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Molecular cloning and sequence analysis of the cDNA for the mouse counterpart to adult hamster liver purified growth inhibitory factor
Biochimica et Biophysica Acta 1355 Ž1997. 205–208
Short sequence-paper
Molecular cloning and sequence analysis of the cDNA for the mouse counterpart to adult hamster liver purified growth inhibitory factor Chieko Hashimoto a,) , Hiroaki Masuda b, Masako Ayaki a , Yasuhiko Suzuki c , Akiko Uenaka d , Tsukasa Seya a , Jun Miyoshi a , Katsuhito Takahashi a , Yukiharu Inui
a
a
b
The Center for Adult Diseases Osaka, 3 Nakamichi, 1-chome Higashinari-ku Osaka, 537, Japan The Second Department of Internal Medicine, Gunma, UniÕersity School of Medicine, 3-39-22, Showa-machi, Maebashi Gunma, 371, Japan c Osaka Prefectural Institute of Public Health, 3-69, Nakamichi 1-chome Higashinari-ku, Osaka, 537, Japan d Okayama UniÕersity School of Medicine, 2-5-1, Shikata-machi Okayama, 700, Japan Received 15 October 1996; revised 10 December 1996; accepted 20 December 1996
Abstract A cDNA clone encoding the mouse counterpart to adult hamster liver purified growth inhibitory factor ŽPGIF. was isolated from a mouse liver cDNA library by using antibodies raised against PGIF and sequenced. It contained a single open reading frame with a coding capacity for a 323 amino acid protein. Sequence analysis showed that it shared high homology with rat- and human liver arginases: the cDNA clone was 92% identical for rat arginase at the nucleotide level and was 93% identical to it at the deduced amino acid level. These results suggest that PGIF derived from adult hamster liver was identical or closely related to an isoform of hamster liver arginases. Keywords: Growth inhibitory factor, purified; cDNA cloning; cDNA sequencing; Arginase
A growth inhibitory factor was purified almost homogenously from adult hamster liver w1x. The factor was found Ž1. to have a molecular mass of 37 kDa, Ž2. to cause reversible arrest of SV40 transformed 3T3 predominantly in the G0rG1 phase of the cell cycle at the concentration of approx. 0.9 m grml, Ž3. to occur exclusively in the nuclear and cytoplasmic fractions of hepatocytes, and Ž4. not to be detected in the nuclei under certain conditions such as regenerated or neonatal hamster liver. ReAbbreviations: PGIF, the adult hamster liver purified growth inhibitory factor; SDS-PAGE, SDS polyacrylamide gel electrophoresis. ) Corresponding author. Fax: q81-6-9727749. 0167-4889r97r$17.00 Published by Elsevier Science B.V. PII S 0 1 6 7 - 4 8 8 9 Ž 9 6 . 0 0 1 8 0 - 2
cently, we have reported a possible intranuclear binding of the factor with underphosphorylated Rb protein w2x. These findings strongly suggest that the inhibitory factor plays important roles in some nuclear events related to cell cycle progression. We decided to clone the cDNA for the inhibitory factor to enable us to study the function of the factor in normal and pathological states and, further, to understand whether this factor could be involved in the progression of cell cycle. In this paper we adopted a mouse liver cDNA library instead of hamster liver cDNA library for its commercial availability. In preliminary experiments the antibodies raised against the adult hamster liver purified growth inhibitory factor ŽPGIF. was found to
206
C. Hashimoto et al.r Biochimica et Biophysica Acta 1355 (1997) 205–208
crossreact with the partially purified mouse liver growth inhibitory factor with a molecular mass of 37 kDa by Western blot analysis Ždata not shown.. Then a mouse liver cDNA library constructed in the lZAPp expression vector Ž Stratagene. was screened with the affinity purified monospecific rabbit polyclonal antibodies to PGIF. Screening of 4 = 10 5 plaques of the lZAPp library resulted in the isolation of 10 clones representing at least 8 distinct classes of overlapping clones on the basis of the insert size. The DNA was purified separately from these clones and sequenced by standard methods w3x. The nucleotide sequence of approx. 1.2 kilobase pair cDNA encoding a mouse counterpart to PGIF is shown in Fig. 1. The open reading frame starts with ATG at nucleotide 121 and terminats with TGA at nucleotide 1090. It encodes a 323 amino acid residues with a predicted molecular weight of 36 880. This value is in agreement with the molecular mass of PGIF Ž37 kDa. determined by SDS-PAGE w1x. The antibodies cross-reacted with the mouse counterpart to PGIF, indicating sequence similarity between the mouse counterpart and PGIF. This was subsequently confirmed in part by comparison of the deduced amino acid sequence for the mouse cDNA clone with the analysed amino acid sequence of the internal peptide fragments ŽFrag. 1: 197–210, Frag. 2: 211– 224, and Frag. 3: 285–323 named for the order from the C-terminal. from PGIF prepared by endoproteinase Lys-C Ž Boehringer. treatments. The analysed amino acid sequences of Frag. 1, 2, and 3 had a 83%, a 100% and a 85% identity, respectively, with the individual regions of the deduced amino acid sequence of the cloned DNA which were supposed to correspond with each fragment ŽFig. 2.. In separate experiments the NH2-terminal of PGIF was found to be blocked. Potential sites for N-linked carbohydrate addition ŽAsn-X-ThrrSer. were located at positions 60 and 279. In addition, a putative nuclear translocation signal w4x was detected at position 222. Sequence analysis showed a high grade of homology to human and rat arginases. Comparison of the deduced amino acid sequences of the mouse cDNA clone, rat and human arginases is shown in Fig. 2. The cDNA clone is 92% identical with that of the rat liver arginase at the nucleotide sequence level. The deduced amino acid sequence homology of the
Fig. 1. Nucleotide and deduced amino acid sequences of the cloned cDNA. The nucleotide sequence of the cloned mouse cDNA is numbered in the 5X to 3X direction and amino acid numbers beginning with the first residue encoded by the open reading frame are indicated at the right side. Potential N-glycosidic linkage sites are underlined and a putative nuclear transport signal is doubly underlined.
cDNA clone to the rat and human arginases is 93% and 87%, respectively. We then determined the arginase activity of PGIF, finding that the factor actually demonstrated the enzyme activity Ž104 Urmg protein, n s 2, 1 U s 1 m mol urea released from argininermin. by the method as previously described w7x. To test the function of the product from the cloned cDNA, we adopted the glutathione-S-transferase gene fusion system ŽPharmacia Biotech. to express the cDNA. The cloned protein thus prepared showed arginase activity Ž26 Urmg protein, n s 2. and almost the same migration velocity on SDS-PAGE as that of PGIF ŽFig. 3.. The protein, also, demonstrated
C. Hashimoto et al.r Biochimica et Biophysica Acta 1355 (1997) 205–208
similar growth inhibitory effect Ž; 97%, n s 2. on SVHF to that of PGIF on the basis of the enzyme activity level Ž0.1 Urml of the growth medium. under the experimental conditions reported previously w1x. The inhibition of proliferation of culture cells from rabbit liver or kidney by the liver cytosol was investigated following the first paper by Lieberman and Ove w8x and the inhibitory principal in the cytosol was identified as arginase w9,10x. Some of the earlier studies related to the effect of arginase on cell growth and cellular function were reported w11–13x. It is conceivable that arginase has some undiscovered additional functions other than the function as a key enzyme in the urea cycle. It remains for future studies to establish whether the arginase of PGIF in nuclei is actually involved in cell cycle progression or not, by using the cDNA clone isolated here as useful tools.
207
Fig. 3. SDS-PAGE of the exprssed protein and PGIF. The proteins were visualized by Coomassie-Blue staining after SDSPAGE. Lane 1: standard proteins, lane 2: the expressed protein, lane 3: PGIF.
Fig. 2. Comparison of the deduced amino acid sequence of the mouse cDNA clone with those of human- and rat liver arginases. Amino acid sequence of human liver arginase is from Haraguchi et al. w5x and that of rat enzyme is from Kawamoto et al. w6x. A gap is introduced to increase the similarity and matching amino acids are boxed. The amino acid sequence of the internal fragments ŽFrag. 1, 2, and 3. from PGIF prepared by the endopeptidase digestion followed by HPLC is shown. The asterisks Ž). indicate amino acid residues which are different from the residues deduced from the cDNA clone.
208
C. Hashimoto et al.r Biochimica et Biophysica Acta 1355 (1997) 205–208
References w1x Hashimoto, C., Ayaki, M. and Inui, Y. Ž1994. Biochim. Biophys. Acta 1220, 107–117. w2x Hashimoto, C., Ayaki, M., Tanaka, K., Yamamoto, R., Fukuda, H., Funai, H., Wada, A. and Inui, Y. Ž1996. Biochim. Biophys. Acta 1310, 309–316. w3x Sambrook, J., Fritsch, E.F. and Maniatis, T Ž1989. Molecular Cloning, A Laboratory Manual. 2nd Edn, Cold Spring Harbor Laboratory Press. w4x Garcia-Bustos, J., Heitman, J. and Hall, M.N. Ž1991. Biochim. Biophys. Acta 1071, 83–101. w5x Haraguchi, Y., Takiguchi, M., Amaya, Y., Kawamoto, S., Matsuda, I. and Mori, M. Ž1987. Proc. Natl. Acad. Sci. USA 84, 412–415.
w6x Kawamoto, S., Amaya, Y., Murakami, K., Tokunaga, F., Iwanaga, S., Kobayashi, K., Saheki, T., Kimura, S. and Mori, M. Ž1987. J. Biol. Chem. 262, 6280–6283. w7x Corraliza, I., Soler, G., Eichmann, K. and Modolell, M. Ž1995. Biochem. Biophys. Res. Commun. 206, 667–673. w8x Liverman, I. and Ove, P. Ž1960. Biochim. Biophys. Acta 38, 153. w9x Holley, R.W. Ž1967. Biochim. Biophys. Acta 145, 525–527. w10x Sasada, M. and Terayama, H. Ž1969. Biochim. Biophys. Acta 190, 73–87. w11x Currie, G.A. Ž1978. Nature 273, 758–759. w12x Currie, G.A., Gyure, L. and Cifuentes, L. Ž1979. Br. J. Cancer 39, 613–620. w13x Mills, C.D., Shearer, J., Evans, R. and Caldwell, M.D. Ž1992. J. Immunol. 149, 2709–2714.
Short sequence-paper
Molecular cloning and sequence analysis of the cDNA for the mouse counterpart to adult hamster liver purified growth inhibitory factor Chieko Hashimoto a,) , Hiroaki Masuda b, Masako Ayaki a , Yasuhiko Suzuki c , Akiko Uenaka d , Tsukasa Seya a , Jun Miyoshi a , Katsuhito Takahashi a , Yukiharu Inui
a
a
b
The Center for Adult Diseases Osaka, 3 Nakamichi, 1-chome Higashinari-ku Osaka, 537, Japan The Second Department of Internal Medicine, Gunma, UniÕersity School of Medicine, 3-39-22, Showa-machi, Maebashi Gunma, 371, Japan c Osaka Prefectural Institute of Public Health, 3-69, Nakamichi 1-chome Higashinari-ku, Osaka, 537, Japan d Okayama UniÕersity School of Medicine, 2-5-1, Shikata-machi Okayama, 700, Japan Received 15 October 1996; revised 10 December 1996; accepted 20 December 1996
Abstract A cDNA clone encoding the mouse counterpart to adult hamster liver purified growth inhibitory factor ŽPGIF. was isolated from a mouse liver cDNA library by using antibodies raised against PGIF and sequenced. It contained a single open reading frame with a coding capacity for a 323 amino acid protein. Sequence analysis showed that it shared high homology with rat- and human liver arginases: the cDNA clone was 92% identical for rat arginase at the nucleotide level and was 93% identical to it at the deduced amino acid level. These results suggest that PGIF derived from adult hamster liver was identical or closely related to an isoform of hamster liver arginases. Keywords: Growth inhibitory factor, purified; cDNA cloning; cDNA sequencing; Arginase
A growth inhibitory factor was purified almost homogenously from adult hamster liver w1x. The factor was found Ž1. to have a molecular mass of 37 kDa, Ž2. to cause reversible arrest of SV40 transformed 3T3 predominantly in the G0rG1 phase of the cell cycle at the concentration of approx. 0.9 m grml, Ž3. to occur exclusively in the nuclear and cytoplasmic fractions of hepatocytes, and Ž4. not to be detected in the nuclei under certain conditions such as regenerated or neonatal hamster liver. ReAbbreviations: PGIF, the adult hamster liver purified growth inhibitory factor; SDS-PAGE, SDS polyacrylamide gel electrophoresis. ) Corresponding author. Fax: q81-6-9727749. 0167-4889r97r$17.00 Published by Elsevier Science B.V. PII S 0 1 6 7 - 4 8 8 9 Ž 9 6 . 0 0 1 8 0 - 2
cently, we have reported a possible intranuclear binding of the factor with underphosphorylated Rb protein w2x. These findings strongly suggest that the inhibitory factor plays important roles in some nuclear events related to cell cycle progression. We decided to clone the cDNA for the inhibitory factor to enable us to study the function of the factor in normal and pathological states and, further, to understand whether this factor could be involved in the progression of cell cycle. In this paper we adopted a mouse liver cDNA library instead of hamster liver cDNA library for its commercial availability. In preliminary experiments the antibodies raised against the adult hamster liver purified growth inhibitory factor ŽPGIF. was found to
206
C. Hashimoto et al.r Biochimica et Biophysica Acta 1355 (1997) 205–208
crossreact with the partially purified mouse liver growth inhibitory factor with a molecular mass of 37 kDa by Western blot analysis Ždata not shown.. Then a mouse liver cDNA library constructed in the lZAPp expression vector Ž Stratagene. was screened with the affinity purified monospecific rabbit polyclonal antibodies to PGIF. Screening of 4 = 10 5 plaques of the lZAPp library resulted in the isolation of 10 clones representing at least 8 distinct classes of overlapping clones on the basis of the insert size. The DNA was purified separately from these clones and sequenced by standard methods w3x. The nucleotide sequence of approx. 1.2 kilobase pair cDNA encoding a mouse counterpart to PGIF is shown in Fig. 1. The open reading frame starts with ATG at nucleotide 121 and terminats with TGA at nucleotide 1090. It encodes a 323 amino acid residues with a predicted molecular weight of 36 880. This value is in agreement with the molecular mass of PGIF Ž37 kDa. determined by SDS-PAGE w1x. The antibodies cross-reacted with the mouse counterpart to PGIF, indicating sequence similarity between the mouse counterpart and PGIF. This was subsequently confirmed in part by comparison of the deduced amino acid sequence for the mouse cDNA clone with the analysed amino acid sequence of the internal peptide fragments ŽFrag. 1: 197–210, Frag. 2: 211– 224, and Frag. 3: 285–323 named for the order from the C-terminal. from PGIF prepared by endoproteinase Lys-C Ž Boehringer. treatments. The analysed amino acid sequences of Frag. 1, 2, and 3 had a 83%, a 100% and a 85% identity, respectively, with the individual regions of the deduced amino acid sequence of the cloned DNA which were supposed to correspond with each fragment ŽFig. 2.. In separate experiments the NH2-terminal of PGIF was found to be blocked. Potential sites for N-linked carbohydrate addition ŽAsn-X-ThrrSer. were located at positions 60 and 279. In addition, a putative nuclear translocation signal w4x was detected at position 222. Sequence analysis showed a high grade of homology to human and rat arginases. Comparison of the deduced amino acid sequences of the mouse cDNA clone, rat and human arginases is shown in Fig. 2. The cDNA clone is 92% identical with that of the rat liver arginase at the nucleotide sequence level. The deduced amino acid sequence homology of the
Fig. 1. Nucleotide and deduced amino acid sequences of the cloned cDNA. The nucleotide sequence of the cloned mouse cDNA is numbered in the 5X to 3X direction and amino acid numbers beginning with the first residue encoded by the open reading frame are indicated at the right side. Potential N-glycosidic linkage sites are underlined and a putative nuclear transport signal is doubly underlined.
cDNA clone to the rat and human arginases is 93% and 87%, respectively. We then determined the arginase activity of PGIF, finding that the factor actually demonstrated the enzyme activity Ž104 Urmg protein, n s 2, 1 U s 1 m mol urea released from argininermin. by the method as previously described w7x. To test the function of the product from the cloned cDNA, we adopted the glutathione-S-transferase gene fusion system ŽPharmacia Biotech. to express the cDNA. The cloned protein thus prepared showed arginase activity Ž26 Urmg protein, n s 2. and almost the same migration velocity on SDS-PAGE as that of PGIF ŽFig. 3.. The protein, also, demonstrated
C. Hashimoto et al.r Biochimica et Biophysica Acta 1355 (1997) 205–208
similar growth inhibitory effect Ž; 97%, n s 2. on SVHF to that of PGIF on the basis of the enzyme activity level Ž0.1 Urml of the growth medium. under the experimental conditions reported previously w1x. The inhibition of proliferation of culture cells from rabbit liver or kidney by the liver cytosol was investigated following the first paper by Lieberman and Ove w8x and the inhibitory principal in the cytosol was identified as arginase w9,10x. Some of the earlier studies related to the effect of arginase on cell growth and cellular function were reported w11–13x. It is conceivable that arginase has some undiscovered additional functions other than the function as a key enzyme in the urea cycle. It remains for future studies to establish whether the arginase of PGIF in nuclei is actually involved in cell cycle progression or not, by using the cDNA clone isolated here as useful tools.
207
Fig. 3. SDS-PAGE of the exprssed protein and PGIF. The proteins were visualized by Coomassie-Blue staining after SDSPAGE. Lane 1: standard proteins, lane 2: the expressed protein, lane 3: PGIF.
Fig. 2. Comparison of the deduced amino acid sequence of the mouse cDNA clone with those of human- and rat liver arginases. Amino acid sequence of human liver arginase is from Haraguchi et al. w5x and that of rat enzyme is from Kawamoto et al. w6x. A gap is introduced to increase the similarity and matching amino acids are boxed. The amino acid sequence of the internal fragments ŽFrag. 1, 2, and 3. from PGIF prepared by the endopeptidase digestion followed by HPLC is shown. The asterisks Ž). indicate amino acid residues which are different from the residues deduced from the cDNA clone.
208
C. Hashimoto et al.r Biochimica et Biophysica Acta 1355 (1997) 205–208
References w1x Hashimoto, C., Ayaki, M. and Inui, Y. Ž1994. Biochim. Biophys. Acta 1220, 107–117. w2x Hashimoto, C., Ayaki, M., Tanaka, K., Yamamoto, R., Fukuda, H., Funai, H., Wada, A. and Inui, Y. Ž1996. Biochim. Biophys. Acta 1310, 309–316. w3x Sambrook, J., Fritsch, E.F. and Maniatis, T Ž1989. Molecular Cloning, A Laboratory Manual. 2nd Edn, Cold Spring Harbor Laboratory Press. w4x Garcia-Bustos, J., Heitman, J. and Hall, M.N. Ž1991. Biochim. Biophys. Acta 1071, 83–101. w5x Haraguchi, Y., Takiguchi, M., Amaya, Y., Kawamoto, S., Matsuda, I. and Mori, M. Ž1987. Proc. Natl. Acad. Sci. USA 84, 412–415.
w6x Kawamoto, S., Amaya, Y., Murakami, K., Tokunaga, F., Iwanaga, S., Kobayashi, K., Saheki, T., Kimura, S. and Mori, M. Ž1987. J. Biol. Chem. 262, 6280–6283. w7x Corraliza, I., Soler, G., Eichmann, K. and Modolell, M. Ž1995. Biochem. Biophys. Res. Commun. 206, 667–673. w8x Liverman, I. and Ove, P. Ž1960. Biochim. Biophys. Acta 38, 153. w9x Holley, R.W. Ž1967. Biochim. Biophys. Acta 145, 525–527. w10x Sasada, M. and Terayama, H. Ž1969. Biochim. Biophys. Acta 190, 73–87. w11x Currie, G.A. Ž1978. Nature 273, 758–759. w12x Currie, G.A., Gyure, L. and Cifuentes, L. Ž1979. Br. J. Cancer 39, 613–620. w13x Mills, C.D., Shearer, J., Evans, R. and Caldwell, M.D. Ž1992. J. Immunol. 149, 2709–2714.