Escolar Documentos
Profissional Documentos
Cultura Documentos
net
108
Department of Biotechnology, Indian Institute of Technology Roorkee-247667, India; 2Medicinal and Process Chemistry Division. CSIR-Central Drug Research Institute; Lucknow-226001, India; 3Plant Molecular Biology Division, CSIRNational Botanical Research Institute, Lucknow-226001, India; Present Address: Department of Biotechnology,
Graphic Era University Dehradun-248002, India
Abstract: A thermotolerant protein with trypsin inhibitory activity designated as CaTI was purified to homogeneity from
seeds of Cassia absus. Gel filtration chromatography and SDS-PAGE analysis showed the apparent molecular mass of
~20 kDa. Partial internal sequences indicate that CaTI belongs to Kunitz-inhibitor family. CaTI inhibits the bovine trypsin
in 1:1 molar ratio and exhibited a competitive-type inhibitory activity with Ki = 5.6 10-9 M. The inhibitory activity was
retained over a broad pH range (2-12). Thermal stability study showed that it is stable up to 80 C and inhibition activity
reduced at and above 90 C which might be due to the presence of predominantly -sheets revealed by the CD study. The
proteolysis studies of CaTI exhibited strong resistance to proteolysis by different proteases tested. The studies show that
CaTI can be used as potential candidates for the development of the transgenic plant against the microbes and insect pests.
Keywords: Cassia absus, Caesalpiniaceae, Kinetic study, Stability studies, Trypsin inhibitor.
INTRODUCTION
Plant Kunitz protease inhibitors (PIs) are widely distributed in plant and are involved in controlling endogenous
proteolysis. These PIs are small proteins and act as potent
natural defence agent against pathogens and the predators [1,
2]. Generally, they are present at higher concentration in the
storage tissues, but also induced in other plant tissues by
biotic stress like herbivory or wounding [3]. The inhibition
activity of the PIs is due to their capacity to form stable
complex with the target protease by blocking, altering or
preventing to access the enzyme active sites leading to unavailability of the amino acid necessary for the growth and
development of the insects and microbes [4,5]. The plant PIs
could also play important endogenous roles to regulate the
uncontrolled proteolysis during seed dormancy, reserve protein mobilization, and protection from foreign proteolytic
enzymes of pests and pathogens. Moreover, PIs may also act
as storage or reserve proteins [6]. Besides blocking the protease action, few PIs play a critical role in the modulation of
the programme cell death in soybean [7], growth factor activities, receptor clearance signalling [8]. It is reported that
consumption of proteins isolated from the plant source constituted with PI, reduces the incidence of breast, colon, prostate, oral and pharyngeal cancers [9].
*Address correspondence to these authors at the Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247 667, India; Tel:
+91-1332-285657; Fax: +91-1332-273560; E-mail: aksbsfbs@yahoo.co.in,
aksbsfbs@iitr.ernet.in
Medicinal and Process Chemistry Division, Central Drug Research Institute,
CSIR, Lucknow, 226001, India; Tel: +9152226124112/ex 4386; Fax:
+9152226123405; E-mail: anilsak@gmail.com
109
Patel et al.
The purified CaTI was subjected to the mass spectroscopic analysis in tandem mass spectrometer. The partial
internal sequencing revealed six different peptides with significant score. Out of these six, first two peptides were found
similar to the sequence present in other known Kunitz trypsin
inhibitors like Acacia confusa trypsin inhibitor [25] and Kunitz-like trypsin inhibitor-like 2 protein from Medicago truncatula (XP_003619553.1) shown in Figure 2. The other four
peptides (FSSXSR, TVVQPVI/LGDR, I/LI/LFI/LHETFR
and I/LVEEGEGTGFVGPFR) did not show sequence similarity to any known trypsin inhibitor. This indicated that the
protease inhibitor discovered in present study is a new entry
in Kunitz family.
Inhibition Properties
The purified CaTI was used for proteolytic stability studies against different proteases. CaTI was incubated with different protease such as proteinase K, bovine trypsin and
chymotrypsin in the molar ratio 20:1(inhibitor: enzyme) in
50 mM Tris-HCl buffer, pH 8.0 while papain in 50 potassium phosphate buffer, pH 7.0 for different time interval (30,
60 and 90 min). After incubation, reducing sample buffer
was added and boiled for 5 min. The stability of CATI
against different protease was analyzed on 15 % reducing
SDS-PAGE gel using BSA as a control.
111
a
b
Figure 1. a) Purity and molecular mass determination of CaTI protein under reducing condition on 12% SDS-PAGE gel. L1, molecular mass
standards; L2, crude extract; L3, 50 mM NaCl eluted fraction; L4, 100 mM NaCl fraction from DEAE; L6, 200 mM NaCl fraction; L5
Eluted fraction from HiLoad 16/60 Superdex-75 column (GE Healthcare).
b) Elution profile of CaTI protein from HiLoad 16/60 Superdex-75pg (120mL) column.
(a) Peptide 1
a
(b) Peptide 2
b
Figure 2. Amino acid sequence similarity of CaTI with other trypsin inhibitors.
cating CaTI exhibits different inhibitor specificity with trypsin and chymotrypsin inhibition activities. The stability study
of CaTI against the protease like trypsin, chymotrypsin and
proteinase K was also determined at different time (30, 60
and 90 min) with BSA as a control protein. The CaTI was
found completely stable against the trypsin and fairly stable
against chymotrypsin but sensitive against the proteinase K
and papain (Fig. 5). The resulted peptide after digestion with
proteinase K and papain show darker region in the gel which
are the less than 14 kDa. Such peptide bands are absent in
case of trypsin indicating stability of CaTI against trypsin
(Fig. 5). The stability of CaTI at pH 10.0 was determined
from 15 min to 20 h and was found stable until 20 h with 5%
loss in inhibition activity.
CD Study of CaTI
The CD spectrum of Far-UV region of a protein is the
most sensitive physical technique to determine and analyze
the secondary structure content of a protein. The result represents the negative maximum at 202 nm in CD spectrum of
CaTI, which is unusual for the most of the proteins but hallmarks for the Kunitz proteins (Fig. 6). The CD data recorded
at pH 7.0 in 20 mM potassium phosphate buffer was analysed by three different methods namely CONTIN, K2D and
CDSSTR. The absence of negative peak at 208 and 222 nm
indicate the lack of helical structure. CD spectrum analysis
of CaTI by CONTIN, K2D and CDSSTR methods showed
that CaTI contains 3.7%, 4% and 10% of -helix respectively while -sheets were found to be 43% , 48% and 32%
by above methods respectively.
Patel et al.
100
80
60
10
12
pH
a
100
Figure 5. Stability of CATI against the different protease at different time interval. L1, molecular mass standards; L2, BSA only; L3,
Incubation of BSA with Proteinase K for 90 min as control, L4-L6,
Incubation of CaTI with Proteinase K for 30, 60 and 90 min respectively; L7, Incubation of BSA with trypsin for 90 min as control;
L8-L10, Incubation of CaTI with Proteinase K for 30, 60 and 90
min respectively. L11, Incubation of BSA with chymotrypsin for 90
min as control; L12-L14, Incubation of CaTI with chymotrypsin for
30, 90 and 60 min respectively; L15, Incubation of BSA with papain for 90 min as control; L16-L19, Incubation of CaTI with papain for 30, 90 and 60 min respectively.
5000
60
80
40
20
-5000
-10000
-15000
0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
-20000
190
b
Figure 3. a) Inhibition activity at different pH (Incubated at different pH for 30 min).
b) Molar Inhibition ratio of CaTI with trypsin.
200
210
220
230
240
Wavelength (nm)
DISCUSSION
Cassia absus is a well known plant and all parts possesses different medicinal properties [17]. The purification
employed simple two steps of ion exchange and gel filtration
chromatography. In the present study we have demonstrated
that CaTI is a single chain Kunitz inhibitor isolated from the
seed of Cassia absus. Reports revealed that single chain Kunitz-type inhibitors are characteristic feature of subfamily
Caesalpiniaceae [13, 27]. CaTI constitutes about 15-18% of
total mature seed proteins indicating to be a storage protein.
This is the main protein of the seed therefore; it may play a
pivotal role in germination, growth, development and defence. A numbers of the trypsin inhibitors have been characterised and possess storage as well as plant defence properties [27-29]. The apparent molecular weight of CaTI was
found to be 20 kDa by both, gel filtration chromatography
and SDS-PAGE analysis under reducing condition. These
113
bovine trypsin and less stable against proteinase K, and papain. CD analysis showed that CaTI primarily consists of sheets. To understand the high stability of CaTI, 3D structure
could be determined by crystallization. Plant Kunitz inhibitors are thought to be the important defence protein particularly against the insect pests as they inhibit the activity of the
digestive gut proteases leading to poor digestion of dietary
proteins and unavailability of important amino acids which
ultimately causes death. The protease inhibitors studied in
present study may be useful in the development of resistant
transgenic plants like the insect resistant cauliflower using
the trypsin inhibitor gene from the sweet potato.
ABBREVIATIONS
PIs
Protease inhibitors
CaTI
BTEE
BAPNA
N-benzoyl-L-arginine-p-nitroanilide
BSA
CONFLICT OF INTEREST
There are none.
ACKNOWLEDGEMENTS
CD studies were performed in NMR facility at Institute
Instrumentation Centre (IIC), IIT Roorkee and Mass Spec
analysis in Proteomics Facility at CSIR-National Botanical
Research Institute, Lucknow. We gratefully acknowledge Dr.
D.C. Saini, Scientist E, Birbal Sahni Institute of Palaeobotany, Lucknow, India for the identification of the
plant. G.K. Patel acknowledges MHRD, A.K. Gupta to
ICMR and M. Mishra to CSIR, Government of India for fellowships. The CDRI communication number allotted to this
manuscript is 8472.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
Ryan, C.A. Protease nhibitors in plants: genes for improving defences against insect and pathogens. Annu. Rev. Phytopathol.,
1990, 28, 425-449.
Koiwa, H.; Bressan, R.A. Hasegawa PM Regulation of protease
inhibitors and plant defense. Trends Plant Sci., 1997, 2, 379-384.
Lee, J.S.; Brown, W.E.; Graham, J.S.; Pearce, G.; Fox, E.A.; Dreher, T.W.; Ahern, K.G.; Pearson, G.D.; Ryan, C.A. Molecular
characterization and phylogenetic studies of a wound-inducible
proteinase inhibitor I gene in Lycopersicon species. Proc. Natl.
Acad. Sci., 1986, 83, 7277-7281.
De Leo, F.; Volpicella, M.; Licciulli, F.; Liuni, S.; Gallerani, R.;
Ceci, L.R. Plant-PIs: a database for plant protease inhibitors and
their genes. Nucl. Acids Res., 2002, 30, 347-348.
Bhattacharjee, C.; Manjunath, N.H.; Prasad, D.T. Purification of a
trypsin inhibitor from Cocculus hirsutus and identification of its
biological activity. J. Crop Sci. Biotech., 2009, 12, 253-260.
Shee, C.; Sharma, A.K. Storage and affinity properties of Murraya
koenigii trypsin inhibitor. Food Chem., 2008, 107, 312-319.
Solomon, M.; Belenghi, B.; Delledonne, M.; Menachem, E.;
Levine, A. The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants.
Plant Cell, 1999, 11, 431-443.
Qi, R.F.; Song, Z.; Chi, C. Structural features and molecular evolution of Bowman-Birk protease inhibitors and their potential application. Biochem. Biophys. Acta, 2005, 37, 283-292.
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
Patel et al.
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]