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The total RNA was obtained from 11 tissues (brain, tentacle, gill, root of the tongue, spinal cord, heart, liver, gut, muscle, skin, and mucous gland) of inshore hagfish using GeneAll® Hybrid-R™ Total RNA (GeneAll Biotechnology Co., Ltd., Korea) as described by previous manuscripts. The RNA was reverse-transcribed by oligo (dT)18 using the PrimeScript™ 1st strand cDNA Synthesis Kit (TaKaRa, Korea) and random hexamer primers. Several PKCβ nucleotide sequences were collected from other species. Degenerated primers were designed around the highly conserved parts of the sequences using BioEdit Sequence Alignment Editor version 5.0.9 (sense primers, DgPKCβ-F1 and DgPKCβ-F2; antisense primer, DgPKCβ-R1 and DgPKCβ-R2,

<Table 1> and employed in order to amplify cDNAs from the inshore hagfish cDNA library.

3′ cDNA library screening was conducted with 3′ GSP (gene specific primer) (sense primer, 3’GSP-EbPKCβ-F1 and 3’GSP-EbPKCβ-F2, Table 1) and Universal primers. In order to isolate the full-length EbPKCβ, 5′ RACE-PCR was conducted using the GeneRacer™ Kit (Invitrogen, Korea). 5′ RACE-PCR clone was amplified with gene specific primer (antisense primer, 5’GSP-EbPKCβ-R1 and 5’GSP-EbPKCβ-R2, <Table 1> and Universal primers. Next, the PCR product was recovered by GeneAll® SV gel (GeneAll, Korea). The purified products were then ligated into pGEM T-Easy vector (Promega, Korea). Finally, database searches were performed using the BLAST (Basic Local Alignment Search Tool) at the NCBI (National Center for Biotechnology Information).

Nucleotide and predicted peptide sequences of E. burgeri PKCβI and βII (EbPKCβI and EbPKCβII) were analyzed using DNASIS for Windows version 2.5 (Hitachi software engineering Co., Japan), BioEdit Sequence Alignment Editor, and BLAST programs in the non-redundant databases of the NCBI (http://www.ncbi.nlm.nih.gov\/BLAST/). Multiple alignments of EbPKCβI and βII amino acid sequences were analyzed using CLUSTAL W version 1.8. The identities and homologies between amino acid sequences were analyzed using BioEdit Sequence Alignment Editor version 5.0.9. The multiple sequence alignment obtained was used in order to generate a phylogenetic tree using neighbor-joining methods, and the reliability of the trees was evaluated using the bootstrap method with 1,000 replications. In order to identify possible phylogenetic clade, a neighbor-joining tree was generated based on this genetic distance matrix using Kimura 2-parameter model included in MEGA 6.

The mRNA distribution of EbPKCβI and βII were measured by qPCR analysis. The total RNA extraction and reverse transcription was obtained through the same process described above (2). For the tissue distributions of EbPKCβI and βII, 18s rRNA was used as an internal control gene. The specific primers Eb18s-rRNA-RT-F, Eb18s-rRNA-RT-R, EbPKCβI-RT-F, EbPKCβI-RT-R, EbPKCβII-RT-F, and EbPKCβII-RT-R were used for qPCR(<Table 1>).

LightCycler® 480 SYBR Green I Master (Roche, Switzerland) was used in order to monitor the quantitative real-time PCR of mRNA transcript abundance on the LightCycler® 480 II Real-Time PCR System (Roche) using the following program: pre-incubation at 95°C for 5 min, 30 cycles at 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. The qPCR mixture was made up of the following components: 10 µl of 2X SYBR (Roche), 7.5 µl of SYBR water (Roche), 1 µl of sense primer, 1 µl of antisense primer, and 0.5 µl diluted first-strand cDNA (diluted at 1:20)

The ^ΔΔCT method was adopted in order to calculate the data and the 2−^ΔΔCt method was adopted in order to calculate the relative quantitative value (Giulietti et al, 2001).

In order to construct an expression vector for the suitable production of recombinant EbPKCβI and βII in E. coli, the open reading frame (ORF) of EbPKCβI was amplified by PCR using the primers (sense primer, EcoRI-EbPKCβI-F; and antisense primer XhoI-EbPKCβI-R, as shown in <Table 1>).

In addition, in the case of EbPKCβII, ORF was amplified by PCR using the primers (sense primer, EcoRI-EbPKCβI-F and antisense primer XhoI-EbPKCβII-R, as shown in <Table 1>). They have the same nucleotide sequence at the beginning of ORF, so the same sense primer was used in both cases. The amplified fragment was cloned into the pET32b (Novagen). The recombinant plasmids (EbPKCβI/pET32b and EbPKCβII/pET32b) were transformed into E. colistrain BL21 (DE3). Transformed cells were grown in LB broth (5 ml) containing ampicillin (100 µg/ml) at 37°C for about 12 h, re-inoculated in two LB broths (5ml) containing ampicillin (100 µg/ml), and grown at 37°C until the OD₆₀₀=0.6. Only one of the two LB broths (5 ml) had IPTG (Isopropyl-β-D-thiogalactopyranoside) added to it to a final concentration of 1 mM and grown at 20°C about 24 h. The induction of the target proteins was checked by SDS-PAGE (10% running gel, 5% stacking gel) and Western blotting. In order to obtain target proteins (EbPKCβI and βII) in large scale, the cells were inoculated in LB broth (500 mL) and grown at 37°C until the OD₆₀₀=0.6.

For the electrophoresis procedures, all samples were denatured in buffer containing 60mM Tris/pH 6.8, 25% glycerol, 2% SDS, 14.4mM 2-mercaptoethanol, and 0.1% bromophenol blue, then boiled for 5min. Purified EbPKCβI and βII were separated by 10% SDS-PAGE (Bio-Rad, USA). Prestained molecular weight markers (Bio-Rad, USA) were run as standards on each gel. Following electrophoresis, the gels were stained with Coomassie brilliant blue R-250.

Western blotting was performed using rabbit polyclonal anti-His antibody (1:2000, Santa Cruz Biotechnology) and rabbit monoclonal anti-His antibody (1:1000, Santa Cruz Biotechnology). Prestained molecular weight markers (Bio-Rad, USA) were run as standards. The electrophoresed samples were transferred to nitrocellulose membranes (Schleicher & Schuell. Co., USA) using a Hoefer transblotting system (Pharmacia. Co., USA). Following this transfer, the membrane was blocked with 3% BSA in TPBS [200 mM Tris (pH 7.0), 1.37 M NaCl, 1% Tween-20] for 30 min at room temperature. Primary antibody was attached to the target proteins at 4℃ for 16 h. Secondary antibody was attached to the target proteins at 4℃ for 1.5 h.

Phosphorylation by PKC of its specific substrate alters the peptide's net charge from +1 to –1. PepTag® Non-Radioactive PKC Assay (Promega) was used to analyze the activities of EbPKCβI and EbPKCβII. For qualitative analysis, proteins were diluted to 1, 2, 5, and 10 ng/μl, and reacted with substrate at 30℃ for 30 min. After reaction, samples underwent electrophoresis on gel, which was made of 50 mM Tris-HCl(pH 8.0) and 0.8% agarose, over 20 min. Phosphorylated peptide was separated for quantitative analysis. At 95℃, it was completely melted and Gel Solubilization Solution, glacial acetic acid, and distilled water were added. Using a spectrophotometer, we assessed the absorbance at A₆₅₀ and calculated activity using the

following equation:

A = εBC,

where: A = absorbance of the sample, ε = the molar absorptivity of the peptide in L/mol • cm^-1, B = the width of the light cell, and C = the concentration of the peptide in mol/L of the sample read.

In order to identify the partial sequences of EbPKCβ, databases of other PKCs were obtained using NCBI sequence data. These sequences were used to design degenerated primers. The initial partial sequences were obtained through PCR amplification of inshore hagfish cDNA, including the brain, tentacle, gill, root of the tongue, spinal cord, heart, liver, gut, muscle, skin, and mucous gland. In order to isolate full-length inshore hagfish PKCβI and βII, the partial sequences were used as bases for gene-specific primers for RACE PCR.

As a result, the full nucleotide sequences of EbPKCβI and EbPKCβII were 2,499 bp and 2,658 bp, respectively. The EbPKCβI sequence was composed of a 238 bp 5’-untranslated region (5’-UTR), a 2,007 bp coding region, and a 254 bp 3’-untranslated region (3’-UTR) [Fig. 1]. 3’-UTR of EbPKCβIhad a miR-199-5p binding site from 73 to 78 (5’-TACTGG-3’). The EbPKCβII sequence was composed of a 235 bp 5’-untranslated region (5’-UTR), a 2,004 bp coding region, and a 419 bp 3’-untranslated region (3’-UTR) [Fig. 2]. 3’-UTR of EbPKCβII had a miR-203a-5p binding site from 328 to 334 (5’-GATCCAT-3’). The EbPKCβI codes 668 amino acids, which the molecular weight is approximately 76.43 kDa, and the EbPKCβII codes 667 amino acids, which the molecular weight is approximately 76.08 kDa. These sequences were submitted to the NCBI database [PKCβI(MH350863), PKCβII(MH350864)].

[Fig. 1] 
Nucleotide sequence and deduced amino acid sequence of EbPKCβI. Shaded sequences indicate C1, C2, Kinase, and V5 domains. The four circles are lipid cofactor binding surface inside the C1 domain. The two squares are calcium-binding Asp and the square brackets are RACK binding sites. The three triangles indicate ATP binding site (GXGXXG). In the Kinase domain, the first circle is invariant Lys and the second circle is Met as a gatekeeper residue. An underlined Trp is the turn motif and an underlined Ser is the hydrophobic motif. Asterisk (*) at the end of amino acid sequence shows the stop codon.

[Fig. 2] 
Nucelotide sequence and deduced amino acid sequence of EbPKCβII. Shaded sequences indicate C1, C2, Kinase, and V5 domains. The four circles are lipid cofactor binding surface inside the C1 domain. The two squares are calcium-binding Asp and the square brackets are RACK binding sites. The three triangles indicate ATP binding site (GXGXXG). In the Kinase domain, the first circle is invariant Lys and the second circle is Met as a gatekeeper residue. An underlined Trp is the turn motif and an underlined Ser is the hydrophobic motif. Asterisk(*) at the end of amino acid sequence shows the stop codon.

EbPKCβI and EbPKCβII have the same sequences in conserved regions (C1, C2, C3, and C4 domain), but not in the C-terminal called the V5 domain. C1 domains were highly conserved DAG/PMA binding sites with a characteristic HX₁₂CX₂CXnCX₂CX₄HX₂CX₇C motif (H, histidine; C, cysteine; X, any other amino acid; n is 13). C2 domains contained a calcium binding loop, which has several highly conserved Asp residues, and a RACK (receptor for activated C-kinase) binding site.

It was confirmed that both EbPKCβI and βII had these highly conserved motifs. Kinase domains include an ATP-binding site (GXGXXG; G is glycine and X is any other amino acid), invariant Lys, and gatekeeper residue. cPKCs and nPKCs have Met as a gatekeeper residue, while aPKCs use Ile. It was confirmed that EbPKCβI and βII have these residues as well. In the case of V5 domains, which are about 50 residues of C-terminus, EbPKCβI and EbPKCβIdiffer significantly. Thus, βI and βII were determined by referencing previous studies [Fig. 3].

[Fig. 3] 
Multiple amino acids sequence alignment analysis of PKC subtypes in various species. The GenBank accession numbers used in the alignment are shown in Table 2. Identical amino acid residues are darkly shaded, similar amino acids are lightly shaded, unrelated residues have a white background, and amino acid numbers are shown on the right. “HX₁₂CX₂CX₁₃CX₂CX₄HX ₂C₇C” in the first square box is C1 motif, that exists in both C1A and C1B. The two circles above the sequence show the Asdp residues binding to calcium. The second square box shows the ATP binding site, “GXGXXG”. The first inverted triangle is invariant Lys and the second inverte triangle is Met as a gatekeeper residue.

In order to determine the evolutionary relationship of EbPKCβI and βII with other families of the PKC, a phylogenetic tree was constructed.

Phylogenetic analysis was performed with the amino acid sequences of human PKCs and the PKCs of other species obtained from GenBank using neighbor-joining methods [Fig. 4]. Based on a comprehensive phylogenetic analysis, cPKCs, nPKCs, and aPKCs were classified. In other species, βI was more closed compared to the βI of another species but not in the case of fish (DrPKCβI and EbPKCβI).

[Fig. 4] 
Phylogenetic relationship of EbPKCβI and βII with other PKC subtypes. In this neighbor-joining phylogram, all individuals are represented and the branches are based on the number of inferred substitutions, as indicated by the bar. The square indicates EbPKCβI and EbPKCβII.

The distributions of EbPKCβI and βII transcripts in different organs were examined by RT-PCR. The results of qPCR indicated that EbPKCβI and βII were expressed in different organs, including the brain, tentacle, gill, root of tongue, spinal cord, heart, liver, gut, muscle, skin, and mucous gland. The expression pattern of EbPKCβI was found at its highest levels in the root of the tongue and spinal cord [Fig. 5A]. The expression pattern of EbPKCβII was found at high levels in the gill, liver, and gut [Fig. 5B].

[Fig. 5] 
Tissue-specific distribution of EbPKCβI and βII Quantitative real-time PCR of EbPKCβI and EbPKCβII in various tissues. Mean of mRNA levels in E. burgeri tissues were analyzed by real-time PCR, and 2−ΔΔCt levels were calculated relative to the tissue with the lowest expression (PKCβI from gut tissue and PKCβII from muscle tissue) set to 1 and normalized against 18s-rRNA expression. Each experiment was done in triplicate.

In order to select recombinants of EbPKCβI and βII, transformed cells were grown in LB broth (5 ml) containing ampicillin (100 µg/ml) and had IPTG added to a final concentration of 1 mM. The sample was checked with induction of target proteins by SDS-PAGE (10% running gel, 5% stacking gel) and Western blotting was performed [Fig. 6A, 6B].

[Fig. 6] 
SDS-PAGE and Western blot analysis of EbPKCβI and βII. 10% SDS-PAGE gel and coomassie R-250 blue was used to perform SDS-PAGE. Anti-His antibody was used for Western blotting. Predicted recombinant EbPKCβs Molecular weight is approximately 92 kDa, as they include Trx-tag, S-tag, His-tag, and other amino acids. M, standard size marker; N, cell lysate from IPTG-not induced EbPKCβ-expressing E. coli strain BL21 (DE3); I, cell lysate from 1 mM IPTG-induced EbPKCβ-expressing E. coli strain BL21 (DE3).

Although the size of EbPKCβI and βII is approximately 76 kDa in both cases, the target size on SDS-PAGE is about 92 kDa, because pET32b vector expresses other proteins, including Trx tag, S tag, and His tag. Recombinant EbPKCβI and βII were purified by affinity column with nickel resin at 4°C. SDS-PAGE was onducted with 10% acrylamide gel [Fig. 7A, 7B]. Purified samples were dialyzed in order to check the EbPKCβ’s activities.

[Fig. 7] 
SDS-PAGE of EbPKCβI and βII purified by affinity chromatography. 10% SDS-PAGE gel and coomassie R-250 blue was used to perform SDS-PAGE. Predicted recombinant EbPKCβs Molecular weight is approximately 92 kDa. M, standard size marker; -10, sample obtained for 10 minutes after passing through an elution buffer; 1~10, sample obtained for the next minute; +10, sample obtained during the last 10 minutes.

In order to check whether recombinant EbPKCβI and βII proteins have PKC activity, we conducted activity assay using substrate which could be phosphorylated by PKC. Consequently, we identified that they can indeed phosphorylate substrate [Fig. 8].

[Fig. 8] 
Qualitative analysis of EbPKCβI and EbPKCβII. Substrate was phosphorylated by purified proteins at 1, 2, 5, and 10 ng/μl, respectively. Lane 1 indicates negative control and lanes 2-5 indicate reactants which contain substrate and diversely diluted purified protein (1, 2, 5, and 10 ng/μl). The upper parts are phosphorylated substrate and the lower parts are non-phosphorylated substrate.

The results of quantitative analysis showed that in the case of EbPKCβI, it phosphorylated substrate at 14.52, 22.36, 29.25, and 40.43 pmol/min at 1, 2, 5, and 10 ng/μl, respectively [Fig. 9A]. EbPKCβII phosphorylated substrate at 0.86, 1.27, 10.32, and 43.87 pmol/mine at 1, 2, 5, and 10 ng/μl, respectively [Fig. 9B].

[Fig. 9] 
Quantitative analysis of EbPKCβI and EbPKCβII. We checked the amount of phosphorylated substrate treated with EbPKCβI and EbPKCβII 1, 2, 5, and 10 ng/μl. (A) Activity of EbPKCβI, (B) Activity of EbPKCβII.