- Open Access
A proteomic analysis of Curcuma comosa Roxb. rhizomes
© Boonmee et al; licensee BioMed Central Ltd. 2011
- Received: 7 January 2011
- Accepted: 29 July 2011
- Published: 29 July 2011
The similarly in plant physiology and the difficulty of plant classification, in some medicinal plant species, especially plants of the Zingiberaceae family, are a major problem for pharmacologists, leading to mistaken use. To overcome this problem, the proteomic base method was used to study protein profiles of the plant model, Curcuma comosa Roxb., which is a member of the Zingiberaceae and has been used in traditional Thai medicine as an anti-inflammatory agent for the treatment of postpartum uterine bleeding.
Due to the complexity of protein extraction from this plant, microscale solution-phase isoelectric focusing (MicroSol-IEF) was used to enrich and improve the separation of Curcuma comosa rhizomes phenol-soluble proteins, prior to resolving and analyzing by two-dimensional polyacrylamide gel electrophoresis and identification by tandem mass spectrometry. The protein patterns showed a high abundance of protein spots in the acidic range, including three lectin proteins. The metabolic and defense enzymes, such as superoxide dismutase (SOD) and ascorbate peroxidase, that are associated with antioxidant activity, were mainly found in the basic region. Furthermore, cysteine protease was found in this plant, as had been previously reported in other Zingiberaceae plants.
This report presents the protein profiles of the ginger plant, Curcuma comosa. Several interesting proteins were identified in this plant that may be used as a protein marker and aid in identifying plants of the Zingiberaceae family.
- Curcuma comosa Roxb
- Superoxide dismutase
Plants in Zingiberaceae family are widely distributed in many countries of Southeast Asia. In Thailand at least two-hundred species of Zingiberaceous plants are found and these include members of various genera, such as Alpinia, Amomum, Curcuma, Etlingera, Kaempferia, and Zingiber . Zingiberaceous plants have been widely used in traditional medicine, as well as a food flavoring and spice agents. Many studies have focused on the bioactive small organic compounds from these plants and have supported the traditional medicinal use of the plant extracts, such as curcumin , sesquiterpene [3–5], and various essential oils [6–8], flavonoids and phenolic compounds [9, 10]. In addition, the biologically active proteins reported from Zingiberaceae plants include, antifungal proteins from Zingiber officinalis  and antioxidant proteins from C. longa  and C. zedoaria . Interestingly, the lectins were also found in many species of this Zingiberaceous plants. The lectins or agglutinin proteins, a class of carbohydrate-binding non-immune origin proteins, have been used as tools in analytical biochemistry [14, 15] including in medical applications, such as drug delivery , blood typing  and potential antineoplastic drugs , amongst others. Their actual physiological functions are likely to be in the defense against phytophagous predators (mostly insects) and phytopathogenic microorganisms [19, 20]. These plant lectins have been found in a variety of plant species, including the ginger family where, for example, the mannose-binding lectin cDNA, Z. officinale agglutinin (ZOA) , was cloned from the rhizomes of Z. officinale. According to the similarity of DNA sequences between ZOA and two other lectins, that is Galanthus nivalis agglutinin (GNA) from the snowdrop, which is highly toxic to sap-sucking insects, and Gastrodia elata antifungal protein (GEAFP), belonging to Orchidaceae lectins, ZOA may have defense based activities along the same lines as these two proteins. Heamagglutination activity was previously determined to be present in fifteen Curcuma plant species when assaying the crude rhizomal protein extract against rabbit erythrocytes , and this array of lectin-like activity positive plants included C. xanthorhiza, which is closely related to C. comosa. Certainly, purified lectins have been reported in a few Zingiberaceae plants. A 32.4 kDa lectin enriched from C. amarissima Roscoe  revealed a growth-inhibitory activity against three plant pathogenic fungi (Fusarium oxysporum, Exserohilum turicicum and Colectrotrichum cassiicola), and showed in vitro cytotoxicity against the BT474 breast cancer cell line. A thermostable lectin of 41.7 kDa isolated from Kaempferia parviflora  showed heamagglutination activity against several different erythrocyte sources, with the strongest activity observed against rabbit red blood cells.
However, most plants in this family have very similar botanical characteristics and this makes it very difficult to clearly identify each species. The mistaken identification of medicinal plant materials is a serious problem for both manufacturers of traditional medicine products and researchers. There are a few methods to distinguish each species of plant, such as botanical characteristics by specialized taxonomists or DNA sequence based methods (e.g. establishing molecular operational taxonomic units with conversion to species by sequence identity to known species in the NCBI database). However, although the latter method is tissue and developmental stage independent, it is time consuming and complicated (due to the problem of discrimination of variety/cultivar polymorphism versus cryptic or sibling species).
Recently, proteomic tools have been used to identify types or isolates in many organisms [25–27] so this technique may be the one of the choices for the classification of Zingiberaceous plants. Of course, with no current baseline database it is far from clear how much the proteome for a specific tissue (e.g. rhizome) may vary within a species due to local genetics (cultivars) or cultivation conditions compared to between species, and so how useful this approach could be, but nevertheless under such a scenario it could still be used for following specific cultivars/cultivation conditions for quality control checking of any given cultivar. Thus, the aim of this report was to perform a preliminary study of the phenol-soluble protein profile from C. comosa as an initial model plant from the Zingiberaceae family.
Because in traditional Thai medicine, the rhizome is generally the part of the plant that is most wildly use and because a higher amount of protein is present in rhizome than in other parts, the protein database study in bulbous plants and those from Curcuma are usually used the rhizomes, respectively. For this reason, we selected C. comosa, an herb with large rhizome, as the model Zingiberaceae plant for proteomic study. C. comosa, commonly known as Waan Chak Mod Look in Thai, has been used as a traditional medicine for the treatment of postpartum uterine inflammation, perimenopausal bleeding and hemorrhoids. The isolated compounds from this plant have been reported to display various biological properties, such as estrogenic , anti-inflammatory , choloretic , antioxidant  and nematocidal  activities. However, the protein profile from this plant has not been reported. Therefore, the proteomic analysis of the rhizomes of C. comosa is expected to be useful for both establishing the potential of protein fingerprints in Zingiberaceae family and for the investigation of its specific proteins in a high throughput manner.
Fresh rhizomes of C. comosa purchased from a local market in Bangkok, Thailand. A voucher specimen (BKF. No. 97298) is deposited at The Forest Herbarium (BKF), Royal Forest Department, Bangkok, Thailand. Grind fresh tissue of this plant to a powder with liquid nitrogen in a mortar and pestle. Base on C. longa proteomic , there are some interference compounds need to remove. Therefore the use of selection extraction method and buffer for C. comosa was similar with C. longa with slightly modification. Briefly, the plant powder (5 g) was extracted by suspension in 20 mL of extraction buffer (0.5 M Tris, 30 mM HCl, 0.1 M KCl, 0.7 M sucrose and 1% (v/v) β-mercaptoethanol) for 30 min at 4°C, whereupon the supernatant was then collected by centrifugation at 4,000 × g for 10 min. The precipitate was extracted twice in extraction buffer and the poled extracts were then extracted with a 1:5 (v/v) ratio of water-saturated phenol at 4°C for 60 min. After phase separation the phenol phase was then harvested and proteins were precipitated from the phenol phase by the addition of a four-volume of 0.1 M ammonium acetate in methanol and left overnight at -20°C. The resulting phenol-soluble protein pellet was collected by centrifugation at 4,000 × g for 10 min, resuspended in cold water with sonication for 3 min and then precipitated again in nine volumes of cold acetone at -20°C for 2 h and centrifuged at 4,000 × g for 10 min. The protein pellet was air-dried to remove the acetone. The amount of protein in each sample was determined by the Bradford assay .
Microscale solution-phase isoelectric focusing (MicroSol-IEF) of the protein extract
Aliquot protein (3 mg) from the isolated proteins (115.5 mg) were dissolved in 0.2 mL of solubilization buffer (7.7 M urea, 2.2 M thiourea and 4.4% (w/v) CHAPS) and then 20 μl of 100 mM iodoacetamide (IAA) was added, mixed and incubated in the dark for 30 min at room temperature. After this incubation, the proteins were then precipitated by the addition of four volumes of cold acetone and harvested by centrifugation, as described above. The protein pellet was resuspended in solubilization buffer and supplemented with 10 mM dithiothreitol (DTT), 0.8% (w/v) ampholine and trace amount of bromophenol blue (The final concentration of protein was approximate 1.5 mg/mL). The Zoom-IEF fractionator (Invitrogen, Carlsbad, CA, USA) was assembled with three disks (pH 3.0, pH 5.4 and pH 10.0). The protein solution (0.65 mL) was loaded between disk pH 3.0-5.4 and pH 5.4-10.0 and focused at 100 V for 20 min, followed by 200 V for 80 min and finally 600 V for 80 min. After separation by Zoom-IEF, the protein solution was kept at 4°C for further analysis.
Two-dimensional polyacrylamide gel electrophoresis (2-DE)
The protein samples (200 μg) were loaded onto immobilized pH gradient (IPG) gel strips (GE Healthcare, Biosciences, Uppsala, Sweden) and left overnight at room temperature. The first dimension was performed on a Pharmacia LKB Multiphor II system at 7,000 Vh. After electrofocusing, the IPG strips were reduced in equilibration buffer (50 mM Tris-HCl buffer, pH 6.8, 6 M urea, 1% (w/v) sodium dodecyl sulfate (SDS), 30% (v/v) glycerol) containing 1% (w/v) DTT and were alkylated with equilibration buffer containing 2.5% (w/v) IAA. After equilibration, the IPG strips were analyzed in the second-dimension on a SDS polyacrylamide gel (15% (w/v) acrylamide resolving gel) performed in a Hoefer system. Coomassie Brilliant Blue R-250 staining was used to visualize the protein bands.
Tryptic in-gel digestion
The protein spots were cut out from the gel and the coomassie blue removed using 0.1 M NH4HCO3 in 50% (v/v) acetonitrile until the gel pieces were colorless. After drying of the gel pieces by Speed Vacuum, the gels were reduced with buffer solution (0.1 M NH4HCO3, 1 mM Ethylenediaminetetraacetic acid (EDTA) and 10 mM DTT) at 60°C for 45 min. The liquid was removed and then the gel slices covered in 100 mM IAA in 0.1 M NH4HCO3 solution and incubated at room temperature in the dark for 30 minutes. The residual IAA solution was then removed and the gel pieces were washed with 50% (v/v) acetonitrile (ACN) in water, and dried in a Speed Vacuum. Next a trypsin solution (0.05 M Tris-HCl buffer pH 8.5, 0.1 μg/μL trypsin in 1% (v/v) acetic acid, 10% (v/v) ACN and 1 mM CaCl2,) was added to the gel pieces and incubated at 37°C overnight. Thereafter, the solution was collected and the gels were extracted three times with 2% (v/v) trifluoroacetic acid, 0.05 M Tris-HCl buffer pH 8.5 containing 1 mM CaCl2 and 2.5% (v/v) formic acid in acetonitrile respectively. The solutions were pooled and dried by Speed Vacuum.
Protein identification by tandem mass spectrometry
The tryptic peptides were analyzed by using LC/MS/MS, a capillary LC system (Waters) coupled to a Q-TOF mass spectrometer (Micromass, Manchester, UK). The database search was performed with ProteinLynx screening. The Mascot http://www.matrixscience.com/search_form_select.html and the Peaks search tools http://www.bioinfor.com:8080/peaksonline/login.jsp were used for samples where proteins were not found by the ProteinLynx screening. Some proteins were interpreted amino acid sequences using the De novo sequencing tool in Masslynx or the Auto De novo sequencing tool in Peaks online 2.0 and then searched by MS BLAST against the NCBI database http://dove.embl-heidelberg.de/Blast2/msblast.html.
Sample extraction and 2-D IEF-SDS-PAGE profile
Phenol-soluble proteins identified from 2-D (IEF-SDS-PAGE) gels of the acidic (pH 3-5.4; spot nos. A2- 40 in figure 2) and basic (pH 5.4-10; spot nos. B4-37 in figure 2) region proteins from C. comos a rhizomes, as analyzed by LC/MS/MS.
Sequence coverage (%)
Guanine nucleotide-binding protein subunit beta
Genomic DNA, chromosome 5, P1 clone
Cytochrome P450 76C2
Nonclathrin coat protein
DNA topoisomerase 2
Myb-related protein P
Physcomitrella patens subsp patens
Similarity to proton pump interactor
Type IIB DNA topoisomerase family protein
Vesicle transport v-SNARE 13
minus agglutinin (SAD1)
NADH dehydrogenase subunit F
Putative F-box protein At4g21240
Mannose-binding lectin precursor
Tulipa hybrid cultivar
ATP-dependent Clp protease proteolytic subunit
glucose 6 phosphate isomerase
UDP glucose pyrophosphorylase
glyceraldehyde 3 phosphate dehydrogenase
glyceraldehyde 3 phosphate dehydrogenase
glyceraldehyde 3 phosphate dehydrogenase
glyceraldehyde 3 phosphate dehydrogenase
glyceraldehyde 3 phosphate dehydrogenase
Putative cytochrome c oxidase subunit II PS17
branched chain alpha keto acid decarboxylase
cysteine protease gp3a
Predicted ATPase (ISS)
cysteine protease COT44
L-ascorbate peroxidase 2
Heat shock protein 16.9C
Superoxide dismutase [Cu-Zn]
Gibberellin 2-beta-dioxygenase 1
BES1/BZR1 homolog protein 2
Endochitinase CH25 precursor
In the acidic region, three mannose-binding lectins with an observed mass range of 14.4 - 17 kDa (spots A20, A27 and A34) were found. Note, however, that the predicted (theoretical) mass of the homologous proteins used to identify these three spots are slightly higher for A27 and A34 (19.5 and 20.3 kDa, respectively) but significantly so for A20 with a predicted mass of 404 kDa. A mannose binding lectin with a molecular mass of 13.4 kDa was also isolated from C. zedoary . In addition, six homologous lectin proteins of various molecular masses (8.84-32.8 kDa) were found in C. aromatica . Most of them are mannose binding lectins. With respect to high throughput protein identification, agglutinin was also found to be present in the C. longa 2-D IEF-SDS-PAGE protein profile  at around 14.4 kDa in the acidic region (pI~4.6) which is similar to spot 27 here.
Eleven of the putatively identified proteins from the basic region (Figure 2B) in the 2-D protein pattern were likely to be involved in plant metabolism. Enolase, a ubiquitous enzyme that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate in the glycolytic pathway, was identified as spot B7. Endochitinase, an enzyme that belongs to the glycosyl hydrolase family and is involved in carbohydrate metabolism and chitin degradation, was present in spot B37. Glyceraldehyde 3 phosphate dehydrogenase, an enzyme that catalyzes the conversion of glyceraldehyde 3 phosphate to D-glycerate 1,3-bisphosphate in the sixth step of glycolysis, was identified as spots B10-14. Four other glycogenesis proteins, phosphoglucomutase, glucose-6-phosphate isomerase, UDP glucose pyrophosphorylase and UGPase, were identified in spots B4, B6, B8 and B9 respectively. Interestingly, two antioxidant proteins were found in the basic region. Superoxide dismutase (SOD), a class of enzymes that convert the reactive superoxide radical into oxygen and hydrogen peroxide, was identified in spot B24. This result is in accord with the recent report of an antioxidant activity and the isolation of a SOD homologue from C. comosa . Indeed, SOD homologues have also been reported in other Zingiberaceae plant species, such as C. longa  and C. zedoaria Roscoe . Their current biotechnological application has mainly been in cosmetic products to reduce free radical levels that otherwise cause skin damage . Ascorbate peroxidase, an enzyme that detoxifies peroxides by using ascorbate as the substrate, was found as spot B21. The main function of this enzyme is control the hydrogen peroxide concentration in cells. The discovery of these two antioxidant enzymes may suggest some benefit for C. comosa for the natural product based cosmetic industry, but this will depend upon their relative specific activity or ease of enrichment. Moreover putative cysteine proteases were identified as spots B17 and B19 at molecular weigh about 20.1 kDa and 14.4 kDa respectively. This enzyme family plays a role in plant growth, development and senescence. Most plant cysteine proteases belong to the papain and legumain families. Recently this enzyme family was reported from three members of the ginger family, in C. longa , C. aromatica  and Z. offinale Roscoe , and this ginger protease is used as a food improver and anti-inflammatory agent. Founding cysteine protease in four members of Zingiberaceae plant, C. comosa, C. longa, C. aromatica and Z. offinale at difference molecular weigh and pI position, the ginger cysteine protease might be a protein marker to classify specific species in this family in the future.
The protein profile of C. comosa was improved by separation by microscale solution-phase isoelectrofocusing, and identified in part by using high throughput two-dimensional IEF-SDS-PAGE together with tandem mass spectrometry. Some proteins were identified as lectins and antioxidant proteins, which appears to be related with their activity and cysteine proteases that are also found in other Zingiberaceae plant species.
The authors thank the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Programme (Grant No. PHD/0224/2548), the 90th Anniversary of Chulalongkorn University fund for financial support of this research and Ratchadapisek Somphot Endowment Fund (AG001B), and (AM1019A), the Thai Government Stimulus Package 2 (TKK2555), the National Research University (AS613A) the Department of Chemistry, the Faculty of Science, and the Laboratory of Biochemistry, Chulabhorn Research Institute, are both acknowledged for support and facilities. We also, thank Dr. Robert Butcher (Publication Counseling Unit, Chulalongkorn University) for his constructive comments in preparing this manuscript
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