a homologue of the mammalian multidrug resistance gene (mdr) is functionally expressed in the...

E L S E V I E R Biochimica et Biophysica Acta 1262 (1995) 113 - 123

BB Biochi]ic~a et Biophysica A~ta

A homologue of the mammalian multidrug resistance gene (mdr) is functionally expressed in the intestine of Xenopus laevis

Gonzalo Castillo, Heng-Jia Shen, Susan Band Horwitz *

Departments of Molecular Pharmacology and Cell Biology, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA

Received 8 December 1994; accepted 16 February 1995

Abstract

P-glycoprotein is an integral membrane protein that functions in multidrug resistance (MDR) cells as a drug efflux pump to maintain intracellular concentrations of antitumor drugs below cytotoxic levels. A homologue of the mammalian mdr gene has been isolated and characterized from Xenopus/aevis (Xe-mdr). The cDNA was isolated from a tadpole cDNA library using the full length mouse mdrlb cDNA as a probe. The Xe-rrutr encodes a protein that is 66% identical to the mouse mdrlb and 68% identical to the human mdrl. The predicted structure of the Xe-mdr gene product identifies twelve membrane spanning domains and two ATP binding sites both of which are the hallmark of the ABC (ATP binding cassette) transporters. Xe-mdr mRNA is expressed as a single message of 4.5 kb and is found predominantly in the intestine. Xe-mdr message is increased 3- to 4-fold in the ileum compared to the rest of the small intestine. In situ hybridization of sequential sections from the small intestine localized the expression of the Xe-mdr to the cells lining the lumenal epithelium. Brush border merabrane vesicles prepared from the small intestine of Xenopus laevis effluxed vinblastine in an ATP-dependent manner. Efflux was decreased by verapamil, a known inhibitor of P-glycoprotein function. These studies indicate that the structure of Xe-mdr has been conserved zLnd suggest that the protein has a role in maintaining the function of the normal intestine in Xenopus.

Keywords: Multidrug resistance; P-glycoprotein; Transport; (X. laevis)

1. Introduct ion

Mammalian cell lines grown in tissue culture often develop the multidrug resistance (MDR) phenotype when being selected for resistance to a single drug. Such cells are resistant to a range of hydrophobic drugs and compounds to which they have not been exposed [1-5]. The MDR phenotype is associated with the overproduction of a family of high molecular weight membrane glycoproteins, referred to as P-glycoproteins. The latter are thought to act as efflux pumps to maintain intracellular drug concentrations below cytotoxic levels in a process mediated by ATP [6-8]. There are two members of the mdr gene family in humans and three members in mouse [5,9-16]. In the

The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number U17608.

* Corresponding author. Fax: + 1 (718) 8298705.

0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0167-4781(95)00056-9

mouse, the class I genes, mdr lb and mdr la that are also referred to as mdrl and mdr3, respectively, confer the MDR phenotype [17-19]. Recent studies in which ho- mozygous disruption of the mdr la gene gave rise to mice that were up to 100-fold more sensitive to the neurotoxic drug ivermectin than mice carrying the normal gene, indicated that the product of the mdrla gene has a role in maintaining the blood-brain barrier. Absence of the gene also induced an increase in vinblastine accumulation in several tissues including the brain [20]. The class II gene, mdr2, is not involved in drug resistance, however it is expressed in the apical membranes of bile canaliculi. Mice with a disruption of the mdr2 gene displayed liver pathol- ogy and their bile had a complete absence of phosphatidyl- choline, the most abundant phospholipid in normal bile [21]. In addition, secretory vesicles prepared from yeast that had been transfected with mdr2, translocated phospha- t idylcholine in an ATP and Mg 2÷ dependent manner that was inhibited by vanadate and verapamil [22].

Analysis of the sequence predicted for P-glycoprotein,

114 G. Castillo et al. / Biochimica et Biophysica Acta 1262 (1995) 113-123

indicated that it was composed of two homologous halves, each encoding six putative transmembrane domains and an ATP binding site [9,11,12,18,23]. The two halves are connected by a linker region that contains the major sites for P-glycoprotein phosphorylation [24,25]. These features define a rapidly expanding family of membrane proteins called the ABC (ATP binding cassette) transporters [26].

The two class I genes in mouse are expressed in a tissue specific manner suggesting that their function is related to their site of expression. The mouse mdrlb is found predominantly in the adrenals and pregnant uterus [27,28]. mdrla is expressed mostly in the intestine, particularly in the villus epithelium of the small intestine [29]. Intestinal expression is a common characteristic of both lower and higher organisms [30].

Xenopus laevis is an aquatic organism that lives in an environment filled with xenobiotics, products of plant and animal degradation that are in direct contact with its gut. The natural habitat of the organism suggested that P-glycoprotein could be an important component of the intestine that may have a role in the survival of Xenopus. A cDNA from Xenopus laevis that is a homologue of mammalian mdr has been cloned and shown to be expressed in the intestine. The structure and function of the Xenopus mdr is highly conserved.

2. Materials and methods

2.1. Screening of a tadpole library

The mouse mdrlb full length cDNA [14] was used to screen an XTC (Xenopus tadpole cell line) oligo(dT) cDNA library constructed in Azap vector, a generous gift of Igor Dawid. Hybridization was performed at 60 ° C in 1 M NaCI, 50 mM Tris-HC! pH 8.0, 1% SDS, and 10% dextran sulfate. Filters were washed at 60°C in 2 × standard saline citrate (SSC; 1 x SCC is 0.15 M NaC1, 0.15 M sodium citrate, pH 7.0). The cDNA fragment, B (Fig. 1), was subcloned into pBluescript SK (Stratagene) and sequenced using the Sequenase system (U.S. Biochem- ical).

2.2. PCR cloning methods

To obtain the 5' end of the gene, total RNA was prepared from intestine of an adult frog with Tri Reagent TM

(Molecular Research Center), per the manufacturer's in- structions. First strand cDNA synthesis was done using avian myeloblastosis polymerase reverse transcriptase (RT) (Boehringer-Mannheim) and an oligo dT(25) primer. The product was extended further with terminal deoxynucleotidyl transferase (TdT) (U.S. Biochemicals). An aliquot of this reaction served as a template in a PCR reaction using primers 1 and 3 (Fig. 1) [31]. The PCR reaction was

performed for 30 cycles in 100 /.d of reaction buffer (10 mM Tris-HC1 pH 8.3, 50 mM KCI, 25 mM MgCI 2, 0.001% (w/v ) gelatin) for 1 min at 94°C, 1 min at 50°C and 1 rain at 72 ° C, using recombinant Taq polymerase (Boebringer-Mannheim).

To obtain the 3' end of the gene, first strand synthesis was performed with RT and primer 2 [32]. The product of this reaction was used as a template in a PCR reaction that was performed as described above, except that primers 2 and 4 were used. The PCR products were cloned into pCR~MII (Invitrogen) and sequenced. Sequence analysis was done with the University of Wisconsin Genetics Com- puter Group Software Package.

2.3. Northern blot analysis

Adult Xenopus laecis were obtained from Nasco Bio- logicals (Fort Atkinson, WI). Total RNA was prepared from tissues of the adult frog with Tri Reagent TM. 10 /zg of total RNA was resuspended in FORMAzol TM (Molecular Research Center), separated on a 1.5% agarose gel containing 0.66 M formaldehyde and Mops buffer (1 X Mops is 40 mM morpholinopropanesulfonic acid, 10 mM sodium acetate and 10 mM EDTA) and transferred by capillary action to GeneScreen Plus (New England Nuclear) in 10 X SSC. The membrane was incubated at 80 ° C for 2 h and hybridized with fragment B (Fig. 1) that was labeled with [32p]dCTP (Amersham) by random priming (Boeh- ringer-Mannheim). The blot was hybridized for 15 h in 1 M NaC1, 10% dextran sulfate (Pharmacia), 50 mM Tris- HCI pH 7.5, 1% SDS and 10 ng /ml herring sperm DNA at 60 °C. The final wash was carried out at 60°C in 2 x SSC. Blots were exposed to X-OMAT AR film (Kodak) at - 80 ° C.

2.4. Western blot analysis

To isolate intestinal epithelial cells, a modification of the distended intestinal sac method was used [33]. In- testines from adult Xenopus laevis were surgically re- moved and were thoroughly washed in Frogs's Ringer Solution (115 mM NaC1, 2.5 mM KCI, 1.8 mM CaCI 2, 5 mM 4-(2-hydroxyethyl)-l-piperazineethane sulfonic acid (Hepes), pH 7.4). Intestinal segments were filled with CMFM buffer (88 mM NaC1, 1.0 mM KC1, 2.4 mM NaHCO 3, 7.5 mM Tris-HC1, pH 7.6) under pressure and incubated for 40 min in CMFM at room temperature. The mucosal cells were resuspended in 300 mM mannitol, 20 mM Hepes and 10 mM Tris-HC1, pH 7.5 and 4 mM MgC1 z. Membranes were prepared by nitrogen cavitation followed by differential centrifugation [34], analyzed by SDS-PAGE on a 10% gel [35] and transferred to nitro- cellulose [17]. The blot was probed with a rabbit poly- clonal antibody made against amino acids 907-924 (peptide

G. Castillo et al. / Biochimica et Biophysica Acta 1262 (1995) 113-123 115

#5) from the mouse mdrlb gene product [36]. The antibody was used at a dilution of 1:20 000 and detection was with the ECL system (Amersham).

and hematoxylin. Photography was done with a Zeiss Axiophot microscope and Ektachrome 400 film.

2.6. Isolation of brush border membrane vesicles

2.5. In situ hybridization

In situ hybridization was carried out as described [37]. Briefly, tissue from the sraall intestine was obtained from adult Xenopus laevis and fixed with paraformaldehyde in phosphate-buffered saline (PBS). Paraffin sections (6 /xm) were mounted on silanized slides (Electron Microscopy Systems), rehydrated, digested with 10 p ,g/ml proteinase K for 15 min at 37°C and washed with glycine in PBS. The slides were treated with 0.1 M triethanolamine, acety- lated in 0.25% acetic anhydride for 10 rain and dehydrated in a graded series of ethanol solutions. Sense and antisense probes were prepared from a pBluescript plasmid containing the B fragment. The plasmid was digested with BamHI (sense) or XhoI (antisense) and transcribed in vitro with T7 or T3 polymerase, respectively, in the presence of [35S]UTP (New England Nuclear). The slides were hybridized at 55°C in 50% formamide, 4 X SSC, 1 X Denhardt's solution (0.1% bovine serum albumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone), 10% dextran sulfate, 10 mM dithiothreitol, 50 ng /ml herring sperm DNA and 25 ng /ml tRNA. The final wash was performed at room temperature in 0.2 X SSC, containing 0.1% /3-mercapto- ethanol. The slides were d!ipped in Kodak NTB-2 emulsion that was diluted 1:1 in water and incubated for 2 weeks at 4 ° C. After development, the slides were stained with eosin

Epithelial cells were isolated as described above and membrane vesicles were prepared by a modification of the magnesium precipitation method [38,39]. The cell suspen- sion was diluted 5-fold with ice cold distilled water containing 100 units/ml aprotinin and 0.1 mM PMSF. The cells were homogenized in a blender for 3 min at high speed and MgC12 was added to a final concentration of 10 mM. The sample was incubated on ice for 15 min prior to centrifugation at 2300 × g for 15 min. The supernatant was centrifuged at 21 000 × g for 30 min. The pellet was resuspended in 60 mM mannitol, 5 mM EGTA, 12 mM Tris-HCl pH 7.5 and homogenized with a Dounce homog- enizer. MgC12 was added to a final concentration of 10 mM and the procedure repeated. The pellet was resuspended in 300 mM mannitol, 20 mM Hepes, 5 mM MgGlu (2,3,4,5,6-pentahydroxy caproic acid, hemimagnesium salt), 12 mM Tris-HC1 pH 7.5, homogenized and centrifu- gated at 21 000 × g for 60 min. The pellet was resuspended in 300 mM mannitol, 20 mM Hepes, 10 mM Tris-HC1, pH 7.5 and 4 mM MgC12.

2.7. Transport of vinblastine in brush border membrane t,esicles

Vesicles were loaded with 1 /zM [3H]vinblastine, 3 mM ATP, 3 mM creatine phosphate and 5 /zg creatine phos-

51

Td'r~dGTP

PCR

A i

B

I RT

~ PCR

A(n ) 3'

4

C

PRIMER 1 5' -GCGGCCGCATGCGAATTCTGCACCCCCCCCCCCCCC- 3' PRIMER 2 5' -CGCGGAA'n'ccCCGGGCGCGC i i i i i t i i i t , t ,- 3' PRIMER 3 5'-TGTGGTCATTGGCCCTGCAGT- 3' PRIMER 4 5' -TGGC'I'rl 'GC'n'GCTCAGAATG- 3'

Fig. I. Cloning strategy used to obtain the three overlapping fragments (A,B,C) for the full length Xenopus laeL'is mdr gene. Fragment B was isolated from a tadpole eDNA library. Fragmeats A and C were isolated by anchored PCR. The numbered arrows indicate the primers used. RT, reverse transcriptase; TdT, terminal deoxynucleotidyl transferase.


phokinase using the freeze-thaw method [40], placed on liquid nitrogen for 5 min and then on ice for 1 h. For the drug transport studies, loaded vesicles were diluted 1:1000

in membrane buffer at time zero. Aliquots were taken at the indicated time points, vacuum filtered, washed and radioactivity determined.

1 tttggttcagcagcatcccaccttgctgcct taaat cggac tttaacaagtgtatcaagtgcaacagtaactctaggataaacctacccgt tggtgaaagaggt tactccagat tctaaa 121 cATGGAGCCAGAGCAGAAGACTC.CGCAGAACGGTTCAGC~GAcATCG~TGTCGCCATTTCAGAcCC~AATrCAAACAGTAAAGAAAAGAAA~T~Tc~~

1 M E P E Q K T A Q N G S A D I A V A I S D P N S N S K E K K G F F S K F K K K K 241 AGAAAAAACGGAGAAACCCCCAAAGGTTC.C~GTGTrTACTATGTTTcC.CTATTCTAGTACAT~C~ACAAGATGCTAATGCTGTT~GGTACCATAGCATCGCTTGC~Ac~T~T 41 E K T E K P P K V G V F T M F R Y S S T S D K M L M L F G T I A S L A H G A A L

361 GC~QCTAATGATGCTTGTGTT~GATGACTC~cAGcTTTGTTAATGTTGGACAGGTTGATACAGGC4~TTTACATGGGAAT~TA~AT~T~~CcA 81 P L M M L V F G E M T D S F V N V G Q V D T G N F T W E S M I N A S R E L Q G Q

481 AATGACCACATATGCCTATTATTA~TCAGGcTTGGGATTTC.GAGTGATGCTTTGT~CCTA~ATTCAGATTTCCTTCTGGACACTCTCAGC~GGTAGACAC4tTTAAAAAAA~ 121 M T T Y A Y Y Y S G L G F G V M L C A Y I Q I S F W T L S A G R Q I K K I R S N 601 CTTTTTTC~GCTGTGCTGCGGCAGGAGATTGGA~GGTTTGATAT~AATC~TGCAGGAGAACTGAACACACGACTCACAG~TGAT~TTTccAAAATT~~TC~T~T 161 F F H A V L R Q E I G W F D I N D A G E L N T R L T D D V S K I N E G I G D K I 721 TGCAATGCTGCTTCAGTCAT TAACGACATTGGTGAC TGGcTTCATTATTGGCTTTATTAAAGGATGGAAGcTGAcTTGGGTTATGGGGG•TATTAGTCCAATTATGGGACTCTCTGCTGC 201 A M L L Q S L T T L V T G F I I G F I K G W K L T W V M G A I S P I M G L S A A 841 TATCTGG~GGTATTGT CTGCATTCACTAACAAAGAGCTCAAAGCCTACGCCAAAGcTGGGGcTGT~GCTGAAGAGGTTCTTTCGTcTATT~GACTGTGTTTGCCTTTGGTGGTCA 241 I W A K V L S A F T N K E L K A Y A K A G A V A E E V L S S I R T V F A F G G Q 961 AAATAAAGAAATAcATAGGTATGAAAAAAATCTAC~%GGAcGCAAAAAAAATAGGAATAAAAAAAGCTATAACTGCCAATGTGTCCATTGC~TT~ TTTC~TATAT~A~CTA 281 N K E I H R Y E K N L E D A K K I G I K K A I T A N V S I G F A F L M I Y A A Y

1081 TTCAC TCGCC TTT~GTATGGTACCACCCTGATTATTGATGGAGGTTATACCAT~GGCTCTGTTcTCACAGTATTCTTTGcAGTCATCATTGGTGC TTTTGCTGTI~GACAAACCTCTCC 321 S L A F W Y G T T L I I D G G Y T I G S V L T V F F A V I I G A F A V G Q T S P

1201 AAATATTGAAGCTTTTGCCAATGCCAGGGGGGCAGCATAcACAATATTCAACATCATTGATAATCAACCCAAAATAGACAGcTTTr CTAAAGAAGGCTTGAAACCGGACAAGATTAAAGG 361 N I E A F A N A R G A A Y T I F N I I D N Q P K I D S F S K E G L K P D K I K G

1321 AGACATTGAATTTAAAAATGTTATATTCACTTATCCATCTAGAAAGGATATTCAGGTTCTGAAGGGCTTGAACCTGAATATACCAAGCGGTAAAAc CGTTGCTTTAGTTG~GCAGTGG 401 D I E F K N V I F T Y P S R K D I Q V L K G L N L N I P S G K T V A L V G S S G 1441 TTGTGGAA~AAGTACAACAGTTCAGCTCATCCAGAGGTTCTATGACCCTC~AGACGGCGTTATCACACTTGATGC~CAAC~CATTCGTTCTTTAAATATCAGGTA~T~T~T 441 C G K S T T V Q L I Q R F Y D P E D G V I T L D G Q D I R S L N I R Y L R E I I

1561 AGGAGTGGTAAGCCAGGA~E CAATCTTATTTGACACCACAATTGcTGATAATATTCGTTATGGTCGGGAAGATGTGACAAAGGAGGAAATTGAAAGAGCAACTAAAGAA~ATA 481 G V V S Q E P I L F D T T I A D N I R Y G R E D V T K E E I E R A T K E A N A Y

1681 CGACT TTATCATGAAAC TGC CAGATAAATTGGAAAC TC TTG TTGC~GACq~G TGGCACGCAG CT C~ TGGGG GACA%AAGCAAAGC~T TG CCATTGC CAGGGCATTGG TT CGCAATCC CAA 521 D F I M K L P D K L E T L V G E R G T Q L S G G Q K Q R I A I A R A L V R N P K

1801 AATCC TCCTTCTAGATGAGGCGACATCTGCTCTGGATACAGAGAGTGAAGC TGTGGTTCAGTCTGC TCTGGATAAGGCAAGAGAGGGCCGTACCACAATTGTAGTTGCCCATCGTTTGTC 561 I L L L D E A T S A L D T E S E A V V Q S A L D K A R E G R T T I V V A H R L S

1921 CACTATACGAAATGCAAA~CAATC GCTGGCTTTGATAATGGTGTCATCGTTGAACAAG~GCCATAAGGAACTAATGGAAAGAGGAGC~GTTTACTTCAACCTGGTCACTCTC-CAGAC 601 T I R N A N A I A G F D N G V I V E Q G S H K E L M E R G G V Y F N L V T L Q T

2041 CGTGGAAACAAGTAAAGACACTGAAGAAGATTTAC.%AAC CCACATA TATC~%AAAGAAAATACCTGT TAC TCATACC CATTC CAACC TGGTCAGGAGGAAATC CAGCC GAA~AC~T~ 641 V E T S K D T E E D L E T H I Y E K K I P V T H T H S N L V R R K S S R N T I K

2161 GAGCAAAGTC CCAGAAACAGAAGATAAAGAAGTGC~TGAAGAGGAGAAGAAAAAGGAGGASGGTCC CCC~C CTGTCTCATTCTTCAAAGTrATGAAGTTGAACAAGC CAGAGTGGCC TTA 681 S K V P E T E D K E V D E E E K K K E E G P P P V S F F K V M K L N K P E W P Y

2281 TTTTGTGGTTGGAGTGATCTGTGCAATGATAAATGGTGCCACTCAGCCTGCATT~GCCATrATCTrCTCCAGGATTATTGGGGTGTTTGcTGGTCCAGTTTCACAAATGAGATCTGAAAG 721 F V V G V I C A M I N G A T Q P A F A I I F S R I I G V F A G P V S Q M R S E S

2401 CTCCATGT~E TCTTTGCTGTTTTTGGCACTr GGTGGGGTATC CTTCATTACATTCTTCCTGCAGGGATTCACCTTr GGGAAAGCTGGAGAAATTCTTACTATGAGACTGCGACTTGGGAG 761 S M Y S L L F L A L G G V S F I T F F L Q R F T F G K A G E I L T M R L R L G S

2521 TTTCAAATCCATGTTAAG~£AGGAAATCGGCTGGTr TGATGACTCCAAGAACAGTACGGGGGCACTGACGACAAGGCTTGCCACTGATGCTT~GT~~ ~G 801 F K S M L R Q E I G W F D D S K N S T G A L T T R L A T D A S Q V Q G A T G T R

2641 ACTGGCTTTGCTTGC TCAC~TGTAGCAAAT CTGGGCAC~J3C CATCATCATATCATTTATTTACGGATGGCAATTGACCCTTCTCATTTTGGCGATTGTTCCAGTCATCGCTGCTGCAGG 841 L A L L A Q N V A N L G T A I I I S F I Y G W Q L T L L I L A I V P V I A A A G

2761 CCTGGTGGAAATGAAAATGTTCGCTGGACATGCAAAAAAGGACAAAAAC-C~CT~GAAAAAGCAGGAAAC~TTTCAACCGATGCTGTTT~GAATATCAGAACTGTTGTGTCCCTGACCCG 881 L V E M K M F A G H A K K D K K E L E K A G K I S T D A V L ~ N I R T V V S L T R

2881 AGAGAGGAAATTTGAAGCGATGTATGAGAAAAGTcTGGAAGGGCcTTACAGGAATTCTATTAAGAAAGcCCATCTCCATGGATTC~CCT~T~GTCT~AT~TGTA~CTG 921 E R K F E A M Y E K S L E G P Y R N S I K K A H L H G L T Y G L S Q A H H V L C

3001 CCTATGCTGGGTGT~TTCCGTTTTGGGAGCC TATTTAGTGGTTGAAGGTTTAATC.KAGTTGGATC~GGTTTTTCTGGTCTCCTCAGCCATTGTGTTGGGTGC CATGGCCTTAGGCCAGAC 961 L C W V F S V L G A Y L V V E G L M K L D E V F L V S S A I V L G A M A L G Q T

3121 CAGCTCCTTTGCCCC TGACTATACTAAAGcCATGATTTCAGCGGCTCATATATTCAGCTTGTTGC~GAGAGTCCCACAGATTGACAGCTACAGCC~CCAGGGCGAC~CCAAAAAACTG i001 S S F A P D Y T K A M I S A A H I F S L L E R V P Q I D S Y S D Q G E K P K N ¢ 3241 CAGTGGGAACGTGGTATTCAAAGGGGTTAAT TTTAACTACC CTACACGGCCAGACATAACAGTGCTGCAGGGACTGGATATCTCCGTAAAGCAAGGGGAG~CCC~TT~G 1041 S G N V V F K G V N F N Y P T R P D I T V L Q G L D I S V K Q G E T L A L V G S 3361 CAGCGGCTGTGGAAAGAGTACGACCGTGTCACTAcTGGAC~GATTCTATGACCCGTTTGAAGGAGAAGTGTTGGTAGACGGCCTT~CTG~GAGGAATCTGAACATcCAGTGGGTGAGGGC 1081 S G C G K S T T V S L L E R F Y D P F E G E V L V D G L S V R N L N I Q W V R A 3481 GCAGATGGGAATCGT CTCCCAGGAGCCCAT~TTGTT TGACTGCAGCATTGGTGACAATA~ GCTTACGGGGATAACAACAGAAAASTGACGCAAC4%AGAAATAGAAACAGCAGCTAA~ 1121 Q M G I V S Q E P I L F D C S I G D N I A Y G D N N R K V T Q E E I E T A A K E 3601 AGCCAACATCCACAGCTTCATCGAATCTCTrACTC~TAAATACA~EACCCGCGTGGGAGATAAAGGCACCCAGCTCTCAGGGGGACAGAA~CAGCGTATA~T~GCT~T 1161 A N I H S F I E S L T D K Y N T R V G D K G T Q L S G G Q K Q R I A I A R A L I 3721 TCGGAAACCCAAAATCCTTCTC-CTGGACGAGGCCAC CTCTGCGCTCGACACAGAAAGTGAAAAGGT TGTGCAAGAAGCACTGGAeAAAGCCAGAATGGGCCGCACGTGTATTGTCATCGC 1201 R K P K I L L L D E A T S A L D T E S E K V V Q E A L D K A R M G R T C I V I A 3841 TCATCGACTATCGAC CATAcAGAATGCAGATAAAATAGCCGTCATACAGAATGGAAAGGTGGTAC~GCAAGGGACACATCAGCAGCTCCT~CAGCTCAAGGGCGTTTACTTTTCTTTAGT 1241 H R L S T I Q N A D K I A V I Q N G K V V E Q G T H Q Q L L Q L K G V Y F S L V 3961 GACTATACAGCTTGGCCACTCCTGAGAGAAGAGCCGATTACAACCTGAAAC CGCCACTCAGGAAACACCACTCACAAAGATGGATCCTGT~TCCTGTCCAGTTGCTGCTAAACACTGAGA 1281 T I Q L G H S * 4081 AATcTACTGCTCGTGTCACTCCTTGTGACTCCGACTACTACGGATAGTGTCAGTcCGTATAACCTGAAAAGAACGAGAAAGATCACAGTTGGGG(~GGAACATATGTTTTTGTTATTTCA 4201 GGAAATGTATACTAATTATTTGCCTTATCAATACTf TTATATACcGTTCATATTTTTCTATGTAcAATAcCTCAATTATT~GATAATTA~TTCTGTGTGATGCGCCTAAGCACTCCTGCC 4321 TTACGTTCCAAGTATTTC~TGCTTTTTATTAATCAGGGCAAGTCCTT~TTGCTTAGCTCTT ~ A ~

Fig. 2. Nucleotide and predicted amino acid sequence of Xenopus laevis mdr gene product. DNA sequence is presented in the 5' to 3' orientation. Untranslated sequences are in lower case letters and translated sequences are in upper case letters. Numbering of the amino acids begins in the first in frame AT(]. Putative N-glycosylation sites are underlined and the polyadenylation signal is boxed.

Hu-Mdr

i I_ ...........

MDLEGDRNGTARR,GAKKKNF

FKLNNKS

EKDKKEKKK

PT

VS

VF

SM

FR

YS

NW

LD

KL

~

D i FANAGNLEDLMSNI

TNRS DI N

DTG FF S -MNL E EDMT,YAYYY

S~

~R

Q

I HKI RKQ F FHAI MRQE I GWFD~/~DVGELNTRLTDDVS

HU-Mar2

............

----AAK

.......

TSAEGD-

ELGI SSKQ-R-

- T-TVKM-G-

-T .

....

D-Q .....

~~

- - -K-VDTAGNFS

- PV- F. S

L- LL -

PGKI

..... - ............

~ ~

T~

,~

- - - R - - - QK .

..............

N-TT ...........

Ha-Pgpl

............

----E-FS-.

..... R-O ......

GR--K

......

.--V .... T .... A

G-

--

R-

--

~

~----S--SV--IPT..

NATN-ATO--ASDI-..GK

......

T ..... T

g,

~V

~~

.......

QK .

.....

N .......................

.a-P~2

................

E-rSA

....

.. ~

-D

.... Ga--,

......

~-,-G--G

.... ~

.......

~<

4~

~i

~-

--

S- T

,- E..TSmP-,--Q

.... N-~W-.CS

..... AT ..... T~ ~

!~

~i

~

- - - "- - - 0~ .

.....

~ .

......................

Mu-Mdrla

............

----E-LK-.

..... RA,---S--GK--K

......

.--A .... T .... A

G-

--

R-

--

~~

--

-S

--

SV

--

VS

K

o. NST. -M--ADKRA/4-..AK

......

T ..... T

~;

~f

@~

,~

!~

.~

.......

QK .

.....

N .......................

Mu-,dr

i b

............

----ENLK-.

..... RAD- --S--GK--K

......

. --A-G--G

.... AD .....

C- ~

5~

q~

~

~N

-

- - S- T

K-E..

ASI-PS---Q-GP-S-LII

-NSS .....

AI .

....

T ~[~ ~<

~ ~ ~%~i~

~ ~

,~

~ .......

QK .

.....

N ........

KGTT ...........

Mu-Mdr2

............

----AA

.......

...LDGD-ELGSISNQGRE-K--VNL-GL-T

.....

D-Q .....

~:~M~g~I~[~,~----K-VDNTGNFS-PV-F.SL-ML-PGRI

..... - ............

~:

~~

:~

--

-K

--

-Q

K

.........

M ..... N

A ...........

Xe-Mdr

MEPEQKTAQNGSA-IAVAISDPNSNSKE--GF-S--

° ° .KK-KE-TE-P-K-G--T

.....

STS .....

~~

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.........

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........

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.......

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{~

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......

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......

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1300

....

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...................................................................

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....................

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........

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.....................

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...................................................................

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..........................

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....................................................................

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....................

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...............

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.....................

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.......

T--L-HS

Fig.

3.

Am

ino

acid

ali

gnm

ent

of t

he m

amm

alia

n m

ulti

drug

res

ista

nce

gene

pro

duct

s an

d th

e X

enop

us la

et,is

hom

olog

ue.

The

am

ino

acid

seq

uenc

es o

f th

e hu

man

(m

drl

and

mdr

2) [9

,55]

, th

e m

ouse

(m

drla

, m

drlb

, an

d m

dr2)

[12,

13,1

8]

and

the

ham

ster

(p

gpl,

pg

p2 a

nd p

gp3)

[56]

, w

ere

alig

ned

wit

h th

e de

duce

d am

ino

acid

seq

uenc

e of

the

Xen

opus

gene

. T

he a

lign

men

t w

as c

reat

ed u

sing

the

PIL

EU

P p

rogr

am

of t

he G

enet

ic C

ompu

ter

Gro

up.

Iden

tica

l am

ino

acid

s ar

e re

plac

ed w

ith

a da

shed

lin

e (-

). T

he e

ntir

e se

quen

ce f

or t

he h

uman

mdr

l gen

e pr

oduc

t is

sho

wn.

Gap

s ar

e in

trod

uced

to

opti

miz

e th

e al

ignm

ent

and

are

indi

cate

d by

a d

ot (

.).

The

gre

y bo

xes

repr

esen

t th

e 12

pre

dict

ed t

rans

mem

bran

e do

mai

ns.

The

whi

te s

olid

box

es r

epre

sent

the

nuc

leot

ide

bind

ing

dom

ains

and

the

whi

te b

roke

n bo

x re

pres

ents

the

lin

ker

regi

on.

2 9 I

t~


2.8. Electron microscopy

Brush border membrane vesicles were fixed with 2.5% glutaraldehyde, postfixed with 1% osmium tetroxide, stained with aqueous uranyl acetate, dehydrated through a graded series of ethanol and embedded in LXI12 resin (LADD industries, Burlington, VT). Thin sections were cut with a Reichiert Ultra cut E ultra microtome, poststained with uranyl acetate followed by lead citrate and examined using a JEOL 1200EX microscope at 80KV.

3. Results

3.1. Cloning of the Xenopus mdr homologue

A homologue of the mdr gene has been identified in Xenopus laevis by screening a tadpole cDNA library with a cDNA probe containing the full length mouse mdrlb cDNA sequence [14]. A clone containing a 2.3 kb insert was isolated (Fig. 1, fragment B) and sequenced. Align- ment to the mdrlb sequence revealed the middle portion of the coding sequence but indicated that the 5' and the 3' ends of the cDNA were missing. Further screening of the library failed to isolate additional clones containing the full length cDNA. To obtain the 5' end of the gene, total RNA from the intestine of Xenopus was reverse transcribed. Anchored PCR [31] was performed using primer 3 (Fig. 1) (an oligonucleotide 86 bp upstream of the start of fragment B) and primer 1. A 490 bp fragment (Fig. 1, fragment A) was amplified and cloned into pCR'~II. The sequence of fragment A overlapped with 86 bp of fragment B, and contained the 5' end of the gene.

To obtain the 3' end of the gene, total RNA from the intestine of Xenopus was reverse transcribed using primer 2. The DNA was amplified subsequently by PCR using primers 2 and 4. A 1.8 kb fragment, C, was obtained (Fig. 1) and the PCR product was cloned into pCRT"II. Se- quence analysis showed that the first 117 bp of the 1.8 kb insert overlapped with the 3' end of fragment B. Fragments (A, B and C) form a contig that contains the full length sequence of the Xenopus mdr homologue.

3.2. Sequence analysis

The contig of 4414 bp contains an open reading frame of 3864 bp, with a 1288 predicted amino acid long polypeptide chain that is sufficient for the full length Xenopus mdr cDNA (Fig. 2). The fragment A contains a putative ATG translation initiation site at position 122 plus a short 5' untranslated region. The open reading frame ends in a TGA at position 3985 followed by a 3' untranslated region of 426 bp. This region contains a noncon- served polyadenylation signal, ATTAAA [41], that is present 22 bp upstream of the polyadenylation start site.

Extensive overall homology is shared throughout the

I En-Pgpl (63/41)

En-Pgp2 (61/40)

Dr-Mdr49 (85/42)

Vr-Mdr65 (83/43)

Ha-Pgp8 (61/67)

Mu-Mdr2 (81/87)

Hu-Mdr2 (02/67)

Mu-Mdrlb (80/65)

Ra-Mdr (79/65)

- - Sa-Pgp2 (79/85)

Mu-Mdrla (8Z/68)

Ha-Pgpl (8Z/68)

- - Hu-Mdrl (82/68)

Xe-Mdr

Ce-PgpA (83/44)

Ce-PgpC (63/41)

Le-Mdrl (59/37)

PI-Mdrt (57/32)

Ye-Ste6 (51/28)

Fig. 4. Dendrogram representing a cluster analysis of members of the multidrug resistance gene family. A multiple analysis was created using the PILEUP program of the Genetic Computer Group. The dendrogram is a tree representation of clustering relationships among the deduced amino acid sequences of the genes used for the analysis. The protein sequences used for the analysis were the hamster pgpl, pgp2 and pgp3 (Ha-pgpl, Ha-pgp2 and Ha-pgp3) [56], the mouse mdrlb, mdrla and mdr2 (Mu- mdrlb, Mu-mdrl and Mu-mdr2) [12,13,18], the rat mdr (Ra-Mdr) [57], the human mdrl and mdr2 (Hu-mdrl and Hu-mdr2) [9,55], the Enta- moeba histolytica pgpl and pgp2 (En-Pgpl and En-Pgp2) [58], the Drosophila mdr49 and mdr65 (Dr-Mdr49 and Dr-Mdr65) [59], the C. elegans cepgpA and cepgpC (Ce-PgpA and Ce-PgpC) [30], the Leishma- nia ldmdrl (Le-Mdrl) [60], the Plasmodium mdr (Pl-Mdrl) [61] and yeast ste6 (Ye-Ste6) [49]. The two numbers in parenthesis represent the percent homology/identity to the Xenopus laevis mdr gene, designated as Xe-mdr.

entire sequence between Xenopus P-glycoprotein and mammalian P-glycoproteins that have been characterized (Fig. 3). The highest region of homology resides in the nucteotide binding sites, consistent with previous observa- tions in the mouse and hamster P-glycoproteins. The great- est divergence among the sequences is seen in the linker region [14]. Dendrogram analysis performed on selected members of the mdr gene family clearly demonstrated that Xenopus mdr is closely related to the human and rodent family of genes (65-68% identity and 79-82% overall total homology) and more distantly related to the nonmam- malian genes (Fig. 4).

Kyte and Doolittle hydropathy analysis [42] of the Xenopus laevis mdr gene product predicted twelve transmembrane domains with a hydrophilic region separating the two halves of the protein [9]. The profile is indistin-


guishable from that of the mouse mdrlb gene (data not

shown). The product of 1:he Xenopus mdr gene contains six putative N-glycosyla~Lion sites, N-X-S or N-X-T, at amino acids 10, 104, 112, 306, 817 and 1039 (Fig. 2). However, only two of these sites (amino acids 104 and 112) are predicted to be e,~tracellular. Based on hydropathy plot analysis both are contained within the first extracellu- lar loop of the protein. Similar positions are glycosylated

in rodent mdr gene products [43].

3.3. The expression of the Xenopus mdr message is confined predominantly to the intestine

To study tissue localization, total RNA from adult frog tissues was prepared and analyzed on northern blots. Frag- ment B was used as the probe for hybridization experi- ments. A single mRNA species was detected with an

apparent size of 4.5 kb sufficient to code for P-glycoprotein (Fig. 5A). The message was highly expressed in the small intestine (Fig, 5A, lane 11) and in lesser amounts in the large intestine (F ig 5A, lane 5). Very low levels of message were observed in the adult kidney (data not shown). Although the Xenopus mdr gene is expressed throughout the small intestine, it is most predominant in the terminal ileum (Fig. 5B).

3.4. The product of the Xenopus gene is recognized by antibodies prepared against murine P-glycoprotein

Since the mRNA coding for P-glycoprotein was highly expressed in the small inLestine, the presence of the gene product in that tissue was investigated. Membranes from the small intestine of an adult frog were isolated as well as membranes from a mouse macrophage cell line, J7-V1,

kD 1 2 3

190

125

88

65 Fig. 6. Expression of endogenous P-glycoprotein in the small intestine of adult Xenopus. Membranes were purified by differential centrifugation and separated by SDS-PAGE on a 10% gel, electroblotted to nitrocellu- lose filters and immunoprobed with antibody 5. Lane 1, membranes from the drug resistance cell line J7-V1; lane 2, membranes from the drug sensitive cell line J7. Lane 3, membranes from the small intestine of Xenopus laetfis. Approx. 1 /zg of membrane protein was loaded in lanes 1 and 2, 20 /zg in lane 3.

that overexpressed p-glycoprotein and from the drug sensitive parental cell line, J774.2. Western blot analysis was performed using an antibody prepared against a synthetic

A

,-IZ=...

1 2 3 4 5 6 7 8 910 111213 Kb

-7.4 -5 .3

-2.8

-1.9 -1.6

B 1 2 3 4 5 6 7 Kb

-7 .4 -5 .3

- 2 . 8

-1.9 -1.6

H4

H4

Fig. 5. Northern blot analysis of total RNA from normal tissues of adult frog. Total RNA was isolated from normal tissue of Xenopus laevis, separated on a 1.5% agarose gel and transfened by capillary action to a Genescreen membrane that was hybridized with fragment B. As a control, the same blot was stripped and hybridized with a probe for the Xenopus histone H4. Lane 1, oocyte; 2, brain; 3, gall bladder; 4, heart; 5, large intestine; 6, liver; 7, lung; 8, muscle; 9, oviduct; 10, skin; 11. small intestine; 12, spleen; 13, stomach. (B) Northern blot analysis of total RNA from sequential segments of the small intestine, starting at the duodenum. Each lane represents approx. 1 cm of the small intestine.

120 G. Castillo et al. / Biochimica et Biophysica Acre 1262 (1995) 113-123

peptide from murine mdrlb P-glycoprotein [36] that is 66% identical in this region to the Xenopus mdr P-glycoprotein. The antibody detected an immunoreactive protein of the expected size, 150-180 Da, in membranes from Xenopus intestine (Fig. 6, lane 3) as well as in membranes from J7-V1 cells that overexpressed mouse P-glycoprotein (Fig. 6, lane 1). Similar results were observed when a second antibody that is specific for P-glycoprotein, Onco- gene Science mdr (Ab-1), was employed (data not shown).

3.5. The frog message is expressed in the villus epithelium of the small intestine

To determine which cells in the small intestine expressed the Xenopus P-glycoprotein mRNA, sequential tissue sections of the small intestine were analyzed by in

situ hybridization, using antisense fragment B as a probe. A strong positive signal was observed in the entire epithelial layer of the small intestine including both villus and crypt epithelial cells (Fig. 7a, c and e). No signal was detectable in the other layers of the small intestine. In contrast, staining with the sense probe of fragment B resulted in no signal (Fig. 7b, d and f). Tissue sections from different parts of the small intestine showed the same expression pattern, but the signal was the strongest in the terminal end of the ileum (data not shown).

3.6. Transport qf uinblastine from brush border membrane uesicles

To determine if the P-glycoprotein that is present in the frog intestine was capable of drug transport, brush border

i

Fig. 7. In situ hybridization of Xenopus intestine. Sequential sections of the small intestine were hybridized with single stranded 35S-labelled antisense (panels a,c and e), and sense (panels b,d and f), RNA probes. Panels a and b show brightfield view of hematoxylin and eosin stained small intestine. Panels c and d represent darkfield views of the same sections (magnification, X 65). Panels e and f represent a greater magnification of a brightfield view (magnification, X 265).


A 4. D i s c u s s i o n

B lOO

C m 90

,~ e0

.o 70

el

60 1 l

I I I I

0 5 10 15 20

T ime (min)

Fig. 8. Vinblastine transport by brush border membrane vesicles. Vesicles isolated from the small intestine were visualized by electron microscopy (A) (magnification, X 50000). Vesicles were loaded with [3H]vinblastine in the presence or absence of ATP or ATP',/S. Efflux was measured (B) in the presence of 3 mM ATP (O), 3 mM ATPyS (zx), ATP+ 10 /xM verapamil (A) and in the absence of ATP (O). Differences in [3H]vinblastine efflux in the absence or presence of verapamil were significant at 5 min (P < 0.05) and at 10 min (P < 0.03).

membrane vesicles from tlhe ileum of adult Xenopus laevis were prepared by the MgC12 method [38,39]. Most of the vesicles were sealed when viewed by transmission electron microscopy (Fig. 8A). The vesicles were loaded with [3H]vinblastine, ATP and an ATP regenerating system. The latter was necessary because in the absence of ATP, minimal drug efflux was observed (Fig. 8). A major increase in the efflux of drug was seen when ATP was present suggesting that drug efflux from the vesicles was ATP dependent. Furtherrnore, when ATPy S, a nonhydro- lyzable analog of ATP was used, no efflux of the drug was observed. When efflux was performed in the presence of 10 /xM verapamil, a known inhibitor of mammalian P- glycoprotein function, drug efflux was partially inhibited.

A homologue of the mammalian mdr gene has been isolated from Xenopus laevis. This gene is highly homologous to its mammalian counterparts, being 68 and 62 percent identical to the human mdrl and mdr2 genes, respectively, in the coding region. Xenopus P-glycoprotein contains all of the hallmarks that characterize the members of the mdr gene family. The Kyte and Doolittle hydropathy profile of the deduced amino acid sequence for the Xenopus gene product predicts six transmembrane domains in each half of the molecule separated by a hydrophilic region referred to as the linker region. This region has been implicated in the regulation of P-glycoprotein function and is the major phosphorylation domain of the protein [24,25]. The Xenopus P-glycoprotein contains two PKA consensus phosphorylation sites (amino acids 671 and 672) and three PKC sites (amino acids 643, 674 and 678) within that region. It also displays an alternate expression of basic/acidic domains as has been described for the murine linker region [25]. The frog gene product has two ATP binding sites (Walker motifs) within the molecule and this is also a common characteristic shared by all members of the p-glycoprotein family [44]. The highest region of homology resides in the area surrounding the ATP binding sites. Based on the similarity of both halves of P-glycoprotein, it has been proposed that the mammalian genes arose by the duplication of an ancestral gene of half the size [9]. The two halves of the frog P-glycoprotein are 43% identical suggesting that Xenopus mdr may also have arisen from the duplication of an ancestral gene.

Abundant expression of mammalian mdr genes has been described in the intestinal tract and the organs associated with it. Xenopus mdr is specifically expressed in the intestine. Consistent with this result is the observation that Xenopus mdr exhibits highest homology to the human mdrl and the mouse mdrla genes, both of which also are expressed in the intestine [27,45,46]. Intestinal expression of P-glycoprotein is not limited to higher organisms. The expression pattern directed by the promoters of the pgpl and pgp3 genes of C. elegans is confined exclusively to the small intestine [47], suggesting that the function of p-glycoprotein is related to its expression in this organ and that it has been conserved throughout evolution. Expres- sion of the frog P-glycoprotein was increased in the terminal ileum by approx. 3- to 4-fold when compared to the rest of the small intestine, indicating that the primary site for the normal function of the Xenopus gene product is the terminal ileum. Since expression of the frog mdr is limited to the epithelial cells of the small intestine, it is clear that expression of this gene is tightly regulated and studies of its promoter should yield new information on the regulation of normal gene expression.

To examine the function of Xenopus P-glycoprotein, brush border membrane vesicles were prepared from the

122 G. Castillo et aL / Biochimica et Biophysica Acta 1262 (1995) 113-123

small intestine and were shown to transport vinblastine, an antitumor drug, in an ATP-dependent manner. Verapamil, a calcium blocker known to inhibit the function of P- glycoprotein [44], decreased the ATP dependent efflux of vinblastine in Xenopus brush border membrane vesicles. The localization of the expression of the Xenopus mdr to epithelial cells suggests that it is involved in transport of a natural substrate present in the gut. The intestine in Xeno- pus is an organ that is in contact with all of the naturally occurring compounds ingested by the frog from its environment. It has been suggested that P-glycoprotein partici- pates in the transport of proteins that lack signal peptides, in a system distinct from the classical secretion pathway [28,48,49]. This hypothesis is supported by the similarity of mdr to the HLY B gene involved in the secretion of hemolysin [23,50] and by studies on the yeast STE6 gene that is responsible for transport of the Yeast mating a-factor, a 12 amino acid famesylated peptide. A mutation in the STE6 gene that abolishes its capability to secrete the a-factor can be complemented by the murine mdr gene, mdrla [51].

Many peptides that are thought to play a role in maintaining the normal physiology of Xenopus are secreted from the frog through its intestine. A variety of peptides of different structure and function, some of which have intrin- sic antibacterial activity are secreted. Interestingly, all of them have mammalian peptide homologues [52,53]. Local- ization of P-glycoprotein to the epithelial membranes of the intestine makes it a candidate for a peptide transporter. The latter could be involved in the secretion of peptides into the lumen of the intestine, thereby providing protec- tion from bacterial invasion or regulation of the normal bacterial flora. Alternatively, the transporter could be involved in preventing toxic compounds or peptides, normally ingested by the frog, from entering the intestinal cells in a manner analogous to the resistance observed to the hydrophobic peptide N-acetyl-leucyl-leucyl-norleucinal [54]. Definitive proof that the product of Xenopus laevis mdr is involved in peptide and/or drug transport will require expression of the gene and functional studies in cells that do not normally produce P-glycoprotein.

Xenopus has proven to be a powerful system for the study of genes during development. Research focused on the expression of the mdr gene during development will be important in understanding the normal physiological role of P-glycoprotein.

Acknowledgements

This study was supported in part by United States Public Health Service Grant CA39821 and 5P30 CA13330.

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a homologue of the mammalian multidrug resistance gene (mdr) is functionally expressed in the...

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