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Molecular cloning of the murine cMOAT ATPase1
Biochimica et Biophysica Acta 1492 (2000) 531^536
www.elsevier.com/locate/bba
Short sequence-paper
Molecular cloning of the murine cMOAT ATPase1 Friederike Fritz a , Jing Chen a , Paula Hayes a , F.M. Sirotnak a
a;b;
*
Laboratory for Molecular Therapeutics, Program of Molecular Pharmacology and Experimental Therapeutics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021, USA b Graduate School of Medical Sciences, Cornell University, New York, NY 10021, USA Received 22 February 2000; accepted 10 May 2000
Abstract cMOAT encodes an ATPase within the family of cMOAT/MRP ATPases that functions as an ATP dependent, multispecific anion transporter within the canalicular surface of hepatocytes that has pharmacologic significance. We describe here the cloning of a murine cMOAT cDNA isolated from mouse liver. The open reading frame of this cDNA incorporates 4627 nucleotides encoding 1309 amino acids with 77.5% and 86.7% identity with the human and rat encoded amino acids, respectively. Northern blotting showed that the expression of cMOAT mRNA occurs primarily in mouse liver in the form of two variants with approximately 5.6 and 7.8 kb of sequence each. cMOAT mRNA was also detected in mouse kidney at a low level but was not detected in other mouse organs or tumors except the Hep 1-6 murine hepatoma where expression was also in the form of the same two mRNA variants. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Canalicular multispeci¢c anion transporter ; Murine cDNA
The multispeci¢c organic anion transporter (cMOAT) is a member of the MRP family of ATPases involved [1^4] in the hepatobiliary transport and excretion of compounds from hepatocytes across the bile canalicular membrane. Mutation in rat cMOAT has been shown [3,5,6] to result in hyperbilerubemia, a model for Dubin^Johnson syndrome in humans [7]. Recent studies have shown [8] that cMOAT is capable of excreting the folate analogue, MTX, in bile. This and other recent ¢ndings associating [9,10] overexpression of cMOAT with cis-platinum resistance in tumor cells establish the pharmacologic relevance of this gene. As a preface to future work using the mouse as a model for studies on di¡erential tissue expression of cMOAT, we sought to isolate and sequence a cDNA clone encoding murine cMOAT. In the present report, we provide the total coding sequence for cMOAT cDNA and a portion of its 3P untranslated region (UTR) that was de-
Abbreviations : ORF, open reading frame; UTR, untranslated region; cMOAT, canalicular multispeci¢c anion transporter * Corresponding author. Fax: +1-212-794-4342; E-mail : [email protected] 1 Sequence data presented in the paper have been submitted to the EMBL/GenBank data libraries under accession number AF227274.
rived from poly(A) RNA isolated from mouse liver. We also compare its homology to the published rat and human cMOAT sequence. Partial length clones of murine cMOAT were obtained from a Lambda gt11 mouse liver cDNA library (Clontech) using a rat cMOAT cDNA probe prepared by PCR. Hybridization and PCR as well as Northern blotting were performed as originally described [11]. In view of the total length of the cMOAT cDNA anticipated from Northern blotting (see below), we only sought to obtain the entire sequence of the open reading frame (ORF). To do this, it was necessary to perform 5PRACE PCR (Gibco BRL) and chromosome walking (Genome Walker, Clontech). Some sequence of the 3PUTR of this cDNA was also obtained. In Fig. 1A, the nucleotide and deduced amino acid sequence of the ORF for mouse cMOAT cDNA is provided along with some sequence of the 3PUTR. The ORF encodes 1543 amino acids with a predicted molecular mass of 171 kDa. Comparison of amino acids for mouse, rat and human orthologs (Fig. 1B and Table 1) revealed 85.2% and 76.5% identity, respectively, between rat and human sequences. Northern blotting of poly(A) RNA from various mouse tissues and tumors showed a high level of expression of cMOAT in liver and low level of expression in kidney (Fig. 2). This expression in liver occurred in the form of two variants of approximately 5.6 and 7.8 kb of sequence. Thus, these transcripts incorporate
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 1 3 2 - 9
BBAEXP 91427 5-7-00
532
F. Fritz et al. / Biochimica et Biophysica Acta 1492 (2000) 531^536
Fig. 1. (A) Nucleotide and deduced amino acid sequence of murine cMOAT isolated from a mouse liver cDNA library and extended by 5PRACE and chromosome walking. (B) Amino acid sequence comparison of human, rat and mouse cMOAT. Alignment was performed with DNAStar software, using the method of Lipman^Pearson. The sequence alignments are designated as non-homologous (white on black), conserved (black on gray, identical (black on white), weakly similar (white on dark gray) and block similar (dark gray on black).
Table 1 Amino acid similarity of cMOAT between di¡erent species cMOAT/hu cMOAT/rat cMOAT/mu
cMOAT/hu
cMOAT/rat
cMOAT/mu
100 77.5 77.5
100 86.7
100
Fig. 1 (continued).
extremely lengthy 3P and (or) 5PUTR sequences since the ORF represents only 4.62 kb of sequence. Interestingly, there was little, if any, expression of cMOAT in all other mouse tissues. Low level expression in kidney occurred in the form of two variants between 5 and 6 kb in length. In summary, cMOAT is highly conserved among three mammalian species with expression occurring primarily in liver. Supported in part by Grants CA08748 and CA56517 from the National Cancer Institute.
Alignment was performed with DNAStar software, using the Lipman^ Pearson protein alignment method.
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F. Fritz et al. / Biochimica et Biophysica Acta 1492 (2000) 531^536
Fig. 1 (continued).
Fig. 1 (continued).
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Fig. 1 (continued).
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Fig. 1 (continued).
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Fig. 1 (continued).
References [1] Z.C. Gatmaitan, I.M. Arias, Physiol. Rev. 75 (1995) 261^275. [2] P.J. Mieir, Am. J. Physiol. 269 (1996) G801^G812. [3] K. Ito, H. Suzuki, T. Hirohashi, K. Kume, T. Shimizu, Y. Sugiyama, Am. J. Physiol. 272 (1996) G16^G22. [4] M.G. Belinsky, L.J. Bain, B.B. Balsara, J.R. Testa, G.D. Kruh, J. Natl. Cancer Inst. 90 (1998) 1735^1741. [5] S. Hosakowa, O. Tagaya, T. Mikami, Y. Nozaki, A. Kawaguchi, M. Shamato, Lab. Anim. Sci. 42 (1992) 27^34. [6] P.L. Jansen, W.H. Peters, W.H. Lamers, Hepatology 5 (1985) 573^ 579. [7] I.N. Dubin, F.B. Johnson, Medicine 33 (1954) 155^172. [8] M. Masuda, Y. I'izuka, M. Yamazaki, R. Nishigaki, Y. Kato, K. Ni'Inuma, S. Hirashi, Y. Sugiyama, Cancer Res. 57 (1997) 3506^ 3510. [9] K. Taniguchi, M. Wada, K. Kohno, T. Nakamura, T. Kawabe, M. Kawakami, K. Kogotani, K. Obumura, S. Alsiyama, M. Kuwano, Cancer Res. 56 (1996) 4124^4129. [10] M. Kool, M. de Haas, G.L. Sche¡er, R.J. Scheper, M.J. Van Eijk, J.A. Juijn, F. Baas, P. Borst, Cancer Res. 57 (1997) 3537^3547. [11] T. Esaki, K. Roy, R. Yao, J. Galivan, F.M. Sirotnak, Gene 219 (1998) 37^44. Fig. 2. Northern blot analysis of cMOAT expression in various mouse tissues. A mouse cDNA probe was prepared by PCR and used to blot poly(A) RNA from each tissue.
BBAEXP 91427 5-7-00
www.elsevier.com/locate/bba
Short sequence-paper
Molecular cloning of the murine cMOAT ATPase1 Friederike Fritz a , Jing Chen a , Paula Hayes a , F.M. Sirotnak a
a;b;
*
Laboratory for Molecular Therapeutics, Program of Molecular Pharmacology and Experimental Therapeutics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021, USA b Graduate School of Medical Sciences, Cornell University, New York, NY 10021, USA Received 22 February 2000; accepted 10 May 2000
Abstract cMOAT encodes an ATPase within the family of cMOAT/MRP ATPases that functions as an ATP dependent, multispecific anion transporter within the canalicular surface of hepatocytes that has pharmacologic significance. We describe here the cloning of a murine cMOAT cDNA isolated from mouse liver. The open reading frame of this cDNA incorporates 4627 nucleotides encoding 1309 amino acids with 77.5% and 86.7% identity with the human and rat encoded amino acids, respectively. Northern blotting showed that the expression of cMOAT mRNA occurs primarily in mouse liver in the form of two variants with approximately 5.6 and 7.8 kb of sequence each. cMOAT mRNA was also detected in mouse kidney at a low level but was not detected in other mouse organs or tumors except the Hep 1-6 murine hepatoma where expression was also in the form of the same two mRNA variants. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Canalicular multispeci¢c anion transporter ; Murine cDNA
The multispeci¢c organic anion transporter (cMOAT) is a member of the MRP family of ATPases involved [1^4] in the hepatobiliary transport and excretion of compounds from hepatocytes across the bile canalicular membrane. Mutation in rat cMOAT has been shown [3,5,6] to result in hyperbilerubemia, a model for Dubin^Johnson syndrome in humans [7]. Recent studies have shown [8] that cMOAT is capable of excreting the folate analogue, MTX, in bile. This and other recent ¢ndings associating [9,10] overexpression of cMOAT with cis-platinum resistance in tumor cells establish the pharmacologic relevance of this gene. As a preface to future work using the mouse as a model for studies on di¡erential tissue expression of cMOAT, we sought to isolate and sequence a cDNA clone encoding murine cMOAT. In the present report, we provide the total coding sequence for cMOAT cDNA and a portion of its 3P untranslated region (UTR) that was de-
Abbreviations : ORF, open reading frame; UTR, untranslated region; cMOAT, canalicular multispeci¢c anion transporter * Corresponding author. Fax: +1-212-794-4342; E-mail : [email protected] 1 Sequence data presented in the paper have been submitted to the EMBL/GenBank data libraries under accession number AF227274.
rived from poly(A) RNA isolated from mouse liver. We also compare its homology to the published rat and human cMOAT sequence. Partial length clones of murine cMOAT were obtained from a Lambda gt11 mouse liver cDNA library (Clontech) using a rat cMOAT cDNA probe prepared by PCR. Hybridization and PCR as well as Northern blotting were performed as originally described [11]. In view of the total length of the cMOAT cDNA anticipated from Northern blotting (see below), we only sought to obtain the entire sequence of the open reading frame (ORF). To do this, it was necessary to perform 5PRACE PCR (Gibco BRL) and chromosome walking (Genome Walker, Clontech). Some sequence of the 3PUTR of this cDNA was also obtained. In Fig. 1A, the nucleotide and deduced amino acid sequence of the ORF for mouse cMOAT cDNA is provided along with some sequence of the 3PUTR. The ORF encodes 1543 amino acids with a predicted molecular mass of 171 kDa. Comparison of amino acids for mouse, rat and human orthologs (Fig. 1B and Table 1) revealed 85.2% and 76.5% identity, respectively, between rat and human sequences. Northern blotting of poly(A) RNA from various mouse tissues and tumors showed a high level of expression of cMOAT in liver and low level of expression in kidney (Fig. 2). This expression in liver occurred in the form of two variants of approximately 5.6 and 7.8 kb of sequence. Thus, these transcripts incorporate
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 1 3 2 - 9
BBAEXP 91427 5-7-00
532
F. Fritz et al. / Biochimica et Biophysica Acta 1492 (2000) 531^536
Fig. 1. (A) Nucleotide and deduced amino acid sequence of murine cMOAT isolated from a mouse liver cDNA library and extended by 5PRACE and chromosome walking. (B) Amino acid sequence comparison of human, rat and mouse cMOAT. Alignment was performed with DNAStar software, using the method of Lipman^Pearson. The sequence alignments are designated as non-homologous (white on black), conserved (black on gray, identical (black on white), weakly similar (white on dark gray) and block similar (dark gray on black).
Table 1 Amino acid similarity of cMOAT between di¡erent species cMOAT/hu cMOAT/rat cMOAT/mu
cMOAT/hu
cMOAT/rat
cMOAT/mu
100 77.5 77.5
100 86.7
100
Fig. 1 (continued).
extremely lengthy 3P and (or) 5PUTR sequences since the ORF represents only 4.62 kb of sequence. Interestingly, there was little, if any, expression of cMOAT in all other mouse tissues. Low level expression in kidney occurred in the form of two variants between 5 and 6 kb in length. In summary, cMOAT is highly conserved among three mammalian species with expression occurring primarily in liver. Supported in part by Grants CA08748 and CA56517 from the National Cancer Institute.
Alignment was performed with DNAStar software, using the Lipman^ Pearson protein alignment method.
BBAEXP 91427 5-7-00
F. Fritz et al. / Biochimica et Biophysica Acta 1492 (2000) 531^536
Fig. 1 (continued).
Fig. 1 (continued).
BBAEXP 91427 5-7-00
533
534
F. Fritz et al. / Biochimica et Biophysica Acta 1492 (2000) 531^536
Fig. 1 (continued).
BBAEXP 91427 5-7-00
F. Fritz et al. / Biochimica et Biophysica Acta 1492 (2000) 531^536
Fig. 1 (continued).
BBAEXP 91427 5-7-00
535
536
F. Fritz et al. / Biochimica et Biophysica Acta 1492 (2000) 531^536
Fig. 1 (continued).
References [1] Z.C. Gatmaitan, I.M. Arias, Physiol. Rev. 75 (1995) 261^275. [2] P.J. Mieir, Am. J. Physiol. 269 (1996) G801^G812. [3] K. Ito, H. Suzuki, T. Hirohashi, K. Kume, T. Shimizu, Y. Sugiyama, Am. J. Physiol. 272 (1996) G16^G22. [4] M.G. Belinsky, L.J. Bain, B.B. Balsara, J.R. Testa, G.D. Kruh, J. Natl. Cancer Inst. 90 (1998) 1735^1741. [5] S. Hosakowa, O. Tagaya, T. Mikami, Y. Nozaki, A. Kawaguchi, M. Shamato, Lab. Anim. Sci. 42 (1992) 27^34. [6] P.L. Jansen, W.H. Peters, W.H. Lamers, Hepatology 5 (1985) 573^ 579. [7] I.N. Dubin, F.B. Johnson, Medicine 33 (1954) 155^172. [8] M. Masuda, Y. I'izuka, M. Yamazaki, R. Nishigaki, Y. Kato, K. Ni'Inuma, S. Hirashi, Y. Sugiyama, Cancer Res. 57 (1997) 3506^ 3510. [9] K. Taniguchi, M. Wada, K. Kohno, T. Nakamura, T. Kawabe, M. Kawakami, K. Kogotani, K. Obumura, S. Alsiyama, M. Kuwano, Cancer Res. 56 (1996) 4124^4129. [10] M. Kool, M. de Haas, G.L. Sche¡er, R.J. Scheper, M.J. Van Eijk, J.A. Juijn, F. Baas, P. Borst, Cancer Res. 57 (1997) 3537^3547. [11] T. Esaki, K. Roy, R. Yao, J. Galivan, F.M. Sirotnak, Gene 219 (1998) 37^44. Fig. 2. Northern blot analysis of cMOAT expression in various mouse tissues. A mouse cDNA probe was prepared by PCR and used to blot poly(A) RNA from each tissue.
BBAEXP 91427 5-7-00