A serine proteinase inhibitor isolated from Tamarindus indica seeds
and its effects on the release of human neutrophil elastase
J.M.S.L.L. Fook
a,b, L.L.P. Macedo
a, G.E.D.D. Moura
a, F.M. Teixeira
a, A.S. Oliveira
a,c,
A.F.S. Queiroz
a, M.P. Sales
a,T
a
Departamento de Bioquı´mica, Centro de Biocieˆncias, Universidade Federal do Rio Grande do Norte, Campus Universita´rio, 59072-970, Natal, RN, Brasil
bDepartamento de Morfologia, Centro de Biocieˆncias, Universidade Federal do Rio Grande do Norte, Natal, RN, Brasil c
Departamento de Bioquı´mica e Biologia Molecular, Centro de Cieˆncias, Universidade Federal do Ceara´, Fortaleza, Ce, Brasil
Received 20 June 2004; accepted 5 October 2004
Abstract
Proteinaceous inhibitors with high inhibitory activities against human neutrophil elastase (HNE) were found in seeds of the Tamarind tree (Tamarindus indica). A serine proteinase inhibitor denoted PG50 was purified using ammonium sulphate and acetone precipitation followed by Sephacryl S-300 and Sephadex G-50 gel filtration chromatographies. Inhibitor PG50 showed a Mrof 14.9 K on Sephadex G-50 calibrated column and a Mrof 11.6
kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PG50 had selective activity while cysteine proteinases (papain and bromelain) and serine proteinases (porcine pancreatic elastase and bovine chymotrypsin) were not inhibited, it was strongly effective against serine proteinases such as bovine trypsin and isolated human neutrophil elastase. The IC50 value was determined to be 55.96 Ag.mL 1. PG50 showed neither cytotoxic nor
haemolytic activity on human blood cells. After pre-incubation of PG50 with cytochalasin B, the exocytosis of elastase was initiated using PAF and fMLP. PG50 exhibited different inhibition on elastase release by PAF, at 44.6% and on release by fMLP, at 28.4%. These results showed that PG50 preferentially affected elastase release by PAF stimuli and this may indicate selective inhibition on PAF receptors.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Tamarindus indica; Human neutrophil elastase; PAF; fMLP
0024-3205/$ - see front matterD 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.10.053
TCorresponding author.
Introduction
Proteinase inhibitors are widely distributed among bacteria, animals and plants. They are present in
their reproductive and storage organs and vegetative tissues of most plant families (
Ryan, 1990; Shewry
and Lucas, 1997
). They have regulatory and defensive roles and act as storage proteins (
Xavier-Filho,
1993
). Among the various groups of proteinase inhibitors, the serine proteinase inhibitors are the most
studied and have been isolated from various leguminous seeds (
Oliveira et al., 2002; Macedo et al.,
2002; Mello et al., 2002; Oliva et al., 2000
). Serine proteinase inhibitors are effective against various
insect enzymes and therefore have been studied as an alternative approach to pest control (
Reckel et al.,
1997
). Other uses of these inhibitors have been suggested, such as in the prevention of carcinogenesis
(
Kennedy, 1998
). Serine proteinase inhibitors have also been shown to control abnormal exocytosis and
secretion of endogenous proteinases which is characteristic of a number of diseases (
Kennedy, 1998
).
The abnormal exocytosis of elastase, a serine proteinase from human neutrophil, is considered to be the
primary source of tissue damage associated with inflammatory diseases as pulmonary emphysema
(
Janoff, 1985; Groutas, 1987; Fujita et al., 1990; Nishi et al., 2003; Ishizawa et al., 2004
), adult
respiratory distress syndrome (
Lee et al., 1981; McGuire et al., 1982; Gossage et al., 1993; Yamazaki et
al., 1999; Carney et al., 2001; Nakayama et al., 2002
), septic shock (
Uchida et al., 1995; Fujie et al.,
1999; Dhainaut et al., 2001; Dhainaut, 2002; Devine, 2003
), cystic fibrosis (
O’Connor et al., 1993;
Hansen et al., 1995; Lee and Downey, 2001; McGarvey et al., 2002; Greer et al., 2004
), Chronic
bronchitis (
Fujita et al., 1990; Llewellyn-Jones et al., 1996; Moodley et al., 2000; Lai et al., 2004
) and
rheumatoid arthritis (
Mohr and Wessinghage, 1983; Odagaki et al., 2001; Tur et al., 2004
). Compounds
which directly inhibit elastase or its release from human neutrophils are of increasing interest in the
development of new anti-inflammatory drugs. In the present study, we have isolated and purified
proteinaceous inhibitors from seeds of Tamarindus indica and examined its effects on human neutrophil
elastase (HNE) release by PAF (Platelet activating factor) and fMLP
(N-formyl-methionyl-leucyl-phenylalanine) stimulus.
Material and methods
Material
Seeds from Tamarindus indica were obtained from IBAMA (Environmental Institute of Brazil) seeds
bank in Natal, RN, Brazil. Leukocytes-rich blood from health adults was obtained from HEMONORTE
(blood bank), Natal, RN, Brazil. HNE substrate N-succinyl-L-alanyl-L-valine-p-nitroanilide (SAAVNA)
was purchased from Bachem Feinchemikalien AG. Cytochalasin B, PAF (Platelet activating factor),
fMLP (N-formyl-methionyl-leucyl-phenylalanine), Dimethylformamide from Sigma. Proteinases:
Porcine pancreatic elastase, bovine trypsin, bovine chymotrypsin, bromelain and papain were purchased
from Sigma. Polidet P-40 was obtained from Polysciences, Inc.
Inhibitor isolation and purification
Finely ground T. indica seed meal was extracted with 0.1 M PBS, pH 7.4, for 30 min at room
temperature. After centrifugation for 30 min at 12000 g at 4 8C, the supernatant (crude extract) was
precipitated with ammonium sulfate at concentrations of 0–30, 30–60, and 60–90%. The F
30–60fraction,
which corresponds to a 30–60% saturation range, showed a high level of inhibitory activity against
HNE. This fraction was precipitated again with acetone at 0.5 vol (F
50), 1.0 vol (F
100) and 2.0 volumes
(F
200). The F
100, which had high level of anti-elastase activity, was applied to a Sephacryl S-300 SH
size-exclusion column (80 2.5 cm), equilibrated with 0.1M PBS, pH 7.4. Fractions of 1.5 mL were
collected at a flow rate of 20 mL.h
1. The ninth anti-elastase activity peak, denoted FS9, was pooled,
concentrated and then applied to a Sephadex G-50 gel filtration column (116 1.5 cm) and fractions
were collected at flow rate 18 mL.h
1. The Sephadex G-50 column was calibrated with molecular
weight protein markers (Mr: 30.1, 20.1, 12.4 and 5.7 kDa). The peak with anti-elastase activity, denoted
PG50, was pooled, concentrated and subjected to further analysis.
Protein determination
Total protein content was measured as described by
Bradford (1976)
with bovine serum albumin as
the standard protein. All chromatographies were monitored at 280 nm.
Polyacrylamide Gel Electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted as described
by
Laemmli (1970)
at room temperature. Protein molecular weight markers (Full-Range Rainbow
Molecular Weight Markers) were purchased from Amersham Pharmacia. Bromophenol blue was used as
a tracking dye.
Human neutrophil elastase activity isolation
Human neutrophils elastase activity was isolated from leukocytes-rich blood units obtained from
healthy volunteers. Erythrocytes were lysed as described by
Grimberg et al. (1989)
with modifications.
Isolation of neutrophils elastase activity followed two steps: In the first step, 150 mL of blood was added
to 350 mL of red blood lyses solution (3.5 mM NH
4HCO
3, 530 mM NH
4Cl). The mixture remained on
ice for 30 min and then the lysate was centrifuged at 1650xg for 5 min at 4 8C. The pellet was washed
again with red blood lyses solution and centrifuged as before. In the second step, the pellet obtained,
denoted white cell mass, was rinsed with cold 0.1M PBS pH 7.4 and cells ( around 1 10
65 10
6cells/mL) were lysed using 0.1M PBS pH 7.4, 12.5% polidet P-40 solution for 24 hours at 5 8C. After
centrifugation for 15 min at 12000g at 4 8C, the supernatant (HNE extract) was used in the elastase
assays with its specific substrate (SAAVNA).
Human neutrophil elastase assay
The elastase assay using SAAVNA as substrate was quantified by photometric measurement of
p-nitroanilide, the colored product of the cleavage of SAAVNA (
Johansson et al., 2002
). Enzymatic
extracts (HNE extract) at 12.5, 25, 50, 75, 100 and 200AL volumes were incubated with 0.1M PBS pH
7.4 adjusted to a final volume of 500AL. Reactions were started with the addition of 2.5 Al of 0.3 M
SAAVNA solution. After 60 min at 37 8C, the reaction was stopped by the addition of 250AL of 2.0%
citric acid solution. These samples were centrifuged and supernatants were measured by absorbance at
405 nm. An elastase activity curve was obtained and 50 Al enzymatic extract was used to further assays.
All assays were performed in triplicate.
Human neutrophil elastase inhibitory assay
The elastase inhibitory assay was performed using SAAVNA as substrate, according to
methodology developed by
Johansson et al. (2002)
, with modifications. 50 Al of human neutrophil
elastase was incubated for 15 min at 37 8C with 50Ag. mL
1of samples and 0.1M PBS pH 7.4
adjusted to a final volume of 500AL. Reactions were started with addition of 2.5 Al of 0.3 M
SAAVNA solution. After 60 min at 37 8C, the reaction was stopped by addition of 250 AL of 2.0%
citric acid solution. These samples were centrifuged and supernatants were measured by absorbance at
405 nm. One unit of inhibitory activity was defined as the amount of inhibitor that decreased
absorbance by 0.01 at 405 nm. All assays were made in triplicate. The results of each series were
expressed as the mean value F SD.
Determination of IC
50value of PG50 to human neutrophil elastase
The inhibitory capacity of PG50 towards HNE activity was determined. Volume of 50 AL of Human
neutrophil elastase was incubated for 15 min at 37 8C with various concentrations of PG50 (10 to 320
Ag.mL
1). Reactions were started after addition of 2.5 Al of 0.3 M SAAVNA solution. After 60 min at 37
8C, the reaction was stopped by addition of 250AL of 2.0% citric acid solution. These samples were
centrifuged and supernatants were measured by absorbance at 405 nm. All assays were made in triplicate.
IC
50was defined as the concentration of PG50 required for achieving 50% inhibition of the enzyme.
Assay of PG50 Inhibitory activity against serine and cysteine proteinases
Inhibitory activity of the PG50 towards closely related serine proteinases (pancreatic elastase, trypsin
and chymotrypsin) and cysteine proteinases (bromelain and papain) was tested. Stock solutions of the
enzymes were prepared: trypsin (0.3 mg.mL
1in 0.0025M HCL), chymotrypsin (0,1 mg.mL
1, 0.1 M
PBS, pH 7.4), pancreatic elastase (0,1 mg.mL
1, 0.1 M PBS, pH 7.4), papain (0,1 mg.mL
1, 0.1 M PBS,
pH 7.4) and bromelain (0,1 mg.mL
1, 0.1 M PBS, pH 7.4). PG50 (0.05mg.mL
1, 0.1 M PBS, pH 7.4)
was used as inhibitor solution. The preincubation reaction with trypsin contained 10 Al of enzyme
solution, 50 Al of inhibitor solution, and 440 Al of 0.1 M PBS, pH 7.4. The preincubation reaction with
chymotrypsin contained 40 Al of enzyme solution, 50 Al of inhibitor solution, and 410 Al of 0.1 M PBS,
pH 7.4. The preincubation reaction with pancreatic elastase contained 40 Al of enzyme solution, 50 Al of
inhibitor solution, and 410 Al of 0.1 M PBS, pH 7.4. The preincubation reaction with papain contained 15
Al of enzyme solution, 40 Al of an activation solution containing 0.02 M ethylenediamine tetroacetic acid
(EDTA) and 0.05 M cysteine, pH 8.0, 40 Al of 0.1 M PBS. The preincubation reaction with bromelain
contained 20 Al of enzyme solution, 40 Al of an activation solution containing 0.02 M ethylenediamine
tetroacetic acid (EDTA) and 0.05 M cysteine, pH 8.0, 40 Al of 0.1 M PBS. After this 50 Al of inhibitor
solution was added with 335 Al of 0.1 M PBS, pH 7.4. All preincubation reactions were performed for 10
min at 37 8C. Reactions were performed 15 min at 37 8C and reactions were started after addition of 200
Al of 1% azocasein for 30 min. After 30 min at 37 8C, the reaction was stopped by the addition of 150 Al
of 20% TCA solution. Samples were centrifuged and supernatants were alkalinized with 0.25 N NaOH.
The soluble peptides were measured by absorbance at 440 nm. One unit of inhibitory activity was
defined as the amount of inhibitor that decreased absorbance by 0.01 at 440 nm. All assays were made in
triplicate. The results of each series were expressed as the mean value F SD.
Cytotoxicity and Haemolytic assay
The effect of PG50, at its IC
50, on blood cell viability, was studied. Cells were incubated for 15 min at
37 8C, with either PG50 or controls (without inhibitor). Viability of cells was checked by flow cytometry.
Effect of PG50 on the release of human neutrophil elastase stimulated by PAF and fMLP
The assay was performed according to
Johansson et al. (2002)
, with modifications. Isolated
neutrophils (1 10
65 10
6ce´lls/mL) and cytochalasin B (2.5 Ag) in PBS with Ca
2+/Mg
2+, 2.5%
BSA were incubated with PG50 at its IC
50and SAAVNA (0.8 mM final concentration) for 5 min at 37
8C. The reaction was initiated by the addition of 50 AM PAF or 50 AM fMLP. After incubated for 10 min
at 37 8C, the reactions were stoped with 2 % acid citric solution and samples were centrifuged and
absorbance of the supernatant was measured at 405 nm. All assays were made in triplicate. The results of
each series were expressed as the mean value F SD.
Statistical analysis
The levels of statistical significance were calculated using a paired Student’s t-test, through GraphPad
InStat program.
Results and discussion
Elastase from human neutrophil granulocytes is a serine proteinase that has the ability to degrade
elastin, fibronectin, proteoglycans and plasma proteins; it is also a modulator of the inflammatory cell
0.0 0.5 1.0 1.5 2.0 2.5 150 200 250 300 350 400 450 Volume(mL) O.D. 280 nm 0 10 20 30 40 50 60 70 80 90 % inhibition
Fig. 1. Eluation profile on Sephacryl 300-SH of HNE inhibitor from Tamarindus indica seeds. The F100 (acetone protein fraction) with high level anti-elastase activity was aplied to a column (80 2.5 cm), equilibrated with 0.1M PBS, pH 7.4. Fractions of 1.5 mL were collected at flow rate of 20 mL.h-1and monitored for protein content at 280 nm ( ). The detached last peak (FS9), with HNE inhibitory activity (E), was detached and used to posterior purification steps.
functions, such as lymphocytes activation and platelet aggregation (
Owen et al., 1997
). Increased release
of elastase result in an imbalance between elastase and its endogenous inhibitor, a-anti-trypsin (
Siedle et
al., 2003
). The abnormal accumulation of elastase causes a number of acute and chronic inflammation
diseases (
Bernstein et al., 1994
); therefore, there is demand for specifics and potent exogenous inhibitors
of proteinases, such as HNE, associated with these inflammatory processes (
Sternlicht and Werb, 1999
).
In this study a serine proteinsase inhibitor from Tamarindus indica seeds with high activity against HNE
was detected, isolated and purified. The F100 acetone protein fraction showed higher anti-elastase
activity than the others acetone, so this fraction was then applied on Sephacryl S-300 column. Peaks
obtained were assayed against isolated HNE. Nine inhibitory activity peaks (
Fig. 1
) were obtained, peak
denoted SP9, was choser for further assays, due to its high anti- HNE activity. SP9 was applied to a
calibrated gel filtration Sephadex G-50 chromatography, an anti-elastase activity peak, denoted PG50,
with a molecular weight of 14.9 kDa (
Fig. 2
) was obtained. In this purification stage, HNE inhibitor was
purified 80 times (
Table 1
). SDS-PAGE analysis revelead the presence of a protein band with a
molecular mass of 11.6 kDa (
Fig. 3
).
Several studies have been performed on plant proteinase inhibitors, especially on those isolated
from leguminous family (
Batista et al., 1996; Macedo et al., 2000; Oliva et al., 2000; Troncoso et al.,
2003
). These seeds possess serine proteinase inhibitors that belong to various inhibitor families such
0.0 0.1 0.1 0.2 0.2 0.3 0.3 80 90 100 110 120 130 Volume (mL) O.D. 280 nm 24 26 28 30 32 34 36 38 40 % inhibition 30.1 20.1 14.9 12.45.7 MW (Kda)
Fig. 2. Eluation profile on Sephadex G-50 of FS9 anti-elastase activity peak was pooled, concentrated and then applied to a column (116 1.5 cm). Fractions of 1.5 mL were collected at flow rate 18 mL.h-1and monitored for protein content at 280 nm (x). The Sephadex G-50 column was calibrated with molecular weight protein markers (Mr: 30.1, 20.1, 12.4 and 5.7 kDa). Bar corresponds to HNE inhibition. PG50 was detached and used to posterior characterization.
Table 1
Sumary of purification of the HNE inhibitor from Tamarindus indica seeds Fractions Total inhibitory
activity
Total protein (mg) Especific Activity (UI/mg protein) Purification (X) Recovery (%) CE 252710.0 1110.0 227.3 1.0 100 F30–60 289929.4 668.0 434.0 1.9 97.7 F100 14305.7 32.2 444.0 2.0 5.7 PS9 642.2 0.3 2451.6 10.8 0.3 PG50 602.8 0.03 18333.3 80.5 0.2
as Kuntiz, Bowman-Birk, Potato I and II, Squash, cereal super family and Thamatin-like types
(
Richardson, 1991
). The Kunitz and Bowman-Birk inhibitor families are involved in many biological
processes and therefore have been studied as potential therapies. In this study we purified an inhibitor
with Mw 11.6~14.9 kDa that inhibited only HNE and trypsin (
Table 2
).
Extracts of several legumes such as Soybean (Glycine max), Lima bean (Phaseolus limensis), Red
kidney bean (Phaseolus vulgaris), Adzuki bean (Phaseolus angularis) and Lentil (Lens sculenta) are
rich sources of HNE inhibitors (
Hojima et al., 1983
). These inhibitors from legumes are part of the
Kuntiz or Bowman-Birk family, however, Bowman-Birk inhibitors, with 6–8 kDa have high activity
to both HNE and trypsin. The Kunitz inhibitor isolated from potato tubers, PSPI-21, with a Mw of 21
kDa, is a potent inhibitor of HNE, and a normal inhibitor of trypsin and chymotrypsin (
Valueva et al.,
1998
). In comparison, PG50 was more similar in activity with Bowman-Birk inhibitors, but with a
molecular mass of 11.6~14.9 kDa. The calculated IC
50value of PG 50 was 55.96 Ag.mL
1(
Fig. 3
),
lower than that found by
Johansson et al. (2002)
who studied 100 plants from 96 species that were
used as exocytosis inducers. No cytotoxicity and haemolytic activity on cells were observed at IC
50value (data not showed) (
Fig. 4
).
Table 2
Inhibitory activity of PG50 toward cysteine and serine proteinases
Enzymes Inhibition (%)
Papain NdT
Bromelain NdT
Trypsin 77.7F2.3
Chymotrypsin NdT
Porcine Elastase pancreatic Nd*
TNot detecble. 250 k 160 k 105 k 75 k 50 k 35 k 30 k 25 k 15 k 10 k 250 k 160 k 105 k 75 k 50 k 35 k 30 k 25 k 15 k 10 k M 1 2 3 4 M 5 A B
Fig. 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein fractions from Tamarindus indica seeds. (A) 1. Crude extract; 2. F30-60; 3. F100; 4. FS9. (B) 5. PG50; M. Protein molecular weight markers (Full-Range
In experiments to detect natural anti-inflammatory products, the agent cytochalasin B is used,
which, together with stimulation by PAF and fMLP, induces the release of Elastase. This enzymatic
activity is detected by the formation of pNA ( p-nitroanilide), degradation product of SAAVNA
substrate (
Johansson et al., 2002
). The inhibition of elastase release is indicative of a candidate to an
anti-inflammatory product. PG50, at a concentration of 55.96 Ag.mL
1, inhibited pNA formation in
44.6% to PAF induction, and 28.4% to fMLP induction (
Fig. 5
). Stimulation with PAF results in
utilization of intracellular release p38 MAPK (p38 mitogen-activated protein kinase) pathway, rather
than by p42/44 pathway as is utilized by the fMLP inducer (
Nick et al., 1997; Partrick et al., 2000
).
These results showed that PG50 preferentially affected the release of elastase by PAF stimuli and this
may indicate selective inhibition by PAF receptors.
0 10 20 30 40 50 60 T1 T2 % Inhibition
Fig. 5. Effect of PG50 on the release of human neutrophil elastase stimulated by PAF and fMLP. T1: Inhibition of PG50 on the release of elastase stimulated by fMLP; T2: Inhibition of PG50 on release of elastase stimulated by PAF. Each column represents mean F S.E. P b 0.05; Student t test for paired samples (bar T1 vs. T2).
0 10 20 30 40 50 60 70 80 90 100 0 50 100 150 200 250 300 350 µg.ml-1 Residual activity (%)
Fig. 4. Inhibitory effect of PG50 against HNE. Enzyme was inhibited by increasing amounts of PG50 and inhibitory activity was monitored using SAAVNA, a HNE substrate, at pH 7.4. The 100% of activity refers to the absorbance of the sample without PG50. IC50was defined as the concentration of PG50 required for achieving 50% inhibition of the enzyme. Each value
Acknowledgments
This work was supported by the following Brazilian Agencies: CAPES, CNPq and PRONEX.
The authors thank Dr Rosana Lucena de Sa´ Leita˜o, Dr Dulce H. Seabra de Souza Silva and Silene
Telma Lima de Santana, pharmacists from Hemonorte, Natal, RN, Brazil, for the rich leukocytes
blood bags donation. Also we thank Dr. Daniel Leung, Dr. Corina F. B. Braga and Dr. Sathyabama
Chellappa for review of manuscript.
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