Identification of proteinase inhibitors in Piper species m p nitha, k johnson George, b chempakam, r sandeep Varma and V a parthasarathy Indian Institute of Spices Research, Calicut, Kerala -673012




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Identification of proteinase inhibitors in Piper species

M P Nitha, K Johnson George, B Chempakam, R Sandeep Varma

and V A Parthasarathy

Indian Institute of Spices Research, Calicut, Kerala -673012.

Presence of trypsin and chymotrypsin inhibitors in three Piper species is described for the first time. These proteins were detected in considerable amounts in two Piper species, namely P. colubrinum and P. chaba, where as in P. nigrum, the protein was detected at low concentrations. These inhibitor proteins were tolerant to heat, acid and alkali but were denatured at extreme conditions of pH and boiling temperature. Preliminary studies by western blotting indicated the presence of a trypsin inhibitor of approximately 58 KDa.


Key words: trypsin inhibitor, chymotrypsin inhibitor, Piper species, western blotting
Introduction

Proteinase inhibitors are one of the abundant and most studied proteins, and are present in almost all life forms. These inhibitors, either specifically or nonspecifically, inhibit a wide range of enzymes. They are found to inhibit nearly all of the four types (serine, sulphydryl, metallo and acidic) of proteolytic enzymes1. Trypsin is an important serine protease of the digestive tract of animals and insects. Therefore, the plant inhibitor proteins against trypsin deserve much attention as far as their role in plant defense mechanism is concerned. Earlier, much of the work on plant proteinase inhbitors was concentrated on trypsin inhibitors. Later, studies were also focussed on other proteinase inhibitors and an important one among them was chymotrypsin inhibitor. It is also a serine protease related to trypsin so that the inhibitors of trypsin are also effective to chymotrypsin in several systems2.

These inhibitor proteins are believed to play many important roles in the physiology of plants. Their significant roles are ascribed to the regulation of endogenous proteinases and amylases, as N- reserve proteins and the biological defense against pests and pathogens3. They are also abundant in plant storage tissues, and serves as storage proteins4, in addition to their response to abiotic and biotic stresses.

Proteinase inhibitors (PI) are known since 1938 and they have been isolated and characterized from a wide variety of economically important crop plants4. Some of the plants in which PI has been detected are alfalfa, tomato, potato, maize, mustard, poplar, tobacco, rice, sweet potato, soybean, amaranthus, cow pea, barley and capsicum. They have regulatory and protective role at different stages of plant development4. In potato, the association of inhibitor I with meristamatic growth and its appearance in other tissues with breakage of apical dominance suggests a specific regulatory role for chymotrypsin inhibitor I5. In insects, trypsin is involved in the developmental processes such as moulting and synthesis of neuro peptides. Thus trypsin inhibitors can disrupt these processes leading to major damage to the growth and development of the insect larvae6. TI such as CpTI (cow pea TI) are reported to be effective against the insects of the families Coleoptera, Lepidoptera and Orthoptera7 .The black eyed pea trypsin/ chymotrypsin inhibitor was reported to be highly inhibitory to the trypsin of adult Anthonomus grandis which is a coleopteran. Soybean trypsin inhibitor was effective against digestive proteinases of Helicoverpa armigera Hubner. and H. virenscens8, 9 .

PI genes from various sources are being cloned to economically important crop plants to make them resistant to attack by insects and also against microbial infections. Transgenic tobacco expressing CpTI genes were reported to be partially resistant to insect damage6.

There have been no reports on identification of proteinase inhibitors in black pepper or related species. The genus Piper contains a very large number of species, distributed mainly in the Central and South America, India, Malaysia, Indonesia and Sri Lanka. Some of the important species of this family include P. nigrum, P. chaba, P. betle, P. galeatum, P colubrinum, P. argyrophyllum, P. longum etc.

The objective of the present study was to identify the inhibitor proteins against trypsin and chymotrypsin enzymes from three Piper species, namely P.nigrum L., P. chaba Hunt., and P.colubrinum L., using wound induced leaf samples.
Materials and methods

Trypsin and Chymotrypsin (3x crystallized; from Bovine pancreas), Soybean trypsin inhibitor and Bovine serum albumin (BSA), (Sisco Research Laboratories, Mumbai, India.), Alpha amylase (EMerck Inc., Germany.), Nitrocellulose membrane (Ambion, USA), standard marker proteins (Banglore Genei, India) were used. All other chemicals were high quality reagent grade products.

Mature leaves of P.colubrinum, P.chaba and P.nigrum (var. Panniyur 1) were collected from the experimental farm of Indian Institute of Spices Research at Peruvannamuzhi, Calicut.

Purification of the inhibitor proteins

Procedure for protein isolation was based on the modified protocol of Sasikiran et al., 2002. The leaf samples were homogenised in 0.01 M phosphate buffer (pH 7.6) containing antioxidants such as L-ascorbic acid, L-cysteine HCl, sodium sulphite and β-mercaptoethanol at 0.1% concentration and Polyvinylpyrrolidone at 5% concentration. After an overnight incubation at 4°C, the homogenates were squeezed through a muslin cloth, centrifuged at 4000 rpm for 20 minutes and the supernatants were subjected to total protein estimation by Lowry’s method11.

The samples were then heated at 70°C for 10 minutes to denature the native proteases and amylases and these were centrifuged off. To remove the quinones (oxidised polyphenols) which were not removed during the extraction, 2 ml of an alkaline solution of 3% casein were added to the sample, gently shaken and mixed well. The casein, other unwanted proteins and phenolic compounds were then precipitated out by adding solid trichloro acetic acid to a final concentration of 5%, followed by a brief centrifugation at 1,500 rpm for 5 minutes. The pH of the supernatant was immediately restored to 7.6 with 5 N NaOH. It was then dialyzed overnight against 0.01 M sodium phosphate buffer (pH 7.6). After each step, inhibitor activity assays for trypsin and chymotrypsin were done.
Assay of inhibitor proteins

Proteinase inhibitor assay was based on the hydrolysis of casein by the protease enzymes, trypsin and chymotrypsin in the presence of the inhibitor extract. For trypsin inhibitor assay, the enzyme used was trypsin, and for chymotrypsin inhibitor assay, the enzyme used was chymotrypsin. Three assay systems viz., control, uninhibited and inhibited systems were followed. The control system consisted of 1 ml of the inhibitor extract, 2 ml of 2% casein, and 1 ml of sodium phosphate buffer. The uninhibited system consisted of 2 ml of 2% casein, 0.2 ml of enzyme and 1.8 ml of sodium phosphate buffer, while the inhibited system consisted of 1 ml of the inhibitor extract, 0.2 ml of the enzyme and 0.8 ml of sodium phosphate buffer. The control and uninhibited systems were allowed to react for 30 minutes and the reactions were stopped by adding 4 ml of 12.5% TCA to the reaction mixture. In the inhibited system, a pre-incubation of the enzyme with the inhibitor extract was carried out for 30 minutes in order to inhibit the enzyme at maximum level. After this incubation period, 2 ml of casein and 0.8 ml of buffer were added to start the reaction. After 30 minutes, the reactions were stopped by adding 12.5% TCA. The unhydrolysed proteins were then allowed to settle for 1 hour and these were removed by centrifugation. The concentration of the hydrolyzed fragments was estimated by Lowry’s method11.

For the calculation of the amount of proteinase inhibitors, a calibration curve was prepared using BSA as standard. One unit of trypsin /chymotrypsin activity is defined as one mg peptide fragments released per minute at 30ºC under assay conditions. Trypsin/chymotrypsin inhibitor unit (TIU/CIU) is defined as the number of trypsin/chymotrypsin units inhibited and is expressed on a fresh weight basis. The effect of free amino acids and phenolic compounds in the samples were nullified by subtracting the readings of the control system from the inhibited system readings.
SDS-PAGE and Western blotting analysis

The crude samples were analysed on SDS-PAGE following the method of Laemmli, 1970. About 40 µl of the homogenates were added to equal volumes of 2x sample buffer and heated at 100ºC for 3 minutes. After this the samples were electrophoresed in 15 % polyacrylamide gels at 60 V for 3 hours. The marker proteins consisted of those having molecular weights ranging from 14.3 to 97.4 KDa .The proteins were detected by staining with Coomassie brilliant blue R 250 and further destaining was done with acetic acid : methanol : water in 4: 4: 2 ratio. Proteins were also transferred to nitrocellulose sheet using tris-glycine buffer (pH 8.3-8.4) and prepared for immunodetection using a polyclonal anti soybean trypsin inhibitor as the primary antibody at 1: 250 ratio for a period of 2 ½ h. The secondary antibody used was enzyme labeled anti- immunoglobulin.



Results and discussion
Purification of the inhibitors

The protein extraction procedure followed was based on the method of Sasikiran et al., 2002 and was modified by adding 0.1 % each of L - ascorbic acid, L - cysteine HCl, sodium sulphite, β- mercaptoethanol and 3% alkaline casein solution in the extraction buffer. The presence of these chemicals in the extraction procedure reduced the interference by polysaccharides and polyphenolic compounds. Also the presence of these reductants in the extraction procedure helped to prevent phenolic oxidation. In plants the high level accumulation of phenolic compounds is also an indication of defense mechanism against microbial infections, and their level increases upon wounding13,14. Therefore successful removal of these phenolic compounds from the protein extract was essential. Polyvinylpyrrolidone (PVP) is known to be a very good adsorbent for the phenolic compounds15, and therefore in the present study it was used at 5% concentration. Further clarification of polyphenols was done with alkaline casein solution. Casein and caseinates are useful for this purpose because they exert a binding action and reduce unwanted polyphenol contents such as tannins16. When solid TCA was added to the extract containing the alkaline casein, the casein lost its counter ion, became less soluble, and the whole complex was precipitated at the bottom.

After the removal of the casein-tannin complex, the pH of the extract was rapidly restored to 7.6 with 5 N NaOH, to avoid any structural alteration to the inhibitor proteins.

Many reports suggest that the levels of PI are elevated by wounding17, 10. They were found to accumulate in the leaves within 4 hours of wounding, remained almost at the same levels up to 16 hours, and were found to decrease after 48 hours. In the present study also mature leaves and vine were injured by random pinpricks in order to elevate the level of inhibitor proteins. After a period of 18 hours, the mature leaves were collected and the protein was isolated.

The inhibitor proteins are reported to be generally resistant to heat and pH variations 10, 18, 19. This principle was used to remove the native proteins from the homogenate. The homogenates were heated at 70°C for 10 minutes in a water bath and the heat denatured native proteins were removed by centrifugation.

The crude supernatant containing the inhibitor proteins were then dialyzed overnight against 0.01 M plain sodium phosphate buffer (pH 7.6) which helped to remove the unwanted ions and other low molecular weight compounds.


Proteinase inhibitor assay

Table 1 shows the results obtained with the inhibitor assay of both trypsin and chymotrypsin. The wild species P.colubrinum and P.chaba showed higher inhibitory activity to both trypsin and chymotrypsin when compared to cultivated P.nigrum.

Upon heating at 80°C for 20 minutes, the trypsin inhibitor was found to be stable and upto 98% activity was retained in the samples (Table 2.) But the TI activity of P.colubrinum sample was rapidly decreased when boiled for 20 minutes, indicating that the inhibitor was denatured upon boiling.

The results of the assays at varying pH indicate their stability over a wide range of pH. Both TI and CI activity were retained even at extreme acidic (pH 3) and alkaline conditions (pH 11). Maximum inhibitory activity was obtained at neutral conditions of pH (Fig 1 & Fig 2). These results corroborate with the findings made by Obidiaro and Akpochafo and Padmaja et al., 18, 4 that maximum inhibitor activity was observed at neutral conditions of pH.


Electrophoresis and immuno blotting

The proteins of crude extracts of the three samples were separated on 15% SDS-PAGE, according to the method of Laemmli12 and the pattern is shown in Fig 3.

Western blotting studies using antibodies raised against pure soybean trypsin inhibitor also indicated a band of molecular weight around 58 KDa. This is the first report that identifies a trypsin inhibitor in Piper species. Low molecular weight proteinase inhibitors of this nature were identified in several crops earlier. A few reports are also available on high molecular weight trypsin inhibitors from crops such as sweet potato19 and oats20.

The identification of TI from Piper species is significant in that, this genus include plants such as P. nigrum and P. chaba which are economically important. A major problem in the production of black pepper in the country is that it is susceptible to a variety of diseases caused by pests like pollu beetle (Longitarsus nigripennis Mots). The wild species P. chaba has been found to be resistant to this pest21 and hence it may be concluded that the presence of TI in these species may aid in the resistance reaction. The present study opens a new path of study on these proteins. The proteins present in these plants may be further purified by using techniques like column chromatography. Their chemical and physical characteristics and biological applications are to be determined. Further, the protein may be sequenced for the development of markers for sequence comparison with similar proteins and for detecting its presence in Piper. These may also be further investigated for the production of transgenic black pepper plants for resistance to insect pests.


Acknowledgement

We thank Dr (Mrs) G Padmaja, Principal Scientist, CTCRI, Trivandrum and Dr. A Ishwara Bhat, Senior Scientist, IISR, Calicut for technical support, helpful advice and useful discussions.


References

  1. Richardson M (1977) Phytochemistry 16, 159-169

  2. Bidlingmeyer U D V, Leary T R  Laskowski M J (1972) Biochemistry 11, 3303

  3. Ryan C A (1973) Ann Rev Plant Physiol 24, 173-196

  4. Padmaja G, Sasikiran K, Rekha M R  Pradeep Kumar S (1999) J Root Crops 25 (2), 99-123

  5. Ryan C A, Huisman O C  Van Denburgh R W (1968) Plant Physiol 43, 589-596

  6. Ghoshal D, Sen S K  Goyal A (2001) J Plant Biochem Biotech 10, 19-24

  7. Ussuf K K, Laxmi N H, Mitra R (2001) Current Sci 80, 847-853

  8. Ferreira C, Capella A N, Sitnik R & Terra W R (1994) Biochem Physiol 107, 631–640

  9. Johnston K A, Lee M, Brough C, Hilder V A, Gatehouse A M R, Gatehouse J A, (1995) Insect Biochem Mol Biol 25, 375 –383

  10. Sasikiran K, Rekha M R  Padmaja G (2002) Bot Bull Acad Sin 43, 291- 298

  11. Lowry O H, Rosebrough N J, Farr A L & Randall R L (1951) J Biol Chem 193, 265-275

  12. Laemmli U K (1970) Nature (London) 227, 680

  13. Campos –Vargas R, Saltveit M E (2002) Physiol Plant 114 (1), 73- 84

  14. Nykanen H & Koricheva J (2004) Oikos 104, 247-268

  15. Pirttila A M, Hirsikorpi M, Kamarainei T, Jaccola L  Hohtola A (2001) Plant Mol Biol Reptr 19, 273

  16. Murdiati T B, McSweeney C S, Lowry J B (1991) J Appl Toxicol 11(5), 333-338

  17. Moura D S  Ryan C A (2001) Plant Physiol 126 (1), 289-298

  18. Obidiaro T K  Akpochafo O M (1984) Enz Micro Technol, 6,132-134

  19. Sugiura M, Ogiso T, Takeuti K, Tamura S,  Ito K (1973) Biochim Biophys Acta 328,407- 417

  20. Mikola J & Kirsi M (1972) Acta Chem Scand 26, 787

  21. Chempakam B, Jnana Rani L J, Devasahayam S, Krishnamurthi K S, and Anandaraj M (2003) Proceedings of New perspectives in spices, Medicinal and Aromatic Plants, Goa, November 2003, pp-185-186

Table 1 – Trypsin and Chymotrypsin inhibitory activity of Piper species




Sample

TIU

CIU

P.colubrinum

0.429

0.4246

P.chaba

0.6355

0.4828

P.nigrum

0.2506

0.3712

TIU/CIU is expressed as the number of trypsin /chymotrypsin units inhibited /min

Table 2- Trypsin inhibitory activity of the heat- treated samples

(For assay of the heat treated samples, the samples were heated at 80ºC for 20 minutes and then assayed)


Sample

TIU

Retention of

original activity(%)



P.colubrinum

0.4191

97.69

P.chaba

0.5732

90.19

P.nigrum

0.2479

98.92

P.colubrinum

(Boiled sample)



0.0077

1.79











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