INTRODUCTION

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THE MULTIDRUG resistance-associated proteins (Mrps) belong to family C of the ATP-binding cassette (ABC) superfamily and function as ATP-dependent export pumps for a variety of organic solutes (2, 11). One member of this family, human MRP2 (ABCC2) and rodent Mrp2 (Abcc2), is localized to the apical membrane of excretory organs, and in particular the canalicular membrane of hepatocytes, where it functions to export a diverse group of compounds, including sulfate, glutathione (GSH), and glucuronide conjugates (10, 12, 18). MRP2 mutations in humans lead to the Dubin-Johnson syndrome (8, 13, 19). However, the functional determinantes of this ABC transporter are not yet known.

Previous studies from our laboratory carried out in the evolutionarily ancient marine skate, Raja erinacea, indicate that a functional homolog of the Mrp2 export pump is present on the liver canalicular membrane of this marine vertebrate (14). Studies in intact skate, isolated perfused skate livers, and isolated hepatocyte cell clusters, demonstrate biliary excretion of the Mrp2 substrates GSH, glutathione S-conjugates, and other organic anions (1, 3, 15, 17). Antibodies directed against mammalian Mrp2-specific epitopes labeled a 180-kDa protein band in skate liver plasma membranes and stained canaliculi by immunofluorescence, indicating that skate livers express a homologous protein (14). Moreover, studies with skate liver plasma membrane vesicles demonstrated ATP-dependent transport of GSH and dinitrophenyl-S-glutathione (14). These previous findings suggest that Mrp2-like transporters arose early in vertebrate evolution. The present study reports the molecular identification, tissue distribution, and cellular localization of this evolutionarily ancient Mrp2 from the little skate.

	EXPERIMENTAL PROCEDURE

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Materials. [-³²P]dCTP was purchased from Amersham-Pharmacia. All other chemicals and reagents were obtained from Sigma, NEN Life Science, J. T. Baker, Invitrogen, and Clontech.

Skate Mrp2 gene fragment preparation. We first analyzed human, rabbit, rat, mouse, Clostridium elegans, and yeast MRP2/Mrp2s from Genbank and aligned their protein sequences with DNAStar Software. Two highly conserved regions were identified with the sequence of VGRTGAGK---DEATAAVD, separated by 120 amino acids. Based on these sequences, two degenerate oligonucleotide primers were made. They are, forward 5'-GTNGGNMGNACNGGNGCNGGNAA-3' and reverse 5'-TCNACNGCNGCNGTNGCYTCRTC-3'. Five micrograms of skate liver or kidney total RNA were used for reverse transcription to generate single-strand cDNA as a PCR template. After touchdown PCR, a 410-bp band was amplified from both skate liver and kidney total RNAs. DNA sequencing, performed by the W. M. Keck Biotechnology Center at Yale University, confirmed that this DNA fragment encoded for a portion of an Mrp2 protein.

Northern blot analysis. Total RNA was isolated from skate tissues as previously described (4). Total RNA (15 µg) was loaded in each lane on a 1% agarose gel. After electrophoresis, the gel was treated with 0.05 N NaOH to partially hydrolyze the RNA. The gel was then neutralized with Tris · HCl buffer (pH 7.4) and transferred to a nylon membrane. The blot was hybridized with either the ³²P-labeled skate Mrp2 gene fragment or skate -actin probe.

Full-length cDNA cloning. A skate liver cDNA library was screened using a [³²P]PCR fragment as probe (16). Several positive clones were selected from a million plaques, with the longest insert of 4 kb encoding for 900 amino acids and the 3'-untranslated region (UTR) of the skate Mrp2 gene. To obtain the full-length skate Mrp2 clone, the 5'-end 300 bp of the 4-kb clone was used as probe to rescreen the library. However, only four clones were obtained from 1.5 million plaques, each with the same size as the original 4-kb clone. Therefore, to obtain the 5' of this skate Mrp2 gene, 5'-rapid amplification of cDNA ends (RACE) was conducted using a SMART RACE cDNA Amplification Kit (Clontech). A 2.1-kb gene fragment was amplified out by PCR and cloned into a pTrcHis vector with a TOPO TA Cloning Kit (Invitrogen) for DNA sequencing. The resulting DNA sequence revealed that this 2.1 kb encoded for the skate Mrp2 NH₂-terminal portion with 200 bp overlapping with the original 4-kb clone.

Antibody preparation and Western blot analysis. A 21-amino acid peptide CGHFYRMAMEAGVTMEKNTAL from the COOH-terminal sequence of skate Mrp2 was made by the W. M. Keck Biotechnology Center at Yale University. The peptide was conjugated to keyhole limpet hemocyanin and sent to Chemicon International (Temecula, CA) to develop a rabbit polyclonal antibody. An Escherichia coli-expressed GST-skate Mrp2 construct was made for titrating skate Mrp2 antisera and purifying the polyclonal antibody. The antibody was affinity purified by conjugating GST-skate Mrp2 protein on glutathione agarose beads. For Western blot analysis, 25 µg skate liver plasma membranes were resolved in 7% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The blot was blocked with 5% nonfat milk and incubated with the purified antibody (1:500 dilution) for 2 h at room temperature or overnight at 4°C. Horseradish peroxidase-conjugated goat anti-rabbit IgG was used as secondary antibody. The blot was visualized using an enhanced chemiluminescence kit.

Immunofluorescence studies. Indirect immunofluorescence was performed on frozen sections from skate liver, kidney, and intestine. Briefly, livers were perfused rapidly with ice-cold elasmobranch Ringer, and small cubes of tissue from various lobes were snap-frozen in liquid nitrogen and then stored in liquid nitrogen until cut. Tissue was placed on a pedestal of optimum cutting temperature embedding medium (Miles, Elkhart, IN), and 5- to 7-µm frozen sections were cut and placed on poly-L-lysine-coated glass slides. Sections were treated with acetone at 20°C for 10 min and air-dried, and nonspecific sites were blocked with 1% BSA in PBS containing 0.05% Triton X-100. Affinity-purified polyclonal antibody to skate Mrp2 or the preimmune serum was diluted to 1:250 in blocking buffer and incubated on the sections for 2 h at room temperature. Secondary antibody was Alexa 594 anti-rabbit IgG (Molecular Probes, Eugene, OR). All fluorescent imaging was performed on a Zeiss LSM 510, and digital images were recorded on an Iomega Zip disc and processed with Adobe Photoshop. This study followed the guiding principles for research involving animals and human beings.

	RESULTS

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A full-length skate Mrp2 was identified that contains 5,870 bp and encodes for 1,564 amino acids, with 197 bp at the 5'-UTR and 1,033 bp at the 3'-UTR (Genbank accession no. AF486830). Genbank BLASTing indicates that this skate Mrp2 is an ABC transporter that shares the highest identities with human and rabbit MRP2/Mrp2 (56%), 54% identity with mouse and rat Mrp2s, 44% identity with C. elegans Mrp2, 49% identity with human MRP1, and 48% identity with human MRP3. Protein sequence analysis, using the Kyte-Doolittle algorithm, reveals that skate Mrp2 has a similar hydrophilicity plot with human MRP2. Computer modeling predicts 17 transmembrane domains, 2 ABCs, 2 N-glycosylation sites, 5 protein kinase C phosphorylation sites, and 1 tyrosine phosphorylation site (Fig. 1A). An apical targeting motif (TAL) has also been found at the COOH terminus of skate Mrp2. A phylogenetic analysis indicates that skate Mrp2 is closely related to mammalian MRP2/Mrp2s (Fig. 1B).

View larger version (53K): [in this window] [in a new window]   Fig. 1.   A: deduced amino acid sequence of skate multidrug resistance protein (Mrp2) derived from the contiged full-length cDNA clones. The predicted 17 transmembrane domains are highlighted, the Walker A and B motifs are underlined, the protein kinase C and tyrosine kinase phosphorylation sites are in bold type, the N-glycosylation sites are labeled with an asterisk (*), and the apical targeting tag is in bold type with an underline. B: phylogenetic analysis of MRP2/Mrp2s and other human MRPs with Clustal W algorithm. The rooted tree was generated by DNAStar Software. The scale numbers indicate the evolution mutation rate.

Northern blot analysis of skate tissue RNA indicates that skate Mrp2 is a 6-kb transcript that is highly expressed in liver, intestine, and kidney (Fig. 2). Skate -actin was probed as a loading control. Western blot analysis, using a purified anti-skate Mrp2 polyclonal antibody, demonstrated that the fully expressed skate Mrp2 was a 180-kDa protein when analyzed from skate liver plasma membranes (Fig. 3). Peptide competition completely eliminated the staining of this 180-kDa band.

View larger version (105K): [in this window] [in a new window]   Fig. 2.   Northern blot of multiple skate tissues for skate Mrp2. Total RNA (15 µg) was loaded for each lane. Lane 1, brain; lane 2, heart; lane 3, intestine; lane 4, kidney; lane 5, liver; lane 6, pancreas; lane 7, rectal gland; lane 8, spleen; lane 9, stomach; lane 10, testes.

View larger version (65K): [in this window] [in a new window]   Fig. 3.   Western blot analysis using purified anti-skate Mrp2 peptide antibody. Skate liver membrane vesicles (25 µg) were loaded in each lane. Lane 1, incubation with preimmune sera; lane 2, incubation with recombinant protein purified antibody; lane 3, incubation with purified antibody that has reacted with immunopeptide.

Immunofluorescence studies indicate that skate Mrp2 is localized at the canalicular membrane of skate hepatocytes, the apical membrane of proximal convoluted tubes of the skate kidney, and the apical membrane of enterocytes in the skate small intestine (Fig. 4). Preimmune serum did not reveal any specific staining pattern in any of the tissues.

View larger version (111K): [in this window] [in a new window]   Fig. 4.   Immunofluorescence staining of skate Mrp2 in skate liver, kidney, and intestine. Preimmune serum demonstrates no specific staining in liver (A), kidney (C), or intestine (E), whereas the affinity-purified anti-skate Mrp2 antibody shows strong reactivity with the canalicular membrane of the hepatocyte (B), the apical membrane of the proximal convoluted tubule of the kidney (D), and the apical enterocyte membrane in the small intestine (F). Bar = 20 µM.

	DISCUSSION

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In this study, we have identified an evolutionarily primitive ABC protein from the small skate, R. erinacea, a marine vertebrate that evolved about 200 million years ago. This skate gene belongs to the Mrp/cystic fibrosis transmembrane receptor family of ABC proteins, and several lines of evidence indicate that it is the ortholog of the MRP2/Mrp2 gene. The full-length skate Mrp2 cDNA is 6 kb, and it encodes for a protein of 1,564 amino acids that exhibits the highest identity (56%) with human MRP2. Phylogenetic analysis indicates that skate Mrp2 is closely related to its mammalian counterparts. Moreover, sequence alignments indicate that amino acids that are mutated in MRP2 in Dubin-Johnson syndrome patients (8, 9, 13, 19) are conserved in skate Mrp2 (Fig. 5), suggesting that the conserved regions between skate Mrp2 and human MRP2 may encode for important functional determinants of this member of the ABCC gene family. Further sequence analysis reveals that transmembrane domains 1, 9, 11, 16, and 17 are the most conserved domains between skate Mrp2 and mammalian MRP2/Mrp2s, suggesting that those domains may play an important role in Mrp2 substrate specificity or function (Table 1). Indeed, previous mutational studies have demonstrated that specific positively charged amino acids in transmembrane domains 9, 11, 16, and 17 play a critical role in Mrp2 substrate recognition and transport activity (7).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Regional alignment of MRP2/Mrp2s from different species using DNAStar software with Clustal W algorithm. MRP2 mutants from Dubin-Johnson Syndrome patients are conserved in skate Mrp2. The mutated and deleted amino acids are in bold type.

The tissue distribution and subcellular localization of skate Mrp2 provide additional evidence that this skate gene is the Mrp2 ortholog. Of various Mrp proteins that have been identified thus far, Mrp2 is the only one that is localized to the apical membrane of polarized epithelial cells, including the apical membranes of liver, intestine, and kidney cells. The present study demonstrates that skate Mrp2 is abundantly expressed in skate liver, intestine, and kidney, as demonstrated by Northern blot analysis, an expression pattern that is similar to that in mammals (2, 11). Immunoblots of skate liver plasma membranes show that skate Mrp2 is a 180-kDa protein. This size is slightly larger than predicted (172 kDa), suggesting that skate Mrp2 may be glycosylated or/and phosphorylated when fully expressed in the intact tissue. Moreover, immunofluorescence studies localized skate Mrp2 to the apical membrane of hepatocytes and to the apical surface of epithelial cells in the kidney and intestine. Furthermore, sequence comparison with mammalian MRP2/Mrp2s indicates that the last three amino acids (TAL) are a PDZ-interacting motif, which may be important in modulating Mrp2 targeting to the apical membrane of hepatocytes (Fig. 6; see Ref. 6).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Alignment of MRPs/Mrps COOH-terminal region using DNAStar software with Clustal W algorithm. The apical membrane targeting sequences are in bold type.

Although functional studies with this skate protein have not yet been carried out, our previous in vivo and in vitro studies in the skate indicate that the substrate preference for this canalicular membrane protein is generally similar to that for mammalian MRP2/Mrp2s. Skate liver is able to transport many organic anions into bile in relatively high concentrations, including sulfobromophthalein, bilirubin, biliverdin, carboxyfluorescein diacetate, S-dintrophenyl glutathione, and lucifer yellow (1, 3, 5, 15, 17). Altogether these studies indicate that skate Mrp2 is most likely the responsible gene product for this canalicular excretion.