Journal of Amino Acids

Volume 2013 (2013), Article ID 569527, 6 pages

http://dx.doi.org/10.1155/2013/569527

Research Article

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

Shamkant B. Badgujar1,2 and Raghunath T. Mahajan²

¹Department of Biotechnology, Faculty of Science, Post Graduate College of Science, Technology and Research, North Maharashtra University, Jalgaon, Maharashtra 425002, India

²Department of Biotechnology, Faculty of Science, Moolji Jaitha College, Jalgaon, Maharashtra 425002, India

Received 24 October 2012; Revised 30 December 2012; Accepted 30 December 2012

Academic Editor: Hieronim Jakubowski

Copyright © 2013 Shamkant B. Badgujar and Raghunath T. Mahajan. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

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.

1. 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.

Euphorbia nivulia Buch.-Ham. is a wild, thorny, xerophytic, succulent plant, found in boundaries of the agricultural field and also in dry barren areas. The secretion of milky juice is a characteristic property of this plant. Phytochemical studies have led to the isolation of ingol diterpenes (3-acetyl-8-methoxyl-7-angolyl-12-hydroxylingol; 3,12-diacetyl-7-hydroxy-8-methoxylingol; 3,12-diacetyl-7-angolyl-8-hydroxylingol; 3,12-diacetyl-8-benzoylingol; and 3,12-diacetyl-7-benzoyl-8-nicotinylingol) along with three macrocyclic ingol diterpenes derivatives (3,7,12-triacetyl-8-benzoylingol; 3,12-diacetyl-7-angeloyl-8-methoxyingol; and 7-angeloyl-12-acetyl-8-methoxyingol) [5]. The latex of E. nivulia has been cited for its antioxidant, immunomodulator, cytotoxic, anti-inflammatory, wound healing, haemostatic, and antiproliferative activity [6]. During the course of screening for biochemical constituents, a substantial amount of proteolytic and milk clotting activity was found in the latex of this plant [7]. Recently, an attempt has been made on peptide sequencing of 31-kDa, Tubulin alpha-1 chain-like protein called nivulian-I, present in the latex of E. nivulia [8]. Very recently, a comparative account on proteolytic activity of E. nivulia and other three plants, namely, Calotropis procera, Carica papaya, and Ficus carica, was reported by us. Additionally, we characterized the glycosylated cysteine protease called Nivulian-II of the latex of E. nivulia [5]. This paper describes the biochemical characterization, of this cysteine like protease with respect to peptide fingerprinting and N-terminal amino acid sequencing.

2. Materials and Methods

2.1. Chemicals

Sigma Chemicals: phenyl isothiocyanate (PITC), N-methylpiperidine, Methanol, Trifluoroacetic acid, Dithiothreitol, Acetonitrile, 20 Amino acid PTH standards, etc. were thankfully gifted by the Department of Biochemistry, Indian Institute of Science, Bangalore (Karnataka), India. Sequencing grade Hybond-PVDF membrane (Amershyam) was gifted by the Department of Biochemistry, TMC, Advanced centre for Treatment, Research and Education in Cancer (ACTREC), Navi Mumbai, India. All other chemicals were of the highest purity, analytical HPLC grade purchased from Himedia Laboratories, Mumbai; SRL Chemicals, Bangalore; Qualigen Fine Chemicals, Mumbai; Merck Chemicals, India; and Bangalore Genie, India.

2.2. 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].

2.3. 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 μL matrix solution and 2 μL protein (enzyme) fraction were spotted on the sample plate, mixed them, and allowed to evaporate to dryness. Proteins of known molecular mass (insulin, ubiquitin I, cytochrome C, and myoglobin) were used as standards for mass calibration.

2.4. 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 . Alkylation was performed by replacing the DTT solution with 55 mM iodoacetamide in 50 mM ammonium bicarbonate. After 30 min incubation at 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 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 α-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].

2.5. 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”.

3. 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 are, respectively, optimum pH and 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].

Figure 1: Mass spectroscopy of Nivulian-II of Euphorbia nivulia latex.

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 (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/).

Figure 2: MALDI-TOF mass spectrum of trypsin-digested peptide map of Nivulian-II.

(a)

(b)

Figure 3: (a) Peptides of Nivulian-II matched with Maturase K (Q52ZV1_9MAGN) of Banksia quercifolia. (b) Peptides of Nivulian-II matched with unknown protein (gi|118485477) of Populus trichocarpa.

The mass spectrum showed several protonated ions [M+H]⁺ of the peptide fragments. As listed in Table 1, the ions at 832.490, 1037.590, 1046.635, 1154.597, 1188.631, 1234.750, 1552.775, 1561.831, 1794.938, 1852.035, 1997.970, 2357.131, and 2872.537 were the thirteen trypsin digested peptides corresponding to residues 115–122, 183–189, 275–282, 283–292, 223–231, 190–199, 287–300, 170–182, 382–397, 350–365, 494–509, 67–86 and 63–86. These were designated as N2T1, N2T2, N2T3, N2T4, N2T5, N2T6, N2T7, N2T8, N2T9, N2T10, N2T11, N2T12, and N2T13, respectively. As depicted in 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 OH-group 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 content of serine (11.65%) and asparagine (10.52%) was abundant in sequenced peptides of Nivulian-II.

Table 1: Calculated and observed ions of trypsin digests of Nivulian-II.

Peak number
Intensity
Amino acid sequence
Sequence
[M+H]⁺

From
To
Calculated
Observed

m/z

02
13226.59
115
122
LVSSLEGK
832.477
832.490

18
3770.272
183
189
FFLYEYR
1037.509
1037.590

19
3540.355
275
282
DPFIHYVR
1046.542
1046.635

27
5762.630
283
292
YQAKSFLAAR
1154.632
1154.597

30
2878.922
223
231
CESMFVFLR
1188.554
1188.631

31
3792.376
190
199
NWNSFITPKK
1234.658
1234.750

52
5565.563
287
300
SFLAARGTPLMMNK
1552.797
1552.775

54
71959.36
170
182
VWVQDVPSLHLLR
1561.885
1561.831

67
3587.283
382
397
AKFCNALGHPISKPVR
1794.980
1794.938

70
3325.145
350
365
SQMLENSFLIDIAINK
1851.952
1852.035

73
5424.503
494
509
VWYLDIIRINDLVNHD
1998.044
1997.970

90
1525.765
67
86
MYQQNHFIISSNSNSSNQNK
2357.057
2357.131

98
1297.007
63
86
FIDRMYQQNHFIISSNSNSSNQNK
2872.343
2872.537

Table 2: Amino acid composition of peptides obtained from Nivulian-II by trypsin digestion.

Amino acid
Number of amino acids per peptide
Total

A
B
C
D
E
F
G
H
I
J
K
L
M

Gly (G)
1
—
—
—
—
—
1
—
1
—
—
—
—
03

Ala (A)
—
—
—
3
—
—
2
—
2
1
—
—
—
08

Val (V)
1
—
1
—
1
—
—
3
1
—
2
—
—
09

Leu (L)
2
1
—
1
1
—
2
3
1
2
2
—
—
15

Ile (I)
—
—
1
—
—
1
—
—
1
3
3
2
3
14

Ser (S)
2
—
—
1
1
1
1
1
1
2
—
5
5
20

Thr (T)
—
—
—
—
—
1
1
—
—
—
—
—
—
02

Cys (C)
—
—
—
—
1
—
—
—
1
—
—
—
—
02

Met (M)
—
—
—
—
1
—
2
—
—
1
—
1
1
06

Asp (D)
—
—
1
—
—
—
—
1
—
1
3
—
1
07

Asn (N)
—
—
—
—
—
2
1
—
1
2
2
5
5
18

Glu (E)
1
1
—
—
1
—
—
—
—
1
—
—
—
04

Gln (Q)
—
—
—
1
—
—
—
1
—
1
—
3
3
09

Phe (F)
—
2
1
1
2
1
1
—
1
1
—
1
2
13

Tyr (Y)
—
2
1
1
—
—
—
—
—
—
1
1
1
07

Try (W)
—
—
—
—
—
1
—
1
—
—
1
—
—
03

Lys (K)
1
—
—
1
—
2
1
—
2
1
—
1
1
10

Arg (R)
—
1
1
1
1
—
1
1
1
—
1
—
1
09

His (H)
—
—
1
—
—
—
—
1
1
—
1
1
1
06

Pro (P)
—
—
1
—
—
1
1
1
2
—
—
—
—
06

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
98
—

A: N2T1; B: N2T2; C: N2T3; D: N2T4; E: N2T5; F: N2T6; G: N2T7; H: N2T8; I: N2T9; J: N2T10; K: N2T11; L: N2T12 and M: N2T13 Peptides.

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)).

Table 3: Comparison of N-terminal sequences of Nivulian-II with other known cysteine proteases.

Source
Enzyme
Sequence*
Identities
Positives

Euphorbia nivulia
Nivulian-II
D
F
P
P
N
T
C
C
C
I
C
C
—
—

Opisthorchis viverrini
Cathepsin F
M
R
P
F
V
C
C
V
L
V
T
T
2/12 (16.66)
3/12 (25.00)

Streptococcus pyogenenes
IdeS
K
S
C
D
K
T
H
T
C
P
P
C
3/12 (25.00)
3/12 (25.00)

Zingiber officinale
GP-I
D
V
L
P
D
S
I
D
W
R
E
K
2/12 (16.66)
3/12 (25.00)

Zingiber officinale
GP-II
D
D
L
P
D
S
I
D
W
R
E
N
2/12 (16.66)
3/12 (25.00)

Pineapple stem
Bromelain
—
A
V
P
Q
S
I
D
W
R
D
Y
1/12 (08.33)
2/12 (16.66)

Rat liver
Cathepsin J
D
T
P
A
N
E
T
Y
P
D
L
L
3/12 (25.00)
3/12 (25.00)

Shared amino acids as bold.

Table 4: Comparison of N-terminal sequence of Nivulian-II with other matched fragments of known cysteine proteases.

Source
Type
Sequence*
Identities
Positives

Euphorbia nivulia
Cysteine
D
F
P
P
N
T
C
C
C
I
C
C
—
—

Naegleria gruberi
Cysteine
D
F
P
P
N
E
T
S
L
G
F
S
5/12 (41.66%)
6/12 (50.00%)

Polysphondylium pallidum
Cysteine
V
I
E
P
N
T
S
C
I
I
I
P
5/12 (41.66%)
5/12 (41.66%)

Ricinus communis
Cysteine
C
P
D
G
N
T
C
C
C
I
Y
E
6/12 (50.00%)
6/12 (50.00%)

Hevea brasiliensis
Cysteine
C
P
E
S
N
T
C
C
C
I
F
E
6/12 (50.00%)
6/12 (50.00%)

Saccoglossus kowalevskii
BP-10
G
P
A
P
N
S
C
C
I
I
A
M
5/12 (41.66%)
7/12 (58.33%)

Mycoplasma hominis
MAP
V
G
F
P
N
T
C
C
I
S
V
N
5/12 (41.66%)
5/12 (41.66%)

Shared amino acids as bold.

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.

Conflict of Interests

None of the authors declare any competing financial interests.

Authors’ Contribution

Both authors contributed equally in the design and performance of research, in the analysis of data, and writing in the paper.

Acknowledgments

The authors are thankful to Dr. Surekha Zingde, Director, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Mumbai, India, for generously allowing the use of MALDI-TOF/MS facility. They are extremely grateful to professor Sathees Raghavan, Department of Biochemistry, Indian Institute of Science (IISc), Bangalore, India, for helping in determination of N-terminal amino acid sequence.

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