International Journal of Biological Macromolecules 62 (2013) 684–690
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Purification and partial characterization of a novel anti-tumor glycoprotein from cultured mycelia of Grifola frondosa Fengjie Cui a,b,∗ , Xinyi Zan a , Yunhong Li a , Yan Yang c , Wenjing Sun a,b,d,∗ , Qiang Zhou b,d , Silian Yu b,d , Ying Dong a a
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China Jiangxi Provincial Engineering and Technology Center for Food Additives Bio-production, Dexing 334221, PR China c National Engineering Research Center of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, PR China d Parchn Sodium Isovitamin C Co. Ltd, Dexing 334221, PR China b
a r t i c l e
i n f o
Article history: Received 27 August 2013 Received in revised form 19 October 2013 Accepted 21 October 2013 Available online 25 October 2013 Keywords: Grifola frondosa Glycoprotein Purification Structural characterization Anti-tumor activity
a b s t r a c t A novel glycoprotein GFG-3a with the molecular weight of 88.01 kDa and potent anti-tumor activity was isolated from the cultured mycelia of Grifola frondosa GF9801. GFG-3a was heat-sensitive with the decreasing anti-proliferative activity after treated from 56 ◦ C to 100 ◦ C for 10–120 min. GFG-3a was a glycoprotein with O-glycosylation and contained 6.20% carbohydrate composed of d-arabinose, d-fructose, d-mannose, and d-glucose with a molar ratio of 1.33:4.51:2.46:1.00. FT-IR and NMR spectra proved that GFG-3a contained protein and carbohydrate portions with 3-O-methyl-galactose residues, (1→4)-linked -galactose residues, and -linked glucose residues. Circular dichroism (CD) revealed that GFG-3a was a predominantly -sheet glycoprotein with a relatively small ␣-helical content. Protein sequencing and 3D model of GFG-3a were finally predicted by using MALDI-TOF-MS, NCBI blast search and online SWISSMODLE Workspace service. Our findings will be a reference for the further structure–activity relationship analysis of the mushroom glycoproteins. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Edible mushrooms are well-known for their nutritional/medicinal values and used worldwide as the dietary supplement and/or traditional medicine [1]. Numerous mushroomoriginated compounds with antitumor, immunomodulatory, antiviral and antimicrobial activities [2] have been isolated and identified including polysaccharides [3], glycoproteins [4], terpenes [5] and phenols [6]. Glycoproteins are the polysaccharide–peptide or polysaccharide–protein complexes with the protein/polypeptide chains covalently and specifically bound with carbohydrate sidechains [7,8]. Generally, glycoproteins have different structures and bioactivity mechanisms compared with those of polysaccharides. The processes for elucidating the polysaccharide structure include the determination of the monosaccharide composition, glycosidic linkage configuration, glycosidic linkage position, monosaccharide sequence, even secondary and 3D structure [9]. As for investigating
∗ Corresponding authors at: School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China. Tel.: +86 511 88780226; fax: +86 511 88780201. E-mail addresses: [email protected], [email protected] (F. Cui), [email protected] (W. Sun). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.025
the glycoprotein structure, the polysaccharide and protein/peptide chains are firstly elucidated, respectively. Then the glycosylated site in the glycoprotein is also an essential item to be determined [7]. Polysaccharides and glycoproteins possibly have significant difference of antitumor mechanisms. In most cases, polysaccharides exert their antitumor action through activating the host immune systems [10], while glycoproteins kill the tumor cells directly by arresting the cell cycle and/or inducing the apoptosis of tumor cells [11,12]. The famous edible mushroom Grifola frondosa belongs to the order Aphyllopherales and family Polyporeceae [13]. It has recently attracted considerable attention for its taste and health value [14]. Polysaccharides including -(1→6)-d-glucan and heteropolysaccharide are obtained from G. frondosa fruiting body and mycelia [15]. Our research group has focused on optimization of fermentation process for maximum G. frondosa mycelia production, structural elucidation, and antitumor mechanism assessment of bioactive polysaccharide–peptide/protein from the cultured mycelia [16,17]. A polysaccharide–peptide GFPS1b from the cultured mycelia of G. frondosa was obtained with significant prohibition of the gastric carcinoma tumor cells SCG-7901 in vitro [17]. However, to date, glycoprotein from the cultured mycelia of G. frondosa has not yet been reported. In the present study, a novel antitumor glycoprotein GFG3a was isolated and purified from the cultured mycelia of
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G. frondosa GF9801. The basic and potential 3D structural characteristics of GFG-3a were investigated, and its thermo-stability during the 37–100 ◦ C treatment was also evaluated. 2. Materials and methods 2.1. Strain and culture conditions The strain G. frondosa GF9801 was obtained and kept in our laboratory [16]. Stock culture was maintained on potato-dextroseagar (PDA) slant at 4 ◦ C, and sub-cultured every two months. The mycelia were grown at 25 ◦ C in a 20-L mechanical stirred fermentor (GRCB, Green Bio-engineering Co., Ltd., Zhenjiang, China) with working volume of 12 L with the optimized medium containing (g/L): glucose 45.2, KH2 PO4 2.97, peptone 6.58, MgSO4 ·7H2 O 1 and corn steep liquor 15 [16]. Mycelia were filtered off, washed with distilled water, and maintained in a frozen condition. 2.2. Isolation and purification of glycoprotein G. frondosa mycelia were homogenized using a blender and then extracted with 1 L of water at 4 ◦ C. The homogenate was centrifuged at 8000 × g for 20 min. The supernatant was precipitated with 80% saturated ammonium sulphate overnight at 4 ◦ C. The precipitate collected by centrifugation (10,000 × g, 20 min) was dissolved in and dialyzed against distilled water. The dialyzed solution was applied to a column (2.5 cm × 25 cm) DEAE-Sepharose fast-flow anionic resin (Pharmacia AP, GE). The column was eluted sequentially with 0.1, 0.3 and 0.5 M NaCl at pH 7.0 (every fraction tube 8 mL). The carbohydrate content of the eluate was determined spectrophotometrically at 490 nm using the phenol–sulfuric acid method. The protein content was determined by measuring the absorption at 280 nm. Three factions GFG-1, GFG-3, and GFG-5 were collected, dialyzed and freeze-dried. GFG-3 was detected to be the main anti-proliferative fraction and was further purified by gel filtration by AKTA purifier 100 (Amersham Biosciences, Sweden) on a SuperdexTM 75 prep grad column (Amersham Biosciences, Sweden) eluted with distilled water (pH 7.0). Three fractions of GFG-3a, GFG3b and GFG-3c were separated and then lyophilized for anti-tumor evaluation and structural analysis. 2.3. Assay of anti-proliferative activity The anti-proliferative activity of G. frondosa glycoprotein fractions was determined as follows. The mouse sarcoma cell line S-180 and human hepatoma cell line Bel-7402 were purchased from American Tissue Culture Collection (ATCC) and maintained in RPMI-1640 (Gibco, America) supplemented with 10% fetal bovine serum (FBS), 4 g/mL insulin, 100 mg/L streptomycin, and 100 IU/mL penicillin at 37 ◦ C in a humidified atmosphere of 5% CO2 . Cell (5 × 105 ) in their exponential growth phase was seeded into each well of a 96-well plate and incubated for 24 h before addition of the samples with the concentration of 5, 10, 20, 40, 80 and 160 g/mL. Incubation was carried out for another 48 h. The viability of cells was assessed in the MTT assay. The absorbance was detected in the microplate reader (Multiskan GO, Thermo Labsystems, America) at 570 nm. The inhibition ratio was calculated as follows: Inhibition ratio (%)=
OD570 nm (control)−OD570 nm (sample) ×100. OD570 nm (control)
2.4. Thermostability of glycoprotein GFG-3a Glycoprotein GFG-3a (20 g/mL) was kept at various temperatures (37, 56, 70, 100 ◦ C) for 30, 60, 90, 120 min, respectively. The
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S-180 and Bel-7402 cells (5 × 105 ) in the exponential growth phase were incubated for 48 h after adding the temperature-treated samples. The untreated sample was used as a positive control. The absorbance was detected at 570 nm for calculating the inhibition ratio. 2.5. SDS-PAGE The purity and molecular mass of GFG-3a were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) using a 12% resolving gel and blue bromophenol as the tracking dye. The molecular weight of glycoprotein GFG-3a was determined by comparison of its electrophoretic mobility with those of molecular weight standard markers. 2.6. Carbohydrate content and monosaccharide compositions Carbohydrate content of GFG-3a was determined by the phenol–sulfuric acid method using glucose as the standard. Monosaccharide compositions and their ratios of GFG-3a were determined by absolute hydrolysis followed by gas chromatography with rhamnose, arabinose, mannose, galactose and glucose as the standards. The hydrolysed glycoprotein and sugar standards were converted to the alditol acetate derivatives and then analyzed by a 6890N GC (Agilent Technologies, Santa Clara, CA, USA) with capillary column (30 m × 0.32 mm) and a flame-ionization. The oven temperature was maintained at 180 ◦ C for 3 min, and then increased gradually to 240 ◦ C at a rate of 2 ◦ C/min. The temperatures of detector and injector were set 260 ◦ C and 250 ◦ C, respectively. The carrier gas, supporter and stationary liquid were nitrogen, Chromosorb WAW DMCS, and 3% OV-1701, respectively. The carrier gas flow rate is 1.5 mL/min. 2.7. Protein content and amino acid composition Protein content of GFG-3a was determined by Micro BCATM Protein Assay Reagent Kit (Shenergy Biocolor BioScience & Technology Co., Shanghai, China) using bovine serum albumin as the standard. For determination of amino acid composition, GFG-3a was subjected to hydrolysis under vacuum in 6 M HCl at 110 ◦ C for 24 h. The hydrolyses were evaporated and the dried residue was re-dissolved in 0.02 M HCl, the amino acid composition was determined using a Hitachi 835-50G automatic amino acid analyzer (Hitachi Ltd., Tokyo, Japan). 2.8. Conformation analysis For determination of the glycosylation type, GFG-3a (5 mg) was dissolved in 0.2 M sodium hydroxide for 3 h followed by UV analysis by using a UV spectrophotometer (Varian cary 100, Varian, USA) ranging from 200 cm−1 to 400 cm−1 . The sample without sodium hydroxide treatment was used as the control. FT-IR spectral analysis of GFG-3a was recorded using the potassium bromide (KBr) pellet method with a Nicolet Fourier transform infrared spectrometer (Thermo Nicolet Co., USA) in the range of 400–4000 cm−1 . CD spectra of glycoprotein GFG-3a (0.5 mg/mL, in distilled water) were obtained by scanning from 190 nm to 300 nm on the JASCO J-810 spectropolarimeter (Jasco Corporation, Japan). Secondary structure percentages were calculated by de-convoluting CD analysis using CD analysis programs including CONTIN, SELCON3 and CDSSTR. A reference protein package consisting of 48 kinds of proteins was used in these three programs. 1 H and 13 C NMR spectra were obtained using with a 400-MHz Bruker model AVANCE II spectrometer. Samples were dissolved in D2 O and mainly examined at 50 ◦ C.
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Fig. 1. Chromatograph isolation and purification of glycoprotein and SDS-PAGE of GFG-3 and GFG-3a. (A) Anion-exchange chromatography of crude extract from the cultured mycelia of Grifola frondosa GF 9801 on DEAE-Sepharose Fast Flow column (2.5 cm × 20 cm). Flow rate: 2 mL/min; fraction size: 8 mL; temperature for chromatography: 20 ◦ C. (B) Gel filtration of fraction GFG-3 from DEAE-Sepharose Fast Flow column on SuperdexTM 75 prep grad column and SDS-PAGE of GFG-3 and GFG-3a.
Chemical shifts are expressed in ı PPM, relative to external MeSi (ı = 0).
using the MASCOT search engine. The search parameters were defined as follows: database, Swiss-Prot; taxonomy, Eucaryotes; enzyme, trypsin; and allowance of one missed cleavage.
2.9. In-gel tryptic digestion 2.11. Protein modeling The band of GFG-3a on the SDS-PAGE gel was excised and rinsed three times with Milli-Q purified water. The gel slices were dried and added to the trypsin solution for incubating at 37 ◦ C overnight. The supernatant was poured out and kept when trypsin hydrolysis was finished. The gel was subsequently extracted with 100 L 0.1% and 5% TFA in 50% ACN by gently mixing for 15 min at 20 ◦ C, respectively. The obtained supernatants were combined and freeze-dried for further MALDI analysis.
A 3D structure model of GFG-3a was obtained using the automatic modeling mode service available in the SWISS-MODEL Workspace. The online procedure gave the 3D model on the basis of multiple-threading alignments. The program automatically selected the template which corresponded to the 3D structures of acetyl-transferase (pdb code: 1Y9W). 3. Results and discussion
2.10. Mass spectrometry and protein identification MALDI mass spectrometric analysis was performed on AXIMA resonance MALDI-TOF-MS (Shimadzu Corp. JP), equipped with a 337 nm nitrogen laser in positive ion detection. Spectra were accumulated until a satisfactory S/N had been obtained. Parent mass speaks with the range from 400 to 1600 m/z were picked out for MS/MS analysis. The peptide mass finger-printings of glycoprotein and MS/MS data were acquired and processed using Mass-Lynx V4.1 software (Micromass) and were converted to PKL files by the Protein-Lynx 2.2.5 software (Waters). The PKL files were analyzed
3.1. Purification and screening of glycoprotein fractions with anti-proliferative activity This study was attempted to screen the antitumor glycoprotein from G. frondosa mycelia by bioactivity-directed purification procedures. The antitumor potency of the fractions GFG-1, GFG-3, and GFG-5 from the DEAE-Sepharose Fast Flow column (Fig. 1A) was evaluated by analyzing the inhibition ratio of the S180 and Bel-7402 cells growth. Fraction GFG-3 eluted with 0.3 M NaCl showed highest antitumor activity on S180 and Bel-7402 cells
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Fig. 2. Inhibition of proliferation of S180 cells (A) and Bel-7402 cells (B) by different concentrations of glycoprotein fractions from the cultured mycelia of Grifola frondosa GF9801.
in a dose dependent manner compared with those of GFG-1 and GFG-5 (Fig. 2). However, GFG-3 appeared to be an unpurified sample based on the presence of three bands in the SDS-PAGE electrophoregram (Fig. 1B). Subsequently, fraction GFG-3 was further separated into three sub-fractions including a larger GFG-3a and two tiny GFG-3b and GFG-3c by using a SuperdexTM 75 prep grad column gel filtration on the AKTA Purifier 100 (Fig. 1B). The fraction GFG-3a appeared as a single band with a molecular weight of 88.01 kDa in SDS-PAGE electrophoregram and exhibited the high percentage and anti-proliferative activity (Fig. 2). 3.2. Thermostability of GFG-3a Thermostability experiment results showed that the GFG-3a (20 g/mL) was heat-sensitive although its anti-proliferative activity was stable at 37 ◦ C with the treatment time from 30 min to 120 min (Fig. 3). The inhibition ratio on S180 cells and Bel-7401 decreased to 33.56% and 8.54% at 56 ◦ C treated for 30 min and to negligible levels treated for over 90 min. GFG-3a was even positive to the tumor cells growth when the treatment temperature kept increasing from 70 ◦ C to 100 ◦ C. Similar results could be found in the hemagglutinating activity of some lectins under different temperature treatments. For example, the hemagglutinating activity of mushroom Xerocomus spadiceus lectin [18], Inocybe umbrinella
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Fig. 3. Inhibition of proliferation of S180 cells (A) and Bel-7402 cells (B) by GFG-3a (20 g/mL) with various temperature treatments.
lectin [19], and Pleurotus citrinopileatus lectin [20] were moderately thermostable and unaffected when temperatures were set between 20 and 60 ◦ C. About 25–50% of their hemagglutinating activity remains at 70 ◦ C. However, the thermostability of GFG-3a was significantly different with those of Fusarium solani lectin [21] and BfL [22] with the temperature tolerance up to 70–100 ◦ C.
3.3. Carbohydrate content and monosaccharide compositions The phenol–sulfuric acid method of GFG-3a indicated that GFG3a contains 6.20% (w/w) of carbohydrate. The result was similar with the carbohydrate content of BfL accounts for 6.24% [22]; however, slightly higher than the 4.9% (w/w) sugar content of MAL [23] and 3.9% (w/w) neutral sugar of F. solani lectin [21], and lower than 24% (w/w) carbohydrates in the soluble protein from green microalgae Tetraselmis sp. [24] and 9.3% (w/w) neutral sugar in the novel 114 kDa hexameric lectin [25]. Monosaccharide analysis showed the presence of d-arabinose, d-fructose, d-mannose, and d-glucose with a molar ratio of 1.33:4.51:2.46:1.00. The origins and purification procedures of glycoprotein or glycopeptide could result in the difference of monosaccharide compositions. For example, Mycoleptodonoides aitchisonii glycoprotein MAL consisted of the GlcNAc, Gal, Man, and
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Fig. 4. Determination of the type of monosaccharide linkage (A), circular dichroism (CD) spectrum of glycoprotein GFG-3a (B), and NMR spectrum of glycoprotein GFG-3a in D2 O: (C) 1 H NMR and (D) 13 C NMR.
Xyl with the molar ratio of 1.56:4.89:3.68:1.00 [23], which were remarkably different with those of GFG-3a. 3.4. Protein content and amino acid composition The BCA assay showed that GFG-3a contains 87.04% of soluble protein portion which proved that GFG-3a was a protein-dominate glycoprotein. Amino acid analysis results (Table 1) revealed that GFG-3a contained 17 kinds of amino acids. It contained high amount of Asp (8.52%), Met (8.07%) and low amount of Lys (2.02%) and His (1.47%), which indicated that GFG-3a was possibly an acidic
Table 1 Amino acid composition of GFG-3a. Amino acid
Mol%
Amino acid
Mol%
Asp Glu Ser His Gly Thr Arg Ala Tyr
8.52 6.78 3.28 1.47 3.03 3.36 2.56 3.96 2.65
Cys Val Met Phe Ile Leu Lys Pro Trp
3.96 3.93 8.07 3.35 2.34 3.85 2.02 2.74 n.d.a
a
Not determined.
glycoprotein. Similar results were found that M. aitchisonii lectin MAL had the high content of acidic amino acid (AsX 12.1%) with its low theoretical pI value (4.3–4.5) [23] and BfL was a glycoprotein from Bauhinia forficate seeds with pI value 5.5 [22]. However, F. solani lectin was possible a basic protein with the high content of alkaline amino acid (Lys, 11.6%) and pI value equal to pH 8.7 [21].
3.5. Conformation analysis The absorption of NaOH-treated GFG-3a at 240 nm was much stronger than that of untreated GFG-3a, which revealed that GFG3a contained O-glycosidic linkage since O-linked glycans could be released by -elimination in the presence of sodium hydroxide (Fig. 4A). The diffuse reflectance Fourier transform infrared spectra of glycoprotein GFG-3a (Fig. 5) was followed for conformation analysis. A strong and broad absorption peak from 3500 cm−1 to 3200 cm−1 was O H stretching vibrations of polysaccharides and N H stretching vibrations of protein. Two peaks from 3000 cm−1 to 2800 cm−1 were C H stretching vibrations and bending vibrations. The absorption peaks from 1600 to 1200 cm−1 were C H bending vibrations of carbohydrates. The peak at 1658 cm−1 was assigned to the absorbance of the C O stretching vibrations and N H bending vibrations of the acylamino. Absorption peak at 1422 cm−1 was C N stretching vibrations and may be due to carboxylate groups of
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Fig. 5. FT-IR spectrum of glycoprotein GFG-3a.
galacturonic acid residues. The wave numbers between 1200 and 800 cm−1 represent the finger print region for carbohydrates. The chemical structure of GFG-3a was also elucidated by 13 C NMR and 1 H NMR spectroscopy (Fig. 4C and D). The 13 C NMR spectrum of GFG-3a showed that it contained carbohydrate and protein, with a signal at 175.9 ppm assigned to the CONH group of protein. GFG-3a also contained acetyl groups
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in the polymer as indicated by the presence of signals at 21.3 and 174.7 ppm, which belonged to methyl carbons and carbonyl groups of acetyl groups, respectively. Signals at 130.79, 129.39, 129.10, and 127.65 ppm were assigned to the C-1, C-2/C6, C-3/C-5 and C-4 of phenylalanine residues. The C-4 signal of (1→3)-linked glucose showed a downfield shift due to the glycosylation effect and appeared at 69.62 ppm. The C-6 signal at ı 66.21 ppm indicated the presence of the d-(1,6) linked glycoside residues. Signals at 60.56, 60.45, 56.48 and 56.19 ppm represented the carbons of OCH3 groups from 3-O-methylgalactose typical chemical shifts for OCH3 carbons fall in the range of 55–61 ppm. The signals of 1 H NMR at 7.3–7.2 ppm were assigned to the benzene ring of phenylalanine residues. Signals of 1 H NMR at 3.7 and 3.8 ppm were assigned to H-2 and H-3 of (1→4)-linked -galactose residues, respectively. The signals at 3.6–3.5 ppm represent H2 of the -linked galactose and 3.4–3.3 ppm were assigned to the H-2 of the -linked glucose. The proton NMR for GFG-3a also showed that a significant amount of acetyl groups in the region 2.2–2.1 ppm. Proton NMR of GFG3a had two signals at 1.4–1.2 ppm representing the methyl group of rhamnose residues. Many antitumor polysaccharide–protein complexes from Basidiomycetes mushroom are characterized as -(1→3) linkages in the main glucan chain and additional (1→6) branch points [25]. For instance, a new water-soluble intracellular polysaccharide PTP from the mycelium of Polyporus albicand consisted of a backbone composed of (1→3)-linked-d-mannopyranodyl, (1→3,6)-linked--d-mannopyranodyl and (1→6)-linked-␣-d-galactopyranosyl residues in the ratio of 3:1:1,
Fig. 6. Peptide mass fragments (A) and the predicted three-dimensional structure (B) of GFG-3a confirmed by a NCBI blast search and online SWISS-MODLE Workspace. The GFG-3a contains two domains maybe linked via hydrophobic interactions.
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and terminated with a single non-reducing terminal (1→3,6)--dmannopyranodyl residues along the main chain [27]. Based on CD data, the secondary structure content of GFG-3a was estimated to be 16.6% of ␣-helix, 44.4% of -sheet, 7.9% of turn and 31.1% of unordered structure, indicating GFG-3a was a predominantly -sheet glycoprotein with a lower amount of ␣helical content (Fig. 4B). Similar secondary glycoprotein structure was found that B. forficate seeds and F. solani lectins had the predominant -sheet structure with the 27% -sheet, 22% -turn and 19% ␣-helices [22], and 26% -sheet, 22% -turn and 9% ␣-helices [21], respectively. 3.6. Protein sequencing and model Generally, few specific glycoprotein or lectins databases are available for searching or comparing the purified fungal and bacterial glycoproteins. The sequence of GFG-3a was analyzed to search for homologous proteins in the current known protein databases and to perform structure predictions. The results revealed that the GFG-3a sequence was similar to GCN5-related N-acetyltransferase (gi|496194480) composed of 191 amino acid residues and a molecular mass of 22,227 Da confirmed by mass spectrometry (MADIF-TOF-MS) and an NCBI BLAST search (Fig. 6A). In order to predict the GFG-3a 3D structure, the online SWISS-MODLE Workspace was used and the GFG-3a 3D model was obtained by selecting the 3D structure of the acetyl-transferase as template. The overall fold of the GFG-3a model was shown in Fig. 6B. The protein core was composed of two domains linked via hydrophobic interactions. Protein Workshop reveals that C-terminal domain of GFG-3a adopted a fold which was usually observed in catalytically active proteins including N-glycanases, N-acetyltransferases, transglutaminases and cysteine proteases [26,27]. In the present study, the potential 3D structural model of G. frondosa glycoprotein GFG-3a was obtained as a necessary reference for the 3D structure and structure–activity relationship analysis of other mushroom glycoproteins. However, structural information obtained by MS still has some limits including incomplete sequence information, lower identity to the known protein, and limited typical mass difference to the polysaccharide portions. Enzymatic digestion, column affinity chromatography, and crystallization for separating and analyzing the protein and polysaccharide portions and glycosylation site are still necessary to provide more data basis for further GFG-3a structural analysis. 4. Conclusions A novel glycoprotein GFG-3a from cultured mycelia of G. frondosa GF9801 was successfully purified and further structureelucidated by GC, FT-IR, NMR and circular dichroism (CD). 3D structure of its protein portion was finally obtained by using MALDI-TOF-MS, NCBI blast search and online SWISS-MODLE Workspace service. Future studies would focus on finding the carbohydrate-binding site of GFG-3a and the growth inhibitory mechanism of GFG-3a for clarifying its antitumor mechanisms and developing GFG-3a as an antitumor agent or an ingredient in health food.
Acknowledgements This work was supported by funding from National Natural Science Foundation of China (NSFC 31101269), China Postdoctoral Science special Foundation (2013T60648), National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2012BAD36B05), the Second Class General Financial Grant from the China Postdoctoral Science Foundation (Grant No. 2012M511702), 2012 Excellent Key Young Teachers Project of Jiangsu University, Graduate Research and Innovation Projects of Jiangsu Province (CXLX12 0670); Advanced Programs of Jiangxi Postdoctoral Science Foundation ([2012]195); the Research Foundation for Advanced Talents of Jiangsu University; Science & Technology Platform Construction Program of Jiangxi Province. References [1] L. He, P.F. Ji, J.W. Cheng, Y.B. Wang, H. Qian, W.Q. Li, X.G. Gong, Z.Y. Wang, Food Chemistry 141 (2013) 946–953. [2] L.B. Zhou, B. Chen, International Journal of Biological Macromolecules 48 (2011) 1–4. [3] N.S. Li, C.Y. Yan, D.H. Hua, D.Z. Zhang, International Journal of Biological Macromolecules 57 (2013) 285–290. [4] M. Zhang, L. Zhu, S.W. Cui, Q. Wang, T. Zhou, H.S. Shen, International Journal of Biological Macromolecules 48 (2011) 5–12. [5] S. Krügener, C. Schaper, U. Krings, R.G. Berger, Bioresource Technology 100 (2009) 2855–2860. [6] G. Pigatto, A. Lodi, B. Aliakbarian, A. Converti, R.M.G.D. Silva, M.S.A. Palma, Bioresource Technology 143 (2013) 678–681. [7] T.D. Butters, D.C.A. Neville, in: J.M. Walker, R. Rapley (Eds.), Molecular Biomethods Handbook, Humana Press, New Jersey, 2008, pp. 495–513. [8] J. Labat-Robert, L. Robert, Pathologie Biologie 60 (2012) 66–75. [9] M. Zhang, S.W. Cui, P.C.K. Cheung, Q. Wang, Trends in Food Science & Technology 18 (2007) 4–19. [10] B. Dey, S.K. bhunia, K.K. Maity, S. Patra, S. Mandal, B. Behera, T.K. Maiti, S.R. Sikdar, S.S. Islam, International Journal of Biological Macromolecules 52 (2013) 312–318. [11] S. Zhao, Y.C. Zhao, S.H. Li, J.K. Zhao, G.Q. Zhang, H.X. Wang, T.B. Ng, Glycoconjugate Journal 27 (2010) 259–265. [12] J. Pohleven, N. Obermajer, J. Sabotic, S. Anzlovar, K. Sepcic, J. Kos, B. Kralj, B. Strukelj, J. Brzin, Biochimica et Biophysica Acta 1790 (2009) 173–181. [13] T.B. Andrea, S.S. Judith, M.H. Robert, L.K. Carl, Proceedings of the Society for Experimental Biology and Medicine 221 (1999) 281–293. [14] I.L. Shih, B.W. Chou, C.C. Chen, J.Y. Wu, C.Y. Hsieh, Bioresource Technology 99 (2008) 785–793. [15] G.T. Chen, X.M. Ma, S.T. Liu, Y.L. Liao, G.Q. Zhao, Carbohydrate Polymers 89 (2012) 61–66. [16] F.J. Cui, Y. Li, Z.H. Xu, H.Y. Xu, K. Sun, W.Y. Tao, Bioresource Technology 97 (2006) 1209–1216. [17] F.J. Cui, Y. Li, Y.Y. Xu, Z.Q. Liu, D.M. Huang, Toxicology In Vitro 21 (2007) 417–427. [18] Q.H. Liu, H.X. Wang, T.B. Ng, Peptides 25 (2004) 7–10. [19] J.K. Zhao, H.X. Wang, T.B. Ng, Toxicon 53 (2009) 360–366. [20] Y.R. Li, Q.H. Liu, H.X. Wang, T.B. Ng, Biochimica et Biophysica Acta 1780 (2008) 51–57. [21] F. Khan, A. Ahmad, M.I. Khan, Archives of Biochemistry and Biophysics 457 (2007) 243–251. [22] M.C.C. Silva, L.A. Santana, R. Mentele, R.S. Ferreira, A.D. Miranda, R.A. SilvaLucca, M.U. Sampaio, M.T.S. Correia, M.L.V. Oliva, Process Biochemistry 47 (2012) 1049–1059. [23] H. Kawagishi, J.I. Takagi, T. Taira, T. Murata, T.C. Usui, Phytochemistry 56 (2001) 53–58. [24] S. Anja, A.W. Peter, G. Harry, Bioresource Technology 102 (2011) 9121–9127. [25] Y.X. Sun, J.C. Liu, Bioresource Technology 100 (2009) 983–986. [26] N. Yang, D.F. Li, L. Feng, Y. Xiang, W. Liu, H. Sun, D.C. Wang, Journal of Molecular Biology 387 (2009) 694–705. [27] A.D. Maro, F. Farisei, D. Panichi, V. Severino, N. Bruni, A.G. Ficca, P. Ferranti, V. Capuzzi, F. Tedeschi, E. Poerio, Planta 234 (2011) 723–735.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Purification and partial characterization of a novel anti-tumor glycoprotein from cultured mycelia of Grifola frondosa Fengjie Cui a,b,∗ , Xinyi Zan a , Yunhong Li a , Yan Yang c , Wenjing Sun a,b,d,∗ , Qiang Zhou b,d , Silian Yu b,d , Ying Dong a a
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China Jiangxi Provincial Engineering and Technology Center for Food Additives Bio-production, Dexing 334221, PR China c National Engineering Research Center of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, PR China d Parchn Sodium Isovitamin C Co. Ltd, Dexing 334221, PR China b
a r t i c l e
i n f o
Article history: Received 27 August 2013 Received in revised form 19 October 2013 Accepted 21 October 2013 Available online 25 October 2013 Keywords: Grifola frondosa Glycoprotein Purification Structural characterization Anti-tumor activity
a b s t r a c t A novel glycoprotein GFG-3a with the molecular weight of 88.01 kDa and potent anti-tumor activity was isolated from the cultured mycelia of Grifola frondosa GF9801. GFG-3a was heat-sensitive with the decreasing anti-proliferative activity after treated from 56 ◦ C to 100 ◦ C for 10–120 min. GFG-3a was a glycoprotein with O-glycosylation and contained 6.20% carbohydrate composed of d-arabinose, d-fructose, d-mannose, and d-glucose with a molar ratio of 1.33:4.51:2.46:1.00. FT-IR and NMR spectra proved that GFG-3a contained protein and carbohydrate portions with 3-O-methyl-galactose residues, (1→4)-linked -galactose residues, and -linked glucose residues. Circular dichroism (CD) revealed that GFG-3a was a predominantly -sheet glycoprotein with a relatively small ␣-helical content. Protein sequencing and 3D model of GFG-3a were finally predicted by using MALDI-TOF-MS, NCBI blast search and online SWISSMODLE Workspace service. Our findings will be a reference for the further structure–activity relationship analysis of the mushroom glycoproteins. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Edible mushrooms are well-known for their nutritional/medicinal values and used worldwide as the dietary supplement and/or traditional medicine [1]. Numerous mushroomoriginated compounds with antitumor, immunomodulatory, antiviral and antimicrobial activities [2] have been isolated and identified including polysaccharides [3], glycoproteins [4], terpenes [5] and phenols [6]. Glycoproteins are the polysaccharide–peptide or polysaccharide–protein complexes with the protein/polypeptide chains covalently and specifically bound with carbohydrate sidechains [7,8]. Generally, glycoproteins have different structures and bioactivity mechanisms compared with those of polysaccharides. The processes for elucidating the polysaccharide structure include the determination of the monosaccharide composition, glycosidic linkage configuration, glycosidic linkage position, monosaccharide sequence, even secondary and 3D structure [9]. As for investigating
∗ Corresponding authors at: School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China. Tel.: +86 511 88780226; fax: +86 511 88780201. E-mail addresses: [email protected], [email protected] (F. Cui), [email protected] (W. Sun). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.025
the glycoprotein structure, the polysaccharide and protein/peptide chains are firstly elucidated, respectively. Then the glycosylated site in the glycoprotein is also an essential item to be determined [7]. Polysaccharides and glycoproteins possibly have significant difference of antitumor mechanisms. In most cases, polysaccharides exert their antitumor action through activating the host immune systems [10], while glycoproteins kill the tumor cells directly by arresting the cell cycle and/or inducing the apoptosis of tumor cells [11,12]. The famous edible mushroom Grifola frondosa belongs to the order Aphyllopherales and family Polyporeceae [13]. It has recently attracted considerable attention for its taste and health value [14]. Polysaccharides including -(1→6)-d-glucan and heteropolysaccharide are obtained from G. frondosa fruiting body and mycelia [15]. Our research group has focused on optimization of fermentation process for maximum G. frondosa mycelia production, structural elucidation, and antitumor mechanism assessment of bioactive polysaccharide–peptide/protein from the cultured mycelia [16,17]. A polysaccharide–peptide GFPS1b from the cultured mycelia of G. frondosa was obtained with significant prohibition of the gastric carcinoma tumor cells SCG-7901 in vitro [17]. However, to date, glycoprotein from the cultured mycelia of G. frondosa has not yet been reported. In the present study, a novel antitumor glycoprotein GFG3a was isolated and purified from the cultured mycelia of
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G. frondosa GF9801. The basic and potential 3D structural characteristics of GFG-3a were investigated, and its thermo-stability during the 37–100 ◦ C treatment was also evaluated. 2. Materials and methods 2.1. Strain and culture conditions The strain G. frondosa GF9801 was obtained and kept in our laboratory [16]. Stock culture was maintained on potato-dextroseagar (PDA) slant at 4 ◦ C, and sub-cultured every two months. The mycelia were grown at 25 ◦ C in a 20-L mechanical stirred fermentor (GRCB, Green Bio-engineering Co., Ltd., Zhenjiang, China) with working volume of 12 L with the optimized medium containing (g/L): glucose 45.2, KH2 PO4 2.97, peptone 6.58, MgSO4 ·7H2 O 1 and corn steep liquor 15 [16]. Mycelia were filtered off, washed with distilled water, and maintained in a frozen condition. 2.2. Isolation and purification of glycoprotein G. frondosa mycelia were homogenized using a blender and then extracted with 1 L of water at 4 ◦ C. The homogenate was centrifuged at 8000 × g for 20 min. The supernatant was precipitated with 80% saturated ammonium sulphate overnight at 4 ◦ C. The precipitate collected by centrifugation (10,000 × g, 20 min) was dissolved in and dialyzed against distilled water. The dialyzed solution was applied to a column (2.5 cm × 25 cm) DEAE-Sepharose fast-flow anionic resin (Pharmacia AP, GE). The column was eluted sequentially with 0.1, 0.3 and 0.5 M NaCl at pH 7.0 (every fraction tube 8 mL). The carbohydrate content of the eluate was determined spectrophotometrically at 490 nm using the phenol–sulfuric acid method. The protein content was determined by measuring the absorption at 280 nm. Three factions GFG-1, GFG-3, and GFG-5 were collected, dialyzed and freeze-dried. GFG-3 was detected to be the main anti-proliferative fraction and was further purified by gel filtration by AKTA purifier 100 (Amersham Biosciences, Sweden) on a SuperdexTM 75 prep grad column (Amersham Biosciences, Sweden) eluted with distilled water (pH 7.0). Three fractions of GFG-3a, GFG3b and GFG-3c were separated and then lyophilized for anti-tumor evaluation and structural analysis. 2.3. Assay of anti-proliferative activity The anti-proliferative activity of G. frondosa glycoprotein fractions was determined as follows. The mouse sarcoma cell line S-180 and human hepatoma cell line Bel-7402 were purchased from American Tissue Culture Collection (ATCC) and maintained in RPMI-1640 (Gibco, America) supplemented with 10% fetal bovine serum (FBS), 4 g/mL insulin, 100 mg/L streptomycin, and 100 IU/mL penicillin at 37 ◦ C in a humidified atmosphere of 5% CO2 . Cell (5 × 105 ) in their exponential growth phase was seeded into each well of a 96-well plate and incubated for 24 h before addition of the samples with the concentration of 5, 10, 20, 40, 80 and 160 g/mL. Incubation was carried out for another 48 h. The viability of cells was assessed in the MTT assay. The absorbance was detected in the microplate reader (Multiskan GO, Thermo Labsystems, America) at 570 nm. The inhibition ratio was calculated as follows: Inhibition ratio (%)=
OD570 nm (control)−OD570 nm (sample) ×100. OD570 nm (control)
2.4. Thermostability of glycoprotein GFG-3a Glycoprotein GFG-3a (20 g/mL) was kept at various temperatures (37, 56, 70, 100 ◦ C) for 30, 60, 90, 120 min, respectively. The
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S-180 and Bel-7402 cells (5 × 105 ) in the exponential growth phase were incubated for 48 h after adding the temperature-treated samples. The untreated sample was used as a positive control. The absorbance was detected at 570 nm for calculating the inhibition ratio. 2.5. SDS-PAGE The purity and molecular mass of GFG-3a were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) using a 12% resolving gel and blue bromophenol as the tracking dye. The molecular weight of glycoprotein GFG-3a was determined by comparison of its electrophoretic mobility with those of molecular weight standard markers. 2.6. Carbohydrate content and monosaccharide compositions Carbohydrate content of GFG-3a was determined by the phenol–sulfuric acid method using glucose as the standard. Monosaccharide compositions and their ratios of GFG-3a were determined by absolute hydrolysis followed by gas chromatography with rhamnose, arabinose, mannose, galactose and glucose as the standards. The hydrolysed glycoprotein and sugar standards were converted to the alditol acetate derivatives and then analyzed by a 6890N GC (Agilent Technologies, Santa Clara, CA, USA) with capillary column (30 m × 0.32 mm) and a flame-ionization. The oven temperature was maintained at 180 ◦ C for 3 min, and then increased gradually to 240 ◦ C at a rate of 2 ◦ C/min. The temperatures of detector and injector were set 260 ◦ C and 250 ◦ C, respectively. The carrier gas, supporter and stationary liquid were nitrogen, Chromosorb WAW DMCS, and 3% OV-1701, respectively. The carrier gas flow rate is 1.5 mL/min. 2.7. Protein content and amino acid composition Protein content of GFG-3a was determined by Micro BCATM Protein Assay Reagent Kit (Shenergy Biocolor BioScience & Technology Co., Shanghai, China) using bovine serum albumin as the standard. For determination of amino acid composition, GFG-3a was subjected to hydrolysis under vacuum in 6 M HCl at 110 ◦ C for 24 h. The hydrolyses were evaporated and the dried residue was re-dissolved in 0.02 M HCl, the amino acid composition was determined using a Hitachi 835-50G automatic amino acid analyzer (Hitachi Ltd., Tokyo, Japan). 2.8. Conformation analysis For determination of the glycosylation type, GFG-3a (5 mg) was dissolved in 0.2 M sodium hydroxide for 3 h followed by UV analysis by using a UV spectrophotometer (Varian cary 100, Varian, USA) ranging from 200 cm−1 to 400 cm−1 . The sample without sodium hydroxide treatment was used as the control. FT-IR spectral analysis of GFG-3a was recorded using the potassium bromide (KBr) pellet method with a Nicolet Fourier transform infrared spectrometer (Thermo Nicolet Co., USA) in the range of 400–4000 cm−1 . CD spectra of glycoprotein GFG-3a (0.5 mg/mL, in distilled water) were obtained by scanning from 190 nm to 300 nm on the JASCO J-810 spectropolarimeter (Jasco Corporation, Japan). Secondary structure percentages were calculated by de-convoluting CD analysis using CD analysis programs including CONTIN, SELCON3 and CDSSTR. A reference protein package consisting of 48 kinds of proteins was used in these three programs. 1 H and 13 C NMR spectra were obtained using with a 400-MHz Bruker model AVANCE II spectrometer. Samples were dissolved in D2 O and mainly examined at 50 ◦ C.
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Fig. 1. Chromatograph isolation and purification of glycoprotein and SDS-PAGE of GFG-3 and GFG-3a. (A) Anion-exchange chromatography of crude extract from the cultured mycelia of Grifola frondosa GF 9801 on DEAE-Sepharose Fast Flow column (2.5 cm × 20 cm). Flow rate: 2 mL/min; fraction size: 8 mL; temperature for chromatography: 20 ◦ C. (B) Gel filtration of fraction GFG-3 from DEAE-Sepharose Fast Flow column on SuperdexTM 75 prep grad column and SDS-PAGE of GFG-3 and GFG-3a.
Chemical shifts are expressed in ı PPM, relative to external MeSi (ı = 0).
using the MASCOT search engine. The search parameters were defined as follows: database, Swiss-Prot; taxonomy, Eucaryotes; enzyme, trypsin; and allowance of one missed cleavage.
2.9. In-gel tryptic digestion 2.11. Protein modeling The band of GFG-3a on the SDS-PAGE gel was excised and rinsed three times with Milli-Q purified water. The gel slices were dried and added to the trypsin solution for incubating at 37 ◦ C overnight. The supernatant was poured out and kept when trypsin hydrolysis was finished. The gel was subsequently extracted with 100 L 0.1% and 5% TFA in 50% ACN by gently mixing for 15 min at 20 ◦ C, respectively. The obtained supernatants were combined and freeze-dried for further MALDI analysis.
A 3D structure model of GFG-3a was obtained using the automatic modeling mode service available in the SWISS-MODEL Workspace. The online procedure gave the 3D model on the basis of multiple-threading alignments. The program automatically selected the template which corresponded to the 3D structures of acetyl-transferase (pdb code: 1Y9W). 3. Results and discussion
2.10. Mass spectrometry and protein identification MALDI mass spectrometric analysis was performed on AXIMA resonance MALDI-TOF-MS (Shimadzu Corp. JP), equipped with a 337 nm nitrogen laser in positive ion detection. Spectra were accumulated until a satisfactory S/N had been obtained. Parent mass speaks with the range from 400 to 1600 m/z were picked out for MS/MS analysis. The peptide mass finger-printings of glycoprotein and MS/MS data were acquired and processed using Mass-Lynx V4.1 software (Micromass) and were converted to PKL files by the Protein-Lynx 2.2.5 software (Waters). The PKL files were analyzed
3.1. Purification and screening of glycoprotein fractions with anti-proliferative activity This study was attempted to screen the antitumor glycoprotein from G. frondosa mycelia by bioactivity-directed purification procedures. The antitumor potency of the fractions GFG-1, GFG-3, and GFG-5 from the DEAE-Sepharose Fast Flow column (Fig. 1A) was evaluated by analyzing the inhibition ratio of the S180 and Bel-7402 cells growth. Fraction GFG-3 eluted with 0.3 M NaCl showed highest antitumor activity on S180 and Bel-7402 cells
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Fig. 2. Inhibition of proliferation of S180 cells (A) and Bel-7402 cells (B) by different concentrations of glycoprotein fractions from the cultured mycelia of Grifola frondosa GF9801.
in a dose dependent manner compared with those of GFG-1 and GFG-5 (Fig. 2). However, GFG-3 appeared to be an unpurified sample based on the presence of three bands in the SDS-PAGE electrophoregram (Fig. 1B). Subsequently, fraction GFG-3 was further separated into three sub-fractions including a larger GFG-3a and two tiny GFG-3b and GFG-3c by using a SuperdexTM 75 prep grad column gel filtration on the AKTA Purifier 100 (Fig. 1B). The fraction GFG-3a appeared as a single band with a molecular weight of 88.01 kDa in SDS-PAGE electrophoregram and exhibited the high percentage and anti-proliferative activity (Fig. 2). 3.2. Thermostability of GFG-3a Thermostability experiment results showed that the GFG-3a (20 g/mL) was heat-sensitive although its anti-proliferative activity was stable at 37 ◦ C with the treatment time from 30 min to 120 min (Fig. 3). The inhibition ratio on S180 cells and Bel-7401 decreased to 33.56% and 8.54% at 56 ◦ C treated for 30 min and to negligible levels treated for over 90 min. GFG-3a was even positive to the tumor cells growth when the treatment temperature kept increasing from 70 ◦ C to 100 ◦ C. Similar results could be found in the hemagglutinating activity of some lectins under different temperature treatments. For example, the hemagglutinating activity of mushroom Xerocomus spadiceus lectin [18], Inocybe umbrinella
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Fig. 3. Inhibition of proliferation of S180 cells (A) and Bel-7402 cells (B) by GFG-3a (20 g/mL) with various temperature treatments.
lectin [19], and Pleurotus citrinopileatus lectin [20] were moderately thermostable and unaffected when temperatures were set between 20 and 60 ◦ C. About 25–50% of their hemagglutinating activity remains at 70 ◦ C. However, the thermostability of GFG-3a was significantly different with those of Fusarium solani lectin [21] and BfL [22] with the temperature tolerance up to 70–100 ◦ C.
3.3. Carbohydrate content and monosaccharide compositions The phenol–sulfuric acid method of GFG-3a indicated that GFG3a contains 6.20% (w/w) of carbohydrate. The result was similar with the carbohydrate content of BfL accounts for 6.24% [22]; however, slightly higher than the 4.9% (w/w) sugar content of MAL [23] and 3.9% (w/w) neutral sugar of F. solani lectin [21], and lower than 24% (w/w) carbohydrates in the soluble protein from green microalgae Tetraselmis sp. [24] and 9.3% (w/w) neutral sugar in the novel 114 kDa hexameric lectin [25]. Monosaccharide analysis showed the presence of d-arabinose, d-fructose, d-mannose, and d-glucose with a molar ratio of 1.33:4.51:2.46:1.00. The origins and purification procedures of glycoprotein or glycopeptide could result in the difference of monosaccharide compositions. For example, Mycoleptodonoides aitchisonii glycoprotein MAL consisted of the GlcNAc, Gal, Man, and
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Fig. 4. Determination of the type of monosaccharide linkage (A), circular dichroism (CD) spectrum of glycoprotein GFG-3a (B), and NMR spectrum of glycoprotein GFG-3a in D2 O: (C) 1 H NMR and (D) 13 C NMR.
Xyl with the molar ratio of 1.56:4.89:3.68:1.00 [23], which were remarkably different with those of GFG-3a. 3.4. Protein content and amino acid composition The BCA assay showed that GFG-3a contains 87.04% of soluble protein portion which proved that GFG-3a was a protein-dominate glycoprotein. Amino acid analysis results (Table 1) revealed that GFG-3a contained 17 kinds of amino acids. It contained high amount of Asp (8.52%), Met (8.07%) and low amount of Lys (2.02%) and His (1.47%), which indicated that GFG-3a was possibly an acidic
Table 1 Amino acid composition of GFG-3a. Amino acid
Mol%
Amino acid
Mol%
Asp Glu Ser His Gly Thr Arg Ala Tyr
8.52 6.78 3.28 1.47 3.03 3.36 2.56 3.96 2.65
Cys Val Met Phe Ile Leu Lys Pro Trp
3.96 3.93 8.07 3.35 2.34 3.85 2.02 2.74 n.d.a
a
Not determined.
glycoprotein. Similar results were found that M. aitchisonii lectin MAL had the high content of acidic amino acid (AsX 12.1%) with its low theoretical pI value (4.3–4.5) [23] and BfL was a glycoprotein from Bauhinia forficate seeds with pI value 5.5 [22]. However, F. solani lectin was possible a basic protein with the high content of alkaline amino acid (Lys, 11.6%) and pI value equal to pH 8.7 [21].
3.5. Conformation analysis The absorption of NaOH-treated GFG-3a at 240 nm was much stronger than that of untreated GFG-3a, which revealed that GFG3a contained O-glycosidic linkage since O-linked glycans could be released by -elimination in the presence of sodium hydroxide (Fig. 4A). The diffuse reflectance Fourier transform infrared spectra of glycoprotein GFG-3a (Fig. 5) was followed for conformation analysis. A strong and broad absorption peak from 3500 cm−1 to 3200 cm−1 was O H stretching vibrations of polysaccharides and N H stretching vibrations of protein. Two peaks from 3000 cm−1 to 2800 cm−1 were C H stretching vibrations and bending vibrations. The absorption peaks from 1600 to 1200 cm−1 were C H bending vibrations of carbohydrates. The peak at 1658 cm−1 was assigned to the absorbance of the C O stretching vibrations and N H bending vibrations of the acylamino. Absorption peak at 1422 cm−1 was C N stretching vibrations and may be due to carboxylate groups of
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Fig. 5. FT-IR spectrum of glycoprotein GFG-3a.
galacturonic acid residues. The wave numbers between 1200 and 800 cm−1 represent the finger print region for carbohydrates. The chemical structure of GFG-3a was also elucidated by 13 C NMR and 1 H NMR spectroscopy (Fig. 4C and D). The 13 C NMR spectrum of GFG-3a showed that it contained carbohydrate and protein, with a signal at 175.9 ppm assigned to the CONH group of protein. GFG-3a also contained acetyl groups
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in the polymer as indicated by the presence of signals at 21.3 and 174.7 ppm, which belonged to methyl carbons and carbonyl groups of acetyl groups, respectively. Signals at 130.79, 129.39, 129.10, and 127.65 ppm were assigned to the C-1, C-2/C6, C-3/C-5 and C-4 of phenylalanine residues. The C-4 signal of (1→3)-linked glucose showed a downfield shift due to the glycosylation effect and appeared at 69.62 ppm. The C-6 signal at ı 66.21 ppm indicated the presence of the d-(1,6) linked glycoside residues. Signals at 60.56, 60.45, 56.48 and 56.19 ppm represented the carbons of OCH3 groups from 3-O-methylgalactose typical chemical shifts for OCH3 carbons fall in the range of 55–61 ppm. The signals of 1 H NMR at 7.3–7.2 ppm were assigned to the benzene ring of phenylalanine residues. Signals of 1 H NMR at 3.7 and 3.8 ppm were assigned to H-2 and H-3 of (1→4)-linked -galactose residues, respectively. The signals at 3.6–3.5 ppm represent H2 of the -linked galactose and 3.4–3.3 ppm were assigned to the H-2 of the -linked glucose. The proton NMR for GFG-3a also showed that a significant amount of acetyl groups in the region 2.2–2.1 ppm. Proton NMR of GFG3a had two signals at 1.4–1.2 ppm representing the methyl group of rhamnose residues. Many antitumor polysaccharide–protein complexes from Basidiomycetes mushroom are characterized as -(1→3) linkages in the main glucan chain and additional (1→6) branch points [25]. For instance, a new water-soluble intracellular polysaccharide PTP from the mycelium of Polyporus albicand consisted of a backbone composed of (1→3)-linked-d-mannopyranodyl, (1→3,6)-linked--d-mannopyranodyl and (1→6)-linked-␣-d-galactopyranosyl residues in the ratio of 3:1:1,
Fig. 6. Peptide mass fragments (A) and the predicted three-dimensional structure (B) of GFG-3a confirmed by a NCBI blast search and online SWISS-MODLE Workspace. The GFG-3a contains two domains maybe linked via hydrophobic interactions.
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and terminated with a single non-reducing terminal (1→3,6)--dmannopyranodyl residues along the main chain [27]. Based on CD data, the secondary structure content of GFG-3a was estimated to be 16.6% of ␣-helix, 44.4% of -sheet, 7.9% of turn and 31.1% of unordered structure, indicating GFG-3a was a predominantly -sheet glycoprotein with a lower amount of ␣helical content (Fig. 4B). Similar secondary glycoprotein structure was found that B. forficate seeds and F. solani lectins had the predominant -sheet structure with the 27% -sheet, 22% -turn and 19% ␣-helices [22], and 26% -sheet, 22% -turn and 9% ␣-helices [21], respectively. 3.6. Protein sequencing and model Generally, few specific glycoprotein or lectins databases are available for searching or comparing the purified fungal and bacterial glycoproteins. The sequence of GFG-3a was analyzed to search for homologous proteins in the current known protein databases and to perform structure predictions. The results revealed that the GFG-3a sequence was similar to GCN5-related N-acetyltransferase (gi|496194480) composed of 191 amino acid residues and a molecular mass of 22,227 Da confirmed by mass spectrometry (MADIF-TOF-MS) and an NCBI BLAST search (Fig. 6A). In order to predict the GFG-3a 3D structure, the online SWISS-MODLE Workspace was used and the GFG-3a 3D model was obtained by selecting the 3D structure of the acetyl-transferase as template. The overall fold of the GFG-3a model was shown in Fig. 6B. The protein core was composed of two domains linked via hydrophobic interactions. Protein Workshop reveals that C-terminal domain of GFG-3a adopted a fold which was usually observed in catalytically active proteins including N-glycanases, N-acetyltransferases, transglutaminases and cysteine proteases [26,27]. In the present study, the potential 3D structural model of G. frondosa glycoprotein GFG-3a was obtained as a necessary reference for the 3D structure and structure–activity relationship analysis of other mushroom glycoproteins. However, structural information obtained by MS still has some limits including incomplete sequence information, lower identity to the known protein, and limited typical mass difference to the polysaccharide portions. Enzymatic digestion, column affinity chromatography, and crystallization for separating and analyzing the protein and polysaccharide portions and glycosylation site are still necessary to provide more data basis for further GFG-3a structural analysis. 4. Conclusions A novel glycoprotein GFG-3a from cultured mycelia of G. frondosa GF9801 was successfully purified and further structureelucidated by GC, FT-IR, NMR and circular dichroism (CD). 3D structure of its protein portion was finally obtained by using MALDI-TOF-MS, NCBI blast search and online SWISS-MODLE Workspace service. Future studies would focus on finding the carbohydrate-binding site of GFG-3a and the growth inhibitory mechanism of GFG-3a for clarifying its antitumor mechanisms and developing GFG-3a as an antitumor agent or an ingredient in health food.
Acknowledgements This work was supported by funding from National Natural Science Foundation of China (NSFC 31101269), China Postdoctoral Science special Foundation (2013T60648), National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2012BAD36B05), the Second Class General Financial Grant from the China Postdoctoral Science Foundation (Grant No. 2012M511702), 2012 Excellent Key Young Teachers Project of Jiangsu University, Graduate Research and Innovation Projects of Jiangsu Province (CXLX12 0670); Advanced Programs of Jiangxi Postdoctoral Science Foundation ([2012]195); the Research Foundation for Advanced Talents of Jiangsu University; Science & Technology Platform Construction Program of Jiangxi Province. References [1] L. He, P.F. Ji, J.W. Cheng, Y.B. Wang, H. Qian, W.Q. Li, X.G. Gong, Z.Y. Wang, Food Chemistry 141 (2013) 946–953. [2] L.B. Zhou, B. Chen, International Journal of Biological Macromolecules 48 (2011) 1–4. [3] N.S. Li, C.Y. Yan, D.H. Hua, D.Z. Zhang, International Journal of Biological Macromolecules 57 (2013) 285–290. [4] M. Zhang, L. Zhu, S.W. Cui, Q. Wang, T. Zhou, H.S. Shen, International Journal of Biological Macromolecules 48 (2011) 5–12. [5] S. Krügener, C. Schaper, U. Krings, R.G. Berger, Bioresource Technology 100 (2009) 2855–2860. [6] G. Pigatto, A. Lodi, B. Aliakbarian, A. Converti, R.M.G.D. Silva, M.S.A. Palma, Bioresource Technology 143 (2013) 678–681. [7] T.D. Butters, D.C.A. Neville, in: J.M. Walker, R. Rapley (Eds.), Molecular Biomethods Handbook, Humana Press, New Jersey, 2008, pp. 495–513. [8] J. Labat-Robert, L. Robert, Pathologie Biologie 60 (2012) 66–75. [9] M. Zhang, S.W. Cui, P.C.K. Cheung, Q. Wang, Trends in Food Science & Technology 18 (2007) 4–19. [10] B. Dey, S.K. bhunia, K.K. Maity, S. Patra, S. Mandal, B. Behera, T.K. Maiti, S.R. Sikdar, S.S. Islam, International Journal of Biological Macromolecules 52 (2013) 312–318. [11] S. Zhao, Y.C. Zhao, S.H. Li, J.K. Zhao, G.Q. Zhang, H.X. Wang, T.B. Ng, Glycoconjugate Journal 27 (2010) 259–265. [12] J. Pohleven, N. Obermajer, J. Sabotic, S. Anzlovar, K. Sepcic, J. Kos, B. Kralj, B. Strukelj, J. Brzin, Biochimica et Biophysica Acta 1790 (2009) 173–181. [13] T.B. Andrea, S.S. Judith, M.H. Robert, L.K. Carl, Proceedings of the Society for Experimental Biology and Medicine 221 (1999) 281–293. [14] I.L. Shih, B.W. Chou, C.C. Chen, J.Y. Wu, C.Y. Hsieh, Bioresource Technology 99 (2008) 785–793. [15] G.T. Chen, X.M. Ma, S.T. Liu, Y.L. Liao, G.Q. Zhao, Carbohydrate Polymers 89 (2012) 61–66. [16] F.J. Cui, Y. Li, Z.H. Xu, H.Y. Xu, K. Sun, W.Y. Tao, Bioresource Technology 97 (2006) 1209–1216. [17] F.J. Cui, Y. Li, Y.Y. Xu, Z.Q. Liu, D.M. Huang, Toxicology In Vitro 21 (2007) 417–427. [18] Q.H. Liu, H.X. Wang, T.B. Ng, Peptides 25 (2004) 7–10. [19] J.K. Zhao, H.X. Wang, T.B. Ng, Toxicon 53 (2009) 360–366. [20] Y.R. Li, Q.H. Liu, H.X. Wang, T.B. Ng, Biochimica et Biophysica Acta 1780 (2008) 51–57. [21] F. Khan, A. Ahmad, M.I. Khan, Archives of Biochemistry and Biophysics 457 (2007) 243–251. [22] M.C.C. Silva, L.A. Santana, R. Mentele, R.S. Ferreira, A.D. Miranda, R.A. SilvaLucca, M.U. Sampaio, M.T.S. Correia, M.L.V. Oliva, Process Biochemistry 47 (2012) 1049–1059. [23] H. Kawagishi, J.I. Takagi, T. Taira, T. Murata, T.C. Usui, Phytochemistry 56 (2001) 53–58. [24] S. Anja, A.W. Peter, G. Harry, Bioresource Technology 102 (2011) 9121–9127. [25] Y.X. Sun, J.C. Liu, Bioresource Technology 100 (2009) 983–986. [26] N. Yang, D.F. Li, L. Feng, Y. Xiang, W. Liu, H. Sun, D.C. Wang, Journal of Molecular Biology 387 (2009) 694–705. [27] A.D. Maro, F. Farisei, D. Panichi, V. Severino, N. Bruni, A.G. Ficca, P. Ferranti, V. Capuzzi, F. Tedeschi, E. Poerio, Planta 234 (2011) 723–735.
