Purification, characterization and developmental expression of pig liver PSP

Comparative Biochemistry and Physiology Part B 134 (2003) 571–578

Purification, characterization and developmental expression of pig liver PSP K. Kaneki, M. Matsumoto, K. Suzuki, M. Akuzawa, T. Oka* Department of Veterinary Medicine, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan Received 17 April 2002; received in revised form 24 September 2002; accepted 14 November 2002

Abstract We have isolated a perchloric acid-soluble protein designated as PL-PSP from the post-mitochondria supernatant fraction of pig liver. It is soluble in 5% perchloric acid and purified by ammonium sulfate fractionation and CMSephadex chromatography. The PL-PSP showed approximately 80–90% homology with PSP isolated from rat liver (RLPSP) with its partial amino acid sequences. The protein has a molecular mass of approximately 14 kDa which was slightly higher than that of RL-PSP. It inhibited protein synthesis in a rabbit reticulocyte lysate system. The expression of PL-PSP was predominant in liver, kidney and duodenum, and was also expressed in stomach, lung and brain. PLPSP expression in liver increased from the 1st day to the 1st month. Thus, our findings are the first report on the presence of a PSP in porcine tissues which may be involved in the regulation of cellular growth and differentiation. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Pig; Perchloric acid-soluble protein; Development

1. Introduction In the recent papers, we reported the isolation and characterization of a perchloric acid-soluble protein (PSP) of the rat liver (RL-PSP) (Oka et al., 1995) and the rat kidney (RK-PSP) (Asagi et al., 1998) and chick liver (CL-PSP) (Nordin et al., 2001) and rat brain (RB-PSP) (Suzuki et al., 2001) which is located in the cytosolic fraction. The PSP is a homodimer consisting of two identical subunits with a molecular mass of 14 kDa. The cDNA of RL-PSP contained a 411 bp, encoding a 137 amino acid protein with a molecular mass of 14 149 Da. The deduced amino acid sequence of RL-PSP was completely identical with that of RK-PSP and RB-PSP but showed the little difference with that of CL-PSP. PSP also inhibited *Corresponding author. Tel.yfax: q81-99-285-8714. E-mail address: [email protected] (T. Oka).

a cell-free protein synthesis in lysate of rabbit reticulocyte in different manner from RNase A (Asagi et al., 1998; Oka et al., 1995). Our recent work has suggested that this inhibition was due to an endoribonucleolytic activity of RL-PSP because RL-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80S ribosomes, even in the presence of cycloheximide (Morishita et al., 1999). Recently, a 14 kDa translational inhibitor protein which shows a remarkable similarity with PSP has been characterized from human monocytes (Schmiedeknecht et al., 1996), mouse liver (Samuel et al., 1997) and goat liver (Ceciliani et al., 1996). The expression of mRNA of the translational inhibitor p14.5, human homologue of RLPSP, becomes significantly up-regulated with the induction of differentiation to macrophages (Schmiedeknecht et al., 1996), and the synthesis of RK-PSP from rat kidney increases from the

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17th fetal day to the 4th postnatal week, and then enters a steady-state level (Asagi et al., 1998). In contrast, the expression of RK-PSP in renal tumor cells was down-regulated (Asagi et al., 1998). Thus the PSP and PSP-like proteins appear to be expressed in a growth and differentiation dependent manner. On the other hand, these cDNA sequences from mammals showed a high similarity to members of a new hypothetical family (YER057cyYJGF family) of small proteins with presently unknown function. The high degree of evolutionary conservation of these proteins may reflect an involvement in basic cellular regulation. Recently, Volz (1999) solved the structure of the YJGF protein from E. coli and refined at a resolution of 1.2 A. The YJGF molecule is a homodimer with exact threefold symmetry. Its tertiary and quaternary structure are related to that of B. subtilis chorismate mutase, although their active sites are completely different. In the present study, we describe the purification and characterization of pig liver PSP (PL-PSP) and partial sequences of its amino acid. Its localization on various tissue and developmental expression in pig liver were also examined. We report for the first time of the isolation, purification and characterization of PL-PSP and also its expression in pig tissues. 2. Materials and methods 2.1. Purification of PL-PSP Liver isolated from pig were homogenized in two volumes of cold 0.25 M sucrose in buffer (50 mM Tris–HCl buffer pH 7.4, 25 mM KCl and 10 mM MgCl2) with an all-glass Potter Elvehjem type homogenizer. After centrifugation at 10 000=g for 30 min, the post-mitochondrial supernatant (PMS) was obtained and treated with the appropriate volume of 60% perchloric acid making a 5% solution of the PMS and it was again centrifuged at 10 000=g for 10 min. The supernatant was then made 25% with respect to trichloroacetic acid, and the precipitate was collected by centrifugation at a similar speed as the later procedure. The precipitate was washed with cold acetone for three times and dried. The dried material was made 0.9% with respect to acetic acid, and the supernatant was dialyzed against 0.1 M sodium phosphate buffer (PBS) pH 7.4 for overnight. After clarification by a 10 min centrif-

ugation at 10 000=g, the proteins in the dialysate were fractionated with saturated ammonium sulfate. The precipitate formed between 30 and 50% saturation was collected by the centrifugation at 10 000=g for 10 min. The precipitate was suspended in 0.1 M sodium phosphate buffer (pH 7.4) and dialyzed against the same buffer. The dialysate was applied through a 2=30 cm2 column of CM-Sephadex C-25. Absorbance readings for protein were taken at 280 nm using an Ultraspec 3000 Spectrophotometer (Pharmacia Biotech) and the flow through fractions were collected and electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, protein fractions which cross-reacted with PSP antibody were pooled and again subjected to electrophoresis. The corresponding protein fractions were used as the purified protein in this study. 2.2. Western blotting The proteins in homogenate from the various tissues were analyzed on polyacrylamide gels (15%) according to the method of Laemmli (1970). After electrophoresis, the proteins on the gels were blotted onto nitrocellulose membranes (Schleicher and Schuell, Germany) and immunoblotted with antisera against pig PSP. 2.3. Proteolysis of PL-PSP and sequencing of the peptide The purified PSP (300 mg) was digested with lysyl endopeptidase (the proteinylysyl endopeptidase, 100:1 (wyw) in 50 mM Tris–HCl buffer (pH 8.0)). A reversed-phase HPLC was used to separate the peptides from the lysyl endopeptidase digestion. The peptides were applied on a Vydac C18 column. It was washed with 0.1% trifluoroacetic acid for the first 5 min, then the peptides were eluted from the column using a linear gradient of 0–60% acetonitrile in 0.1% trifluoroacetic acid for the second 60 min at a flow rate of 200 mlymin. The peptides eluted were detected at the absorbance of 206 and 280 nm. Eight peptides were obtained. The peptides from No. 1, 3, 4, 5, 7 and 8 were left to dry and then used for the amino acid sequencing analysis. 2.4. Immunohistochemistry Three different ages of male Clawn strain miniature pigs, postnatal 1st day, 1 and 3 month of

K. Kaneki et al. / Comparative Biochemistry and Physiology Part B 134 (2003) 571–578

age, were purchased from Japan Farm Co. Ltd., Kagoshima, Japan and sacrificed under ketamine hydrochloride anesthesia. The liver, kidney, stomach, duodenum, lung and brain were dissected out and were fixed with 10% neutral buffered formalin solution, embedded in paraffin, and serially sectioned at 4 mm thickness. The sections were deparaffined and soaked in 0.3% H2O2 in 100% ethanol for 30 min at room temperature. After hydration and rinsing in PBS (10 mM sodium phosphate buffer pH 7.2, containing 0.85% NaCl), the sections were blocked with a normal goat serum at 1:60 dilution for 20 min at room temperature to reduce nonspecific staining, and then incubated at 4 8C over night with polyclonal antibodies against PL-PSP at 1:2000 dilution in PBS containing 0.1% BSA in a moist chamber. The sections were washed extensively with PBS and then incubated for 50 min at room temperature in a 1:200 dilution of biotinylated goat anti-rabbit IgG. After washing with PBS, the sections were incubated for 30 min at room temperature in a avidin–biotin complex (Vectastain ABC kit, Vector Laboratories, Inc., USA), rinsed in PBS, stained for 5 min with 50 mM Tris–HCl (pH 7.6), containing 0.1% diaminobentizine and 0.02% H2O2. After washing with PBS, the sections were counterstained with hematoxylin for 20 s, dehydrated, and mounted. Negative controls were prepared by using nonimmunized rabbit IgG as a primary antibody. 3. Results 3.1. Purification and characterization of the PLPSP The PSP-like protein extracted from pig liver was predominantly present in the PMS fraction (data not shown). The protein obtained by extraction with 5% perchloric acid and 25% trichloroacetic acid from the PMS fraction was almost pure at this step. The PSP-like protein appeared in the flow through fractions of CM-Sephadex chromatography and was shown to be homogenous by SDS-PAGE (Fig. 1a). Approximately 1 mg of the purified protein was obtained from 100 g of pig liver. Under the reducing condition, the purified protein showed the same mobility as well as RLPSP, but its molecular mass was slightly higher than that of RL-PSP ()14 kDa) (Fig. 1a). The immunoblotting result showed a strong cross-reac-

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Fig. 1. SDS-PAGE of the purified PL-PSP. SDS-PAGE (a) and immunoblot analysis (b) of the purified protein from pig liver and the purified RL-PSP. M, Molecular weight marker; 1, PLPSP; 2, RL-PSP.

tion with rat PSP antibody suggesting that the purified protein is the prominent protein in the PMS and is PSP-like protein (Fig. 1b). Here we refer to the purified PSP-like protein from the pig liver as PL-PSP in order to distinguish from the RL-PSP, RK-PSP, RB-PSP and CL-PSP. In the previous study (Oka et al., 1995), we showed that PSP inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner than RNase A. The ability of the PL-PSP to inhibit cell-free protein synthesis in the rabbit reticulocyte lysate system was examined and compared with RL-PSP. The PL-PSP exhibited a dose-dependent inhibition of protein synthesis similar to RL-PSP, and a 50% inhibition (IC50) was found to 100 nM (data not shown). The PL-PSP was shown to be insensitive to automated Edman degradation, indicating that the N-terminal amino acid residue may be blocked such like that of the RL-PSP. Therefore, PL-PSP was digested with lysyl endopeptidase, and the products were separated by reverse-phase HPLC and compared with that of RL-PSP (Fig. 2). The peptide-mapping by HPLC showed that the amino acid sequences of PL-PSP and RL-PSP were quite different (Fig. 2, top and bottom). Finally, eight peptides were isolated from PL-PSP, and six (No. 1, 3, 4, 5, 7 and 8) of them were directly sequenced. The amino acid sequences of these six peptides were aligned with that of PL-PSP (Table 1). The peptide 1 of PL-PSP showed 83%

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Now we are undertaking extensive screening of the PL-PSP cDNA. 3.2. Tissue distribution of the PL-PSP The PL-PSP in the homogenate samples obtained from various tissues was examined by immunoblotting with the antisera raised against PL-PSP in New Zealand White rabbit. The PLPSP was found to be prominently in liver, kidney and intestine, but stomach, spleen, lung and brain were weakly cross-reacted with antisera (Fig. 3). One extra band was observed on muscle, but the physiological significance of this extra band is presently unknown. At present, we are undertaking extensive purification of this protein. The sections of main organs which were stained immunohistochemically with anti-PSP antibody were observed by a light microscope (Fig. 4). Immunoreactive products were shown in the hepatocytes, epithelium of renal tubules, chief and parietal cells of gastric gland, absorptive epithelial cells of intestinal villi, alveolar and bronchiolar epithelial cells of lung, and astrocytes of cerebellum. No reaction products were observed in any control sections prepared using nonimmunized rabbit IgG as a primary antibody (data not shown). These results suggested that the major tissues of the PL-PSP expression are the liver kidney and intestine, but are weakly expressed in the other tissues.

Fig. 2. HPLC of the peptides obtained from PL-PSP and RLPSP by digestion with lysyl endopeptidase. The PL-PSP and RL-PSP were separated by HPLC under Section 2. The separated peptides were collected and evaporated by a centrifugal evaporator. The dried materials were used as the isolated peptides. The elution of the peptides was followed by monitoring the absorbance at 206 and 280 nm.

sequence identity with that of RL-PSP, peptide 3: 91% identity, peptide 4: 82% identity, peptide 5: 90% identity, peptide 7: 80% identity and peptide 8: 91% respectively. Although the result of the peptide-mapping suggested that the amino acid sequences of the PL-PSP and the RL-PSP are different, the identified amino acid sequences of the PL-PSP are similar to the RL-PSP-like protein.

Table 1 Amino acid sequences of peptides obtained from PL-PSP with lysyl endopeptidase and comparison with RL-PSP Species

Peptide number

Amino acid sequence

Pig

Peptide 1

Rata Pig

Peptide 2 Peptide 3

Rat Pig

Peptide 5 Peptide 4

Rat Pig

Peptide 1q4 Peptide 5

Rat Pig

Peptide 6 Peptide 7

Rat Pig

Peptide 8 Peptide 8

Rat

Peptide 9

V * V A * A Q * Q Q * T G * G A * A

I * I A * A A * A Y * Y G S P * P

S * S G * G L * L F * F R * R A * A

*Identical residues. a Peptide numbers obtained from Fig. 2.

T * T A C T K Q * Q V I A * A

V * S D * D N * N G * G E * E I * I

Identity K * K F * F M L N * N I * I G * G

83% 5y6 T * T G * G F * L E * E F A

N * N E * E P * P A * A Y * Y

V * V I * I A * A I * I S * S

V * V L * L R * R A * A Q * Q

K * K K * K A * A

97% 10y11 82% 9y11 A * A

Y * Y

Q * Q

V * V

A * A

A * A

L * L

P * P

K

90% 18y20

K 80% 8y10

A * A

V * V

L * L

V * V

D * D

R * R

T V T

I * I

Y * Y

I V

S * S

G * G

91% 20y22

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Fig. 3. Immunoblot analysis of the PL-PSP from various pig tissues. Twenty micrograms of proteins in the homogenates from various tissues were electrophoresed on 15% SDS-PAGE. Immunoblot analysis was carried out as described under Section 2.

3.3. Developmental expression of PL-PSP The expression of PL-PSP at various postnatal stages of the pig was investigated by immunoblotting (Fig. 5). Interestingly, the expression of PLPSP protein in the liver gradually increased from the 1st day to 1st month, and then it remained almost the same until the 3rd month. These results indicated that PL-PSP expression depend on the cellular development of the pig liver. 4. Discussion In the present study, a PL-PSP was isolated from pig liver and partially sequenced. PL-PSP was shown to consist of a single polypeptide chain, and its molecular mass was slightly higher than that of RL-PSP (14 kDa). This was confirmed when the protein was analyzed by SDS-PAGE. Immunoblotting study showed that the PL-PSP

cross-reacted with RL-PSP antibody, suggesting that the amino acid sequences of the PL-PSP closely resemble with that of RL-PSP. The amino acid sequences of PL-PSP peptides which were obtained after digestion with lysyl endopeptidase are shown to be 80–90% homology with that of RL-PSP. PL-PSP showed a very remarkable change in its expression during various developmental stages of the pigs. The intensity of immunoblot-bands increased from newborn pig to month 1 and remained almost the same until the 3rd month. During the pig developmental life, liver growth is accomplished by cell proliferation, and liver function appears to be completed at the 1st month of life. Therefore, PL-PSP can exert a regulatory role in the synthesis of proteins because it has been found to act as a strong inhibitor of cell-free protein synthesis. The level of PL-PSP is low during the early ages of the pig, and thus the liver

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Fig. 4. Immunohistochemical localization of PL-PSP in liver (a); kidney (b); stomach (c); duodenum (d); lung (e) and cerebellum (f) of the miniature pig at 3 month of age. Immunoreactive products are shown in the hepatocytes (a); epithelial cells of renal tubules (b); chief and parietal cells of gastric gland (c); absorptive epithelial cells of duodenal villi (d); alveolar and bronchiolar epithelial cells of lung (e) and astrocytes of cerebellum. Original magnifications are =25 (a, b, c, d) and =50 (e, f), respectively.

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but is significantly up-regulated when these expression of the p14.5 was suppressed in HepG2 cells derived from human hepatomas and in oncocyte stromal cells from renal neoplasms. On the other hand, the expression of RK-PSP was examined in normal rat kidney epithelial NRK-52E cells (Kanouchi et al., 2000). The expression of RK-PSP was low under the proliferating phase and high in the stationary phase, and shown to have a negative relationship with the protein synthesizing activity of the cells. Furthermore, we examined the effect of overproduction of PSP on the cell-proliferation (Kanouchi et al., 2001). When PSP cDNA was transfected in NRK-52E, the PSP mRNA and its protein were over-expressed. As a result, cell proliferation of the transfected cells was suppressed compared with that of cells transfected the vector only. Thus, the present data together with the above mentioned results indicated that PSP and PSP-like proteins may regulate the growth and the differentiation of cells. Fig. 5. Immunoblot analysis of the PL-PSP from postnatal pig liver. Protein (20 mg) in the homogenate from postnatal pig liver was electrophoresed on a 15% SDS-PAGE. Immunoblot analysis was carried out as described under Section 2. Lane 1: purified PL-PSP, lane 2: 1st day, lane 3: 1st month, lane 4: 3rd month.

can synthesize a large amount of protein which is necessary for growth. Later, when the pig grows to 1st month and more, the level of PL-PSP rises, resulting in slowing down of the synthesis of proteins (Fig. 4). This result was confirmed by the presence of immunopositive deposits in the differentiated-hepatocyte cells of the liver and the differentiated tubular cells of the kidney of 3rd month pig. The same development-dependent expression of PSP was observed in the expression of RK-PSP of the developing rats (Asagi et al., 1998). The immunopositive deposits of RK-PSP were intense only in the differentiated tubular cells. On the other hand, the expression of RK-PSP in tumor cells was suppressed as compared with the normal part of LEC rat kidney (Asagi et al., 1998). Recently, Schmiedeknecht et al. (1996) have isolated and characterized a 14.5 kDa trichloroacetic acid-soluble translational inhibitor protein (p14.5) from human monocytes which shows a remarkable similarity to RL-PSP and RK-PSP. Interestingly, these authors demonstrated that the p14.5 mRNA is weakly expressed in freshly isolated monocytes,

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