Ascidians have the unusual physiological ability to accumulate high levels of vanadium and reduce it to the 3 oxidation state (VIII) in vanadocytes, the vanadium-containing blood cells. We are characterizing several polypeptides specific to vanadocytes that may participate in this. This study revealed that a 100-kDa antigen, recognized by a newly prepared monoclonal antibody, S8E4, is exclusively localized in vanadocytes, and identified the antigen as glycogen phosphorylase (EC 2.4.1.1) by sequencing the encoded cDNA. Since two enzymes, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44), both in the pentose phosphate pathway, have already been identified in vanadocytes, at least three enzymes involved in carbohydrate metabolism are localized in vanadocytes in huge amounts.
INTRODUCTION
Ascidian species, commonly known as tunicates, selectively accumulate high levels of vanadium in their blood cells (coelomic cells), especially ascidians belonging to the suborder Phlebobranchia. The highest recorded concentration of accumulated vanadium is 350 mM, which is 107 times its concentration in seawater (Michibata et al., 1991). Almost all of the vanadium ions accumulated are reduced to the +3 oxidation state (VIII) via the +4 oxidation state (VIV) in the blood cells (Hirata and Michibata, 1991). Studies of this phenomenon are summarized in two recent review articles (Michibata and Kanamori, 1998; Michibata et al., 1998).
Ascidian blood cells can be grouped into six categories on the basis of their morphology: hemoblasts, lymphocytes, leukocytes, vacuolated cells, pigment cells, and nephrocytes (Wright, 1981). The vacuolated cells can be further divided into at least four different types: morula cells, signet ring cells, compartment cells, and small compartment cells (Kaneko et al., 1995; Wuchiyama and Michibata, 1995). Of these, the signet ring cells have been identified as the vanadocytes, which contain high levels of vanadium, sulfate ions, and protons in their vacuoles and are thought to play a central role in the accumulation of vanadium (Michibata et al., 1987; Michibata et al., 1991; Kanamori and Michibata, 1994; Uyama et al., 1994). We are characterizing several polypeptides specific to vanadocytes that may participate in the accumulation and reduction of vanadium, with the ultimate goal of elucidating this unusual physiological function in ascidians. So far, large amounts of two different polypeptides, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44), both in the pentose phosphate pathway, have been found to be localized in vanadocytes (Uyama et al., 1998a, b).
In this experiment, we discovered that a 100-kDa antigen recognized by a newly prepared monoclonal antibody, S8E4, specific to vanadocytes, is glycogen phosphorylase (EC 2.4.1.1), an enzyme that catalyzes the phosphorolysis of glycogen to produce glucose 1-phosphate. The glucose 1-phosphate is interconverted into glucose 6-phosphate, the initial substrate in both the pentose phosphate and the Embden-Meyerhof pathways.
MATERIALS AND METHODS
Ascidians
Specimens of the vanadium-rich ascidian, Ascidia sydneiensis samea, were collected in the vicinity of the Asamushi Marine Biological Station of Tohoku University at Asamushi, Aomori Prefecture, and the Otsuchi Marine Research Center, Ocean Research Institute, the University of Tokyo, Otsuchi, Iwate Prefecture, Japan. The ascidians were maintained in an aquarium that contained circulating natural seawater at 18°C.
Preparation of monoclonal antibodies
Ascidian blood was centrifuged at 300 × g for 10 min to separate the blood cells from the serum. The blood cells were suspended in Ca2+- and Mg2+-free artificial seawater containing 0.2 M sucrose, 368 mM NaCl, 7.2 mM KCl, 26.4 mM Na2SO4, 4.8 mM NaHCO3, and 4 mM HEPES, at pH 7.0, to prevent clotting and were centrifuged at 100 × g for 10 min at 4°C. The centrifuged blood cells formed two layers. The upper layer, consisting of a subpopulation of giant cells predominantly, was discarded. The lower one, consisting of signet ring cells (vanadocytes), morula cells, compartment cells, and pigment cells, was used to prepare antigens.
An aliquot of 200 mg wet weight of blood cells was homogenized in 6 ml of 0.2 M Tris-HCl buffer (pH 8.0) containing protease inhibitors [leupeptin, pepstatin A, chymostatin, phenylmethylsulfonyl fluoride (PMSF), each at a concentration of 10 μg/ml], using a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 10,000 × g for 10 min. The supernatant was further centrifuged at 100,000 × g for 1 hr to obtain the microsome fraction. The precipitate was suspended in phosphate buffered saline (PBS), which consisted of 136.9 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.2. An aliquot of 0.5 ml of the suspension, which contained approximately 250 μg protein, was mixed with Freund's complete/incomplete adjuvant and injected intraperitoneally into female BALB/c mice. Monoclonal antibody was prepared in the same manner as described previously (Uyama et al., 1991).
Western blot analysis
In order to identify the antigen recognized by S8E4 monoclonal antibody, Western blot analysis was performed as described previously (Uyama et al., 1997, 1998b). In brief, samples containing approximately 30 μg protein were dissolved in a sample dissociation buffer solution consisting of 62.5 mM Tris-HCl at pH 6.8, 5% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, and 2.3% (w/v) SDS. The dissolved sample was electrophoresed by 10% uniform SDS-PAGE and subsequently subjected to Western blot analysis to detect the antigen recognized by the monoclonal antibody S8E4. The antigen-antibody reaction was visualized by ECL Western blotting detection system (Amersham Pharmacia Biotech, Uppsala, Sweden).
Immunoscreening the cDNA library
The cDNA library, prepared using the Uni-ZAP XR vector (Stratagene, La Jolla, CA, USA) as described previously (Uyama et al., 1998b), was screened using S8E4 monoclonal antibody as a probe. The one positive clone obtained was purified by two rounds of screening and subcloned by in vivo excision in accordance with the protocol provided by Stratagene. The resulting cDNA was inserted into the EcoRI-XhoI site of the plasmid vector pBluescript SK(−). The plasmid clone, which contained the 3 kbp-cDNA of the S8E4 antigen gene, was sequenced on both strands using the dideoxy chain-termination method with a ThermoSequenase Kit for the ALFexpress DNA sequencer (Amersham Pharmacia Biotech) using Cy5 labeled primers and resolving the samples on denaturing 6% polyacrylamide gels.
RESULTS
Immunological detection of S8E4
As shown in Fig. 1, the newly prepared monoclonal antibody, designated S8E4, specifically recognized signet ring cells, which are the so-called vanadocytes. No immunoreactivity was observed in blood cells other than the vanadocytes, although A. sydneiensis samea has about ten types of blood cells. After the homogenate was subjected to SDS-PAGE, many proteins were visualized with Coomassie Brilliant Blue staining (Fig. 2, lane 1). Of these proteins, Western blot analysis showed that S8E4 monoclonal antibody clearly recognized a 100-kDa band (Fig. 2, lane 2).
Fig. 1
Immunocytological detection of S8E4 monoclonal antibody in blood cells of the vanadium-rich ascidian, Ascidia sydneiensis samea. The blood cells shown in panels A and a were reacted with S8E4. The blood cells in panels B and b were reacted with nonimmune mouse serum as a negative control. The upper (A and B) and lower (a and b) panels were visualized by Nomarski differential-interference and fluorescence microscopy, respectively. Vanadocytes (signet ring cells) were exclusively recognized by S8E4 and fluoresced with FITC. No immunoreactivity was observed in the other types of blood cells. Morula cells faintly emitted autofluorescence. s, vanadocytes (signet ring cells). Scale bar indicates 10 μm.
Fig. 2
SDS-PAGE and Western blot analysis. Blood cells of A. sydneiensis samea were homogenized and separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue (lane 1). The separated proteins were blotted onto nitrocellulose paper and reacted with S8E4 monoclonal antibody (lane 2). A positive band corresponding to a 100-kDa protein was observed. Lane 1, homogenate of blood cells; Lane 2, Western blot analysis with S8E4.
cDNA cloning and sequence analysis
One cDNA clone was isolated as the result of screening a cDNA library prepared from ascidian blood cells for the gene encoding the 100-kDa antigen, using S8E4 monoclonal antibody as a probe. The insert, which contained a full-length cDNA designated asgp, was subcloned into the plasmid vector pBluescript SK(−) using the sequencing strategy shown in Fig. 3. Consequently, asgp was found to include 116 bp of the 5′ untranslated region, a 2,598 bp open reading frame (ORF), and 327 bp of the 3′ untranslated sequence, as shown in Fig. 4. The ORF encoded an 865 amino acid protein. A search of the SwissProt sequence database for proteins similar to asgp detected matches with glycogen phosphorylase (GP) for both the nucleotide and amino acid sequences. The match was closest for the amino acid sequence. The 865 amino acid protein shares 71.4%, 70.9%, and 69.6% identity with GP derived from the liver, brain, and muscle of the rat, respectively (Schiebel et al., 1992; Hudson et al., 1993). The calculated molecular mass of the predicted protein was 99 kDa, which correlated well with the expected molecular weight determined by Western blot analyses using S8E4 monoclonal antibody (Fig. 2). In addition, it was preliminarily revealed that S8E4 monoclonal antibody specifically recognized a recombinant 100-kDa asgp protein as well as the 100-kDa peptide from vanadocytes (data not shown).
Fig. 3
Subcloning and nucleotide sequencing strategy for the cDNA encoding the 100-kDa antigen recognized by S8E4 monoclonal antibody. A diagram illustrating the structure of a cDNA clone asgp encoding the 100-kDa cDNA. asgp cDNA (3 kbp) was inserted in pBluescript SK(−) between the EcoRI and XhoI sites. The solid line represents part of the vector. The open boxes represent non-coding regions. The closed box represents the coding region. The restriction enzymes were used to subclone asgp. Arrows indicate the extent and direction of the sequenced strands. bp, base pairs.
Fig. 4
Sequence alignments of the asgp gene encoding the 100-kDa antigen (A) and the deduced amino acids (B). The sequence of the functional cDNA isolated from clone asgp has an open reading frame (ORF) 2,598 nucleotides long-including the termination codon. The ORF extends from the first methionine codon at nucleotide 117 of the fragment to a TGA codon at nucleotide 2,714. The stop codon is indicated by an asterisk (A). Alignments between the amino acid sequence (AsGP) deduced from the nucleotide sequence of the asgp gene and those of GP derived from rat liver, brain, muscle, and the yeast Saccharomyces cerevisiae were compared. Amino acids that are identical in the four sequences are marked by an asterisk, while those that are similar are marked by a dot (B). The ORF encoded an 865 amino acid protein with 71.4% identity and 83.4% similarity to rat liver GP (Schiebel et al., 1992), while asgp and yeast GP only share 45.7% amino acid identity and 63.3% similarity (Hwang and Fletterick, 1986). RLGP, rat liver GP; RBGP, rat brain GP; RMGP, rat muscle GP; YGP, yeast GP.
DISCUSSION
In this study, an antigen specific to vanadocytes, recognized by the newly prepared S8E4 monoclonal antibody (Fig. 1), was revealed to be a 100-kDa peptide (Fig. 2). The predicted amino acid sequence of the cDNA clone asgp encoding the 100-kDa antigen shares approximately 70% identity with the amino acid sequence of GP derived from the rat (Fig. 4). These results demonstrate that the 100-kDa peptide from vanadocytes is GP, which is the third enzyme involved in carbohydrate metabolism to be found in vanadocytes. In addition to GP, massive amounts of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), which are both involved in the pentose phosphate pathway, are localized in vanadocytes (Uyama et al., 1998a, b).
Since almost all of the vanadium ions dissolved in the +5 oxidation state (VV) in seawater are reduced to VIII via VIV in ascidian vanadocytes (Hirata and Michibata, 1991), reducing agents must participate in the accumulation process. We have already proposed that the NADPH produced in the pentose phosphate pathway in vanadocytes reduces VV to VIV in the cytoplasm of the vanadocytes, for two reasons. First, VV is reduced to VIV by the addition of NAD(P)H in vitro (Liochev and Fridovich, 1990; Shi and Dalal, 1993). Second, massive amounts of two enzymes in the pentose phosphate pathway, the major supplier of reducing agents in the form of NADPH, are localized in vanadocytes (Uyama et al., 1998a, b). GP, newly identified to be localized in vanadocytes, is an enzyme that catalyzes the phosphorolysis of glycogen to produce glucose 1-phosphate. Glucose 1-phosphate is interconverted into glucose 6-phosphate, which is the initial substrate in both the pentose phosphate and Embden-Meyerhof pathways. Therefore, the discovery of GP provides strong additional evidence for the participation of the pentose phosphate pathway in the reduction of vanadium accompanying the accumulation of vanadium in vanadocytes. It may be safely said that the abundant expression of enzymes in the pentose phosphate pathway reflects a requirement for the NADPH produced in the pathway.
The amino acid sequence of the asgp gene encoding the 100-kDa antigen is highly homologous with that of GP derived from the rat (Fig. 4). Although the amino acid sequence was highly conserved, the asgp gene exhibits a remarkable divergence in G + C content. For some unaccountable reason, only 48.4% of the nucleotides at the third codon position in the ORF of asgp are deoxyguanosine or deoxycytidine residues, while approximately 70% are deoxyguanosine or deoxycytidine residues in the rat GP sequence (Schiebel et al., 1992; Hudson et al., 1993).
Acknowledgments
The authors express their heartfelt thanks to Mr. M. Washio and Mr. S. Tamura of the Asamushi Marine Biological Station of Tohoku University, Aomori Prefecture and Mr. K. Morita of the Otsuchi Marine Research Center, Ocean Research Institute of the University of Tokyo, Iwate Prefecture, Japan. Thanks are also due to Mr. N. Abo in our laboratory, who collected some of the animals and kept them healthy in an aquarium. This work was partially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (#09440278, #09874178 and #09839017).
