Isolation, characterization, molecular cloning and modeling of a new lipid transfer protein with antiviral and antiproliferative activities from Narcissus tazetta

peptides 29 (2008) 2101–2109

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Isolation, characterization, molecular cloning and modeling of a new lipid transfer protein with antiviral and antiproliferative activities from Narcissus tazetta Linda S.M. Ooi a, Li Tian a,b,c, Miaoxian Su a, Wing-Shan Ho a, Samuel S.M. Sun a,c, Hau-Yin Chung a, Henry N.C. Wong d, Vincent E.C. Ooi a,* a

Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China c Life Science Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China d Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China b

article info

abstract

Article history:

A fetuin-binding peptide with a molecular mass of about 9 kDa (designated NTP) was

Received 25 July 2008

isolated and purified from the bulbs of Chinese daffodil, Narcissus tazetta var. chinensis L.,

Received in revised form

by gel filtration and high-performance liquid chromatography, after removing the man-

27 August 2008

nose-binding proteins by mannose–agarose column. Molecular cloning revealed that NTP

Accepted 28 August 2008

contained an open reading frame of 354 bp encoding a polypeptide of 118 amino acids which

Published on line 9 September 2008

included a 26-amino-acid signal peptide. An analysis of the deduced amino acid sequence of NTP shows considerable sequence homology to the non-specific lipid transfer proteins

Keywords:

(nsLTPs) of certain plants. Model of the three-dimensional (3D) structure of NTP exhibits an

Narcissus tazetta

internal hydrophobic cavity which can bind lipid-like molecules and transfer a wide range of

Non-specific lipid transfer proteins

ligands. As a member of the putative non-specific lipid transfer protein of N. tazetta, NTP did

Respiratory syncytial virus

not possess hemagglutinating activity toward rabbit erythrocytes. In a cell-free system, it

H1N1 virus

could arrest the protein synthesis of rabbit reticulocytes. Using the in vitro antiviral assays,

HL-60 cells

NTP could significantly inhibit the plaque formation by respiratory syncytial virus (RSV) and the cytopathic effect induced by influenza A (H1N1) virus, as well as the proliferation of human acute promyelocytic leukemia cells (HL-60). # 2008 Elsevier Inc. All rights reserved.

1.

Introduction

In the early literature, the extract of Narcissus tazetta var. chinensis L. (family Amaryllidaceae) was shown to exert antitumor [6] and antiviral activities [29]. The alkaloids of Narcissus extracts were believed to play an important role in the inhibitory effects in vitro, such as through inhibition of the DNA polymerase of avian myeloblastosis virus [22] and suppression of cancer via blocking the peptide bond formation by eukaryotic

ribosomes [4]. Nevertheless, it has been shown that the mannose-binding lectins isolated from N. tazetta also have antiviral activity against bovine immunodeficiency virus (BIV) through inhibition of syncytium formation [17,18], as well as against influenza A (H1N1) virus and respiratory syncytial virus (RSV) through inhibition of cytopathogenicity (unpublished data). However, they do not display antitumor activity in vitro up to the concentration of 5.4 mM (140 mg/mL) [18]. Some interesting proteins which are similar to the mannose-binding lectins in

* Corresponding author. Tel.: +852 2609 6353; fax: +852 26035745. E-mail address: [email protected] (Vincent E.C. Ooi). 0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2008.08.020

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molecular mass have also been isolated and purified from N. tazetta. A comparative analysis of N-terminal amino acid sequences of these proteins using NCBI Blast searching shows that they have considerable sequence homology to the known non-specific lipid transfer proteins (nsLTP) in some plants. The non-specific lipid transfer proteins are known to be able to bind and carry fatty acids and phospholipids across cell membrane [13,25]. Although no definite role for non-specific lipid transfer proteins is defined in plants, they may be important in the defense of plants against invasion of bacteria and fungi [7,15,24]. In this study we report the purification, characterization, molecular cloning and three-dimensional (3D) modeling as well as antiviral and antiproliferative activities of a new putative non-specific lipid transfer protein from N. tazetta var. chinensis L.

2.

Materials and methods

2.1.

Isolation of proteins

Fresh bulbs of N. tazetta var. chinensis were purchased from the local flower market in Hong Kong during the month of February, near to the date of the Chinese Lunar New Year. They were stored in cold chest after arrival until use. The whole bulbs were skinned, chopped and extracted with ice-cold 0.2 M NaCl (2 mL per gram of material) in a Waring blender (about 2 min with intermittent 1 min). The homogenate was centrifuged at high speed (11,279g, 30 min), and (NH4)2SO4 (561 g per litre of supernatant) was added to the resulting supernatant to 80% saturation. The precipitate from this saturated solution was retrieved again by high-speed centrifugation (11,279g, 30 min) and redissolved in distilled water, which was dialyzed extensively through a membrane with a molecular cut-off at 6000–8000 against distilled water (at least 10 volume, four changes) and the dialysate was re-centrifuged at 11,279g, for 10 min to remove all water-insoluble material and then lyophilized to yield a crude powder. The crude powder was dissolved in Mes buffer (20 mM, pH 6.5) and applied on a mannose–agarose (Sigma) column that was pre-equilibrated with the same buffer. After the breakthrough-peak (M1) was eluted with the equilibrating buffer the M1 fraction was dialyzed against water to remove salt and freeze-dried to yield a M1 powder, which was re-suspended in Tris–HCl buffer (50 mM, pH 8.15), and loaded unto a column packed with fetuin–agarose (Sigma) which was pre-equilibrated with the Tris–HCl buffer. After the breakthrough-peak (F1) was completely eluted, the bounded material was desorbed with an eluting buffer (pH 3.0) containing glycine–HCl (50 mM) in NaCl (0.5 M). The eluted F2 fraction was freeze-dried and desalted using PD 10 column (Pharmacia). The resultant F2 fraction was gel filtered through Superose 12 column using AKTA-FPLC (fast protein liquid chromatography) system (Pharmacia Amersham Biotech) with an eluting buffer of ammonium bicarbonate (0.1 M, pH 8) (Fig. 1). Fraction numbers 39–42 (the major peak at retention time 40  0.11 min) was pooled and re-fractionate on FPLC-Superose 12 column. The eluted peak with retention time at 41.2  0.19 min was collected as a fetuin-binding protein (FBP) (Fig. 2).

Fig. 1 – Gel filtration of the peak of F2 fraction by the AKTAFPLC system on a Superose 12 HR 10/300 column. The column was eluted with NH4HCO3 buffer (0.1 M, pH = 8) at a flow rate of 0.4 mL/min. The major peak with an average retention time (in min) of 41.2 W 0.19 (mean W S.D., n = 4) was collected as fetuin-binding nsLTP-NTP, which has a molecular mass of about 9 kDa.

2.1.1. Molecular size estimation by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) The molecular weight of the proteins was estimated by gel filtration on Superose 12 column (Pharmacia) using AKTAFPLC system (Pharmacia-Amersam Biotech.). The column had been calibrated with bovine serum albumin (molecular mass 66 kDa), carbonic anhydrase (29 kDa), cytochrome C (12.4 kDa),

Fig. 2 – SDS-PAGE profile of the putative non-specific lipid transfer protein of Narcissus tazetta var. chinensis. Lane M, markers (from top to bottom); lanes 1–3, the purified NTP (0.8 mg, 1.1 mg, and 1.4 mg, respectively, were loaded).

peptides 29 (2008) 2101–2109

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aprotinin (6.5 kDa), and cytidine (246 Da). The eluting buffer is 100 mM ammonium bicarbonate (pH 8), and the flow rate is 0.4 mL/min. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out throughout the process of separation to monitor the purity. The molecular mass of sample was deduced from the standard curve [19].

After agarose gel electrophoresis was completed, the targeted DNA band was recovered using QIAquick Gel Extraction Kit (Qiagen), ligated to pGEM-T Easy vector (Promega) and then transformed into Competent cells (E. coli strain DH5a). The PCR positive clone was sequenced using T7 and SP6 primers.

2.1.2. Analyses of N-terminal and C-terminal amino acid sequences

The SMARTTM RACE cDNA Amplification Kit (Clontech) was used for 30 -end and 50 -end of cDNA cloning. According to the sequencing result of RT-PCR, a pair of PCR primers, NLTPR-1 (50 -GCCAGGGTTTTGAGGCAGCCGCACG-30 ) and NLTPR-2 (50 AGACCACGCCCGACCGTCAGACAGC-30 ), was designed for 50 RACE and 30 RACE. Reverse transcription (RT) was conducted by using Powerscript Reverse Transcriptase (Clontech) according to the protocols in the Kit for both 50 RACE and 30 RACE operations. The cDNA sequencing was performed using RTPCR as mentioned above except that NLTP-1 primer was substituted by SP6 primer for 50 RACE.

The isolated protein on gel was blotted onto a polyvinylidene difluoride membrane (PVDF) in a modified Dunn’s Buffer at a constant voltage (30 V) in a mini trans-blot cell (Bio-Rad) at 4 8C for 50 min. The target proteins were cut off and analyzed by Procise1 protein sequencing system (Model 491, Applied Biosystems, USA), which included Edman degradation unit and a capillary HPLC device [19]. Peptides (C-terminal peptide ladder) which were generated by enzymatic digestion with a mixture of carboxypeptidases CPP and CPY, could be detected by MALDI-MS (matrix-assisted laser desorption/ionizationmass spectrometry) and the C-terminal amino acid sequence could thereby be determined by calculation of the differences between consecutive peaks (performed by the Protein and Nucleic Acid (PAN) Facility in the Beckman Center at Stanford University Medical Center, California, USA).

2.2.3. 30 RACE (rapid amplification of 30 cDNA ends) and 50 RACE

2.2.4.

Full-length cDNA amplification

2.2.

Molecular cloning of full length cDNA encoding NTP

A pair of primers, NLTP-3 (50 -ATGGTTCGTTCATCCGTCCTCGTTT-30 ) and NLTP-4 (50 -TCACTTCACCTTGGTGCAGTCCGT-30 ), was designed to amplify the full-length cDNA, which were obtained from the sequences of 50 cDNA and 30 cDNA. PCR was carried out using a total volume of 50 mL of the reaction solution containing 5 mL of HiFi buffer (10, plus 20 mM MgSO4), 1 mL comprising 10 mmol/L each of dNTPs, 1 mL of LP-3 (10 mmol/L), 1 mL of LP-4 (10 mmol/L), 2.5 mL of cDNA (from 30 RACE RT product) and 1 unit of Platinum Taq High Fidelity (Invitrogen) using the following protocol: 94 8C for 3 min followed by 30 cycles of amplification (94 8C for 30 s, 58 8C for 30 s, 68 8C for 1 min). The cDNA sequencing was performed with RT-PCR as mentioned above.

2.2.1.

Extraction of total RNA

2.2.5.

2.1.3. Glycoprotein detection using periodic acid-Schiff (PAS) staining The presence of carbohydrate in the molecule was detected by periodic acid-Schiff (PAS) staining after Western blotting onto an immobilized membrane (PVDF) as described previously [19].

Modeling of three-dimensional (3D) structure of NTP

Total RNA was extracted using Tripure Isolation Reagent (Roche) from 10-day-old leaves of N. tazetta var. chinensis grown in the Green House, Department of Biology, The Chinese University of Hong Kong, and purified using RNeasy Plant Mini Kit (Qiagen). Total RNA sample was diluted with RNase-free water, stored in 80 8C ultra low temperature refrigerator.

Based on the sequencing result for full-length cDNA amplification, the amino acid sequence of NTP was deduced. Molecular model of the three-dimensional (3D) structure of NTP was simulated using SWISS-MODEL programme provided online by ExPASy, and the 3D model (Fig. 6) was drawn using Rasmol software.

2.2.2. RT-PCR (reverse transcription-polymerase chain reaction)

2.3.

Assays for bioactivities

A pair of degenerate primers, NLTP-1 (50 -GCNGTNACNTAYGGNACNGT-30 ) and NLTP-2 (50 -YTTNCCNGGNACNCCRTANGC30 ), were designed on the basis of the N-terminal amino acid sequence (AVTYGTV) and C-terminal amino acid sequence (AYGVPGK). The synthesis of the first-strand cDNA (reverse transcription) was performed with Super Script II (Invitrogen) and 2 mg of total RNA following the product protocol. The second-strand was synthesized using a total volume of 25 mL of the reaction solution containing 2.5 units of Taq DNA polymerase (Promega), 1 mL of NLTP-1 (10 mmol/L), 1 ml of NLTP-2 (10 mmol/L), 2 mL comprising 2 mmol/L each of dNTPs, 1.5 mL of MgCl2 (25 mmol/L) and first-stranded cDNA (2 mL). PCR (polymerase chain reaction) was performed as follows: cDNA was denatured at 94 8C for 3 min followed by 30 cycles of amplification (94 8C for 1 min, 55 8C for 1 min, 72 8C for 2 min).

2.3.1.

Assay for cell-free protein synthesis inhibitory activity

The NTP protein obtained from the aforementioned procedure was tested for the ability to inhibit incorporation of [4,5-3H] Lleucine into protein. Ten microliter (mL) of the test sample was added to 10 mL of hot mixture containing KCl (500 mM), MgCl2 (5 mM), phosphocreatine (130 mM) and [4,5-3H] L-leucine (1 mCi) as a working mixture which was then added to 30 mL of untreated rabbit reticulocyte lysate (Promega, Madison) containing hemin (0.1 mM) and creatine kinase (5 mL) as a reaction mixture. After incubated at 37 8C for 30 min, a 5-mL aliquot of the reaction mixture was added to 1 mL of distilled water to stop the reaction, and this was followed by addition of 0.5 mL of H2O2 (10%) in NaOH (1 N) and further incubation at 30 8C for 30 min. The resultant proteins were precipitated with 3 mL of 2% casein hydrolysate in 25% trichloroacetic acid (TCA)

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at 4 8C and collected by filtration through a Whatman GB/F filter paper using a cell harvest. The filter paper was finally washed with ethanol (absolute) once and dried in order to be measured the radioactivity of [4,5-3H] L-leucine which was incorporated into protein by liquid scintillation counting.

2.3.2.

Cell culture and cell proliferation assay

HL-60 cells (human acute promyelocytic leukemia cell line) were grown in RPMI-1640 medium (Gibco) supplemented with fetal bovine serum (FBS, 10%) (Gibco), gentamicin (25 mg/mL, Sigma) and L-glutamine (200 mM, Sigma). Cultures were maintained in a humidified atmosphere with CO2 (5%) at 37 8C. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay was used to determine and monitor the number of viable cells and the rate of cell survival as affected by the test samples [16,27]. 4  103 cells/well were incubated in 96well plates in the presence or absence of the tested samples for 72 h in a final volume of 200 mL. At the end of treatment, 20 mL of MTT (5 mg/mL in PBS, Sigma) was added to each well and incubated for an additional 4 h at 37 8C. The purple–blue MTT formazan precipitate was dissolved in DMSO (150 mL). The activity of mitochondria, reflecting cellular growth and viability, was evaluated by measuring the optical density (OD) at 570 nm on microtiter plate reader. At each concentration of the tested samples, two different experiments were carried out in three replicates. The inhibitory effect of the samples was calculated by the following formula: % inhibition = (1  Absorbancetreatment group/Absorbancecontrol)  100%. The concentration of the sample required to inhibit 50% of the proliferation of the cell line is abbreviated as EC50.

2.3.3. Antiviral assay 2.3.3.1. Cells and viruses. Madin-Darby canine kidney (MDCK) cells, human larynx epidermoid carcinoma (HEp-2) cells, influenza A (H1N1) virus and respiratory syncytial (RSV, Long strain) virus were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured in Eagle’s minimum essential medium (EMEM, Invitrogen, Carlsbad, CA) supplemented with sodium bicarbonate (1.5 g/ L), sodium pyruvate (1 mM), gentamycin sulphate (24 mg/L), Lglutamine (0.29 g/L) (Sigma, St. Louis, MA), and fetal bovine serum (10%, Hyclone, Logan, UT, USA) at 37 8C in a humidified atmosphere with 5% CO2. For H1N1 virus culture, 2  105 MDCK cells per well were seeded into 12-well plates and incubated for 24 h. After washing the cells with phosphate-buffered saline (PBS), H1N1 viruses were diluted using trypsin–EMEM, which is a serumfree EMEM supplemented with trypsin (12.5 mg/mL), and added to the MDCK cell monolayers (1 mL/well). When more than 90% of the cells died, supernatant were collected and centrifuged at 4 8C, 2000g for 10 min to remove cell debris. Aliquots of supernatant were frozen at 70 8C. RSV was propagated in confluent HEp-2 cell monolayers in 96-well plates (4  104 cells/well) using EMEM with 1% FBS (maintenance medium, MM). Medium was collected after 3 days of incubation and centrifuged at 2000g, 4 8C to remove cell debris and the supernatant were stored at 70 8C.

2.3.3.2. Virus titre determination. Virus titre was determined by plaque assay. For the determination of H1N1 virus titre,

MDCK cells were seeded into a 12-well cell culture plate at 2.5  105 cells/well and incubated for 24 h. After rinsing the cell monolayers with PBS, serially 10-fold diluted viruses in trypsin–EMEM (0.2 mL) were added and the cells were further incubated for 2 h. The unattached viruses were removed by rinsing the cells with PBS, and EMEM containing 1.5% agarose (1 mL) was added to each well. After the overlay medium was solidified, 1 mL of EMEM containing trypsin (25 mg/mL) was added. The plate was further incubated for 3 days at 37 8C until formation of plaques. The overlay media were carefully removed and the cells were fixed with 10% formalin overnight and stained with 1% crystal violet solution. Plaques were then counted under light microscope. RSV titer was determined using similar procedure but MM and 1% agarose were used instead of trypsin–EMEM and 1.5% agarose, respectively.

2.3.3.3. Anti-H1N1 virus using MTT assay. MDCK cells were seeded into 96-well cell culture plates at 1.5  104 cells/ well and incubated for 24 h at 37 8C in a humidified atmosphere with CO2 (5%). After removing the culture medium, the cell monolayer was washed with PBS. NTP (dissolved in distilled water) and the positive control drug, ribavirin (dissolved in DMSO) were diluted into appropriate concentrations using assay media, with the final concentration of DMSO not exceeding 0.5%. Diluted samples (50 mL) of each dilution were added to each of the corresponding wells, whereas 50 mL EMEM was added to the cell control and virus control wells. For the antiviral assay, 50 mL of H1N1 virus at 1  104 plaque forming units/mL (PFU/mL) in trypsin–EMEM was added to each virus control and sample well. For the cytotoxicity assay, trypsin–EMEM without virus was added. For both assays, cell controls, which include trypsin–EMEM (50 mL) in the well, were also run in parallel. After further incubated for 48 h, the dead cells of the plates were removed by rinsing the cells twice with PBS. The cellular growth and viability was evaluated by MTT method as described in the previous section (see cell proliferation assay). The percentage of inhibition of H1N1 virus-induced cytopathic effect (CPE) by the samples were calculated according to the following equation: Percentage of inhibition = [(mean optical density of sample wells  mean optical density of virus controls)/ (mean optical density of cell controls  mean optical density of virus controls)]  100. The half maximal effective concentration of the sample on the inhibition of H1N1 virus-induced cytopathic effect was defined as the effective concentration (EC50). 2.3.3.4. Anti-RSV plaque reduction assay. For plaque reduction assay, HEp-2 cells were seeded into a 12-well plate at 3  105 cells/well and incubated for 24 h. Then the media were aspirated, and RSV (0.2 mL, at about 140 PFU) was added to the wells. After 2 h of incubation, each well were rinsed with PBS (1 mL), and 1 mL of EMEM containing 1% agarose was added, followed by the addition of 1 mL of MM (for virus control), 1 mL of NTP (100 mg/mL) or 1 mL of the positive control drug ribavirin (7.81 mg/mL). The plate was incubated for 3 days to allow plaque formation, which was then fixed and stained, and the number of plaques was counted.

peptides 29 (2008) 2101–2109

3.

Results

3.1.

Purification of NTP

A fetuin-binding protein was isolated using fetuin–agarose affinity column from the bulbs of N. tazetta var. chinensis (designated NTP), after removing the mannose-binding proteins by mannose–agarose column, which was then subjected to FPLC-Superose 12 to obtain the major peak with the retention time at about 40.7 min (Fig. 1). NTP is a protein of about 9 kDa as revealed by both gel filtration and SDS-PAGE (Fig. 2). The yield of the defined NTP protein was about 5.69 mg per 1.25 kg of the fresh bulbs by weight, i.e., about 4.6 mg/kg. The first 25 amino acids of the N-terminal sequence of these proteins showed significant similarity to the known nonspecific lipid transfer proteins (nsLTPs) with 55–68% identity to those isolated from various plants, such as cotton tree [20], lily [10], wall cress [2], and sunflower [21].

3.2.

Molecular cloning of cDNA encoding NTP

Based on the N-terminal amino acid sequence of NTP and Cterminal amino acid sequence of a homologous nsLTP, a pair of degenerate primers (NTLP-1 and NTLP-2) was designed for amplification of cDNA. Agarose gel analysis revealed that amplification with primers NTLP-1 and NTLP-2 resulted in a clear specific DNA band of about 220 bp (Fig. 3A). Based on the sequencing result, full-length amino acid sequence was deduced, and the C-terminal of the deduced amino acid sequence matched perfectly with the C-terminal amino acid sequence of nsLTP previously obtained, but the N-terminal sequence was not identical. NCBI Blast result with the deduced amino acid sequence showed that NTP had wide similarities to the known lipid transfer proteins of Oryza sativa (ABA91223), Lilium longiflorum (ABK41612), Arabidopsis thaliana (AAF76929), Nicotiana glauca (AAT68263), Gossypium hirsutum (AAF35186), Helianthus annuus (CAA63340), Vitis vinifera (ABA29446) and

Fig. 3 – Agarose gel electrophoresis for full-length cDNA amplification of NTP: (A) lane M, 1 kb plus DNA ladder (Invitrogen); lane a, RT-PCR of NTP; (B) lane M, 1 kb plus DNA ladder (Invitrogen); lane a, 30 RACE product; lane b, 50 RACE product; lane c, full-length cDNA amplifications of NTP.

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Davidia involucrate (AAL27855), implying that it was a part of the lipid transfer protein gene family. To make sure the N-terminal amino acid sequence is putative and to extend the 50 -end and 30 -end of the cDNA, two new primers, NLTPR-1 and NLTPR-2, were designed on the basis of the sequencing result of RT-PCR, and RACE was performed using SMART RACE kit. Agarose gel analysis revealed that the amplification with primers NLTPR-1 and NLTPR-2 resulted in a specific DNA band of about 410 bp (Fig. 3B, Lane a), whereas there was about 370 bp band in 50 RACE result (Fig. 3B, Lane b). Based on the overlapped sequence of 30 RACE and 50 RACE, the predicted full-length cDNA of NTP consisting of 639 bp was aligned with the 357 bp open reading frame (ORF) from 73 bp to 429 bp and 23 bp of poly-A tail in the end (Fig. 4). As anticipated, the specific 357 bp DNA band was solely amplified in cDNA PCR (Fig. 3B, Lane c) using a pair of primers for the amplification of full-length cDNA encoding for NTP precursor. The sequencing result of this fragment completely coincided with the aligned cDNA sequence (Fig. 4).

3.3.

Characterization of the predicted NTP protein

The ORF was translated on Primer Premier 5.0 and the predicted NTL protein was 118 amino acids in length with a molecular weight of 11.88 kDa and a pI value of 9.28. Signalp analysis (www.cbs.dtu.dk/services/SignalP/) showed a most likely cleavage site between S26 and A27. Cutting away the 26amino acid signal peptide, the resultant proprotein is 92 amino acids with a molecular weight of 9.3 kDa and a pI of 9.24. Based on the amino acid analysis by Protparam software in Expasy (http://ca.expasy.org/cgi-bin/protparam), negatively charged residues (Asp + Glu) and positively charged residues (Arg + Lys) constituted 3.3% and 12.0% of the polypeptide, respectively. The amino acid sequence of deduced NTP proprotein showed similarity of 51–56% identities to the other plant nsLTP-like proteins by NCBI BLAST search (http://www. ncbi.nlm.nih.gov/BLAST). Multiple sequence alignment of NTP to the reported nsLTP-like proteins was shown in Fig. 5. Molecular model of the three-dimensional (3D) structure of NTP was simulated using SWISS-MODEL program provided online by ExPASy, and the 3D model was drawn using Rasmol software (Fig. 6). Based on the 3D model, the structure of NTP was similar to that of the known nsLTPs from many plants, suggesting that they share a common fold structure comprising a flexible hydrophobic cavity which is able to bind lipid-like molecules and accommodate a variety of ligands. The four helixes involving residues Cys4-Lys20 (H1), Pro26 to Ala39 (H2), Thr42 to Lys58 (H3), and Tyr64 to Cys74 (H4) were stabilized by four disulfide bridges between the eight conserved cysteine residues. For NTP proprotein, four disulfide bridges were formed between cysteine 4 with cysteine 51, cysteine 14 with 28, cysteine 29 with 74, and cysteine 49 with 88 (Fig. 6).

3.4. Biological, antiviral and antiproliferative activities of NTP When isolated NTP was tested for inhibiting protein synthesis in a cell-free system (the reticulolysate of rabbit), an effective

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Fig. 4 – The full-length cDNA sequence and deduced NTP amino acid sequence of N. tazetta var. chinensis. The start codon (ATG) was underlined and the stop codon (TAG) was in italic and underlined. Cysteines were boxed. The 72 bp 50 UTR leader sequence and the polyadenylation signal sites were underlined with - - - and   , respectively. The 357 bp open reading frame (from 73 bp to 429 bp as shown in capital letters) encoded a 118-amino acid NTP precursor with a signal peptide of 26 amino acids. The arrow indicated the cleavage site of signal peptide between S26 and A27.

Fig. 5 – Multiple sequence alignment of the deduced amino acid sequence of NTP with eight nsLTPs from other plants, available in the data banks. Gene bank numbers corresponding to these sequences are as follows: Oryza sativa (ABA91223), Lilium longiflorum (ABK41612), Arabidopsis thaliana (AAF76929), Nicotiana glauca (AAT68263), Gossypium hirsutum (AAF35186), Helianthus annuus (CAA63340), Vitis vinifera (ABA29446) and Davidia involucrate (AAL27855), which are indicated as Os, Ll, At, Ng, Gh, Ha, Vv and Di, receptively. The completely identical amino acids were shaded black with eight cysteines in rectangular boxes. The lines over the sequences indicate the pattern of the disulfide bounds connectivity.

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Fig. 7 – Inhibitory effect of the putative non-specific lipid transfer protein of N. tazetta var. chinensis (NTP) on the proliferation of HL-60 cells. The 3 day’s EC50 is about 168 mg/mL by MTT method.

Fig. 6 – Cartoon diagram of NTP simulated using SwissModel (supplied by ExPASy proteomic tools, http:// swissmodel.expasy.org/) and drawn by RasMol software. Sulfide atoms were showed in yellow ball. Cx–Cy indicated the disulfide bond formed by two cysteines, and numbers were the sequence number of cysteines in the amino acid. C-Ter indicated C-terminal and N-Ter indicated Nterminal. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Schiff’s reagent to give pink color as the positive control did (ovalbumin). NTP had inhibitory activity on the proliferation of HL-60 cells with an EC50 of about 168 mg/mL (Fig. 7). The half maximal effective concentration of NTP (EC50) that inhibited the replication of influenza A (H1N1) virus was about 4.47 mg/ mL by MTT method (Fig. 9). NTP was also able to inhibit the plaque formation by respiratory syncytial virus (RSV) by a reduction of 37.55% at a concentration of 50 mg/mL (Fig. 8).

4.

Discussion

In this study, we have isolated and purified a new fetuinbinding peptide with a molecular mass of about 9 kDa (designated NTP) from the bulbs of Chinese daffodil, N. tazetta var. chinenesis. Molecular cloning reveals that NTP contains a

concentration of 7.1 mg/mL of NTP could inhibit 50% protein synthesis, i.e. in the presence of about 0.7 (M of this NTP, the protein translation of the rabbit reticulocytes at tRNA level (during the process of peptide elongation at ribosome) could be arrested at 50% (Table 1). The crude protein isolated from fetuin–agarose affinity column was initially a weak agglutinin toward rabbit erythrocytes with a specific hemagglutinating activity of 443 units per mg of protein when compared to 12,800 units per mg of Concanavalin A (the positive control). After NTP was purified, it showed no hemagglutinating activity for rabbit red blood cells. NTP was not a glycoprotein, as it did not react with

Table 1 – The protein-synthesis inhibitory concentrations of nsLTP isolated from Narcissus tazetta in a cell-free system nsLTP of N. tazetta

Scintillation count

Protein-synthesis

Protein concentration (mg/mL)

CPMa (n = 3)

Inhibition (%)

0.702 0.0702 0.00702 0 (negative control)

841 1,350 5,218 10,526

92 87 50 0

a CPM—net mean value, after subtracting the median value of non-specific binding (284).

Fig. 8 – Inhibitory effect of nsLTP of N. tazetta var. chinensis (NTP) on plaque formation by RSV-infected cells. HEp-2 cells were infected by RSV and NTP at final concentration of 50 mg/mL or ribavirin at final concentration of 3.91 mg/ mL were added. The number of plaques was counted after 3 days of incubation and NTP led to a 37.55% reduction of plaque numbers. The results were the mean value from two replicates in a representative experiment.

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Fig. 9 – Inhibitory effects of nsLTP of N. tazetta var. chinensis on influenza-induced cytopathic effect (CPE). H1N1 viruses and different concentrations ranging from 3.125 mg/mL to 100 mg/mL of NTP were added to MDCK cells. MTT assays were carried out at 48 h post-infection. The percentage of inhibition was calculated and the dose–response curve was generated by GraphPad Prism 4.0 software. The EC50 of NTP on H1N1 virus is about 4.47 mg/mL. Data were obtained from three independent experiments and expressed as mean W S.D.

polypeptide of 92 amino acids and a signal peptide of 26 amino acids. An analysis of the deduced amino acid sequence of NTP shows considerable sequence homology to the non-specific lipid transfer proteins (nsLTPs) of certain plants, such as Oryza sativa [1], Lilium longiflorum [10], Arabidopsis thaliana [2], Gossypium hirsutum [20], and Helianthus annuus [21]. This wide similarity in amino acid sequences of NTP and other nonspecific lipid transfer proteins (nsLTPs) implies that it is a part of the lipid transfer protein gene family [12]. Our preliminary results of isolation and purification of nsLTPs from various developmental stages and parts of N. tazetta var. chinensis, such as bulb, leaves of different days of growth and flowers, also show that the amino acid sequences determined at N-terminus and C-terminus of these purified peptides have very high homology, even though they might be isoforms expressed by the multigene family at the genome organization level [5]. Molecular model of the three-dimensional (3D) structure of NTP exhibits a flexible hydrophobic cavity which can bind lipid-like molecules and transfer a wide range of ligands, which is in line with the global fold structure of most of the known non-specific lipid transfer proteins from various plants. The non-specific lipid transfer proteins are known to be able to bind and carry fatty acids and phospholipids across cell membrane [13,25]. The unique characteristics of these proteins may be useful for the development of drug delivery system. Non-specific lipid transfer proteins have been suggested as one of the plant defense peptides. It is known that the expression of non-specific lipid transfer proteins in plants may be induced by both biotic factors, such as pathogens and

aging [8,11], and abiotic stress, such as cold temperature, high salinity and chemicals [9,28]. Although no definite role for non-specific lipid transfer proteins is defined in plants, several biological functions have been suggested for them [5], such as a role in plant defense signaling [14], and defensive role in inhibiting plant bacteria and fungi [7,15,24]. In addition to alkaloids (such as narciclasine and pretazettine) of N. tazetta, which had antiviral and antitumor activities [4,6,22,29], the proteins including the previous reported mannose-binding lectin(s) [18] also contributed to the antiviral activity of this plant. In this study we further report the fetuin-binding non-specific lipid transfer proteins (nsLTPs) isolated from the bulbs of N. tazetta var. chinensis (designated NTP), which could inhibit the proliferation of both cancer cells and viruses. In the in vitro assay, it possesses the ability to inhibit protein synthesis through terminating the elongation of polypeptides in eukaryotic cell-free system, which in a certain extent, is similar to the action of antitumoric Narcissus alkaloid, narciclasine [4]. Similar to the nsLTP of P. amaryllifolius, NTP of N. tazetta is not a glycoprotein. However, it does not agglutinate rabbit red blood cells up to the concentration of 1.34 mg/mL whereas that of P. amaryllifolius does [19]. Comparison of the Nterminal amino acid sequences of fetuin-binding nonspecific lipid transfer proteins isolated from P. amaryllifolius (Pandanaceae) and N. tazetta (Amaryllidaceae) shows about 44% similarity. Furthermore, both NTP and nsLTP of P. amaryllifolius [19] exhibit inhibitory activity on the proliferation of HL-60 cells with EC50 values of about 168 mg/mL and 277 mg/mL, respectively. The potencies for antiproliferation of the human leukemia cancerous cell line (HL-60 cells) may be due to its cytotoxic disposition possibly by inhibiting protein translation once it gets into the cells, through terminating the elongation of polypeptides similar to common eukaryotic cells as mentioned above. The half maximal effective concentration of NTP (EC50) that inhibits the replication of influenza A (H1N1) virus is about 4.47 mg/mL by MTT method. NTP was also able to inhibit the plaque formation by respiratory syncytial virus (RSV). It is logic to speculate that the inhibition of influenza A (H1N1) virus might be associated with its fetuin-binding property by blocking the neuramidase on the viral envelope of this virus, because from the MTT assay the survival rate of MDCK cell is nearly 100% throughout the assay after adding NTP (up to 100 mg/mL). By the same token, the reduction of the plaque formation by RSV would be in the similar way blocking the entrance of this virus to the host cells. For example, NTP might interfere with RSV spreading by binding to viral glycoproteins or inhibit other events during the viral replication or assembly processes. The true mechanisms should be confirmed by further study. The capability of NTP to suppress the proliferation of human leukemia cancerous cells (HL-60) may be due to its cytotoxic disposition possibly by inhibiting the protein translation once it gets into the cells. Both the properties of antiproliferative and antiviral activities of NTP perhaps make it more interesting and worthy to be further investigated. Some allergic reactions are attributed to non-specific lipid transfer proteins as major allergens in fruit and food [30] due to their high resistance to pepsin digestion and stability in

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cooking process [3,23,26]. Whether or not the non-specific lipid transfer proteins of N. tazetta and food plants could cause allergy is also an issue worththy of our attention.

Acknowledgements This study was supported by a grant from UGC-Area of Excellence in Plant and Fungal Biotechnology, and the Research Fund for the Control of Infectious Diseases of the Food and Health Bureau, the Hong Kong Special Administrative Region Government.

references

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