubated in renaturing buffer for 1 h at room temperature, followed with incubation in devel-oping buffer at 37◦C overnight. We used Mini-Protean®II Cell Bio-Rad (Hercules, CA, USA).
2.4. Enzyme activity
Proteinase activity was measured on Z–Phe–Arg–MCA (Calbiochem Ltda, Darm-stadt, Germany) and Bz-Arg-pNan (BAPA) (Sigma–Aldrich Company, St. Louis, USA) as substrates. Baupain was incubated at 37◦C in a microtiter plate in 250l final
volume of assay buffer [0.1 M sodium phosphate buffer, pH 6.3 containing 0.4 M NaCl, 10 mM EDTA, and 2.0 mM DTT (dithiothreitol)]. The reaction was followed for 10–30 min and the reaction was stopped by the addition of 50l acetic acid 30% (v/v). The fluorescence release was measured on a FluoroCount PackardTM, spectrofluorometer set at 355 nm for excitation and 460 nm for emission.
In the case of BAPA (0.8 mM) as substrate, the reaction was followed by mea-suring the absorbance of releasedp-nitroaniline at 405 nm in a spectrophotometer PackardTMwith a 50 mM Tris/HCl buffer, pH 8.0[36].
2.5. Effects of activators on enzyme activity
Baupain (0.23M) was preincubated with DTT,-mercaptoethanol, andl -cysteine (2 mM) in 0.1 M sodium phosphate buffer, pH 6.3 containing 0.4 M NaCl, 10 mM EDTA, at 37◦C for 30 min. The enzyme activity was determined as described,
using Z–Phe–Arg–MCA (0.4 mM) as substrate.
2.6. Effect of pH on the enzyme activity
Prior to the addition of the substrate Z–Phe–Arg–MCA 40l of baupain (0.23M) was pre-incubated at 37◦C for 30 min with 100l of the following buffer
systems: 0.2 M sodium citrate, pH 4.0; 0.2 M sodium acetate, pH 5.0; 0.2 M sodium phosphate, pH 6.0; 0.2 M Tris/HCl, pH 7.0 and pH 8.0 and 0.2 M sodium bicarbonate, pH 9.0 and pH 10.0 in a final volume of 250l by adding 90l of distillated water. The enzymatic activity was measured using 20l of Z–Phe–Arg–MCA (5 mM) as substrate.
2.7. Proteinase inhibition studies
Effect of low molecular weight inhibitors: Baupain (0.23M) was incu-bated with 1.0M E-64 (l-trans-epoxysuccinyl-leucylamido [4-guanidino] butane), 1.0 mM benzamidine, 2.0 mM phenylmethanesulfonyl fluoride (PMSF), 2.0 mM ortho-phenantroline, 5.0 mM ethylenediamine tetraacetic acid (EDTA), 1.0 mM N-tosyl-l-phenylalanylchloromethyl ketone (TPCK), and 1.0 mM N-tosyl-l-lysyl chloromethyl ketone (TLCK), for 10 min at 37◦C, before the addition of the substrate
Z–Phe–Arg–MCA (0.4 mM). The assay concentration of each inhibitor was chosen based in suppliers information and inhibitor mode of action[37]. Enzyme activity was expressed in percent of residual activity on Z–Phe–Arg–MCA compared to the control.
Effect of high molecular weight (proteinaceus) inhibitors: baupain (0.23M) was pre-incubated for 10 min in assay buffer with the serine proteinase inhibitors SbTI (Soy beans trypsin inhibitor)[38]1.39, 2.78, 5.56, 8.35M; EcTI (Enterolobium contortisiliquumtrypsin inhibitor)[39]0.2, 0.7, 1.50, 2.0, 2.7M and with the cys-teine proteinase inhibitors HMWK (High Molecular Weight Kininogen) 0.08, 0.16, 0.24, and 0.32M; canecystatin[5]0.06, 0.12, 0.18, 0.24 and 0.34 nM and BbCI (Bauhinia bauhinioidescruzain inhibitor)[40]1.2, 1.8, 2.4, 3.6, and 4.8M. Enzyme activity was expressed in percent of residual activity on Z–Phe–Arg–MCA compared to the control.
2.8. Determination of baupain substrate specificity
The hydrolysis of two series of FRET peptides derived from Abz-KLRSSK-Q-EDDnp (Q-Abz-KLRSSK-Q-EDDnp is the fluorescence acceptor and Abz is the fluorescence donor that corresponds to glutamine-[N-(2,4-dinitrophenyl)-ethylenediamine] and ortho-aminobenzoic acid, respectively), in which the residues L and R and S were substituted by natural amino acids were quantified in a Hitachi F-2500 spectroflu-orimeter at 37◦C. Baupain concentration was fixed as 11 nM and the substrates
as 4M. Fluorescence changes were monitored continuously atex= 320 nm and em= 420 nm. The enzyme concentrations were chosen so that less than 5% of the substrate was hydrolyzed over the course of the assay. The reaction rate was con-verted into nanomoles of substrate hydrolyzed per second based on a calibration curve obtained from the complete hydrolysis of each peptide. The scissile bond of hydrolyzed peptides were identified by isolation of the fragments using analyti-cal HPLC followed by determination of their molecular mass by LC/MS using an LCMS-2010 equipped with an ESI-probe (Shimadzu, Japan).
2.9. CD experiments
Circular Dichroism (CD) measurements were taken on a Jasco J-810 spectropo-larimeter (Jasco, Japan). Far UV-CD spectra were recorded at 25◦C in a cuvette of
1 mm pathlength with a 10M protein solution in the presence of 10 mM sodium phosphate buffer, pH 6.3. The spectra were recorded in the 190–250 nm wave-length range. The CD intensities were expressed as mean residue ellipiticities [] (deg cm2dmol−1) using the formula [] =
e/10.C.l.N, whereeis the experimental ellipticity in millidegrees, MRW is the mean residue weight,Cis the concentration of the protein in molar,lis the cuvette pathlength in centimeters, andNbeing the average number of residues adopted as 110 residues baupain. For the analysis of baupain CD spectrum the CDPro program was used. CDPro software package con-sists of three programs for analyzing the protein CD spectra for determining the secondary structure fractions (SELCON3, CDSSTR and CONTIN) and a program for determining tertiary structure class (CLUSTER)[41,42]. The estimation of baupain secondary structure was performance using 43 proteins in the reference set.
2.10. Fluorescence experiments
Fig. 1.Size exclusion chromatography on Sephadex G-25 column of B. forfi-cateleaves extract.Eluting buffer: 0.1 M sodium phosphate buffer, pH 6.3. Flow rate: 16 ml/h. The arrow indicates activity on Z–Phe–Arg–MCA (0.4 mM). Insert: Canecystatin-Sepharose column (2 mL). Sample: 0.4 mg of baupain from Sephadex G-25 column. (A) Unbound fractions were eluted with 0.1 M sodium phosphate buffer, pH 6.3 buffer. (B) The bound fraction (16) with activity on Z–Phe–Arg–MCA substrate was eluted with 0.1 M sodium phosphate buffer, pH 6.3 containing 0.001 M
l-cysteine.
set a 5.0 and 2.5 nm, respectively. Baseline correction was carried out with buffer without protein.
2.11. Radioimmunoassay
Baupain bradykinin release ability was assayed with HMWK[43]. The enzyme, in concentrations of 0.019, 0.038, and 0.057 A280 in 0.1 M sodium phosphate, pH 6.3 containing 10 mM EDTA, 0.4 M NaCl, and 2.0 mM DTT was incubated with HMWK (1.4M) in 40l for 2 h at 37◦C. Formed kinin was extracted in ethanol (1:4 v/v) for
10 min at−70◦C. The solution was concentrated and dissolved in 200l of 0.01 M
phosphate buffer, pH 7.0, NaCl 0.14 M, NaN3, 0.03 M EDTA, 0.003 M 1,10 phenan-throline and ovalbumin 0.1%. 50l of the sample was incubated with 100l anti-BK antibody[44](1: 80.000) and 100l tyrosyl-bradykinin probe with125I, for 20 h at 4◦C, 400l the BSA (2 mg/ml) 0.01 M phosphate buffer, pH 7.0, 0.1% NaCl 0.14 M,
NaN30.1% and 800l with polyethylene glycol 6000 25% solution were added to the samples and incubated by 10 min at 4◦C. Samples were centrifuged at 2000×g
for 20 min at 4◦C. The solution obtained was removed and the pellet was submitted
to the radiation counting and the bradykinin released was calculated.
3. Results and discussion
3.1. Proteinase extraction and purification
The protocol used for isolation and purification of the
endopro-teinase from
B. forficata
consists of three steps. Freshly prepared
leaves saline extracts were treated with 80% acetone in order to
precipitate the protein content. The precipitate containing
prote-olytic activity was submitted to a size exclusion chromatography
in a Sephadex G-25 column (
Fig. 1
) and the fractions with enzyme
activity on Z–Phe–Arg–MCA were polled and concentrated by
Ami-con ultrafiltration.
The strategy to apply affinity chromatography with
thiopropyl-Sepharose have been successfully used in the cysteine proteinase
purification since this resin presents strong binding 2-pyridyl
disul-fide
[45,46]
. In our case, the procedure resulted in low yield of
purification (data not shown), probably due to the pH 6.3 used
where the interaction to the activated group is not efficient. Further
purification was obtained using a canecystatin-Sepharose column
(
Fig. 1
, insert) being the enzyme activity detected in the broad
peak eluted with 1.0 mM
l
-cysteine. The peak showing activity
on Z–Phe–Arg–MCA was polled, concentrated and submitted to
a Concanavalin A-Sepharose affinity chromatography to complete
removal of leave pigments. Proteolytic activity was detected only
in the unbound fraction, depleted of leave pigments. The specific
Fig. 2.(A) SDS-PAGE (12%) of the cysteine proteinase baupain fromBauhinia forficata leaves. (1) Baupain (10g) from Con A-Sepharose under reducing conditions, Coomassie blue staining; (S), standard proteins,-lactoglobulin A (25 kDa), triosephosphate isomerase (32 kDA), aldolase (47 kDa, glutamic dehydro-genase (62 kDa), MBP-paramyosin (83 kDa) and MBP--galactosidase (175 kDa). (B) Gelatin-PAGE proteinase activity analysis of baupain 10g (1), and 20g (2). Clear band with dark background indicate sites of protein degradation in 10% acrylamide and 0.04% gelatin in non-reducing conditions and incubated in 0.1 M sodium phosphate, pH 6.3 for 24 h. (S) standard proteins,-lactoglobulin A (25 kDa), triosephosphate isomerase (32 kDA), aldolase (47 kDa, glutamic dehydrogenase (62 kDa), and MBP-paramyosin (83 kDa).
activity of the endopeptidase was increased more than 79-fold by
this isolation procedure and both SDS and non-denaturing PAGE of
the final preparation stained with Coomassie brilliant blue showed
a homogeneous preparation (data not shown). The purification
steps are summarized in
Table 1
.
The molecular weight of the purified proteinase was determined
by SDS-PAGE as 30 kDa (
Fig. 2
). The same result was obtained by
size exclusion chromatography on calibrated Superdex-200
col-umn, where the proteolytic activity was found essentially in the
main peak eluted with a volume that corresponds to a molecular
mass of 33
±
4 kDa (data not shown).
3.2. N-terminal sequence
Reverse phase chromatography in a C
18column was
per-formed with acetonitrile gradient, and the N-terminal sequences
of reduced and pyridylethylated inhibitors determined by
auto-mated Edman degradation allowed the identification of the first
10 amino acid residues (IPEYVDWRQQ). Using NCBI database,
PSI-BLAST
[47]
baupain shows similarity to those of the CA family of
cysteine proteinases, being 90% and 40% identical to papain and
bromelain, respectively.
3.3. Effect of metal ions, selective inhibitors and sulfhydryl
reagents on proteinase activity
Table 1
Purification of cysteine proteinase fromB. forficata.
Steps Volume (mL) Protein (mg mL) UA Specific activity (UA mg) Purif. Yield (%)
Saline extraction (50 g) 700 – – – – –
Acetone fractionation (80%) 30 1.0 – – – –
Sephadex G-25 5 0.32 0.2 0.6 1.0 100
Canecystatin-Sepharose 2.2 0.48 0.4 0.8 2.0 88
Con A-Sepharose 1.6 0.43 0.5 1.2 2.5 79
Enzyme activity was determined on Z–Phe–Arg–MCA 0.4 mM as substrate. UA –mols of Z–Phe–Arg–MCA hydrolyzed/min mL. Protein determinated by Bradford[32]assay.
(dithiothreitol) and
l
-cysteine) enhanced the hydrolytic activity of
baupain (data not shown), all further assays where performed with
of 2.0 mM DTT.
3.4. Circular dicroism spectra
The tertiary structure of the enzyme was assessed by far UV
circular dichroism. The CD spectrum (
Fig. 3
) shows two negative
CD bands at 222 nm and 208–210 nm and one positive band near
190 nm. The cluster analysis showed that the peptidase belongs to
the
␣
+

class of proteins
[48]
and the percentages of secondary
structure, calculated as 44%
␣
-helix, 16%

-sheet and 12%

-turn.
These data are consistent with the reported data on papain like
cysteine proteinases
[2]
.
The intrinsic fluorescence spectra of the proteinase was
obtained at pH 7.0 and the maximum of fluorescence intensity
was observed at 343 nm, a value characteristic of solvent accessible
tryptophan residues. This data is in agreement with the
fluores-cence emission maximum value of 345 nm obtained with papain
(data not shown)
[49,50]
. All together, these results suggest that
the enzyme belongs to the group of cysteine proteinases of the clan
CA of the papain family, and the name baupain was proposed for
this new endopeptidase.
3.5. Proteolytic activity characterization
The baupain hydrolytic activity remains after preincubation of
the enzyme at different pH values in the range of 4.5–9 for
30-min at 37
◦C. In the same experiments performed in the pH range
190 200 210 220 230 240 250
-40 -20 0 20 40
[
θ
] (10
3
deg.cm
2.dmol
-1)
Wavelength (nm)
Fig. 3.CD spectrum of baupain (0.05 mg/ml), in 0.001 M sodium phosphate buffer, pH 7.0. The spectrum was recorded over the range 190–250 nm, at 25◦C, in 1 mm
cell pathlength. CD Pro program was used for estimation of secondary structure of baupain. The calculated fractions were 44%␣-helix, 16%-sheet and 12%-turn.
3–4.5 and 9–10 the enzyme became irreversibly inactivated (data
not shown).
The pH-profile of the hydrolytic activity of baupain on
Z–Phe–Arg–MCA was measured in the pH range from 4 to 10
(
Fig. 4
A). The baupain maximum activity was achieved at pH 6.0
and very low activity observed at extreme acid and basic pHs
sim-ilar to that obtained with papain like cysteine proteinases
[4]
, we
used the following assay buffer for further kinetic studies: 0.1 M
sodium phosphate buffer, pH 6.3 containing 10 mM EDTA, 0.4 M
NaCl, and 2.0 mM DTT.
3.6. Substrate specificity of baupain using FRET substrates
Seven residues intramolecularly quenched fluorogenic
Abz-peptidyl-EDDnp substrates, containing systematic substitutions at
the carboxyl-side of the scissile bond, were used to evaluate the
contribution of these positions in baupain substrates hydrolytic
rate. Two series of peptides were generated with variations at P2
and P1 positions to explore the specificity at the S2 and S1 subsites
that usually define substrate specificity among papain-like cysteine
proteinases
[51]
.
3.6.1. Hydrolysis of Abz-KLXSSKQ-EDDnp series, for mapping S1
specificity
Fig. 5
A shows the baupain relative hydrolysis of the peptide
series Abz-KLXSSKQ-EDDnp with different amino acids at X
posi-tion. All substrates in this series were cleaved only at the X-Ser
bond, indicating that X occupied the P1 position. Baupain did not
present a defined specificity over the assayed peptides, being the
peptide containing Gly at P1 hydrolyzed at the same rate as the
one containing Phe and Arg. Deviations were observed only with
the peptides containing negatively charged residues (Glu and Asp)
and the bulky Trp. This apparent lack of specificity at S1 contrasts
Abz-KL
X
SSKQ-EDDnp
R* K D E G I F W
Activity (nM.s
-1
)
Activity (nM.s
-1
)
0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200
Abz-K
X
RSSKQ-EDDnp
R K E G A L* V M F Y W 0
200 400 600 800 1000 1200 1400 1600 1800 2000 2200
Z-FR -MCA Z-LR-MCA
A
B
C
Fig. 5.(A) Baupain S1 substrate specificity. All substrates in this series were cleaved only at the X-Ser bonds, indicating that X occupied the P1 position in this series. Assay conditions are as described in Section2. (B) Baupain S2 substrate specificity. All substrates of this series were exclusively cleaved by the enzyme at the R–S bond with X occupying the P2position. (C) Baupain activity on Z–Phe–Arg–MCA and Z-Leu-Arg-MCA is included for comparison. Assay conditions are as described in Section2.
with that of papain, bromelain and human cathepsin L that show a
preference for basic amino acids in P1
[29,51]
.
3.6.2. Hydrolysis of Abz-KXRSSKQ-EDDnp series, for mapping S2
specificity
The relative hydrolysis of the peptide series
Abz-KXRSSKQ-EDDnp is shown in
Fig. 5
B. All peptides from this series, under the
assay conditions used, were exclusively cleaved by the enzyme at
the Arg-Ser bond with X occupying P2 position. The best hydrolyzed
peptides were those containing Tyr or Phe at P2 position, whereas
those containing positively or negatively charged amino acids (Arg,
Lys, His and Asp) as well as polar groups (Ser, Thr) were poorly
hydrolyzed. It is interesting to observe that FRET substrates having
aliphatic amino acids Val and Leu were hydrolyzed with 50% and
30% of the efficiency obtained with the larger aromatic residues.
This preference was corroborated by higher hydrolytic efficiency
of baupain over Z–Phe–Arg–MCA, in comparison to Z-Leu-Arg-MCA
(
Fig. 5
C). In this sense, baupain specificity differs from papain, that
prefers Leu and Val at P2 and from bromelain, that accepts Arg at
this position
[51]
but is similar to human cathepsin L and V
pro-teinases (as well as cruzain and
L. mexicana
cathepsin L) that also
prefers aromatic to aliphatic amino acids interacting at S2 subsite
[2,29,51]
.
3.7. Effect of proteinaceus peptidase inhibitors on baupain
activity
Inhibitors from the Bowman–Birk and plant Kunitz-type family
have been characterized by proteinase specificity, primary
struc-ture and reactive site residues
[52]
. Since they present differences in
proteinase inhibition profiles, they are valuable tolls for proteolytic
enzyme characterization. In this sense, the distinctive substrate
preference observed with baupain was further studied accessing
the effect of both plant and mammals proteinaceous peptidase
inhibitors on its hydrolytic activity.
Plant Kunitz type inhibitors STI (Soybean trypsin inhibitor) and
EcTI (Enterolobium contortisiliquum
trypsin inhibitor), as well as
the cysteine proteinase inhibitor isolated from
Bauhinia
bauhin-ioides
(BbCI) do not inhibit baupain at the assayed concentrations
(0.005–0.5
M). While the absence of inhibition observed with
both trypsin like inhibitors would be a expected feature due to
baupain cysteine proteinase nature, it is interesting to observe
that BbCI does inhibits cathepsin L, cruzain and cruzipain, but it
also fails to inhibit papain
[40]
, sustaining a close relationship
between both enzymes. Canecystatin, a recombinant cysteine
pro-teinase inhibitor from sugar cane, efficiently inhibits baupain (K
iapp10
−8M) as well as papain (K
iapp
= 3.3
×
10
−9M) and cathepsin L
(K
iapp= 0.6
×
10
−9M) but no inhibitory effect was observed on ficin
or bromelain
[53]
.
As observed with papain
[54]
, a strong inhibition of baupain
(K
iapp= 1.9
×
10
−8M) was also obtained with human high
molec-ular weight kininogen (HMWK) (
Fig. 6
), a plasma multifunctional
glycoprotein that besides their role as precursor of the vasoactive
peptides kinin, posses the sequence motif QXVXG in cysteine
pro-teinase inhibitor domains
[55]
that is reported to inhibit cysteine
proteinases papain family
[4]
. Is worth to point out to the fact that
canecystatin, a baupain inhibitor also encompass the conservative
QXVXG motif in contrast to BbCI which does not affect the enzyme
activity.
3.8. Baupain kininogenase activity
The kininogenase activity of some plant cysteine proteinases
(papain, ficin and bromelain) on human plasma was long ago
reported by Prado
[56]
, more recently this characteristic was
reported for cathepsin L but not for cathepsin B. The kininogenase
activity of cathepsin L was reinforce by the release of bradykinin
from HMWK
[57]
and also from synthetic bradykinin-containing
fragment of kininogen
[58]
. This property was also detected for
baupain. The baupain kininogenase activity was observed using
stoichiometric amounts of enzyme and kininogen. The kinin release
was quantified by radioimmunoassay (
Fig. 7
A). This activity was
abolished by the addition of the irreversible cysteine proteinase
inhibitor E-64, demonstrating the specificity of assay. Baupain
cleavage of HMWK generates one major band of 45 kDa and that
0.4 0.3
0.2 0.1
0 1
0.8
0.6
0.4
0.2
0
HMWK (uM)
Residual Activity
(%)
Kiapp = 1.9 x 10-8 M
Fig. 7.(A) Effect of baupain on bradykinin release. Bradykinin levels were determined by radioimmunoassay[44]in triplicate determinatives. (B) Degradation of HMWK (10g) by baupain (0.23M) demonstrated by SDS-PAGE 12%. (S) Standard range of molecular weight protein markers 20–94 kDa. (A) Control HMWK in 0.1 M sodium phosphate buffer, pH 6.3, containingl-cysteine 2 mM. (B) Control HMWK in 0.1 M sodium phosphate buffer, pH 6.3. (C) Baupain and HMWK were incubated for 10 min at 37◦C. (D) Baupain and HMWK incubated for 60 min at 37◦C. (E) DTT reduced HMWK.
hydrolysis profile is not modified in the period of 10 at 60 min of
incubation (
Fig. 7
B), probably by the impairment of the enzyme
activity by the HMWK cysteine proteinase inhibitor domains. It is
worth to emphasize that this impairment was not observed using
other proteinaceous substrate such as insulin B-chain and the
ser-ine proteinase inhibitor isolated from the seeds of
Bauhinia forficata
(BfTI) (data not shown). As baupain digests proteins effectively and
rapidly yielding numerous proteolytic fragments, suggesting that
baupain is involved in endogenous protein degradation.
Bradykinin excised from HMWK requires the cleavage of
the
. . .
MK-R
. . .
and
. . .
FR-S
. . .
bonds at the N- and C-terminal
bradykinin insertion sites, respectively. In this sense, while
bau-pain specificity data support both cleavages (Phe and Met were
accepted at P2; Lys and Arg at P1; Ser at Pˇı1 –
Fig. 5
), the
kinino-genase activity data indicate that baupain also accepts Arg at Pˇı1,
as observed with cathepsin L
[59]
.
These results provide structural and biochemical information
on the cysteine proteinase isolated from the leaves of
B. forficata
which is utilized in popular therapy for diabetes mellitus.
Bau-pain is similar to other cathepsins and closer to cathepsin L shown
to be essential for the development of type I diabetes in
non-obese diabetic mice
[60]
. Indeed, it is important to consider the
kininogenase activity of baupain that releases bradykinin (BK) from
HMWK. Bradykinin modulates the release of insulin
in vivo
[61,62]
and the stimulation of insulin secretion by beta cells occurs through
activation of B2 receptor
[63,64]
. These results were confirmed
using kininogen-deficient rats in which the release of insulin
stim-ulated by glucose administration was lower than in normal rats
[65–68]
. Our results suggest that the kininogenase activity of
bau-pain may be involved in the hypoglycemic property of
Bauhinia
forficata
leaves. However, further studies are necessary to
demon-strate the effectiveness of baupain in diabetes.
4. Conclusion
By acetone precipitation and different chromatographic steps an
endoproteinase named baupain detected in the
B. forficata
leaves
was purified to homogeneity. While the N-terminal sequence
sim-ilarity, molecular mass, circular dicroism spectra and intrinsic
fluorescences profiles points to a close structural relationship to
papain, its activity on FRET and MCA peptides indicates substrate
specificity more related to the mammalian cathepsin L enzyme. The
proteinase also shares with cathepsin L and papain the capacity of
releasing bradykinin from HMWK.
In conclusion, this work point out the kininogenase activity
specificity profile of baupain that is distinct from other cysteine
proteinases and similar to cathepsin L
[57]
. We believe that this
property may be relevant to regroup cluster papain-like cysteine
proteinases.
Acknowledgments
This work was supported in Brazil by Fundac¸ão de Amparo à
Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq).
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