Study on the function of P-gp protein in florfenicol

metabolism of Portunus tritructus 

Abstract: Portunus trituberculatus is a famous swimming crab with the highest output in the north of China. With the rapid development of aquaculture industry, the environment has become increasingly deteriorated, resulting in the increase of pathogens and high incidence of disease in cultivation environments, which may cause huge economic losses to the mariculture industry and seriously hinder the sustainable and healthy development of mariculture. At present, aquatic drugs are extensively applied as effective measures to prevent and control aquatic animal disease because of their remarkable efficacy, easy production and utilization, and low cost. Florfenicol (FLR) is one of the most commonly used antimicrobial agents in aquaculture. This chemical ingredient is characterized with broad spectrum, easy absorption, wide distribution and low residue in the body after administration. FLR can bind closely to the 50S subunit of 70S ribosome of bacteria, which plays a killing role by effectively inhibitting the proteins’ biosynthesis in bacteria, and is widely used in the prevention and treatment of infections caused by Vibrio. In the actual breeding process, there are unreasonable uses of drug and even abuse of drugs. When entering the breeding environment, the antimicrobial agents will induce the opportunistic pathogens of the breeding organisms themselves; microorganisms in aquatic environments including aquaculture sediment can develop drug resistance; FLR will also remain in the cultured subjects, leading to a series of adverse responses of the body, including the induced oxidative stress, inhibited metabolism and growth process in organisms, disrupted behavior and immune response, altered transcriptional expression of key genes in related metabolic pathways, and destroyed balance of the ecological environment in aquaculture. ATPbinding cassette (ABC) transmembrane transporters (ABC transporters) are primarily involved in the active, ATPdependent transport of biological molecules across plasma membranes. The most toxicologically relevant ABC transporter is Pglycoprotein (Pgp, ABCB1), a membrane detoxification protein that can transport and expel exogenous toxic substances and harmful metabolites from the cell. To study the transport function of Pgp protein in P. trituberculatus, the fulllength sequence of ATP binding cassette transporter subfamily b1 (abcb1) was cloned via race amplification technique, named Ptabcb1. The fulllength cDNA was 4 923 bp, and the open reading frame (ORF) was 3 522 bp, encoding 1 172 amino acids. The predicted molecular weight was 127.96 kDa (GenBank ID: KY487995). Two hydrophobic transmembrane regions (TMD) and two nucleotide binding regions (NBD) were found in the cloned sequence, which were consistent with the basic structural characteristics of ABC transporters. Its primary sequence was highly conserved, displaying a typical phosphatebinding loop: Walker A and two magnesium binding sites: Walker B. In addition to these two regions, three other conserved motifs were present in the ABC cassette: the switch region, which contained a histidine loop, postulated to polarize the attaching water molecule for hydrolysis, the signature conserved motif specific to the ABC transporter, the Qloop, which interacted with γ phosphate through a water bond, the aspartic acidcontaining Dloop, and the Hloop, where the first amino acid was either isoleucine. The Walker A, Walker B, Qloop, Hloop, Dloop and switch region formed the nucleotide binding site. Sequence alignment showed that the homology between Ptabcb1 and abcb1 of Exopalaemon carinicauda was the highest (73.32%), followed by Homo sapiens (51.83%) and Mus musculus (51.31%). Phylogenetic tree analysis revealed that the amino acid sequence of Ptabcb1 was closely clustered with that of E. carinicauda (BP=100), which belonged to the monophyletic group of species clustering of the phylum Arthropoda (BP=97). The results of tissue distribution showed that Ptabcb1 was expressed in all examined tissues, with the highest expression in the hepatopancreas (7.49 folds compared with the heart tissue), followed by the intestine (5.89 folds) and the muscle (3.65 folds). Using fluorescence in situ hybridization, and fluorescence staining, Ptabcb1 was mainly expressed in the cytoplasm of the hepatopancreas. The effects of intramuscular injection of 20 mg·kg-1, 40 mg·kg-1 and 80 mg·kg-1 FLR on the transcriptional expression of Ptabcb1 in P. trituberculatus were studied by realtime fluorescence quantitative PCR. Moreover, P. trituberculatus were treated by the injection of 10 μM verapamil (VER) for 1 h, followed by the injection of 40 mg·kg-1 FLR. The effects of combined injection of VER and FLR on P. trituberculatus were explored by evaluating the transcriptional expression and transport activity of Ptabcb1 in P. trituberculatus. The results showed that FLR significantly promoted the transport activity of PtPgp protein at the transcriptional level by promoting the upregulation of Ptabcb1 in a concentration dependent manner within 24 h. It was also found that the combined injection of 40 mg·kg-1 FLR and VER significantly inhibited the efflux of FLR within 12 h, resulting in the decrease of PtPgp protein transport activity. After 12 h, PtPgp protein transport activity gradually returned to that of the control group. In addition, the transcriptional level of Ptabcb1 in P. trituberculatus was highly expressed in the hepatopancreas of P. trituberculatus. Hepatopancreas was the most important organ for detoxification and metabolism in P. trituberculatus. The high expression of Ptabcb1 in hepatopancreas further suggested that PtPgp could be used as the first line of defense against foreign pollutants, and could directly transport substrates or their metabolic end products out of cells. P. trituberculatus was also found to contain a conservative Pgp protein mediated defense system as the first line of defense against exogenous pollutants, which could directly transport substrates or their metabolic end products out of the cell. Our findings provide a basis for further understanding the molecular defense mechanism of Pgp protein mediated detoxification of crustaceans.

Keywords: Portunus trituberculatus; Pglycoprotein; florfenicol; transport activity