Cloning and heterologous expression of the ovine (Ovis aries) P-glycoprotein (Mdr1) in Madin-Darby canine kidney (MDCK) cells.
P-glycoprotein (P-gp) plays a crucial role in the multidrug resistance of pathogenic helminths in sheep (Ovis aries) as well as in antiparasitic drug pharmacokinetics in the host. We cloned sheep P-gp cDNA and expressed it stably in Madin-Darby canine kidney (MDCK) cells. The open reading frame consists of 3858 nucleotides coding for a 1285 amino acids containing protein. The sequence shows high homology to the orthologs of other mammalian species, especially cattle. Both ruminant DNA sequences show a 9 bp insertion that is lacking in all other investigated sequences. Expressed in MDCK cells, the protein displays a size of 170 kDa on Western analysis. Transfection of MDCK cells with sheep P-gp resulted in 10- to 50-fold resistance to the cytotoxic P-gp substrates colchicin and daunorubicin, and in reduced digoxin accumulation.
Zahner
D
D
Faculty of Veterinary Medicine, Institute of Pharmacology and Toxicology, Giessen, Germany. daniel.zahner@vetmed.uni-giessen.de
Alber
J
J
Petzinger
E
E
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004077:Digoxin; D004285:Dogs; D015870:Gene Expression / Q000235:genetics; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D034561:Sheep, Domestic; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018076:DNA, Complementary; D004077:Digoxin