Peptide Mass Fingerprinting and N-Terminal Amino Acid Sequencing of Glycosylated Cysteine Protease of Euphorbia nivulia Buch.-Ham.

A new cysteine protease named Nivulian-II has been purified from the latex of Euphorbia nivulia Buch.-Ham. The apparent molecular mass of Nivulian-II is 43670.846 Da (MALDI TOF/MS). Peptide mass fingerprint analysis revealed peptide matches to Maturase K (Q52ZV1_9MAGN) of Banksia quercifolia. The N-terminal sequence (DFPPNTCCCICC) showed partial homology with those of other cysteine proteinases of biological origin. This is the first paper to characterize a Nivulian-II of E. nivulia latex with respect to amino acid sequencing.


Introduction
Proteases are enzymes which potentially hydrolyze anything that contains peptide bond, from a dipeptide up to a large protein, containing thousands of amino acids and, thus, it comprises a group of hydrolases that are the most relevant in technological terms. Proteolytic enzymes have been the subject of intensive studies for a number of years because proteases have great therapeutic and industrial applications as well [1]. Peptidases in which the nucleophile that attacks the scissile peptide bond is the sulfhydryl group of a cysteine residue are known as cysteine-type peptidase. Many plant proteases have been isolated from the lattices and most of them belong to this catalytic type. Plant cysteine proteases belong to a class which has been widely studied over the years. These enzymes are also used in industries owing to their high stability [2]. Plant cysteine proteases play a major role in the intracellular and extracellular process such as development and ripening of fruits, degradation of storage proteins in germinating seeds, activation of proteins, and degradation of defective proteins [3]. Besides, latex proteases are also involved in the protection of the plants against predator attack [4]. Many researchers have isolated and characterized the plant origin proteases of diverse group.

Plant Material, Collection of Latex, Crude Enzyme Preparation, and Purification of Cysteine Protease.
The detail information about identification, collection, preservation, preparation of crude enzyme and its proteolytic activity of Euphorbia nivulia latex is described in our previous communication [7]. Method of purification of protease was done using acetone precipitation, DEAE cellulose chromatography, dialysis and followed by rechromatography on DEAE cellulose column as described in our earlier communication [5].

Mass Spectroscopy.
Matrix-assisted laser desorption ionization/time of flight mass spectroscopy (MALDI/TOF MS) was used for the determination of the molecular mass, as well as the degree of purity of active enzyme. MALDI/TOF mass spectra were acquired on a Bruker Daltoncs model Ultraflex II Spectrometer, Germany, equipped with a pulsed Neodymium yttrium-aluminium garnet (Nd-YAG) smart beam solid state laser (337 nm), in reflectron positive-ion mode, using a 19-kV acceleration voltage. Enzyme samples were prepared by mixing equal volumes of a saturated solution of the matrix that is, sinapinic acid (prepared in 0.1% TFA (aq.) and 50% acetonitrile (2 : 1)). 2 L matrix solution and 2 L protein (enzyme) fraction were spotted on the sample plate, mixed them, and allowed to evaporate to dryness. Proteins of known molecular mass (insulin,  Total  08  07  08  10  09  10  14  13  16  16  16  20  24  171  Peak number  02  18  19  27  30  31  52  54  67  70  73  90    ubiquitin I, cytochrome C, and myoglobin) were used as standards for mass calibration.

Peptide Analysis.
For peptide mass fingerprinting, the targeted protein band (spot) was manually excised from the gel and was processed for MS and MS/MS analysis as described previously [9]. In brief, gel pieces were washed twice with 50% (v/v) acetonitrile in 50 mM ammonium bicarbonate, shrunk by dehydration in acetonitrile and dried in vacuum centrifuge. Disulfide bonds were reduced by incubation with 10 mM dithiothreitol (DTT) in 50 mM ammonium bicarbonate for 45 min at 56 ∘ C. Alkylation was performed by replacing the DTT solution with 55 mM iodoacetamide in 50 mM ammonium bicarbonate. After 30 min incubation at 25 ∘ C in the dark, the gel pieces were washed with 50% (v/v) acetonitrile in 50 mM ammonium bicarbonate, shrunk by dehydration in acetonitrile, and dried in vacuum centrifuge. The dried gel pieces were incubated with sequencing-grade modified trypsin at 37 ∘ C overnight.
To extract the tryptic digested peptides, 0.1% trifluoroacetic acid in acetonitrile was added, the samples were sonicated for 5 min and the separated supernatant was dried under vacuum. Then, the samples (peptides) were desalted using a Ziptip C 18 (Millipore, USA) according to the manufacturer's protocol. MALDI-MS measurements were performed using MALDI TOF/MS in reflectron mode using -cyano-4-hydroxycinnamic acid as a matrix. All mass spectra were  internally calibrated with trypsin autolysis peaks. Peptide mass fingerprinting (PMF) was carried out using MASCOT Matrix Sciences (London, UK) program for protein identification (http://www.matrixscience.com/). MALDI-TOF/TOF fragment ion analysis (see Figure S1 in supplementary Material available online at http://dx.doi.org/10.1155/2013/569527) of selected individual parent peptide ( Figure S2) was carried out in the LIFT mode of the instrument. Then, in order to further confirm the identification, all MS/MS data from LIFT TOF/TOF spectra were combined with the corresponding MS peptide mass fingerprinting data for database searching. The spectra were analyzed with flex-Analysis software (Version 3.2, Bruker-Daltonics, Germany) and searched against two taxonomies: (i) other green plants and (ii) species information unavailable in the NCBI (NCBInr) and MSDB database using MASCOT software (Version 2.2). The PMF search parameters were: 100 ppm tolerance as the maximum mass error, MH + monoisotopic mass values, allowance of oxidation (M) modifications, allowed for 1 missed cleavage, and fixed modification of cysteine by carboxymethyl, that is, Carbamidomethylation (C) [10].

N-Terminal
Amino Acid Sequence Analysis. The purified protease enzyme was adsorbed onto a PVDF membrane and washed several times with deionised water. The N-terminal sequence was determined by Pehr Edman's automated degradation method using a Procise protein sequencing system (Applied Biosystem) composed of four integrated modules. This system sequentially cleaves N-terminal amino acids from protein and analyzes the resulting phenylthiohydantoin (PTH) amino acid residues. Protein homology searches were performed using the BLAST [11], indicating the specific residues which are identical "identities" as well as those which are nonidentical, but nevertheless have positive alignment scores "Positives".

Results and Discussion
Euphorbia nivulia Buch.-Ham. belongs to the Euphorbiaceae family, whose members are characterized by secretory tissues (laticifers) which frequently include proteolytic and milk clotting enzymes. The young stem latex of E. nivulia possesses proteolytic and milk clotting enzyme in more quantity as compared with other investigated laticiferous plants of Northern region of Maharashtra, India [5]. E. nivulia latex contains thermostable glycosylated cysteine protease having 6.6 and 45 ∘ C are, respectively, optimum pH and Journal of Amino Acids temperature, molecular weight is about 43.42 kDa (SDS-PAGE), cysteine hydrochloride activator, and inhibitor is mercuric chloride [5]. According to protease nomenclature, this protein is designated as Nivulian-II due to previously Nivulian-I (31486.985 Da) was already characterized from E. nivulia latex [8].
Mass spectroscopy (Figure 1) revealed that Nivulian-II had a molecular mass of 43670.846 Dalton (43.67 kDa), which is relatively matched with molecular mass determined by SDS-PAGE, that is, 43.42 kDa [5,12]. Similar molecular pattern was also recorded in the molecular mass measurement of Funastrain CII [13]. The resulted molecular mass, that is, 43.67 kDa is very close to those of other peptidase belonging to cysteine protease family for example cysteine proteases of Vigna mungo, 'Curcuma longa, Taenia crassiceps, Allomyces aebuscula, Triticum arstivum, Haemonchus contortus, and Gymnophalloides seoi [12].
Out of total trypsin-digested peptide fragments ( Figure 2) of Nivulian II, only thirteen peptides that is, 28.50% sequence was hit in MSDB (mass spectrometry protein sequencing database) by Mascot peptide fingerprint search engine with Maturase K (Q52ZV1 9MAGN) of Banksia quercifolia (Family: Proteaceae) with 67.1 score and < 0.05 (Figure 3(a)). An additional match of the mascot peptide mass fingerprint of Nivulian-II with other existing protein sequence from NCBI (National Center for Biotechnology Information) database was also performed. One of the unknown protein (gi|118485477) of Populus trichocarpa (Family: Salicaceae), matches 7 peptides (i.e., 25.2% sequence) of Nivulian-II as shown in Figure 3(b) (http://www.matrixscience.com/).
The mass spectrum showed several protonated ions [M+H] + of the peptide fragments. As listed in Table 1 Table 1, peptide mass profiles were obtained from the database search engine and amino acid sequence of individual peptides were identified from known sequence of Maturase K of Banksia quercifolia from desired spot of Nivulian-II protein of SDS-PAGE. Table 2 summarizes the amino acid composition of obtained peptide fragments (N2T1 to N2T13) of Nivulian-II by trypsin digestion. Only two residues of OHgroup and S-containing amino acid, that is, threonine (T) and cysteine (C), were recorded in sequenced peptides. A total of 171 amino acids were sequenced from Nivulian-II protein.
The N-terminal amino acid sequence of cysteine protease named as Nivulian-II was deposited in the universal protein resource database, "Uniprot" under the accession number P86837. A comparative study of homology of N-terminal sequences of various cysteine proteases with P86837 using BLAST algorithm network services (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was conducted. The N-terminal sequence of Nivulian-II was aligned with those of other plant cysteine proteases and showed 25-50% identity with other cysteine peptidases (Tables 3 and 4). The isoleucine (I) is present on the 10th position from N-terminal side of Nivulian-II, which is fully homologous with isoleucine of Maturase K, as it is present on the 10th position from amino terminal side (Figure 3(a)).
N-terminal sequence of Nivulian-II shows only 25% homology sequence with plant origin cysteine proteases, that is, GP-I and GP-II of Zingiber officinale rhizome [14]. It shows 16.66% similarity with bromelain of pineapple stem protease [15]. It is also homologous with some animal origin cysteine protease, that is, cathepsin F [16] and cathepsin J [17] and bacterial cysteine protease, that is, IdeS of Streptococcus pyogenes [18]. This sequence showed higher identity (50%) with other cysteine endopeptidase isolated from the latex of rubber tree, Hevea brasiliensis Muell, Arg [19], and a member of the Euphorbiaceae family, that is, Ricinus communis. It is also homologous (41.66%) with some microbial origin cysteine proteases of Naegleria gruberi [20] and Polysphondylium pallidum, sulphur-containing protease, that is, Methionine protease (MAP) of Mycoplasma hominis and blastula-like protease (BP-10) of Saccoglossus kowalevskii. Nivulian-II shared the motifs surrounding the catalytic cysteine (NT and CCI) that also occurred in most of the sequences compared ( Table 4). The asparagine (N) is notably conserved and it is present in all the cases as well as the cysteine residue located in position 8.
In the light of all these experimental observations it is envisaged that the present cysteine protease, Nivulian-II, is characterized for the first time. N-terminal amino acid sequence and tryptic digestion profile of nivulian infer the exclusive nature of enzyme and it may be a novel protease of the cysteine family.