Despite potential medical, economical, and agronom- ical importance, the bioprocessing of mistletoe cell cultures, from callus cultures to mass production of.
Biosci. Biotechnol. Biochem., 77 (4), 884–887, 2013
Note
The Identification of in Vitro Production of Lectin from Callus Cultures of Korean Mistletoe (Viscum album L. var. coloratum) Keun Pyo L EE* and Dae Won L EEy Department of Life Science, Dongguk University, Kyongju 780-714, Republic of Korea Received December 14, 2012; Accepted January 12, 2013; Online Publication, April 7, 2013 [doi:10.1271/bbb.120962]
Despite potential medical, economical, and agronomical importance, the bioprocessing of mistletoe cell cultures, from callus cultures to mass production of high-value products (e.g., lectins and viscotoxins), has been unsuccessful to date. In this study, we confirmed the potential of in vitro lectin production from callus cultures of Korean mistletoe (Viscum album L. var. coloratum). Key words:
Viscum album L. var. coloratum; lectin; mistletoe; callus; affinity chromatography
Mistletoe is a name that covers many species of semiparasitic plants that grow on most deciduous trees worldwide. In Europe, mistletoe extracts are widely used in cancer therapy.1,2) They contain lectins and viscotoxins, which are believed to have immunostimulating and cytotoxic effects respectively, as well as alkaloids and polysaccharides.3–7) The composition of mistletoe lectins varies depending on the host tree, mistletoe subspecies, mistletoe tissues, and harvesting time.8,9) The European mistletoe lectins (ML-I, ML-II, ML-III) are type-2 ribosome-inactivating proteins (RIPs) composed of two different polypeptide chains, a cytotoxic A chain and a sugar-binding B chain, connected by a disulfide bond.10) The A chains of the three lectins show strong homology in their amino-acid sequences,11) suggesting that they originated from the same gene, but they show differences in their sugarbinding specificities. ML I is -galactose specific, whereas ML II and ML III bind preferentially to galactose/N-acetyl-D-galactosamine and N-acetyl-D-galactosamine respectively, and have molecular masses ranging from 50 to 60 kDa.10,11) A previous study found that Korean mistletoe, a different subspecies of Viscum album, has no lectin-like cytotoxic proteins, unlike European mistletoe. The primary anticancer activity of Korean mistletoe might be attributable to cytotoxic alkaloids.12) In recent years, many studies have found that Korean mistletoe lectins show anti-cancer activities in vitro and in vivo.13–26) Korean mistletoe lectins show clear differences from European lectins in terms of molecular mass, protein and nucleic acid sequence, glycosylation patterns, and the existence of isotypes.15,27) Mistletoe’s slow growth rate and the difficulty of harvesting it limit its use in cancer therapy. Indeed, the y
mistletoe life cycle, from infection to initial seed production, commonly spans 6 to 8 years and is dependent on host specificity. Hence, the need for artificial cultivation of mistletoe has increased proportionally to its therapeutic importance and rarity. Although there have been reports of in vitro callus cultures of mistletoe,28–31) this is the first report of in vitro lectin production from callus cultures of Korean mistletoe. The effects of different concentrations of 2,4-dichlo rophenoxyacetic acid (2,4-D; Sigma-Aldrich, St. Louis, MO) were evaluated with regard to callus induction in mistletoe explants (leaf and stem). Korean mistletoe tissues (leaves and stems) growing on oak trees were collected from Deokyusan National Park, Jeollabuk-do, Korea. They were sterilized by dipping them in 70% (v/v) ethanol for 1 min prior to a 10 min exposure to 1.5% (v/v) sodium hypochlorite solution. Subsequently they were rinsed 5 times with sterile distilled water for 10 min each. Then the sterilized leaf and stem tissues (about 50 g each) were cut into approximately 1.5– 2.0 cm segments. A total of 450 pieces of sterilized explants were used for callus induction on Murashige and Skoog (MS) medium32) supplemented with 3.0% (w/v) sucrose, 0.4% (w/v) phytagel (Sigma-Aldrich), 0.1% (w/v) casamino acid (BD Biosciences, San Jose, CA), and various concentrations (0, 1.0, 2.0, 3.0, 4.0, and 5.0 mg/L) of 2,4-D. The cultures, after adjustment to pH 5.7, were incubated at 24 2 C for 20–25 d in the dark. Callus was induced only from stem explants: no callus induction was observed from leaf explants. Moreover, callus induction was observed only in MS medium containing 5.0 mg/L of 2,4-D, but the frequency of callus induction was still very low (4.89%, 22 out of 450 stem explants). A previous report indicated that the frequency of callus formation was greater (27.3%) than our results when mistletoe flower buds were cultured on B5 medium containing 0.1 mg/L of indole-3-acetic acid (IAA).31) Further investigation is thus necessary to improve the frequency of callus induction from mistletoe tissues. Some calli were initially produced at the cut edges of stem explants after 15 to 18 d (Fig. 1A) and changed to watery callus 5 d later (Fig. 1B). Afterwards, callus pieces 1 to 1.5 cm in diameter were subcultured every 3 weeks on fresh MS medium containing 5.0 mg/L of 2,4-D, and this morphological callus structure was maintained until needed for protein analysis (Fig. 1C).
To whom correspondence should be addressed. Fax: +82-54-770-2214; E-mail: dwleebio@dongguk.ac.kr Present address: Department of Botany and Plant Biology, University of Geneva, Geneva, CH-1211, Switzerland Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; CBB, Coomassie Brilliant Blue; MALDI-TOF, matrix-assisted laser desorption/ionizationtime-of-flight; Mr, molecular mass; PVDF, polyvinylidene difluoride; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis *
Identification of Mistletoe Lectin from Callus Cultures
Mistletoe lectins from leaves, stems, and callus cultures were isolated by affinity chromatography consisting of asialofetuin bound to Affigel-15. Fifty g of each material was cut into pieces, ground to a fine powder by mortar and pestle under liquid nitrogen, and then crushed in a Waring blender for 3 min with 100 mL of 50 mM Tris-hydrochloride (pH 7.0). The mixture was centrifuged at 4 C for 1 h at 2,000 rpm. Total proteins were precipitated from the supernatants with solid ammonium sulfate to 80% saturation. The solution was stirred slowly for 5 h at 4 C and then centrifuged at 4 C for 15 min at
A
B
C
Fig. 1. Callus Induction from Stem Explants of Korean Mistletoe. As the arrows indicate, calli formed after 15 d (A) and 22 d (B) when cultured on MS medium supplemented with 5.0 mg/L of 2,4D. (C) The callus was subcultured and maintained for protein analysis as shown in the photograph. Bars ¼ 5 mm.
A Lectin levels (µg/g.f.w.)
35
kDa 150
25
100 75
20
50
30
885
10,000 rpm. The pellet was dissolved in 50 mL of 50 mM Tris-hydrochloride (pH 7.0) and extensively dialyzed against dialysis buffer (0.1 M Tris-hydrochloride pH 7.0 and 0.5 M sodium chloride) using a Spectra/Por 6 membrane MWCO 25,000 (Spectrum Laboratories, Rancho Dominguez, CA). The dialyzed extract was passed through an 18 mL affinity column consisting of asialofetuin (Sigma-Aldrich), a glycoprotein that has three asparagine-linked triantennary complex carbohydrate chains with terminal N-acetylgalactosamine residues, bound to Affigel 15 (Bio-Rad, Hercules, CA) following the manufacturer’s recommendations. In brief, the column was first equilibrated with equilibration buffer (0.1 M Tris-hydrochloride pH 7.0 and 0.5 M sodium chloride), and then the dialyzed extract (50 mL) was applied at a flow rate of 4 mL/min. The column was washed with equilibration buffer until absorption of the eluate at 280 nm was undetectable. Then the adsorbed proteins were eluted with 0.1 M glycin-hydrochloride buffer (pH 3.0). The pH of the eluate was immediately raised to neutral with 1 M Tris-hydrochloride (pH 7.0). The eluate (250 mL) was concentrated to 1 mL with Vivaspin 20 mL centrifugal concentrators 30,000 MWCO (Sartorius, Goettingen, Germany). The resulting protein eluates (mainly lectins) were quantified with protein assay kit II (Bio-Rad). Although lectin was present in the callus cultures at low levels (17% and 57%) relative to the leaves and stems respectively, it was clearly detected in the callus cultures (Fig. 2A). Afterwards, the molecular masses of the purified lectins from the leaves, stems, and callus cultures were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). The same volume of the eluate (20 mL) was loaded and separated on 10% SDS–PAGE in the absence and the presence of a reducing agent (2-mercaptoethanol, Sigma-Aldrich). Staining was performed with Coomassie Brilliant Blue (CBB) R-250 (Bio-Rad), and the results were analyzed using the Lab Works 4.0 program on an image analysis system (UVP, Upland, CA). SDS–PAGE investigations in the absence and the presence of a reducing agent showed that the mistletoe lectins from leaves, stems, and callus cultures consisted of two different chains connected by disulfide bonds, consistently with the results of previous reports.10,16) In the absence of the reducing agent, a clear band corresponding to an approximate
B
C
*
15 37
10
*** **
5 25
0 Leaf Stem Callus
Mr
Leaf Stem Callus
Mr
Leaf Stem Callus
Fig. 2. Quantification and SDS–PAGE Patterns of Mistletoe Lectins. (A) Mistletoe lectin was isolated by affinity chromatography consisting of asialofetuin bound to Affigel-15. Histograms show lectin levels measured in eluates from leaves, stems, and callus cultures. Data represent averages of two biological replicates, and error bars represent standard deviations. SDS–PAGE patterns of mistletoe lectin in the absence (B) and the presence (C) of a reducing agent. Equivalent eluate volumes (20 mL) were subjected to 10% SDS–PAGE. Asterisks indicate hololectin ( ), lectin A chain ( ), and lectin B chain ( ). Mr, molecular mass.
K. P. L EE and D. W. L EE
A % Intensity
molecular mass (Mr) of 60 kDa was observed (Fig. 2B). In the presence of the reducing agent, two major bands, corresponding to Mr ¼ 32 kDa and 27 kDa, were detected (Fig. 2C), suggesting that the light band (Mr ¼ 27 kDa) was a lectin A chain and the heavy band (Mr ¼ 32 kDa) was a lectin B chain. In addition, other protein bands below 50 kDa were also detectable in the eluates from leaves, stems, and callus cultures (Fig. 2B), but the relative intensities of the protein bands in the absence and the presence of the reducing agent did not change drastically (Fig. 2B and C), suggesting that they were probably not related to the mistletoe lectins. Thus, as found in previous studies,16,33) further precise research is required to optimize the purification process in order to obtain highly purified mistletoe lectin from callus cultures. To verify that the eluate from the callus cultures consisted of mistletoe lectin, N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) analyses were performed. For the N-terminal amino acid sequencing analyses, the A and B chains of lectin were separated by SDS–PAGE under reducing conditions and blotted onto a polyvinylidene difluoride (PVDF) membrane (BioRad). Picomole quantities of mistletoe lectin A and B chains were sequenced by automated Edman degradation on an ABI 476A protein sequencer (Applied Biosystems, Foster City, CA). The sequences obtained were compared to the sequences reported in protein databanks using BLASTp at the National Center for Biotechnology Information (NCBI, http://www.ncbi. nlm.nih.gov/). The N-terminal amino acid sequence of the light band (Mr ¼ 27 kDa), YERLKLRVTHQTTGD, revealed 100% homology to amino acids 34 to 48 of the A chain of the lectin precursor in Viscum album subsp. coloratum. The accession number is AAM46934 in the NCBI. Sequencing of the heavy band (Mr ¼ 32 kDa) was not successful, and requires further work. The light (Mr ¼ 27 kDa) and heavy bands (Mr ¼ 32 kDa) were analyzed by MALDI-TOF mass spectrometry. The A and B chains of lectin were first separated by SDS–PAGE under reducing conditions. The Coomassie-stained bands were excised, cut into approximately 1 mm3 cubes, and completely destained. Mass spectral analyses of the samples were subjected to trypsin digestion and then analyzed by MALDI-TOF mass spectrometry (Applied Biosystems 4700 proteomics analyzer). The peptides (10–50 pmol) were dissolved in 0.5% (v/v) TFA/H2 O. Samples were prepared with an -cyano-4-hydroxy cinnamic acid (CHCA) matrix in 50% (v/v) acetonitrile and 0.1% (v/v) TFA. The MASCOT search program (www.matrixscience. com) was used to identify peptide sequences from the mass spectra. The fragmentation patterns and molecular masses are shown in Fig. 3A and B. Peptide masses were used to identify peptide sequences using the MASCOT program (Matrix Science), as described previously.34) The Mascot program reported that the light band had a significant match to mistletoe lectin A chain isoform 2 (GIj21314414), and the heavy band was identified as mistletoe lectin B chain isoform 3 (GIj21314422). In conclusion, callus was successfully induced from stem explants of Korean mistletoe, and in vitro
Mass (m/z)
B % Intensity
886
Mass (m/z)
Fig. 3. MALDI-TOF Mass Spectra of Mistletoe Lectin Purified from Callus Cultures. Mistletoe lectin A chain (A) and lectin B chain (B). The samples were prepared with CHCA Matrix in 50% acetonitrile and 0.1% TFA.
production of lectin was confirmed in the callus cultures, indicating a potential for mass production of lectin through in vitro cultivation of mistletoe callus. Although lectins are the main components of mistletoe that exhibit anticancer activity, synergistic effects of other components cannot be excluded. Indeed, the heat-treated mistletoe extracts, in which the cytotoxicity of the lectins disappeared completely, still showed highly cytotoxic activity in tumor MSV cells.35) Thus further investigation is necessary to establish the biological activities of other components, including viscotoxins, polysaccharides, and alkaloids, in the callus extracts.
Acknowledgment We thank the Korea Basic Science Institute for performing N-terminal amino acid sequencing and MALDI-TOF MS analysis. This work was supported by the Dongguk University Research Fund.
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