Multidrug resistance-associated protein 3 (Mrp3/Abcc3/Moat-D) is expressed in the SAE Squalus acanthias shark embryo-derived cell line

Abstract

The multidrug resistance-associated protein 3 (MRP3/Mrp3) is a member of the ATP-binding cassette (ABC) protein family of membrane transporters and related proteins that act on a variety of xenobiotic and anionic molecules to transfer these substrates in an ATP-dependent manner. In recent years, useful comparative information regarding evolutionarily conserved structure and transport functions of these proteins has accrued through the use of primitive marine animals such as cartilaginous fish. Until recently, one missing tool in comparative studies with cartilaginous fish was cell culture. We have derived from the embryo of Squalus acanthias, the spiny dogfish shark, the S. acanthias embryo (SAE) mesenchymal stem cell line. This is the first continuously proliferating cell line from a cartilaginous fish. We identified expression of Mrp3 in this cell line, cloned the molecule, and examined molecular and cellular physiological aspects of the protein. Shark Mrp3 is characterized by three membrane-spanning domains and two nucleotide-binding domains. Multiple alignments with other species showed that the shark Mrp3 amino acid sequence was well conserved. The shark sequence was overall 64% identical to human MRP3, 72% identical to chicken Mrp3, and 71% identical to frog and stickleback Mrp3. Highest identity between shark and human amino acid sequence (82%) was seen in the carboxyl-terminal nucleotide-binding domain of the proteins. Cell culture experiments showed that mRNA for the protein was induced as much as 25-fold by peptide growth factors, fetal bovine serum, and lipid nutritional components, with the largest effect mediated by a combination of lipids including unsaturated and saturated fatty acids, cholesterol, and vitamin E.

FIG. 1

Amino acid and nucleotide sequences of shark Mrp3. Transmembrane regions are shaded. Walker A and B regions are underlined. The ABC protein signature sequences are underlined and bolded. The two predicted extracellular N-glycosylation sites are bolded.

FIG. 1

Amino acid and nucleotide sequences of shark Mrp3. Transmembrane regions are shaded. Walker A and B regions are underlined. The ABC protein signature sequences are underlined and bolded. The two predicted extracellular N-glycosylation sites are bolded.

FIG. 1

Amino acid and nucleotide sequences of shark Mrp3. Transmembrane regions are shaded. Walker A and B regions are underlined. The ABC protein signature sequences are underlined and bolded. The two predicted extracellular N-glycosylation sites are bolded.

FIG. 2

Schematic of Mrp3. MSD indicates membrane-spanning domains (1, 2, and 3). NBD indicates nucleotide-binding domains (1 and 2).

FIG. 3

Multiple alignments of MRP3/Mrp3 amino acid sequences among representative species. S. acanthias, spiny dogfish shark; H. sapiens, human; G. gallus, chicken; X. tropicalis, frog; T. nigroviridis, pufferfish. Transmembrane regions in shark are underlined. Amino acids common with shark are shaded.

FIG. 3

Multiple alignments of MRP3/Mrp3 amino acid sequences among representative species. S. acanthias, spiny dogfish shark; H. sapiens, human; G. gallus, chicken; X. tropicalis, frog; T. nigroviridis, pufferfish. Transmembrane regions in shark are underlined. Amino acids common with shark are shaded.

FIG. 3

Multiple alignments of MRP3/Mrp3 amino acid sequences among representative species. S. acanthias, spiny dogfish shark; H. sapiens, human; G. gallus, chicken; X. tropicalis, frog; T. nigroviridis, pufferfish. Transmembrane regions in shark are underlined. Amino acids common with shark are shaded.

FIG. 3

Multiple alignments of MRP3/Mrp3 amino acid sequences among representative species. S. acanthias, spiny dogfish shark; H. sapiens, human; G. gallus, chicken; X. tropicalis, frog; T. nigroviridis, pufferfish. Transmembrane regions in shark are underlined. Amino acids common with shark are shaded.

FIG. 4

Induction of Mrp3 mRNA in SAE shark embryo cells in vitro. Cultures were incubated with the indicated compounds, and mRNA abundance was determined by qPCR as described in “Materials and Methods” section. Briefly, SAE cells were incubated for 4 days in basal LDF nutrient medium containing amino acids, 10 nM selenous acid, 10 μg/mL transferrin, and 2 mM GlutaMax. Following this, other medium supplements (2% FBS, insulin, 50 ng/mL FGF, 50 ng/mL EGF, and 1:1000 CDL) were added to individual wells. A control was also included, in which none of the medium supplements were added. After incubation for 24 h, total RNA was isolated from each well, and cDNA was synthesized. The expression of shark Mrp3 was compared by qPCR with shark EF-1α as the normalizer. Data were analyzed with MX pro software (Stratagene).