To determine the N-terminal amino acid sequence of PYP1 (18), the P. yezoensis expressed sequence tag (EST) database of the Kazusa DNA institute (Chiba, Japan) was surveyed. Since the resultant information indicated that both mature and immature mRNAs were present, cDNA encoding these variants was cloned. Briefly, the cultivation of P. yezoensis gametophytes and the amplification of cDNA from total RNA were performed as previously described by Uji et al (19). DNA fragments corresponding to the open reading frames (ORFs) of mature and immature variants were then amplified by polymerase chain reaction (PCR) using the following primer sets: PYP1-F and PYP1-R, PYP1-F and PYP1-AC-R, and PYP1-F and PYP1-B-R (Table I). The PCR conditions were as follows: 30 cycles at 98°C for 10 sec and 68°C for 2 min using PrimeSTAR HS DNA polymerase with GC buffer (Takara Bio., Otsu, Japan). Separation, purification, cloning and sequence analysis were performed as previously described by Uji et al (19), with the exception of the pENTR/SD/D-TOPO vector (Invitrogen/Life Technologies, Carlsbad, CA, USA), which was used for the cloning and construction of plasmids that were expressed in bacteria as entry plasmids. To isolate the genomic fragment containing PYP ORF information, genomic DNA was prepared from gametophytes using a DNeasy Plant Mini kit (Qiagen, Hilden, Germany), and genomic PCR was performed as described above, using the PYP1-F and PYP1-R primers. The amplified fragment was inserted into a pCR-Blunt II-TOPO cloning kit (Clontech Laboratories, Inc., Mountain View, CA, USA) and sequenced.

To analyze the expression profile of the PYP1 gene in both gametophytes and sporophytes of P. yezoensis, several generations were cultured and used for total RNA extraction in order to amplify the cDNA, as previously described by Uji et al (19). Following the synthesis of the first-strand cDNA using a PrimeScript II First Strand cDNA Synthesis kit (Takara Bio), reverse-transcription PCR (RT-PCR) was performed using Phusion High-Fidelity DNA polymerase (New England BioLabs, Inc., Beverley, MA, USA) with the primer sets described above, under the following conditions: 98°C for 1 min and 30 cycles at 98°C for 10 sec, 55°C for 30 sec, and 72°C for 1 min.

Gateway Technology (Invitrogen/Life Technologies) was employed to construct the expression plasmids for the PYP1 and PYP1 variants in Escherichia coli (E. coli). To produce the destination vector, pQE-82L (Qiagen) was digested with SmaI in the multi-cloning site and ligated using Restriction fragment analysis (RFA; Invitrogen/Life Technologies). The resultant plasmid was designated as pQE80L-DES. LR recombination reactions were then performed with entry plasmids and pQE82L-DES according to the manufacturer’s instructions, thereby, producing pQE82L-PYP1, pQE82L-PYP1-AC and pQE82L-PYP1-B.

Plasmids (pQE82L-PYP1, pQE82L- PYP1-AC and pQE82L-PYP1-B) were transformed into the E. coli strain DH5α and incubated on ice for 30 min. S.O.C medium (200 µl; Invitrogen Life Technologies) was added followed by incubation for 1 h at 37°C. The mixture was then spread on a plate with LB medium containing 100 µg/ml ampicillin and incubated for 16 h at 37°C. After confirming the transformation in E. coli DH5α cells, the plasmids were transformed into the E. coli strain BL21 (DE3) in a similar manner.

For the induction of the expression of PYP1, PYP1-AC and PYP1-B, LB medium (20 ml) containing 100 µg/ml ampicillin was inoculated and grown at 37°C overnight. Cultures were inoculated into 1 ml of LB medium containing ampicillin (100 µg/ml; 1:50) with fresh LB culture. The cultures were grown at 37°C until an optical density (OD)₆₀₀ of 0.5 was reached. One milliliter of sample was taken immediately prior to induction, and expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The cells were harvested by centrifugation at 4,000 × g for 20 min, resuspended in binding buffer (20 mM Tris-HCl, pH 8.0) and lysed by ultrasonic disruption (Sonics & Materials Inc., Newtown, CT, USA). The sonicated extracts were then separated into soluble and insoluble fractions by centrifugation at 4,000 × g for 20 min at 4°C.

To purify the recombinant proteins, soluble fractions containing the PYP1s were loaded onto an Ni-NTA column (Qiagen) with Ni-NTA His•Bind Resin (Merck Millipore, Darmstadt, Germany) using lysis buffer (50 mM NaH₂PO₄·H₂O, 300 mM NaCl and 10 mM imidazole). After complete loading, weakly bound proteins were removed with wash buffer (50 mM NaH₂PO₄, 300 mM NaCl and 20 mM imidazole). Proteins were subsequently eluted from a Ni-NTA spin kit (Qiagen) with elution buffer (50 mM NaH₂PO₄, 300 mM NaCl and 250 mM imidazole), according to the manufacturer’s instructions. The concentrations of the protein samples collected during purification and purified samples were measured using the BCA method using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA).

All protein samples were resolved by 17% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For N-terminal sequencing analysis, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) using an electrophoresis power supply (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

The N-terminal 11-residue sequence, which was a common sequence in all the PYP1s (ALEGGKSSGGG), was synthesized by Peptron (Daejeon, Korea). Purification of the SP was performed on a Shimadzu Prominence HPLC system and controlled using the software package Class-VP, 6.14 with a C18 column (Shiseido Capcell Pak; Shiseido, Tokyo, Japan) in 0.1% trifluoroacetic acid (TFA)/water and a gradient of 10–70% acetonitrile in 0.1% TFA, at a flow rate of 1 ml/min and ultraviolet (UV) detection at 220 nm. The molecular mass was confirmed at 918 kDa (it matched the sequence mass) using mass analysis (HP 1100 Series LC/MSD; Agilent Technologies, Inc., Santa Clara, CA, USA).

The Chang liver cell line (HPV-18) was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 100 U/ml penicillin and 100 mg/ml streptomycin. The cultures were maintained in a humidified incubator at 37°C in 5% CO₂. The medium was replaced every 2 days.

The effects of PYP1, PYP1-AC, PYP1-B and SP treatment on cell proliferation in the cells treated with 15 mM APAP were colorimetrically determined by MTS assays using CellTiter 96 AQueous One Solution reagent (Promega, Madison, WI, USA). The cells were seeded in 96-well plates at a density of 1.5×10⁵ cells/well. Following incubation for 24 h, the attached cells were maintained in serum-free medium (SFM) for 6 h, and this was followed by treatment with PYP1, PYP1-AC, PYP1-B or SP (0–1,000 pg/ml) for an additional 24 h. The cells were then incubated with MTS solution at 37°C for 30 min, and the absorbance was measured at 490 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The OD₄₉₀ values of the control cells were designated as 100%.

The cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde. The fixed cells were incubated at 37°C for 20 min and washed twice with PBS. The cells were then stained with 1 µg/ml DAPI and incubated at room temperature for 20 min in the dark. The stained cells were observed under a fluorescence microscope (ECLIPSE TS100-F; Nikon Corp., Tokyo, Japan).

Data were evaluated by one-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences version 10.0 (SPSS, Inc., Chicago, IL, USA). Significant differences between means were identified using Duncan’s multiple range test (P<0.05). A P-value <0.05 was considered to indicate a statistically significant difference.

The SP, ALG EGKSSGGGEATRDPEPT, corresponding to the N-terminus of mature PYP1, has been shown to exhibit chemoprotective activity against APAP-induced Chang liver cell death (18). In order to obtain a full-length cDNA encoding PYP1 for use in inducing expression in bacteria, the P. yezoensis EST database of the Kazusa DNA Research Institute (http://est.kazusa.or.jp/en/plant/porphyra/EST/) was surveyed. A comparison of nucleotide sequences from the genomic PYP1 gene revealed the presence of non-spliced introns, which were determined to cause the various lengths of PYP1 mRNAs (Fig. 1A), indicating that alterative splicing produces mature and immature PYP1 mRNAs.

As shown in Fig. 1A, the PYP1 gene contains 2 introns; thus, the coding region is divided into 3 exons. Three types of immature cDNA contain either the first or second intron, or both introns. Proteins derived from the cDNA containing the first, second, or both introns were designated as PYP1-A, PYP1-B and PYP1-C, respectively (Fig. 1A). PYP1-A and PYP1-C encode the same protein, as they both contain the first intron, resulting in 3 PYP1 proteins: one is mature and the other two are variants, but all share an amino acid sequence corresponding to the first exon (Fig. 2). We refer to this mixture of PYP1-A and PYP1-C as PYP1-AC.

RT-PCR was employed to examine the expression of the PYP1 gene in gametophytic and sporophytic generations of P. yezoensis. Since a homology search of P. yezoensis in the EST database resulted in ESTs derived from mRNAs of the gametophytic generation being found (data not shown), the generation-specific expression of the PYP1 gene was speculated. However, Fig. 1B (left panels) clearly illustrates the expression of the PYP1 gene in the gametophytic and sporophytic generations. Moreover, the presence of transcripts corresponding to PYP1-AC and PYP1-B was confirmed by RT-PCR (Fig. 1B, right panel), thus indicating that P. yezoensis-treated cells may contain all 3 PYP1 proteins.

To evaluate the functional activities of the 3 PYP1 proteins, PYP1, PYP1-AC and PYP1-B plasmids for the induction of expression in bacteria were constructed, which produced proteins with a 6xHis tag.

The results of SDS-PAGE revealed purified soluble proteins (Fig. 3). PYP1, PYP1-AC and PYP1-B were first purified with Ni-NTA His•Bind Resin (Merck Millipore) (Fig. 3A-a, B-a and C-a). PYP1, PYP1-AC and PYP1-B were then further purified using a Ni-NTA Spin kit (Qiagen), which produced proteins with the following molecular weights: 15, 12 and 5 kDa, respectively (Fig. 3A-b, B-b and C-b). This two-step process used to extract PYP1s was effective.

The effects and toxicity of PYP1, PYP1-AC and PYP1-B on Chang liver cells were determined by MTS assays. As shown in Fig. 4A, PYP1 was non-toxic. Moreover, treatment with 125–500 pg/ml PYP1 increased cell viability from 114.2±12.5 to 145.0±28.2% (P<0.05) compared with the control. To determine whether PYP1 protects the Chang liver cells against APAP-induced death, cell viability was examined following treatment with APAP. The cells treated with 15 mM APAP demonstrated a viability of 76.6±13.3%. By contrast, when the cells were treated with 125–500 pg/ml PYP1, cell viability increased significantly to 127.0±7.8, 110.2±15.6 and 91.0±4.6% that of the control, respectively (Fig. 4B, P<0.05). The same result was noted in the cells stained with DAPI, according to fluorescence microscopy (Fig. 4C). Similar results were obtained for both PYP1-AC and PYP1-B (Figs. 5 and 6). No significant differences were observed with regard to cytotoxicity by PYP1-AC (P<0.05, Fig. 5A); however, cell viability increased significantly following treatment with 125–500 pg/ml PYP1-AC to 102.7±14.5, 103.5±7.6 and 111.7±9.3% compared to that of the control, respectively (P<0.05, Fig. 5B). These results are presented as photomicrographic images in Fig. 5C. Cell viability increased significantly to 151.4±13.6% following treatment with 125–1000 pg/ml PYP1-B (P<0.05, Fig. 6A). In addition, treatment with PYP1-B resulted in the highest value (151.4±12.6%) of all the APAP treatment groups (P<0.05, Fig. 6B). The effect of PYP1-B was also demonstrated by examining cellular morphology (Fig. 6C). These results suggest that recombinant PYP1s may be used to protect liver cells against APAP-induced cytotoxicity.

To determine whether SP, which contains the N-terminal 11-residue sequence, protects Chang liver cells against APAP-induced death, cell viability was examined following treatment with APAP. As shown in Fig. 7A, SP was not toxic to the cells and resulted in cellular proliferation following treatment with 125–500 pg/ml SP. Notably, following treatment with both APAP and SP, cell viability decreased significantly compared with that of the control (P<0.05, Fig. 7B). Lower values upon SP treatment (64.9±7.5 to 75.9±7.5%, 125–1,000 pg/ml SP) compared to those observed with PYP1, PYP1-AC and PYP1 were observed in the APAP-treated cells. However, treatment with SP led to a significant increase in cell viability compared to treatment with APAP alone (61.8±8.6%; P<0.05, Fig. 7B). The cells treated with SP appeared to increase in number compared to the cells treated with APAP only (Fig. 7C).