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.

Fish as Reference Species in Different Water Masses

“…These transport experiments allow the identification of a wide range of compounds that may interact with the transporters thus limiting their toxicological effects or, by overwhelming or blocking the transporters, increasing the effects. This can be best identified in cell lines that have been characterized for their ABC transporters expression such as the SAE Squalus acanthias shark embryo-derived cell line (Kobayashi et al, 2007) or some rainbow trout cell lines (Fischer et al, 2011). Several studies provided evidence for a regulation of teleost ABC drug efflux transporters by heavy metals (Long et al, 2011;Della Torre et al, 2012).…”

Cell and molecular biology of the spiny dogfish Squalus acanthias and little skate Leucoraja erinacea: insights from in vitro cultured cells

“…The latter can be explored at the transcriptional, post‐transcriptional, translational or post‐translational levels. Cell cultures expressing such proteins also allow examination of the means by which xenobiotics, drugs, drug metabolites and extracellular effectors influence protein function (Kobayashi et al , 2007; Hwang et al , 2008).…”

“…Expression of mRNA for Mrp3, also referred to as ABCC3 or multi‐organic anion transporter‐D, was identified by reverse transcription PCR using as primers sequences derived from cloned L. erinacea mRNA for the related Mrp2 transporter (Cai et al , 2003; Kobayashi et al , 2007). In addition to Mrp2, the subfamily of proteins to which Mrp3 belongs (ABCC) also includes the cystic fibrosis transmembrane regulator.…”

“…Molecular cloning established that the gene for L. erinacea Mrp3 codes for a protein of 1544 amino acids (5048 bp). The mRNA contains a short 5′ untranslated region of 37 bp and a longer 3′ untranslated region of 380 bp (Kobayashi et al , 2007). The predicted protein structure includes 15 membrane‐spanning regions that can be organized into three transmembrane domains.…”

Analysis and functional annotation of expressed sequence tags from in vitro cell lines of elasmobranchs: Spiny dogfish shark (Squalus acanthias) and little skate (Leucoraja erinacea)

“…The little skate has been targeted by the National Institutes of Health Human Genome Research Institute as a species designated for full genome sequencing (http://www.genome.gov/Pages/Research/Sequencing/SeqProposals/SharkSkateSeq.pdf; http://www.sciencedaily.com/releases/2005/03/050307100237.htm). To develop additional tools for cellular, molecular and genomic studies of cartilaginous fish, we derived cell lines from embryos of S. acanthias and L. erinacia (Parton et al, 2007; Forest et al, 2007; Kobayashi et al, 2007; Hwang et al, 2008). We refer to these as the SAE cell line from the dogfish shark and LEE-1 cell line from the little skate.…”