316
Biochimica et Biophysica A cta, 1087 (1990) 316-322 Elsevier
BBAEXP 92173
Characterization of liver-specific expression of rat uricase using monoclonal antibodies and cloned cDNAs Kiyoto Motojima and Sataro Goto Department of Biochemistry, School of Pharmaceutical Sciences, Toho University, Funabashi, Chiba (Japan) (Received 12 June 1990)
Key words: Uricase; Monoclonal antibody; Peroxisome; Liver-specific gene expression; (Hepatocyte); (Rat liver)
Tissue distribution of uricase (urate oxidase, EC 1.7.3.3) was studied by immunobiotting and RNA slot blot analysis. For immunoblotting, highly specific monoclonal antibodies against rat liver uricase were obtained, and for mRNA detection, a cloned uricase cDNA was used. Among seven tissues studied, uricase was immunologically detected only in the liver. The contents of uricase in other tissues, i.e., brain, thymus, heart, spleen, kidney and lactating mammary gland, were estimated to be less than 2% of that in the liver. Uricase mRNA was also detected only in the liver. The steady-state level of the mRNA in the isolated hepatocytes was relatively constant during the 8-day culture period when compared with those of other mRNAs expressed in the liver, suggesting a unique control mechanism of its expression.
Introduction Uricase (urate oxidase, EC 1.7.3.3) is a copper-containing oxidase responsible for the hydrolysis of uric acid to allantoin in the purine degradation pathway. With phylogenetic evolution, some of the enzymes in the pathway have been lost and the extent of degradation differs among species [1]. In contrast to most mammals, human and some primates lack uricase and excrete uric acid as the end product of purine degradation [2]. Birds, some reptiles and most insects also lack uricase and this varied distribution among species cannot be explained by the phylogenetic tree alone. In rat liver, uricase is localized in the peroxisome and is associated with large electron-dense paracrystalline cores [3]. The cores are observed in some, though not all of the peroxisomes in the liver only [4]. To our knowledge, however, detailed studies on the cores have been carried out only for rat liver. Neither absence of the cores in extra-hepatic tissues nor identity of the uricase protein complex with the core has been established, while uricase has been purified not only from rat liver [5] but also from bovine kidney [6]. To determine the tissue specificity of uricase in rat, we produced monoclonal antibodies against the uricase
Correspondence: K. Motojima, Department of Biochemistry, School of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274, Japan.
and immunoblot analyses of various tissue samples were performed. We also characterized the tissue specificity by determining the steady-state levels of the m R N A using a uricase cDNA [4,7] in various tissues and in cultured hepatocytes with several control cDNAs for liver-specific and common mRNAs.
Materials and Methods Materials Materials were obtained from the following sources: Alkaline phosphatase-conjugated goat anti-mouse F(ab')2 was purchased from Helix Biotech (Vancouver, Canada). Affinity purified subclass-specific rabbit antimouse immunoglobulins were from Tago (Burlingame, CA). DNA polymerase I and other enzymes were obtained from Takara Shuzo (Kyoto, Japan). [a-32p]dCTP (700 Ci/mmol) was a product of ICN (Irwin, CA). 5-Bromo-4-chloro-3-indolyl phosphate, p-toluidine salt was purchased from Sigma (St. Louis, MO). Nitrocellulose filters were from Schreicher and Schuell (Dassel, F.R.G.) and X-ray films were from Eastman Kodak (Rochester, NY). Purification of rat liver uricase The uricase was purified from the lysosome-rich fraction obtained from the liver of rats (Wistar, male) treated with Triton WR-1339 as described [8]. After aspirating off the lysosome fraction, the remaining fraction on the 40% sucrose cushion was diluted 4-fold with
n167/4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
317 ice-cold PBS and Triton X-100 was added to a final concentration of 1% (v/v). The suspension was left for 10 min on ice and centrifuged at 10 000 rpm for 10 min. The pellet was washed with PBS containing 1% Triton X-100 and then with PBS and suspended in PBS by sonication.
Amino acid sequence analysis For amino acid sequence analysis, the sample was solubilized with 6 M guanidine hydrochroride, reduced and then carboxymethylated. No PTH-amino acid was detected from the non-digested sample by sequence analysis using automated Edman-degradation with 470A gas-phase sequencer and PTH-analyzer (Applied Biosystem, Foster, CA, U.S.A.). N-terminal amino acid sequence was determined by chymotrypsin digestion of the lysylendopeptidase digested peptide which did not release PTH-amino acid, followed by carboxypeptidase Y treatment and amino acid analysis. Production of monoclonal antibodies Female Balb/c mice were immunized with 20/~g of the purified uricase in complete Freund's adjuvant twice a month by intraperitoneal injection. 3 days prior to fusion of spleen cells to NS-1 cells, mice were injected with the enzyme without adjuvant. The procedure for fusion has been described previously [9]. Hybrid clones were screened by enzyme immunoassay using alkaline phosphatase-conjugated anti-mouse immunoglobulins. Positive hybrids were cloned twice by limiting dilution on fibrin gels. After cloning, the hybridomas were grown and the supernatant fluids were harvested and used as monoclonal antibody preparations. The subclass specificity of monoclonal antibodies was determined by enzyme immunoassay using a microtiter plate coated with affinity purified, subclass-specific rabbit antimouse immunoglobulins and alkaline phosphatase-conjugated anti-whole mouse imrnunoglobulins, followed by enzyme reaction. SDS polyacrylamide gel electrophoresis and immunoblot analysis For immunoblotting, proteins were electrophoresed on 10% acrylamide-SDS gel as described [10], transferred to nitrocellulose in 6 M urea [11], incubated with anti-uricase antibodies and then with alkaline phosphatase-conjugated second antibodies. The phosphatase activity was then visualized as described [12]. Proteins were stained by the method of Fairbanks et al. [13]. Protein was determined by a Bio-Rad protein assay kit. In preparing the postnuclear supernatant fractions, the various tissues were minced well with scissors, homogenized in 3 vol. of sucrose buffer (0.25 M sucrose/1 mM EDTA/0.1% ethanol) by a Potter-Elvehjem homogenizer, and the homogenates were centrifuged for 5 min at 340 x g.
Isolation and culture of rat hepatocytes Parenchymal hepatocytes were isolated from adult male Fischer 344 rats by perfusion of the liver in situ with collagenase as described [14]. The isolated cells were plated in 60 mm plastic dishes at a concentration of 2.106 cells/dish and cultured in medium 199 supplemented with 10% calf serum, 1 ~tM insulin and 10/~M dexamethasone. The medium was changed 2 h after cell inoculation to wash out the cells which did not adhere to the dishes. The culture was continued for 8 days. RNA slot blot analysis mRNA levels in hepatocytes cultured for 0, 1, 3, 6 and 8 days were analyzed by a total extract dot blot hybridization procedure as published [15] using a slot blot device (Milli Blot System, Millipore). Homogenization was carried out in Buffer A containing 7.5 M guanidine hydrochloride, 0.1% sarkosyl, 0.1 M 2-mercaptoethanol and 50 mM sodium citrate (pH 7) using Polytron (Kinematica, Switzerland) and the amounts of the extracts to load onto nitrocellulose filters were normalized by the DNA contents. For DNA estimation, the Hoechst 33258 dye method [16] was employed using calf thymus DNA as a standard. The extracts were diluted in a solution of 2.5 M formaldehyde in 6 x SSC and heated at 65°C for 15 min. After cooling on ice, they were centrifuged at 12000 rpm for 10 rain to remove the denatured proteins and some DNA which otherwise may cause non-specific hybridization. The supernatant was applied on the nitrocellulose filter and the filter was baked at 65 °C in vacuo for 2 h. cDNA probes and hybridization conditions The cDNA probes were labeled by nick translation [17]. Prehybridization and hybridization were carried out at 42°C in the presence of 50% formamide in a mixture of 5 x Denhardt's solution, 5 x SSPE, 0.1% SDS and 100 /tg/ml denatured salmon sperm DNA [17]. Filters were washed with 2 x SSC, 0.1% SDS and finally with 0.1 x SSC, 0.1% SDS at 50°C, followed by autoradiography at - 8 0 ° C with an intensifying screen. Autoradiograms were quantified by densitometry using a Helena Auto Scanner (Texas, USA). cDNA clones for rat uricase [5], albumin and transthyretin [18] were isolated in our laboratory. Mouse apo E, rat metallothionein and /3-actin clones were obtained from C. Heinzman (UCLA, USA), R.D. Andersen (UCLA, USA) and M. Obinata (Tohoku University, Sendai, Japan), respectively. Results
Purification of rat liver uricase Treatment of the lysosome-rich fraction with 1% Triton X-100 solubilized most of the proteins and about 50% of the activity of the uricase was recovered in the
318
1
2
3
4
5
6
A
B
1 2 3 4 5 ~ 200
K
-.=116
K
"=
~
!
C 3 4
1 2 3 4
93K
"1 6 6 K
~
45K
~
31
K
22K
Fig. 1. SDS-PAGE analysis of the purified rat liver uricase. Lane 1: rat liver 'lysosome-rich' fraction (30 #g of protein); Lanes 2-4: the purified uricase of different preparations (3-5 pg of protein); Lanes 5 and 6: molecular weight standards (Bio-Rad).
A
12
Fig. 2. lmmunoblot analyses of the specificities of the monoclonal antibodies against rat uricase. (A) Coomassie blue stained SDS-PAGE pattern of the purified uricase (lanes 1 and 2; 1 /xg and 10 #g of protein), the 'lysosome-rich' fraction (lanes 3 and 4; 50 #g and 500 #g), and molecular weight standards (lane 5). (B) and (C) Immunoblotting of the triplicate sets of gels with monoclonal antibodies 22 (B) and 46 (C).
pellet by centrifugation with an approx. 120-fold increase in purification. The effect was not specific to Triton X-100 but 1% of NP40 or octylglucoside gave a
B
lb-
Fig. 3. Immunoblot analysis of the tissue distribution of the uricase. (A) Coomassie blue-stained SDS-PAGE pattern of the proteins in the post-nuclear fractions of adrenal gland (AG), brain (B), heart (H), kidney (K), liver (L), lactating mammary gland (MG), spleen (S) and thymus (T). The purified uricase (0.2 ~g, URI) and molecular weight standards (MW) are also shown. (B) Immunoblotting of the duplicated gel with monoclonal antibodies No. 22. Arrowheads indicate the position of the uricase.
A1
similar result, while Tween 20 was less effective. Starting from 200 mg protein of the lysosome-rich fraction, 0.9 mg of the uricase was obtained by a single step. The purity was estimated to be about 90% by densitometry of the Coomassie blue-stained gel of SDS-polyacrylamide gel electrophoresis (Fig. 1). The amino acid sequence analysis of this preparation gave the exact Nterminal sequence of the uricase [5], suggesting its near homogeneity. Monoclonal antibodies and their specificities
Nine clones in the four 96-well microtiter plates survived to yield stable cultures. They were positive for antibody activities against the uricase as judged by enzyme immunoassay. Their specificities were further examined by immunoblotting; four of them predominantly recognized the uricase; all of them were in the IgM class. Among them, two (22 and 46) gave strong signals and the results of immunoblotting are shown in Fig. 2. They were highly specific to uricase and cross-reacted little with other proteins in the light mitochondria fraction of the rat liver. The difference in the faint band patterns other than the main band corresponding to uricase suggested that each monoclonal antibody recognized a different epitope on uricase.
2
3
B
1
2
I 1.1
C
D
E
Immunoblot detection of uricase in various tissues
The relative amount of the uricase in brain, thymus, heart, spleen, kidney, liver, or lactating mammary gland of a female rat was examined by immunoblotting using postnuclear fractions of each tissue and a specific monoclonal antibody (22). As shown in Fig. 3, the uricase was detected only in the liver. Essentially the same results were obtained with another monoclonal antibody (46) and with polyclonal anti-uricase antibodies (not shown). Prolonged incubation of the sheet developed a very faint band of molecular weight of about 34000 on the lane of the mammary gland sample (not shown), but other faint bands also appeared and they have not been identified. The content of uricase in extrahepatic tissues was, at any rate, less than 2% of that in the liver. Uricase m R N A levels in various tissues
The mRNA levels of uricase in various tissues were measured by slot blot analyses together with the levels of albumin, transthyretin, apo E and fl-actin mRNA as controls. The results of autoradiography are shown in Fig. 4. The amounts of apo E mRNA in various tissues (Fig. 4, C) were quantified by densitometry of the film. The intensities of the images were in linear relationship to the amounts of extracts applied to the filter in every tissue (not shown). The relative amounts of apo E mRNA in the liver, brain, kidney, heart, lung, spleen and thymus were 100, 37, 24, 22, 13, 6 and 5, respec-
Fig. 4. Slot blot analysis of the tissue distribution of the uricase mRNA. Uricase (A), albumin (B), apo E (C), fl-actin (D), and transthyretin (E) mRNA levels in various tissues are analyzed using total extracts. Total extracts were normalized by DNA contents and columns 1-3 show extracts containing 0.4, 0.2, and 0.1 #g DNA. Columns B - L G show extracts from brain (B), heart (H), thymus (T), spleen (S), liver (L), kidney (K) and lung (LG), respectively.
tively. This result was good consistent with the published data [19]. Albumin mRNA was detected as highly specific and abundant in the liver and transthyretin mRNA in both the liver and the brain as previously reported [20,21]. Under these conditions where control mRNA levels in various tissues were accurately quantitated, the uricase mRNA was detected in the liver. The signals were weak but other faint signals in extrahepatic tissues were understood to be non-specific, because signal patterns of uricase and albumin mRNAs in extrahepatic tissues appearing after long exposure were almost indistinguishable (Fig. 4, B). These were prob-
320 ably caused analyses, in only in the extrahepatic
by DNAs in total conclusion, uricase fiver and the level tissues was less than
extracts. By slot blot mRNA was detected of uricase mRNA in 5% of that in the fiver.
Changes in the levels of uricase and other mRNAs in cultured hepatocytes To characterize liver-specific expression of uricase mRNA, the changes in the steady-state levels of the mRNA expressed in cultured hepatocytes over the period of a few days were compared with those of mRNAs for liver specific and common genes by quantitative slot blot analysis. The results of slot blot autoradiographs are shown in Fig. 5. Under the culture conditions employed, hepatocyte morphology was gradually altered (not shown) and the steady-state levels of three liver-specific mRNAs dropped to various extents. These decreases were not due to general functional deterioration, because the levels of some other mRNAs were maintained at higher levels than those at the start of culture. The level of metallothionein mRNA was increased about 7-fold of the start level after 3-day culture, probably because of the induction by relatively high level of dexamethasone into the culture medium [22]. Elevation of the level of actin mRNA (3-5-fold) during the culture period was well consistent with the previous observation by Isom et al. [23]. However, the rates of decrease in levels of the fiver-specific mRNAs differed among the three: that of albumin mRNA dropped rapidly after 3-day culture to about 5% of the start level and that of transthyretin mRNA to about 50%, but the level of uricase m R N A was maintained almost constant at the start level. It has not yet been
A
B
1 2312
C 3
determined whether the differences were in the rate of transcription a n d / o r in their stabilities, but it is evident that the •er-specific expression of uricase is uniquely controlled. Discussion The method employed to purify rat liver uricase was very similar to that used for the isolation of the peroxisomal core [24]. SDS-PAGE analysis of proteins in this fraction showed one major band of uricase (Fig. 1) and amino acid sequence analyses of this total fraction showed that the N-terminus was blocked alanine followed by the exact sequence of uricase [5], suggesting that the major portion, if not all, of the protein in the core is uricase; whether there are any other components is not known. Liver-specific expression of uricase has been demonstrated using specific monoclonal antibodies against uricase and a uricase c D N A with various control cDNAs for liver-specific or common mRNAs. Although our antibodies against uricase is highly specific, our present results do not exclude the possibility that uricase is also expressed in extrahepatic tissues but the levels are so low as to be undetectable by immunoblotting. The results of slot blot analysis of the m R N A levels using total extracts were consistent with the above conclusion. The method employed could not detect very low levels of specific mRNA sequences as compared with Northern blot using poly(A) RNA, and further detailed analyses are necessary to determine whether uricase mRNA is expressed solely or predominantly in the liver. However, in slot blot analysis using total extracts, the
D
1 23123
E 1231
F 23
0 "O "O-1 O ~D
_.-6 u
8 Fig. 5. Slot blot analysis of the changes in the steady-state levels of various m R N A s during a culture period in isolated rat hepatocytes. The steady-state levels of albumin (A), transthyretin (B), uricase (C), metallothionein (D), fl-actin (E) and apo E (F) m R N A were measured using the total extracts prepared from the hepatocytes at the start of the culture, 1 day, 3 days, 6 days, and 8 days after the start of the culture. C o l u m n s 1 - 3 show extracts containing 0.4, 0.2 and 0.1 ~g DNA, respectively.
321 samples were loaded according to DNA content and the signals of hybridization should be proportional to the amounts the mRNA per cell. Determination of DNA concentration by the Hoechst 33258 dye method will be more specific and accurate than photometric determination of the RNA content in the samples which may contain interfering materials and different levels of rRNA. The procedure was well suited to the analysis of various mRNA levels in multiple small tissue samples and especially in primary cultured hepatocytes. Usuda et al. [4] and Reddy et al. [26] reported liver-specific expression of uricase in rat using polyclonal antibodies, and a cloned cDNA for rat uricase, respectively. Thus, our studies, which have been completed before their works' appearance, confirmed their conclusion using highly specific monoclonal antibodies and a cloned cDNA with various control cDNAs. On the contrary, uricase is also found in the kidney of other species [4,6]. The unique tissue-specificity of rat uricase will be related to the structure of the promoter of the gene as suggested by cDNA [5] and genomic [27] cloning and comparison with cDNAs of other species [7,28]. The N-terminal structure of rat uricase is different from those of other species and this was probably produced by insertion of an intron [28]. The different orgaization of the rat uricase gene around the first exon may be related to the unique transcriptional regulation of the rat gene. To characterize liver-specific expression of uricase in rat, we compared the steady-state levels of its mRNA with those of other liver-specific and common mRNAs in primary cultured rat hepatocytes. The levels of uricase mRNA were largely maintained during the 8-day culture of hepatocytes while those of other liver-specific mRNAs, especially that of albumin mRNA dropped remarkably. In serum-supplemented standard tissue culture medium, which we used, it was reported that the levels of liver-specific mRNAs drop rapidly as the result of a decrease in the rates of transcription [29]. Thus, the steady-state level of uricase mRNA was suggested to be maintained by the unchanged rate of transcription, but our present study does not exclude the possibility that the level of uricase mRNA was maintained by its unusual stability in hepatocytes. Furthermore, Isom et al. [23] suggested that the stabilities of some liver-specific mRNAs are different between in the liver and in the cultured hepatocytes. Further study to determine whether the differences are in the rate of transcription a n d / o r in their stabilities is clearly necessary and should be carried out very carefully. Though the level of regulation was not evident from this study and further study is clearly necessary, our results do suggest that the mechanism of liver-specific expression of uricase is unique and that the steady-state level of the uricase mRNA is regulated differently from those of other typical liver-specific mRNAs.
Acknowledgments We acknowledge Professor T. Takano (Teikyo University, Kanagawa, Japan), in whose laboratory a part of this work was done and whose continuous enthusiasm and helpful advice were greatly appriciated. We are grateful to Drs. C. Heinzman (UCLA, USA), R.D. Andersen (UCLA, USA) and M. Obinata (Tohoku University, Sendai, Japan) for their generous gifts of mouse apoE, rat metallothionein, and fl-actin done, respectively. Thanks are also due to Dr. A. Ishigami for isolation of rat hepatocytes, to Dr. S. Kanaya (Mitsubishi Chemical Industries Ltd.) for amino acid sequence analysis, and to Miss N. Harada for her help in slot blot analyses.
References 1 Lehninger, A.L. (1975) in Biochemistry, 2nd Edn., Worth, New York. 2 Christen, P., Peacock, W.C., Christen, A.E. and Wacker, W.E. (1970) Eur. J. Biochem. 12, 3-5. 3 Tsukada, H., Mochizuki, Y. and Fujiwara, S. (1966) J. Cell Biol. 28, 449-460. 4 Usuda, N., Reddy, M.K., Hashimoto, T., Rao, M.S. and Reddy, J.K. (1988) Lab. Invest. 58, 100-111. 5 Motojima, K., Kanaya, S. and Goto, S. (1988) J. Biol. Chem. 263, 16677-16681. 6 Mahler, H.R., Hubscher, G. and Baum, H. (1955) J. Biol. Chem. 216, 625. 7 Motojima, K. and Goto, S. (1989) Biochim. Biophys. Acta 1008, 116-118. 8 Ohkuma, S., Moriyama, Y. and Takano, T. (1982) Proc. Natl. Acad. Sci. USA 79, 2758-2762. 9 Kimura, K., Nakagami, K., Amanuma, K., Ohkuma, S., Yoshida, Y. and Takano, T. (1986) Virchows Arch. A 410, 159-164. 10 Laemmli, U.K. (1970) Nature 227, 680-685. 11 EMBO, SKMB Course 1980, Barsel in Hybridoma Techniques, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 12 Knecht, D. and Diamond, R.L. (1984) Anal. Biochem. 136, 180184. 13 Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606-2617. 14 Seglen, P.O. (1976) Methods Cell Biol. 13, 29-83. 15 Grimes, A., McArdle, H.J. and Mercer, J.F.B. (1988) Anal. Biochem. 172, 436-443. 16 Labarca, C. and Paigen, K. (1980) Anal. Biochem. 102, 344-352. 17 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 18 Motojima, K. and Goto, S. (1989) FEBS Lett. 258, 103-105. 19 Elshourbagy, N.A., Liao, W.S., Mahley, R.W. and Tayler, J.M. (1985) Proc. Natl. Acad. Sci. USA 82, 203-207. 20 Sargent, T.D., Yang, M. and Bonner, J. (1981) Proc. Natl. Acad. Sci. USA 78, 243-246. 21 Dickson, P.W., Aldred, A.R., Marley, P.D., Guo-Fen, T., Howlen, G.J. and Schreiber, G. (1985) Biochem. Biophys. Res. Commun. 127, 890-895. 22 Andersen, R.D., Birren, B.W., Ganz, T., Piletz, J.E. and Herschman, H.R. (1982) DNA 2, 15-22. 23 Isom, H., Georgoff, I., Salditt-Georgoff, M. and Darnell, Jr., J.E. (1987) J. Cell Biol. 105, 2877-2885.
322 24 Fujiwara, S., Ohashi, H. and Noguchi, T. (1987) Comp. Biochem. Physiol. 86B, 23-26. 25 Gould, S.J., Keller, G-A. and Subramani, S. (1988) J. Cell Biol. 107, 897-905. 26 Reddy, P.C., Nemall, M.R., Reddy, M.K., Reddy, M.N., Yuan, P.M., Yuen, S., LaMer, T.G., Shiroza, T., Kuramitsu, H.K., Usuda, N., Chisholm, R.L., Rao, M.S. and Reddy, J.K. (1988) Proc. Natl. Acad. Sci. USA 85, 9081-9085.
27 Motojima, K. and Goto, S. (1990) FEBS Lett. 264, 156-158. 28 Nguyen, T., Zelechowska, M., Foster, V., Bergmann, H., and Verma, D.P. (1985) Proc. Natl. Acad. Sci. USA 82, 5040-5044. 29 Clayton, D.F. and Darnell, Jr., J.E. (1983) Mol. Cell. Biol. 3, 1552-1561.
Biochimica et Biophysica A cta, 1087 (1990) 316-322 Elsevier
BBAEXP 92173
Characterization of liver-specific expression of rat uricase using monoclonal antibodies and cloned cDNAs Kiyoto Motojima and Sataro Goto Department of Biochemistry, School of Pharmaceutical Sciences, Toho University, Funabashi, Chiba (Japan) (Received 12 June 1990)
Key words: Uricase; Monoclonal antibody; Peroxisome; Liver-specific gene expression; (Hepatocyte); (Rat liver)
Tissue distribution of uricase (urate oxidase, EC 1.7.3.3) was studied by immunobiotting and RNA slot blot analysis. For immunoblotting, highly specific monoclonal antibodies against rat liver uricase were obtained, and for mRNA detection, a cloned uricase cDNA was used. Among seven tissues studied, uricase was immunologically detected only in the liver. The contents of uricase in other tissues, i.e., brain, thymus, heart, spleen, kidney and lactating mammary gland, were estimated to be less than 2% of that in the liver. Uricase mRNA was also detected only in the liver. The steady-state level of the mRNA in the isolated hepatocytes was relatively constant during the 8-day culture period when compared with those of other mRNAs expressed in the liver, suggesting a unique control mechanism of its expression.
Introduction Uricase (urate oxidase, EC 1.7.3.3) is a copper-containing oxidase responsible for the hydrolysis of uric acid to allantoin in the purine degradation pathway. With phylogenetic evolution, some of the enzymes in the pathway have been lost and the extent of degradation differs among species [1]. In contrast to most mammals, human and some primates lack uricase and excrete uric acid as the end product of purine degradation [2]. Birds, some reptiles and most insects also lack uricase and this varied distribution among species cannot be explained by the phylogenetic tree alone. In rat liver, uricase is localized in the peroxisome and is associated with large electron-dense paracrystalline cores [3]. The cores are observed in some, though not all of the peroxisomes in the liver only [4]. To our knowledge, however, detailed studies on the cores have been carried out only for rat liver. Neither absence of the cores in extra-hepatic tissues nor identity of the uricase protein complex with the core has been established, while uricase has been purified not only from rat liver [5] but also from bovine kidney [6]. To determine the tissue specificity of uricase in rat, we produced monoclonal antibodies against the uricase
Correspondence: K. Motojima, Department of Biochemistry, School of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274, Japan.
and immunoblot analyses of various tissue samples were performed. We also characterized the tissue specificity by determining the steady-state levels of the m R N A using a uricase cDNA [4,7] in various tissues and in cultured hepatocytes with several control cDNAs for liver-specific and common mRNAs.
Materials and Methods Materials Materials were obtained from the following sources: Alkaline phosphatase-conjugated goat anti-mouse F(ab')2 was purchased from Helix Biotech (Vancouver, Canada). Affinity purified subclass-specific rabbit antimouse immunoglobulins were from Tago (Burlingame, CA). DNA polymerase I and other enzymes were obtained from Takara Shuzo (Kyoto, Japan). [a-32p]dCTP (700 Ci/mmol) was a product of ICN (Irwin, CA). 5-Bromo-4-chloro-3-indolyl phosphate, p-toluidine salt was purchased from Sigma (St. Louis, MO). Nitrocellulose filters were from Schreicher and Schuell (Dassel, F.R.G.) and X-ray films were from Eastman Kodak (Rochester, NY). Purification of rat liver uricase The uricase was purified from the lysosome-rich fraction obtained from the liver of rats (Wistar, male) treated with Triton WR-1339 as described [8]. After aspirating off the lysosome fraction, the remaining fraction on the 40% sucrose cushion was diluted 4-fold with
n167/4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
317 ice-cold PBS and Triton X-100 was added to a final concentration of 1% (v/v). The suspension was left for 10 min on ice and centrifuged at 10 000 rpm for 10 min. The pellet was washed with PBS containing 1% Triton X-100 and then with PBS and suspended in PBS by sonication.
Amino acid sequence analysis For amino acid sequence analysis, the sample was solubilized with 6 M guanidine hydrochroride, reduced and then carboxymethylated. No PTH-amino acid was detected from the non-digested sample by sequence analysis using automated Edman-degradation with 470A gas-phase sequencer and PTH-analyzer (Applied Biosystem, Foster, CA, U.S.A.). N-terminal amino acid sequence was determined by chymotrypsin digestion of the lysylendopeptidase digested peptide which did not release PTH-amino acid, followed by carboxypeptidase Y treatment and amino acid analysis. Production of monoclonal antibodies Female Balb/c mice were immunized with 20/~g of the purified uricase in complete Freund's adjuvant twice a month by intraperitoneal injection. 3 days prior to fusion of spleen cells to NS-1 cells, mice were injected with the enzyme without adjuvant. The procedure for fusion has been described previously [9]. Hybrid clones were screened by enzyme immunoassay using alkaline phosphatase-conjugated anti-mouse immunoglobulins. Positive hybrids were cloned twice by limiting dilution on fibrin gels. After cloning, the hybridomas were grown and the supernatant fluids were harvested and used as monoclonal antibody preparations. The subclass specificity of monoclonal antibodies was determined by enzyme immunoassay using a microtiter plate coated with affinity purified, subclass-specific rabbit antimouse immunoglobulins and alkaline phosphatase-conjugated anti-whole mouse imrnunoglobulins, followed by enzyme reaction. SDS polyacrylamide gel electrophoresis and immunoblot analysis For immunoblotting, proteins were electrophoresed on 10% acrylamide-SDS gel as described [10], transferred to nitrocellulose in 6 M urea [11], incubated with anti-uricase antibodies and then with alkaline phosphatase-conjugated second antibodies. The phosphatase activity was then visualized as described [12]. Proteins were stained by the method of Fairbanks et al. [13]. Protein was determined by a Bio-Rad protein assay kit. In preparing the postnuclear supernatant fractions, the various tissues were minced well with scissors, homogenized in 3 vol. of sucrose buffer (0.25 M sucrose/1 mM EDTA/0.1% ethanol) by a Potter-Elvehjem homogenizer, and the homogenates were centrifuged for 5 min at 340 x g.
Isolation and culture of rat hepatocytes Parenchymal hepatocytes were isolated from adult male Fischer 344 rats by perfusion of the liver in situ with collagenase as described [14]. The isolated cells were plated in 60 mm plastic dishes at a concentration of 2.106 cells/dish and cultured in medium 199 supplemented with 10% calf serum, 1 ~tM insulin and 10/~M dexamethasone. The medium was changed 2 h after cell inoculation to wash out the cells which did not adhere to the dishes. The culture was continued for 8 days. RNA slot blot analysis mRNA levels in hepatocytes cultured for 0, 1, 3, 6 and 8 days were analyzed by a total extract dot blot hybridization procedure as published [15] using a slot blot device (Milli Blot System, Millipore). Homogenization was carried out in Buffer A containing 7.5 M guanidine hydrochloride, 0.1% sarkosyl, 0.1 M 2-mercaptoethanol and 50 mM sodium citrate (pH 7) using Polytron (Kinematica, Switzerland) and the amounts of the extracts to load onto nitrocellulose filters were normalized by the DNA contents. For DNA estimation, the Hoechst 33258 dye method [16] was employed using calf thymus DNA as a standard. The extracts were diluted in a solution of 2.5 M formaldehyde in 6 x SSC and heated at 65°C for 15 min. After cooling on ice, they were centrifuged at 12000 rpm for 10 rain to remove the denatured proteins and some DNA which otherwise may cause non-specific hybridization. The supernatant was applied on the nitrocellulose filter and the filter was baked at 65 °C in vacuo for 2 h. cDNA probes and hybridization conditions The cDNA probes were labeled by nick translation [17]. Prehybridization and hybridization were carried out at 42°C in the presence of 50% formamide in a mixture of 5 x Denhardt's solution, 5 x SSPE, 0.1% SDS and 100 /tg/ml denatured salmon sperm DNA [17]. Filters were washed with 2 x SSC, 0.1% SDS and finally with 0.1 x SSC, 0.1% SDS at 50°C, followed by autoradiography at - 8 0 ° C with an intensifying screen. Autoradiograms were quantified by densitometry using a Helena Auto Scanner (Texas, USA). cDNA clones for rat uricase [5], albumin and transthyretin [18] were isolated in our laboratory. Mouse apo E, rat metallothionein and /3-actin clones were obtained from C. Heinzman (UCLA, USA), R.D. Andersen (UCLA, USA) and M. Obinata (Tohoku University, Sendai, Japan), respectively. Results
Purification of rat liver uricase Treatment of the lysosome-rich fraction with 1% Triton X-100 solubilized most of the proteins and about 50% of the activity of the uricase was recovered in the
318
1
2
3
4
5
6
A
B
1 2 3 4 5 ~ 200
K
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K
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!
C 3 4
1 2 3 4
93K
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~
31
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Fig. 1. SDS-PAGE analysis of the purified rat liver uricase. Lane 1: rat liver 'lysosome-rich' fraction (30 #g of protein); Lanes 2-4: the purified uricase of different preparations (3-5 pg of protein); Lanes 5 and 6: molecular weight standards (Bio-Rad).
A
12
Fig. 2. lmmunoblot analyses of the specificities of the monoclonal antibodies against rat uricase. (A) Coomassie blue stained SDS-PAGE pattern of the purified uricase (lanes 1 and 2; 1 /xg and 10 #g of protein), the 'lysosome-rich' fraction (lanes 3 and 4; 50 #g and 500 #g), and molecular weight standards (lane 5). (B) and (C) Immunoblotting of the triplicate sets of gels with monoclonal antibodies 22 (B) and 46 (C).
pellet by centrifugation with an approx. 120-fold increase in purification. The effect was not specific to Triton X-100 but 1% of NP40 or octylglucoside gave a
B
lb-
Fig. 3. Immunoblot analysis of the tissue distribution of the uricase. (A) Coomassie blue-stained SDS-PAGE pattern of the proteins in the post-nuclear fractions of adrenal gland (AG), brain (B), heart (H), kidney (K), liver (L), lactating mammary gland (MG), spleen (S) and thymus (T). The purified uricase (0.2 ~g, URI) and molecular weight standards (MW) are also shown. (B) Immunoblotting of the duplicated gel with monoclonal antibodies No. 22. Arrowheads indicate the position of the uricase.
A1
similar result, while Tween 20 was less effective. Starting from 200 mg protein of the lysosome-rich fraction, 0.9 mg of the uricase was obtained by a single step. The purity was estimated to be about 90% by densitometry of the Coomassie blue-stained gel of SDS-polyacrylamide gel electrophoresis (Fig. 1). The amino acid sequence analysis of this preparation gave the exact Nterminal sequence of the uricase [5], suggesting its near homogeneity. Monoclonal antibodies and their specificities
Nine clones in the four 96-well microtiter plates survived to yield stable cultures. They were positive for antibody activities against the uricase as judged by enzyme immunoassay. Their specificities were further examined by immunoblotting; four of them predominantly recognized the uricase; all of them were in the IgM class. Among them, two (22 and 46) gave strong signals and the results of immunoblotting are shown in Fig. 2. They were highly specific to uricase and cross-reacted little with other proteins in the light mitochondria fraction of the rat liver. The difference in the faint band patterns other than the main band corresponding to uricase suggested that each monoclonal antibody recognized a different epitope on uricase.
2
3
B
1
2
I 1.1
C
D
E
Immunoblot detection of uricase in various tissues
The relative amount of the uricase in brain, thymus, heart, spleen, kidney, liver, or lactating mammary gland of a female rat was examined by immunoblotting using postnuclear fractions of each tissue and a specific monoclonal antibody (22). As shown in Fig. 3, the uricase was detected only in the liver. Essentially the same results were obtained with another monoclonal antibody (46) and with polyclonal anti-uricase antibodies (not shown). Prolonged incubation of the sheet developed a very faint band of molecular weight of about 34000 on the lane of the mammary gland sample (not shown), but other faint bands also appeared and they have not been identified. The content of uricase in extrahepatic tissues was, at any rate, less than 2% of that in the liver. Uricase m R N A levels in various tissues
The mRNA levels of uricase in various tissues were measured by slot blot analyses together with the levels of albumin, transthyretin, apo E and fl-actin mRNA as controls. The results of autoradiography are shown in Fig. 4. The amounts of apo E mRNA in various tissues (Fig. 4, C) were quantified by densitometry of the film. The intensities of the images were in linear relationship to the amounts of extracts applied to the filter in every tissue (not shown). The relative amounts of apo E mRNA in the liver, brain, kidney, heart, lung, spleen and thymus were 100, 37, 24, 22, 13, 6 and 5, respec-
Fig. 4. Slot blot analysis of the tissue distribution of the uricase mRNA. Uricase (A), albumin (B), apo E (C), fl-actin (D), and transthyretin (E) mRNA levels in various tissues are analyzed using total extracts. Total extracts were normalized by DNA contents and columns 1-3 show extracts containing 0.4, 0.2, and 0.1 #g DNA. Columns B - L G show extracts from brain (B), heart (H), thymus (T), spleen (S), liver (L), kidney (K) and lung (LG), respectively.
tively. This result was good consistent with the published data [19]. Albumin mRNA was detected as highly specific and abundant in the liver and transthyretin mRNA in both the liver and the brain as previously reported [20,21]. Under these conditions where control mRNA levels in various tissues were accurately quantitated, the uricase mRNA was detected in the liver. The signals were weak but other faint signals in extrahepatic tissues were understood to be non-specific, because signal patterns of uricase and albumin mRNAs in extrahepatic tissues appearing after long exposure were almost indistinguishable (Fig. 4, B). These were prob-
320 ably caused analyses, in only in the extrahepatic
by DNAs in total conclusion, uricase fiver and the level tissues was less than
extracts. By slot blot mRNA was detected of uricase mRNA in 5% of that in the fiver.
Changes in the levels of uricase and other mRNAs in cultured hepatocytes To characterize liver-specific expression of uricase mRNA, the changes in the steady-state levels of the mRNA expressed in cultured hepatocytes over the period of a few days were compared with those of mRNAs for liver specific and common genes by quantitative slot blot analysis. The results of slot blot autoradiographs are shown in Fig. 5. Under the culture conditions employed, hepatocyte morphology was gradually altered (not shown) and the steady-state levels of three liver-specific mRNAs dropped to various extents. These decreases were not due to general functional deterioration, because the levels of some other mRNAs were maintained at higher levels than those at the start of culture. The level of metallothionein mRNA was increased about 7-fold of the start level after 3-day culture, probably because of the induction by relatively high level of dexamethasone into the culture medium [22]. Elevation of the level of actin mRNA (3-5-fold) during the culture period was well consistent with the previous observation by Isom et al. [23]. However, the rates of decrease in levels of the fiver-specific mRNAs differed among the three: that of albumin mRNA dropped rapidly after 3-day culture to about 5% of the start level and that of transthyretin mRNA to about 50%, but the level of uricase m R N A was maintained almost constant at the start level. It has not yet been
A
B
1 2312
C 3
determined whether the differences were in the rate of transcription a n d / o r in their stabilities, but it is evident that the •er-specific expression of uricase is uniquely controlled. Discussion The method employed to purify rat liver uricase was very similar to that used for the isolation of the peroxisomal core [24]. SDS-PAGE analysis of proteins in this fraction showed one major band of uricase (Fig. 1) and amino acid sequence analyses of this total fraction showed that the N-terminus was blocked alanine followed by the exact sequence of uricase [5], suggesting that the major portion, if not all, of the protein in the core is uricase; whether there are any other components is not known. Liver-specific expression of uricase has been demonstrated using specific monoclonal antibodies against uricase and a uricase c D N A with various control cDNAs for liver-specific or common mRNAs. Although our antibodies against uricase is highly specific, our present results do not exclude the possibility that uricase is also expressed in extrahepatic tissues but the levels are so low as to be undetectable by immunoblotting. The results of slot blot analysis of the m R N A levels using total extracts were consistent with the above conclusion. The method employed could not detect very low levels of specific mRNA sequences as compared with Northern blot using poly(A) RNA, and further detailed analyses are necessary to determine whether uricase mRNA is expressed solely or predominantly in the liver. However, in slot blot analysis using total extracts, the
D
1 23123
E 1231
F 23
0 "O "O-1 O ~D
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8 Fig. 5. Slot blot analysis of the changes in the steady-state levels of various m R N A s during a culture period in isolated rat hepatocytes. The steady-state levels of albumin (A), transthyretin (B), uricase (C), metallothionein (D), fl-actin (E) and apo E (F) m R N A were measured using the total extracts prepared from the hepatocytes at the start of the culture, 1 day, 3 days, 6 days, and 8 days after the start of the culture. C o l u m n s 1 - 3 show extracts containing 0.4, 0.2 and 0.1 ~g DNA, respectively.
321 samples were loaded according to DNA content and the signals of hybridization should be proportional to the amounts the mRNA per cell. Determination of DNA concentration by the Hoechst 33258 dye method will be more specific and accurate than photometric determination of the RNA content in the samples which may contain interfering materials and different levels of rRNA. The procedure was well suited to the analysis of various mRNA levels in multiple small tissue samples and especially in primary cultured hepatocytes. Usuda et al. [4] and Reddy et al. [26] reported liver-specific expression of uricase in rat using polyclonal antibodies, and a cloned cDNA for rat uricase, respectively. Thus, our studies, which have been completed before their works' appearance, confirmed their conclusion using highly specific monoclonal antibodies and a cloned cDNA with various control cDNAs. On the contrary, uricase is also found in the kidney of other species [4,6]. The unique tissue-specificity of rat uricase will be related to the structure of the promoter of the gene as suggested by cDNA [5] and genomic [27] cloning and comparison with cDNAs of other species [7,28]. The N-terminal structure of rat uricase is different from those of other species and this was probably produced by insertion of an intron [28]. The different orgaization of the rat uricase gene around the first exon may be related to the unique transcriptional regulation of the rat gene. To characterize liver-specific expression of uricase in rat, we compared the steady-state levels of its mRNA with those of other liver-specific and common mRNAs in primary cultured rat hepatocytes. The levels of uricase mRNA were largely maintained during the 8-day culture of hepatocytes while those of other liver-specific mRNAs, especially that of albumin mRNA dropped remarkably. In serum-supplemented standard tissue culture medium, which we used, it was reported that the levels of liver-specific mRNAs drop rapidly as the result of a decrease in the rates of transcription [29]. Thus, the steady-state level of uricase mRNA was suggested to be maintained by the unchanged rate of transcription, but our present study does not exclude the possibility that the level of uricase mRNA was maintained by its unusual stability in hepatocytes. Furthermore, Isom et al. [23] suggested that the stabilities of some liver-specific mRNAs are different between in the liver and in the cultured hepatocytes. Further study to determine whether the differences are in the rate of transcription a n d / o r in their stabilities is clearly necessary and should be carried out very carefully. Though the level of regulation was not evident from this study and further study is clearly necessary, our results do suggest that the mechanism of liver-specific expression of uricase is unique and that the steady-state level of the uricase mRNA is regulated differently from those of other typical liver-specific mRNAs.
Acknowledgments We acknowledge Professor T. Takano (Teikyo University, Kanagawa, Japan), in whose laboratory a part of this work was done and whose continuous enthusiasm and helpful advice were greatly appriciated. We are grateful to Drs. C. Heinzman (UCLA, USA), R.D. Andersen (UCLA, USA) and M. Obinata (Tohoku University, Sendai, Japan) for their generous gifts of mouse apoE, rat metallothionein, and fl-actin done, respectively. Thanks are also due to Dr. A. Ishigami for isolation of rat hepatocytes, to Dr. S. Kanaya (Mitsubishi Chemical Industries Ltd.) for amino acid sequence analysis, and to Miss N. Harada for her help in slot blot analyses.
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