Purification and identification of an IgE suppressor from strawberry in an in vitro immunization system

Abstract

We purified and identified an IgE suppressor from the strawberry ‘Toyonoka’, based on the decrease of IgE production in in vitro immunization (IVI). Gel filtration experiment indicated that fractions in a 15–48 kDa range and <10 kDa have an IgE suppressive activity. Furthermore, the fraction in 15–48 kDa was subjected to chromatofocusing and found to have activities at isoelectric points, pI 6.0, 7.0, and 8.0–9.2. We focused on the active fractions of pI 8.0–9.2 and the purified a large amount of strawberry extracts by cation exchange resins in batch. A purified 39 kDa protein showed homology to plant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in N-terminal amino acid sequence and had GAPDH enzymatic activity. Nucleotide sequence and deduced amino acid sequence of the obtained cDNA clone of the protein matched with the sequence of Fragaria x ananassa GAPDH in the GenBank with >98% identical nucleotides and >99% identical amino acids, respectively. The purified strawberry GAPDH suppressed total IgE production in IVI in a dose-dependent manner. From these results, we identified GAPDH as IgE suppressor in the strawberry. Our study may be applicable to the development of new methods to relieve allergic conditions using GAPDH and the screening of other functional factors for human health.

Introduction

IgE antibodies are well known as a trigger of acute hypersensitivity such as atopic dermatitis, asthma, food allergy and pollinosis, and their blood level is involved in the severity of these diseases (Holgate 1999; Corry and Kheradmand 1999). They enhance allergic reactions such as vasodilation, smooth muscle contraction, and mucosal edema through the release of chemical mediators by their binding to the FcεRI on mast cells and basophils, followed by their cross-link to the allergen (Metzger et al. 1986). These studies suggest that allergic symptom can be palliated by suppression of IgE antibodies.

There are some reports on IgE suppression by food components such as flavones (Yano et al. 2007; Hassanain et al. 2010). We also found that some species of strawberries (Fragaria x ananassa) suppressed total IgE production in in vitro immunization (IVI) where a population of human peripheral blood lymphocytes was cultured with the ceder pollen antigen, Cry j 1, in a medium containing human plasma, fetal bovine serum (FBS), IL-2, IL-4, IL-6, and muramyl dipeptide (Mitsuda et al. 2010). But, we failed to determine the difference of the Cry j 1-specific IgE production between tested samples because their production level was very low. The IgE suppression by strawberry extract was accompanied a decreased expression of the suppressor of cytokine signaling-3 that evokes Th2 cytokines and IgE antibodies (Kubo and Inoue 2006). Strawberries have been studied for antioxidation and antitumor properties (Aaby et al. 2007; Zhang et al. 2008), but their anti-allergic effect was not fully understood yet. In this study, based on the results of IgE suppression in the IVI we purified an IgE suppressor from the strawberry ‘Toyonoka’, and identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the IgE suppressor.

Materials and methods

Preparation of strawberry extract

The strawberry ‘Toyonoka’ fruits were homogenized after the addition of equal weight volume of phosphate buffered saline (PBS). The homogenate was centrifuged at 9,400g for 60 min and the supernatant was sterilized by a 0.22 μm filter. The filtrate was used for the experiments.

Purification of an IgE suppressor in the strawberry

The strawberry extract was purified by the gel filtration column, HiLoad 26/60 Superdex 200 prep grade (Amersham Biosciences, UK) with 20 mM phosphate buffer (pH 7.0) and by the Chromatofocusing column, Mono P 5/200 GL (GE Healthcare, Sweden) with Polybuffer 96-acetic acid (pH 6.0), respectively, at a flow rate of 1.0 mL/min using the fast protein liquid chromatography (FPLC) system, ÄKTA™ explorer 10S (Amersham Biosciences, UK). Suppressive effect of each fraction on IgE production was examined by the IVI system.

To obtain an IgE suppressor in a large amount, the strawberry extract was newly prepared and applied to the cation exchange resins, TOYOPEAL SP-550C (TOSOH, Japan) with 100 mM acetate buffer (pH 5.0) in batch. The active fraction eluted with acetate buffer containing 0.1 M NaCl was concentrated and subjected to SDS-polyacrylamide gel electrophoresis, followed by electroblotting to PVDF membrane and staining with Coomassie dye. A main band of 39 kDa in the membrane was cut out and subjected to the N-terminal protein sequence analysis.

The GAPDH enzymatic activity of the purified 39 kDa protein was examined according to the method described by Ferdinand (1964). The strawberry GAPDH concentration was determined by the Bio-Rad protein assay (Bio-Rad, Japan) with rabbit muscle GAPDH as a standard.

In vitro immunization (IVI) system

Peripheral blood lymphocytes (PBL) were separated from heparinized blood of healthy donors by using Ficoll-Paque Plus (GE Healthcare, Sweden). To induce IgE production of PBL, IVI was done as described by Kawahara et al. (2000). Briefly, 2.0 × 10⁶ cells/mL of PBL were cultured in the 5% fetal bovine serum (FBS) and 10% human plasma containing ERDF medium (Kyokuto Pharmaceuticals, Japan) supplemented with 10 ng/mL of recombinant human IL-2, IL-4, and IL-6 (R&D systems, USA), 10 μg/mL of muramyl dipeptide (SIGMA, USA) and 100 ng/mL of the ceder pollen antigen Cry j 1 (HAYASHIBARA, Japan) with or without 5% (v/v) of strawberry extract. After 10 days, total IgE concentration in the supernatant was measured by an enzyme-linked immunosorbent assay (ELISA). Noteworthily, IgE production level was different between donors.

ELISA

Total IgE concentration in culture medium was measured by sandwich ELISA. 96 well microplates were coated with anti-human IgE (Biosource, USA) diluted with PBS. The antibody-coated wells were blocked with 1.0% BSA/PBS, and then, each sample was added to them. After washing with 0.05% Tween 20 containing PBS (TPBS) three times, biotin-conjugated anti-human IgE antibody (Biosource, USA) and horseradish peroxidase-conjugated streptavidin were added to them. After washing with TPBS three times, a substrate solution (0.1 M citrate buffer (pH 4.0) containing 0.003% H₂O₂ and 0.3 mg/mL p-2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) was added to them. After 15 min, the absorbance was measured at 414 and 490 nm by a microplate reader.

cDNA cloning by PCR

Total RNA was extracted from ‘Toyonoka’ strawberry fruits using ISOGEN (NIPPON GENE, Japan). First strand cDNA was synthesized from 5 μg of total RNA using Thermoscript™ cDNA synthesis kit (Invitrogen, USA) according to the product instruction manual. For cloning the gene of the target protein, the cDNA fragment was amplified by polymerase chain reaction (PCR). A forward primer (5′-AAGAATCAGATCGGAATCAACGGATTC-3′) was designed from N-terminus protein sequence and optimized strawberry codon usage. A reverse primer (5′-TGACACAAGCTTGACAAAGTTCT-3′) was designed from the mRNA partial sequence of strawberry GAPDH (GenBank, accession number: AF421145.1). PCR was performed with a 50 μL reaction mixture containing 1 μL of cDNA, 5 μL of 2 mM dNTPs, 3 μL of 25 mM MgSO₄, 10 pmol of each primer, KOD plus DNA polymerase, and 5 μL of 10 × KOD FX buffer (TOYOBO, Japan) using the following program: 94 °C for 2 min, then 40 cycles each with 98 °C for 10 s, 58 °C for 30 s, 68 °C for 2 min 30 s, and finally 68 °C for 10 min. The amplified product was analyzed by electrophoresis on a 1% agarose gel and stained with ethidium bromide. The corresponding DNA band was recovered and cloned into the pTargeT™ vector for sequencing (Promega, Japan).

Statistics

Data are means ± standard deviations (SD) from triplicate samples. Statistical significance was determined by the Student’s t test.

Results and discussion

Purification of an IgE suppressor in the strawberry

Among 18 species of strawberries tested, we selected the strawberry ‘Toyonoka’ as the material to purify an IgE suppressor because they showed relatively strong IgE suppression (Mitsuda et al. 2010). As shown in Fig. 1a and b, the separation of the strawberry extract by gel filtration indicated IgE suppressive activity in fraction number 4 (15–48 kDa) and 7 (<10 kDa). In this experiment, IgE suppression was weak, but it was confirmed that the position of the suppressive activity was almost the same between experiments. Low molecular weight constituents such as linocinnamarin and cinnamic acid in the strawberry ‘Nohime’ were reported to inhibit allergic reactions (Ninomiya et al. 2010). But, vitamin C, which is rich in strawberry, had little IgE suppressive activity in the IVI system (Mitsuda et al. 2010). This means that the IgE suppression is not necessarily caused by only antioxidation. Thus, the 15–48 kDa fraction was subjected to chromatofocusing and was found to have the following isoelectric point: pI 6.0, 7.0, and 8.0–9.2 (Fig. 2). These results suggest that the strawberry may include some IgE suppressors..

Fig. 1.

Effect of gel filtration fractions on IgE production in IVI. a Elution pattern and fractions of strawberry extract in gel filtration. MWnumbers represent the molecular weight (kDa) of standard proteins. b IgE production in IVI. Data show the average values of two measurements

Fig. 2.

Effect of chromatofocusing fractions on IgE production in IVI. Chromatofocusing was done in the pH gradient range between 9 and 6. The pIvalue shows isoelectric point of eluate. Data show the average values of two measurements

Identification of an IgE suppressor in the strawberry

We focused on the active fractions of pI 8.0–9.2 and newly purified a large amount of strawberry extract using cation exchange resins in batch. SDS-PAGE analysis of eluted active fraction showed a main band of 39 kDa in molecular size. The 39 kDa protein band was observed in the strawberry species with IgE suppressive activity at a relatively higher level than others (data not shown). Subsequently, the protein band was subjected to N-terminal protein sequence analysis. The resulting first N-terminal amino acid sequence, Ala Lys Ile Lys Ile Gly Ile Asn Gly Phe, showed homology to the sequence of rice and maize glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that is a key glycolytic enzyme that plays a crucial role in energy production. In addition, the purified protein had GAPDH enzymatic activity. Based on the result of this protein sequence and the GAPDH mRNA sequence from the GenBank, a cDNA clone of the protein was obtained by RT-PCR cloning technique. Figure 3 shows its nucleotide sequence and deduced amino acid sequence. These sequences matched with that of strawberry GAPDH in the GenBank (accession number: AF421145.1 and AB363963.1) with >98% identical nucleotides and >99% identical amino acids, respectively. Strawberry GAPDH was purified by cation exchange resins and its purity was examined by SDS-PAGE. The protein bands of strawberry extract were almost invisible, but after purification, a single band of GAPDH was clearly confirmed (Fig. 4a). The purified strawberry GAPDH suppressed total IgE production in IVI in a dose-dependent manner (Fig. 4b). In addition, rabbit muscle GAPDH that exhibits about 67% similarity of strawberry GAPDH in amino acid sequence also showed a IgE suppressive activity (data not shown). Yamaji et al. (2005) reported that GAPDH could bind to mammalian cells in time- and dose-dependent manners and its binding was involved in the cysteine at position 151 of this enzyme. From these findings, we identified GAPDH as the IgE suppressor in the strawberries. Interestingly, Sugahara et al. (1995) reported that human and rabbit muscle GAPDH increased IgM production in human hybridomas and lymphocytes. IgM antibodies are known to be class-switched to IgE antibodies by IL-4 in activated B lymphocyte. Therefore, GAPDH may influence the process of the immunoglobulin class switching in addition to the production level. Recently, the multifunction of GAPDH in apoptosis and NO stress has been reported (Sirover 1999; Hara et al. 2006). Our study indicates an additional new function of GAPDH.

Fig. 3.

Nucleotide sequence and deduced amino acid sequence of the obtained cDNA clone. Numbers above refer to the nucleotide position. The primer sites for PCR are underlined

Fig. 4.

Effect of purified strawberry GAPDH on IgE production in IVI. a SDS-PAGE of strawberry GAPDH purified by cation exchange resins. Lane 1: molecular weight marker, lane 2: rabbit muscle GAPDH, lane 3: strawberry extract, lane 4: purified strawberry GAPDH. b Effect of strawberry GAPDH on IgE production in IVI. Data are means ± SD (n = 3). *p < 0.05 versus control

Conclusions

We reported here that strawberry GAPDH suppressed IgE production in IVI. But, further study is needed to understand the mechanism underlying the IgE suppression by the GAPDH. At present, anti-allergic drugs are used to improve allergic symptoms. They inhibit the release and activity of chemical mediators from mast cells, but produce occasionally harmful side effects. Our study may lead to the development of new methods to relieve allergic conditions using GAPDH and the screening of other functional factors for human health.