Characterization of a Stellate Cell Activation-associated Protein (STAP) with Peroxidase Activity Found in Rat Hepatic Stellate Cells*

A proteome approach for the molecular analysis of the activation of rat stellate cell, a liver-specific pericyte, led to the discovery of a novel protein named STAP (stellate cellactivation-associated protein). We cloned STAP cDNA. STAP is a cytoplasmic protein with molecular weight of 21,496 and shows about 40% amino acid sequence homology with myoglobin. STAP was dramatically induced in in vivo activated stellate cells isolated from fibrotic liver and in stellate cells undergoingin vitro activation during primary culture. This induction was seen together with that of other activation-associated molecules, such as smooth muscle α-actin, PDGF receptor-β, and neural cell adhesion molecule. The expression of STAP protein and mRNA was augmented time dependently in thioacetamide-induced fibrotic liver. Immunoelectron microscopy and proteome analysis detected STAP in stellate cells but not in other hepatic constituent cells. Biochemical characterization of recombinant rat STAP revealed that STAP is a heme protein exhibiting peroxidase activity toward hydrogen peroxide and linoleic acid hydroperoxide. These results indicate that STAP is a novel endogenous peroxidase catabolizing hydrogen peroxide and lipid hydroperoxides, both of which have been reported to trigger stellate cell activation and consequently promote progression of liver fibrosis. STAP could thus play a role as an antifibrotic scavenger of peroxides in the liver.

The molecular mechanism underlying the activation of hepatic stellate cells has been extensively studied during the last decade because activated stellate cells are thought to be a key player in the development of liver fibrosis (1)(2)(3)(4)(5). This activation is characterized by a transdifferentiation from a vitamin Astoring quiescent phenotype to a myofibroblast-like cell and accompanied by the expression of various genes of extracellular matrix matrices, cell growth factors, inflammatory cytokines, and receptors for growth factors (1)(2)(3)(4)(5). Analysis of the difference of gene expression between quiescent and activated stellate cells has provided profound insights into the cell activation mechanism. For instance, subtractive hybridization revealed the expression of Zf9, a Kruppel-like transcription factor, at the initial step of the cell activation (6) and that of cellular prion protein in fully activated stellate cells (7).
We have undertaken a proteomics approach to obtain a deeper knowledge on the molecular mechanism of hepatic stellate cell activation at the protein level (8). Protein populations, proteomes, expressed in quiescent and activated stellate cells were separated by two-dimensional polyacrylamide gel electrophoresis (PAGE) 1 and subsequently analyzed using electrospray ionization mass spectrometry (9,10). Such a protein level "differential display" identified 43 proteins that altered their expression levels during the activation process (8). These included the up-regulation of collagen ␣1(I), collagen ␣1(III), ␥-actin, neural cell adhesion molecule (N-CAM), and smooth muscle ␣-actin in accordance with previous reports (11)(12)(13), and that of calcyclin, calgizzarin, and galectin-1, representing new findings (8).
In the present study, we report a novel protein that was discovered by proteome analysis of the activation process of rat hepatic stellate cells. The protein was found to be heavily up-regulated both in in vitro and in vivo activated stellate cells and was accordingly named STAP (stellate cell activation-associated protein). Expression of STAP and its gene (stap) was dramatically augmented in fibrotic liver tissues induced by thioacetamide (TAA) administration, indicating an important role of STAP in the development of liver fibrosis.

MATERIALS AND METHODS
Induction of Liver Fibrosis-Pathogen-free male Wistar rats (SLC, Shizuoka, Japan) were administered with 50 mg/body of TAA (WAKO Pure Chemical Co., Osaka, Japan) intraperitoneally twice a week for 8 weeks (14). The protocol of experiments was approved by the Animal Research Committee of Osaka City University (Guide for Animal Experiments, Osaka City University).
Preparation of Hepatic Constituent Cells-Hepatic constituent cells were isolated from rat livers as previously described (8,15). Hepatocytes, Kupffer cells, and endothelial cells were used immediately after the isolation. Stellate cells were plated for 3 h in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Gaithersburg, MD) and supplemented with 10% fetal bovine serum (Life Technologies, Inc.), and the cultures were subsequently washed to remove dead cells and cell debris. Stellate cells isolated from normal or fibrotic livers were referred to as quiescent or in vivo activated stellate cells, respectively, in the present study (8). Likewise, stellate cells isolated from normal liver and cultured for 7 days were referred to as in vitro activated stellate cells (8).
Tryptic In-gel Digestion of Two-dimensional PAGE Resolved Proteins and Mass Spectrometry-Protein spots of interest were excised from the two-dimensional gels and in-gel-digested with trypsin (8,17). After extraction and purification from the tryptic digests (17), peptides were sequenced by electrospray ionization mass spectrometry on a quadrupole-time-of-flight mass spectrometer (8,17). The proteins were identified by matching the obtained amino acid sequences against the SwissProt and GenBank TM data bases.
Reverse Transcription-Polymerase Chain Reaction-Total RNA was extracted from stellate cells and liver tissues using Isogen (Nippon Gene, Tokyo, Japan). Messenger RNA expression in each sample was determined by reverse transcription-polymerase chain reaction (RT-PCR) using GeneAmp RNA PCR Core Kit (PerkinElmer Life Sciences). The following primers were used: STAP, ATGGAGAAAGTGC-CGGGCGAC (forward) and TGGCCCTGAAGAGGGCAGTGT (reverse); and glyceraldehyde-3-phosphate dehydrogenase, ACCACAGTCCATG-CCATCAC (forward) and TCCACCACCCTGTTGCTGTA (reverse). cDNA Cloning-Degenerative PCR was performed using a primer pair, CCXGGXGAYTTYGARATHGA and GCXACXCCXACRTCYTC, designed from two amino acid sequences, PGDMEIER and ANCEDVGVA, which were derived by electrospray ionization mass spectrometric analysis of one protein spot later named STAP from two-dimensional gels of activated stellate cells. The template used was a cDNA library that was reverse-transcribed from total RNA of activated stellate cells using oligo(dT) primer, reverse transcriptase (SU-PERSCRIPT II, Life Technologies, Inc.), and GeneAmp RNA PCR Core Kit (PerkinElmer Life Sciences). The obtained 120-bp product was ligated into pGEM-T Easy vectors (Promega), and the sequence of the inserted DNA was determined using ABI PRISM 310. A rat-activated stellate cell cDNA library was inserted into Lambda Zap II vectors (Stratagene, La Jolla, CA) and screened using this 120-bp PCR product as a probe. Positive clones were in vivo excised to pBluescript SK(Ϫ) following the manufacturer's protocol.
Production of Polyclonal Antibodies for STAP-A synthetic NH 2terminal polypeptide of STAP, NH 2 -MEKVPGDMEIERRERNEEϩCys-COOH, was used as an immunogen. This peptide fragment (0.2 mg) was immunized in rabbits first with complete Freund's adjuvant and then twice with incomplete Freund's adjuvant. After the third immunization Proteins of these cells were extracted and analyzed by two-dimensional PAGE as detailed previously (8). One protein spot (arrow) with pI value of 6 and molecular mass of 21 kDa was heavily up-regulated in both in vivo and in vitro activated stellate cells. B, the amino acid sequence deduced from 120-bp cDNA obtained by degenerative PCR. C, STAP cDNA and its deduced amino acid sequence. STAP consisted of 190 amino acids producing molecular weight of 21,496. was finished, the rabbits were sacrificed, and the serum was harvested. The antibody was affinity-purified against the synthetic peptide. The antibody produced a single band at 21 kDa in Western blot analysis of stellate cell homogenates and recognized monospecifically recombinant rat STAP the preparation of which is described below.

Generation of Recombinant Rat STAP-
The open reading frame of cloned rat STAP cDNA was ligated into pTrcHis2 TOPO vectors (Invitrogen, Carlsbad, CA). His 6 -tagged STAP was generated in TOP10 cells cultured overnight in LB medium supplemented with 1 mM isopropyl-1-thio-␤-D-galactopyranoside (Wako Pure Chemical Co., Osaka, Japan). STAP was purified under native conditions using nickel-NTA agarose resins (Qiagen, Valencia, CA).
Identification of Recombinant Rat STAP as a Heme Protein-Absorption spectra of rat recombinant STAP dissolved in PBS at pH 7.4 were obtained at 25°C using a Hitachi spectrophotometer U-2001 (Hitachi, Tokyo) in the presence or absence of Na 2 S 2 O 4 . A peroxidase activity of recombinant STAP was measured as follows. Purified STAP was separated by 15% native PAGE and then transferred electrically onto Immobilon-P membranes. After washing with PBS twice, the membranes were treated with either 3,3Ј-diaminobenzidine (DAB)/hydrogen peroxide (H 2 O 2 ) solution or ECL chemiluminescence solution (Amersham Pharmacia Biotech). Peroxidase-dependent reaction products were detected either directly on the membranes or on the Kodak XAR5 films. The presence of protoheme in STAP was determined by the pyridine hemochrome method (21). In brief, the purified STAP was converted to a pyridine hemochrome by the addition of 0.5 ml of pyridine and 0.5 ml of 0.5 N NaOH solution to 4.0 ml of the protein solution. Absorption spectra of this mixture were scanned at 25°C using a Hitachi spectrophotometer U-2001 (Hitachi, Tokyo) in the presence or absence of Na 2 S 2 O 4 .
Peroxidase Activity-Catalase activity of STAP was determined spectrophotometrically by measuring the decrease of H 2 O 2 at 240 nm in 50 mM PBS buffer in the absence or presence of STAP (22). Fatty acid hydroperoxide peroxidase activity was determined according to a modified version of the Kharasch's method (23). Conjugated diene formation was initiated in the presence or absence of recombinant STAP by adding 30 mM FeCl 2 into a reaction mixture of 30 mM NaCl, 1 mM linoleic acid, 20 mM linoleic acid hydroperoxide, and 0.16% Lubrol PX, pH 7.0. The reaction was monitored at 234 nm.

Identification and cDNA Cloning of a Protein Heavily Upregulated in Activated Stellate
Cells-We investigated the overall protein expression pattern of quiescent, in vitro activated, and in vivo activated stellate cells to obtain a general insight into changes of the activation-associated proteins and to identify them (8). As depicted in Fig. 1A, one protein spot with a pI value of 6 and a molecular mass of 21 kDa was found to be remarkably up-regulated in both in vivo and in vitro activated stellate cells. The protein was digested with trypsin and the peptides produced were sequenced by quadrupole-time-offlight flight mass spectrometry. As a result two partial sequences were obtained: PGDME(I/L)ER and ANCEDVGVA. Neither was listed in SwissProt or GenBank TM , indicating that this was a novel protein. Therefore, we undertook cDNA cloning of the gene.
Degenerative PCR was performed using cDNA produced from total RNA of activated stellate cells as a template. Primer pairs were designed from the two obtained amino acid sequences: CCXGGXGAYTTYGARATHGA and GCXACXCCX-ACRTCYTC. The PCR amplified a single band at 120 bp (data not shown). DNA sequencing revealed that the deduced amino acid sequence actually contained the two above-mentioned pep-  ). B, quiescent, in vivo, and in vitro-activated stellate cells were prepared as detailed previously (8). The expression of STAP was determined at the protein (W.B.) and mRNA level (RT-PCR). Quiescent stellate cells were those freshly prepared from normal liver. In vivo activated stellate cells were those freshly prepared from fibrotic liver treated with TAA for 8 weeks. In vitro activated stellate cells were those prepared from normal liver and cultured for 7 days. C, twodimensional PAGE analysis of STAP expression in stellate cells, liver tissues (total liver), endothelial cells, Kupffer cells, and hepatocytes isolated from either normal control or fibrotic liver treated with TAA for 8 weeks. Normal stellate cells faintly expressed STAP (arrow) and its expression was up-regulated in the fibrotic liver. STAP was not seen in the normal total liver tissues but detectable in the fibrotic liver tissues. Other liver cells tested did not express STAP in both normal and fibrotic conditions. Arrows point to the positions where STAP could be seen if STAP is expressed. D, immunoelectron microscopic identification of STAP-expressing cells. Normal liver tissues were subjected to the immunoelectron microscopic observation. a, stellate cells (S) with lipid droplets showed the black-colored immunoreaction homogeneously in the cytoplasm. Endothelial cells (E) forming the sinusoidal wall were negative for STAP. b, Kupffer cells (K) in the sinusoidal lumen attaching to endothelial cells were negative for STAP. c, myofibroblasts (M) in a portal area were positive for STAP, but bile duct epithelial cells (Bi) and hepatocytes (H) were negative. Magnification, ϫ5,000. tide fragments (Fig. 1B). A rat-activated stellate cell cDNA library was screened using the 120-bp DNA as a probe, which yielded a 2028-bp-long cDNA clone. The open reading frame consisted of 570 bp that encoded 190 amino acids (Fig. 1C). The calculated molecular weight was 21,496. The amino acid sequence showed about 40% homology with myoglobin and indicated that this protein was a cytoplasmic protein according to the Reinhardt's criterion (24). We named this novel protein STAP after stellate cell activation-associated protein.
Expression of STAP in Stellate Cells and Other Hepatic Constituent Cells-Polyclonal antibodies against STAP were raised using its synthetic NH 2 -terminal 18 peptides. Western blot analysis with these antibodies revealed that STAP protein was time dependently induced in culture-stimulated stellate cells in a manner similar to the expression pattern of N-CAM (11), smooth muscle ␣-actin (12), and PDGF receptor-␤ (25) in contrast to the constant expression of glial fibrillary acidic protein ( Fig. 2A). The augmented STAP expression was also seen in in vivo activated stellate cells isolated from fibrotic liver induced by TAA administration for 8 weeks (Fig. 2B). RT-PCR showed that the expression of STAP mRNA was consistently augmented in the process of the stellate cell activation (Fig. 2, A  and B).
Two-dimensional PAGE analysis showed that neither endothelial cells, Kupffer cells, nor hepatocytes isolated from normal or TAA-induced fibrotic liver expressed detectable amounts of STAP (Fig. 2C). Immunoelectron microscopy not only supported these observations (Fig. 2D, a and b), but also revealed a negligible expression in bile duct epithelial cells and a significant expression in myofibroblasts located in the portal area (Fig. 2D, c).
Expression of STAP in Fibrotic Liver-The treatment of rats with TAA induces the liver fibrosis and finally cirrhosis (14). The parenchymal architecture underwent distinct changes, developing thick bundles at 8 weeks of the treatment (Fig. 3A, a  and d). Immunohistochemistry confirmed the previous report (4,11,12) that the expression of smooth muscle ␣-actin, a marker of activated stellate cells, was augmented in fibrotic liver particularly along the fibrotic septum (compare Fig. 3A, b  and e). STAP immunoreactivity was strong around the portal area and also weakly seen sporadically in the normal parenchyma (Fig. 3A, c). These weakly positive cells were identified as stellate cells by their locations and morphology. The reactivity was significantly increased particularly in the fibrotic septa in the TAA-treated animals (Fig. 3A, f). In situ hybridization revealed that STAP mRNA expression was augmented In normal liver, STAP expression was seen along the sinusoid and around portal areas. In A, c, an area enclosed by a square is enlarged 10 times and is presented in the inset, which shows positive staining of STAP in a stellate cell in intact parenchyma. In fibrotic liver, its expression was greatly augmented along the fibrotic septum as that of smooth muscle ␣-actin. g-j, in situ hybridization of STAP mRNA. g, hematoxylin-eosin staining; h, in situ hybridization of albumin mRNA; i and j, in situ hybridization of STAP mRNA using antisense probes (i) and sense probes (j) in serial sections. Note that STAP mRNA was expressed exclusively along the fibrotic septum (arrows) but not seen in the albumin mRNA expressing hepatocytes. k and l, STAP mRNA-expressing cells (k, arrows) were identified as stellate cells because these cells showed the positive signals in desmin immunostains (l, arrows). k and l were photographs obtained from the same section. Magnification, ϫ200 (a-j) and ϫ600 (k and l). B, expression of STAP protein and its mRNA in normal and fibrotic liver tissues induced by TAA administration for 4 and 8 weeks. Liver tissues obtained from two individual animals were tested for each group of experiments. Note that the expression of STAP protein (W.B.) and mRNA (RT-PCR) was increased as the treatment with TAA was prolonged.
in fibrotic liver exclusively along the septum but not in the albumin mRNA-expressing hepatocytes (Fig. 3A, g-j). A section of fibrotic liver tissues was used for in situ hybridization of STAP (Fig. 3A, k) and was subsequently subjected to immunohistochemistry of desmin, a stellate cell marker (Fig. 3A, l). These double staining experiments showed a specific expression of STAP in stellate cells in in vivo. Analyses using Western blot and RT-PCR confirmed that the expression of STAP protein and mRNA was augmented with increased severity of liver fibrosis (Fig. 3B).
Peroxidase Activity of STAP-His 6 -tagged recombinant rat STAP was purified under native conditions using a nickel-NTA agarose column. Aliquots of samples at each step of the purification were run on 15% SDS-PAGE gels as depicted in Fig. 4A. The eluted protein from the column made a single band at the predicted position of molecular weights and the solution was brown-colored. Absorption spectra of rat recombinant STAP under non-reducing conditions exhibited peaks at 415 and 531 nm (Fig. 4B). When the protein was suspended in a solution containing sodium dithionite, its absorption spectra exhibited peaks at 427, 531, and 560 (Fig. 4B), indicating that STAP was a heme protein (26). Characterization of STAP by the pyridine hemochrome method (21) indicated that protoheme was a component of STAP protein (data not shown). The presence of heme was further supported by the fact that the recombinant STAP showed a peroxidase activity as revealed by the development of brown color in DAB/H 2 O 2 reaction and chemiluminescence in the ECL reaction (Fig. 4C). The actual presence of peroxidase activity was determined by a H 2 O 2 degradation assay wherein the recombinant STAP was tested whether it has an H 2 O 2catabolizing activity. STAP was found to have 4.5 munits/mg of catalase activity. Furthermore, STAP was found to suppress conjugated diene formation in a dose-dependent manner when the protein was added in the fatty acid peroxidation reaction, the half-maximal inhibition occurring at ϳ50 nM STAP (Fig.  4D). These results conclusively demonstrated that STAP is a novel cytosolic protein with a peroxidase activity. DISCUSSION The activation of hepatic stellate cells is a key step for the development of liver fibrosis (1)(2)(3)(4)(5). Growth factors, such as PDGF, TGF-␣, and TGF-␤, and reactive oxygen species as well as lipid peroxides produced by intoxicated hepatocytes are thought to trigger the stellate cell activation (1)(2)(3)(4)(5). This activation is initiated by the activation of transcription factors, such as Sp-1 (27), Zf-9/KLF6 (6), and AP-1 (28), leading to the mRNA expression of extracellular matrix matrices and tissue inhibitor of matrix metalloproteinase-1 and -2 (29,30). A variety of factors are up-regulated in the activated stellate cells and thought to contribute to the development of fibrosis in a highly orchestrated manner, including receptors of growth factors such as PDGFR-␤ and insulin-like growth factor receptor type I (31), contractility related molecules such as smooth muscle ␣-actin, endothelin-1, and endothelin receptors (32,33), cell adhesion molecules such as intercellular adhesion molecule 1 and integlins (34), and cytokines such as monocyte chemotactic protein-1 (35) and interleukin-10 (36).
These stellate cell activation-related genes have been identified hitherto using subtractive hybridization (6, 7) and differential display (37). In addition to these conventional methods recent advancements of mass spectrometry have permitted us to analyze proteins in cells and organs on a large scale (9,10). Previously our proteome analysis of the stellate cell activation identified a total of 43 proteins/polypeptides that consistently altered their expression levels when the cells were activated (8). These included the up-regulated proteins such as galectin-1, calcyclin, and calgizzarin, and the down-regulated proteins such as liver carboxylesterase 10 and serine protease inhibitor 3 (8). Thus, protein level "differential display" using proteomics is evidently a powerful approach for displaying protein expression patterns in activated stellate cells.
In addition to the known proteins mentioned above, the proteome analysis performed in the present study revealed an unknown protein, dubbed STAP, which was highly up-regulated during the stellate cell activation. STAP represents a novel protein because it has little homology with existing entries in the protein data bases. According to the PSORT II protein analysis STAP has no endoplasmic reticulum retention motif, membrane retention signal, peroxisomal targeting signal, RNA-binding motif, actin-binding motif, DNA-binding motif, and ribosomal protein motif. STAP is a cytoplasmic protein according to the Reinhardt's criterion for cytoplasmic/nuclear discrimination (24). This is supported by the immunoelectron microscopic observations, which showed that STAP immunoreactivity is ubiquitously seen in the cytoplasm of stellate cells. A further proof hereof was the fact that STAP was only detected in cellular lysates and not in the medium wherein stellate cells had been cultured (data not shown).
During the preparation of this manuscript, a protein whose amino acid sequence is almost identical to that of rat STAP has been listed in the protein data base (NCBI accession number BAB31709). The corresponding cDNA was cloned from the brain of 13-day mouse embryo. The amino acid sequence of this protein (the mouse homologue of STAP) has a 97% identity to rat STAP. We have already cloned the cDNA of human STAP, which showed a 90% identity to rat STAP. 2 Immunohistochemistry in the present report showed that STAP expression is restricted to stellate cells in the liver parenchyma and myofibroblast-like cells around the portal vein in the normal liver. These cells are known to be able to express smooth muscle ␣-actin and to store vitamin A (38,39). In this context, STAP might be a suitable new marker for retinol metabolizing myofibroblasts.
Our spectrophotometric analysis of recombinant rat STAP clearly demonstrated that STAP is a heme protein. In general, heme proteins act as one of the following three: (i) oxygen transporters like hemoglobin and myoglobin, (ii) electron transporters like cytochrome P450, and (iii) enzymes with peroxidase activity like horseradish peroxidase. Although we have not proved it yet, STAP might be an intracellular oxygen transporter because this protein has a 40% amino acid sequence homology with myoglobin. This speculation is reasonable because the oxygen-dependent cellular contractility is augmented in activated stellate cells that are enriched in contractile proteins such as actin and myosin (1)(2)(3)(4)(5)12). This hypothesis is also supported because the induction kinetics of STAP after the treatment with a fibrosis inducer were similar to those of smooth muscle ␣-actin as shown in the present study.
From a functional point of view, the induction of STAP in activated stellate cells is understandable because stellate cells are increasingly exposed to endogenous H 2 O 2, lipid hydroperoxides and trans-4-hydroxy-2-nonenal during chronic liver trauma (36,40,41,43). Scavenging of radical-derived organic peroxides by STAP could be an adaptive reaction to normalize the cellular redox status during the cell activation. STAP as well as glutathione (43) could compensate the redox imbalance caused by a down-regulation of glutathione S-transferases in the activated stellate cells, which counteracts the toxic effects of lipid peroxidation (42).
From a clinical point of view, STAP could be a suitable marker for liver fibrosis and activated stellate cells in the histochemical study. However, STAP is not a secreted protein and, therefore, cannot be used as a serum marker. Our preliminary study reveled that STAP is evidently expressed in stellate cells and also in diseased liver tissues of human (data not shown).
In summary, we have isolated a novel protein named STAP from hepatic stellate cells using proteomics. STAP was expressed in stellate cells and myofibroblasts in the normal liver and is dramatically up-regulated in the course of the stellate cell activation at both mRNA and protein level. This suggests a close relationship between the STAP induction and the liver fibrosis. Functional analysis using a recombinant rat STAP revealed that this protein is a new heme protein with a peroxidase activity against H 2 O 2 and linoleic acid hydroperoxides.