Cathepsin B-like protease from chili pepper revealed by in silico approach (original) (raw)

Cathepsin B-like protease from chili pepper revealed by in silico approach

Sanjog T. Thul 1{ }^{1}, Feroz Khan 2{ }^{2}, Suman P. S. Khanuja 1,3∗{ }^{1,3 *}
1{ }^{1} Genetic Resources and Biotechnology Division, Central Institute of Medicinal & Aromatic Plants, Lucknow (CSIR), India-226015
2{ }^{2} Metabolic and Structural Biology Department, Central Institute of Medicinal & Aromatic Plants, Lucknow (CSIR), India-226015
3{ }^{3} Present address: NutraHelix Biotech (P) Ltd, C41-42, Double Storey, Ramesh Nagar, New Delhi 110 015, India

*Corresponding author: khanujazy@yahoo.com

Abstract

The cathepsin B-like proteases of higher plants are mostly related to stress / damage. The expression of cathepsin B-like transcript in the plant system is regarded the response towards abiotic stimuli, wounding of tissues, organ abscission. We isolated a putative cathepsin B-like proteases partial cDNA from chili pepper (Capsicum frutescens). A cDNA library of wound induced placental tissue transcripts was constructed in the phage vector system. Partial sequencing and in silico analysis revealed high levels of sequence homology to cathepsin B-like cysteine protease from the plants of solanaceae family, but much lower levels with other plant cysteine proteinases. Sequence alignment using ClustalW revealed the consensus sequences of the family solanaceae for cathepsin B-like proteases. Further, translated amino acid sequences by BLASTx revealed the conserved domains among the unrelated families. Name assignment to this cDNA as cathepsin B-like protease was based on nucleotide and translated amino acid sequence similarity which is of 91%91 \% and 97%97 \% respectively with cathepsin B-like cysteine proteinase of Nicotiana rustica. Our current hypothesis towards the function of this cDNA is that it encodes cathepsin B-like proteases in response to mechanical wounding in plant tissues.

Keywords: In silico analysis, Solanaceae, Wound induction, ESTs, Conserved domains
Abbreviations: ABA _ Abscisic acid, cDNA _ Complementary DNA, EST _ Expressed sequence tags, GA_ Gibberellic acid

Introduction

The cathepsin B proteases were originally identified in mammalian systems as lysosomal, hydrolytic enzymes as it can degrade a wide range of peptide/protein substrates (Bond and Butler, 1987). More recently, a role for cathepsin B has been demonstrated in cellular apoptosis, where it activates caspase-11 by processing the pro-form, and can also directly induce nuclear apoptosis (Vancompernolle et al., 1998). Cathepsin B is an ancient family of eukaryotic cysteine proteases. In the process of storage protein utilization, it has been shown that many proteinases are involved in the degradation of storage proteins for nutrient mobilization. They are synthesized as pre-proteins that are processed either auto-catalytically or with the aid of a processing enzyme, and are stored in the vacuole or the lysosome, or are externally secreted. Among the cysteine proteases, the K, S, L, O, B, C and H cathepsins have been widely studied in mammals (Wex et al., 1999) and more recently H and L in plants (Ueda, et al., 2000, and references therein). By contrast, only a few cathepsin B-like proteases of plant origin have been described so far. Hansen and Hannapel (1992) reported a cathepsin D inhibitor cDNA, p749, which were identified in the genomic DNA of tomato (Lycopersicon esculetum) by southern hybridization and of two non-tuber-bearing potato species (Solanum tuberosum and S. brevidens). To date, there have been few reports of cathepsin B-like sequences in plants (Ward et. al., 1997). A cDNA encoding a thiol protease similar to cathepsin B from mammalian cells was isolated from aleurone layers of wheat (Cejudo et al., 1992a). The corresponding mRNA accumulated in the scutellum and the aleurone layers of germinating grains where it was under the
regulation of gibberellin (GA) and abscisic acid (ABA). The analysis of the promoter from the corresponding gene showed that the regulation of its expression was at the level of transcription (Cejudo et al., 1992b and Gubler et al., 1999). In Nicotiana rustica, a wounding-responsive mRNA was isolated from roots and was shown to be expressed in most plant organs (Lidgett et al., 1995). In addition, the sequences of cathepsin B-like proteases from Ipomoea batata and Arabidopsis thaliana are available in the data banks, but there is no record of cathepsin B-like proteases from the genus Capsicum.

Materials and methods

Plant material

Fruits of chili pepper collected from Assam (North-east India) were used as an experimental material. Fruits were dissected to induce wounding and separate out the placental tissues from seeds and fruit wall. The dissected placental tissues were further used for RNA extraction.

RNA extraction and cDNA library construction

mRNA was isolated from placental tissues according to published method (Shukla et al., 2005). cDNA synthesis of mRNA and cloning was performed using ZAP Express cDNA synthesis and ZAP Express cDNA Gigapack III Gold cloning kit (Stratagene, USA). Amplified phage library was screened for recombinant bacterial colony by blue/white

Table 1. Details of identified protein domain, family and active sites in C. frutescens.

Fig 1. Multiple sequence alignment of cDNA of different solanaceae plants showing conserved residues.
colony selection as per recommended procedure of manufacturer. Recombinant white colonies were selected and grown in 5 ml LB media with selective antibiotic. The plasmids were isolated by modified polyethylene glycol precipitation method (Sambrook et al., 1989). Isolated plasmids were screened for the inserts by restriction digestion using Eco RI and Hind III restriction enzymes.

Nucleotide sequencing and in silico analysis

Single-pass sequencing was performed to obtain partial sequences by BigDye Terminator cycle sequencing (Applied Biosystems, Foster City, CA, USA). Each sequence obtained was assessed manually to determine sequence quality. Significant numbers of clones were found to hold the signature sequence of cathepsin gene when searched for
homology in NCBI database. Out these, the clone having longest stretch of cDNA nucleotide of candidate gene was selected for further confirmation of sequence. The amplification of candidate gene was carried out by growing respective single recombinant cloned bacterial colony in liquid LB medium. No mixed or overlapping sequence was observed from sequencing of plasmids. Sequence homology search was performed using BLASTn program (Altshcul et al., 1997) at the DNA sequence databases of NCBI (www.ncbi.nlm.nih.gov.). The sequences then were analyzed using clustalW software package (www.ebi.edu.uk/EMBL) for consensus sequence among the homologous sequences of different plant origin [Nicotiana rustica (CAA57522.1), Petunia X hybrida (AAU81590.1), Solanum tuberosum (AAR25800.1), Ipomia batatas (AAK69541.1)], which were derived from datatabase. Further, the cDNA sequences were

Table 2. Similarity of N. rustica Cathepsin B-like cysteine proteinase with C. frutescens.

Fig 2. Sequence alignment of translated amino acid residues revealing conserved domain of cysteine peptidase active site.

Fig 3. Neighour joining tree using PID showing grouping of plants based on the amino acid sequence similarity.

Fig 4. Three dimensional structure of peptidase C1A Cathepsin-B showing conserved domain recognized by amino acids of CC. frutescens (highlighted by yellow stick).
translated using BLASTx and the amino acid sequences were compared with the available amino acid sequences in the database of plants of different families like poaceae [Hordeum vulgare (AJ310426), Triticum aestivum (X66013) and Oryza sativa (AY916493)] fabaceae [Pisum sativum (AJ251536), Medicago truncatula (AY336982)], brassicaceae [A. thaliana (NM_178950)] and pinaceae [Picea sitchensis (ABK23329)]. The obtained EST sequences were submitted to the EST database of NCBI under the accession number DR741973. The 3D structure of candidate protein was elucidated using Cn3D version-4.2 software (www.ncbi.nlm.nih.gov) to align the proposed stretch of translated amino acid sequences.

Results and discussion

The discovery of proteinase inhibitor transcripts and proteins accumulates systemically in the leaves of wounded plants has led to the investigation of possible signal pathways (Ryan, 1988). Partial sequences of 253 base of cDNA clone obtained from dissected placental tissues of C. frutescens were analyzed in silico to identify the putative homologous sequences related to wound inducible genes. In search of novel promoter region, Fei et al., (2009) used bioinformatics-
based approach towards identification of short 256 bp genomic DNA fragment from Solanum lycopersicum. The used approach revealed the presence of several motifs for plant transcription factors such as circadian, TGA-element and motifs involved in light responsive control. In present study, sequence homology of 253 bp of cDNA clone was searched using BLASTn program and revealed the high levels of homology with cathepsin B-like cysteine protease of N. rustica with 91%91 \% similarity. Further, the comparative analysis of translated amino acid showed highest positive identity with cathepsin B-like cysteine protease (Table 2). The nucleotide sequence shared significant homology with plants like N. rustica, Pitunia, S. tuberosum, I. batatas, with high score value 226, 202, 99.6 and 83.8, respectively. Interestingly, all these plants were belongs to the family solanaceae that also comprises genus Capsicum. Besides, results showed diversity in sequence similarity within studied sequences such as mismatches and gaps. Since mismatches indicate substitution mutation and gaps indicate probability of addition or deletion mutation, therefore we hypothesized that mutation might evolve them as a separate entity of the family solanaceae. To validate this hypothesis, the homologous sequences from different plant origin were analyzed for consensus nucleotide sequences or patterns (Fig. 1). Further nucleotide sequences were translated to amino

Fig 5. Three dimensional structure of peptidase C1A Cathepsin-B, the active site (a) and S2 subsite (b) shown by yellow sticks.
acid sequences using BLASTx (NCBI) and compared with the amino acid sequences obtained from database belonging to plants of different families through ClustalW (Fig. 2). The comparative analysis revealed that conserved protein domains are not only shared by these plants ( NN. rustica, Pitunia, S. tuberosum, I. batatas and C. frutescens), but also by plants of other families (poaceae, fabaceae, brassicaceae and pinaceae). Although PP. sitchensis belongs to family pinaceae (Conifers), but still a major portion of the conserved protein domains shared the same amino acid sequences, based on which these were delineated as separate species (Fig. 3). Similarly, cis-acting upstream regulatory elements of polygalacturonase inhibitory protein (PGIP) encoding genes have been detected using bioinformatics based sequence analysis in seven different plant species (Kumar et al., 2009). Further, the domain/motif wide search was made (Table 1) and exhaustive comparative study was done with NN. rustica indicating a significant similarity (Table 2). Hansen and Hannapel (1992) demonstrated the expression of p749 genes (similar to cathepsin-D) in leaves which was induced at the RNA level in response to wounding. High levels of p749 transcripts were detected in polyadenylated RNA extracted from locally wounded leaves 12 h after wounding. Interestingly, no sequences showed homology with the probe of p749 cDNA upon southern hybridization with genomic DNA from egg plant, pepper or tobacco (Hansen and Hannapel, 1992). As in present in silico analysis, it was quite interesting that the isolated partial cDNA sequences showed a very prominent consensus region among the nucleotide sequences and conserved domains in case of amino acid sequences, this inferred that the cathepsin-B proteases might be more conserved in family solanaceae compared to that of cathepsin-D. Also it was pointed out that the cathepsin gene showed rhythmic expression and its expression increased in response to wounding (Lidgett et al., 1995). The dissection of fruits for the separation of placental tissues from seeds and fruit wall induced the expression of mRNA and by this means the present investigation supports the study of expression of cathepsin like proteinase on wound induction (Hansen and Hannapel, 1992; Lidgett et al., 1995). It is also inferred that likewise the cathepsin variants in human ( K,S\mathrm{K}, \mathrm{S}, L,O,B,C\mathrm{L}, \mathrm{O}, \mathrm{B}, \mathrm{C} and H ), the variants of cathepsin also present in plant kingdom (B, D, H and L). Upon the conserved domain search in NCBI database elucidate the homology with peptidase C1 superfamily. Within the conserved domain of
cathepsin-B (MGGHAVKLIGWGTS) from various origin (Fig. 4), which was elucidated using Cn3D ver. 4.2 (www.ncbi.nlm.nih.gov) showed that the His (hydrophilic amino acid) is the part of active site (Fig. 5a). Whereas the hydrophobic amino acids Gly and Ala (Italicized letters) is the part of S2 subsite (Fig. 5b), which is the dominant substrate specificity subsite of papain-like cysteine proteases. The site prefers for bulky hydrophobic or aromatic residues at the P2 side chain of the substrate to occupy the S2 subsite, indicates the perfect prediction of the function of EST isolated from the C. frutescens. In conclusion, despite the unfurnished work using plant host system for the identification of the role and function of the cDNA in genome of CC. frutescents, the in silico analysis yields a considerable results to hypothesize the function of cDNA as a probable candidate for cathepsin B-like protease. The approach used for the achievement of aim to elucidate the identification of partial cDNA sequence was seems to be most appropriate.

Acknowledgment

The authors are thankful to Central Institute of Medicinal and Aromatic Plants, Lucknow and Council of Scientific and Industrial Research, New Delhi for providing financial support. Authors are also grateful to Dr. A. K. Shasany for extending help during cDNA library construction and sequencing.

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