| Background and Purpose: The liver is the critical organ to maintain the human body normal function. One of its main functions is absorbing some toxic organic compound from portal vein, processing the transformation and metabolism processes in the liver cell, further secreting into the bile, finally removing by the intestinal tract along with the excrement, and thus prevents the toxic matter gathering in body, displays its detoxication. The function of liver cell absorption organic compound is mainly mediated by a group of transport protein with similar structure and function lying on the liver cell membrane, i.e. the Organic anion transporting polypeptides (OATPs). OATPs is a super-family made up of the membrane proteins called transporters of the organic anion substrate characteristic. At present, there are about 11 kinds of rat, 8 kinds of mouse and 9 kinds of human OATPs members that have been found. OATP-C (SLC21A6) is the liver-specific expression of organic anion transport peptides, an important member of the OATPs family, distributing in liver sinus-like space adjacent to the basement membrane of cells, uptaking in a variety of endogenous and exogenous organic solutes (such as bilirubin, a some drugs and exogenous organic toxins, etc.). Research has revealed that OATP-C affects the liver medicine metabolism, especially, in the deactivation. Therefore, the study of its molecular mechanism of transportation manifest a important significance to clarify the transit of organic solutes in the liver and to understand the participation of OATP-C in the pharmacokinetics, liver function of detoxification,and also for further screening and designation its inhibitors or agonists for the clinical treatment.This experiment was based on our previous study of amino acid similarity comparison of skate with rodent or human OATP-C. Skate, or"Rajia"is an ancient low-degree small marine vertebrates fish which is origin from about 2 billion years ago, and its liver exists an organic anion -transit system which is similar to the organic anion transport protein of higher grade mammals such as mice and human. A skate organic anion transporting peptide gene, soatp, was cloned, which is exclusively expressed in liver tissue. We compared the high homology regions of sOatp with the rodent or human liver specific OATP-C cDNA, combining with its organic anion substrate transportation characteristic, found 7 positive charge amino acids of OATP-C are highly conserved (namely 49 lys (k), 57 arg (r), 58 arg (r), 115 his (h), 181 arg (r), 490 lys (k), 580 arg (r)). We postulated that these amino acids are determinant amino acids in its uptake function of transport the organic anion substrate. To test this hypothesis, we have cloned OATP-C gene from human liver mRNA, and individually made 7 mutation of above positive charge amino acids into neutral amino acids, further subcloned the wt and mutants into a eukaryotic expression vector with its C–terminal GFP fusion protein. All the wild type and mutant plasmids were transfected into HEK293 cells respectively, to observe the membrane localization ability and the changes of its uptake function.Material and Method: Wilde type(wt) and 7 mutants (Mut1~7) of the pcDNA-OATP- C-GFP fusion gene eukaryotic expression plasmids were made previously. These plasmids were transformed into coli DH5α, after amplification, plasmids were extracted by PureLinkTM Quick Plasmid Kit. All the plasmids were transfected into adherent cultured HEK293 cells. GFP expression in HEK293 cells was observed by the laser scanning confocal microscope. Equal amount of specific substrate [3H]ES (Estrone sulfate) was added into the culture medium without serum. Uptake assay was undergone at 5 min time point. Additionally, Km and Vmax value of substrate uptake ability was calculated by adding different concentrations of [3H]ES to Wt and Mut1, Mut6 transfected cells at different time point.Result1. Eight fusion plasmids(Wt, Mut1~Mut7)were successfully amplified in the E.coli DH5α, and purified with PureLinkTM Quick plasmid reagent kit (invitrogen). Plasmid DNA OD value was measured with the spectrophotometer to calculated the concentration and purity.2. Wt, Mut1~Mut7 OATP-C -GFP fusion plasmids were transfected into HEK293 cells. No significant difference of green fluorescence expression level on 8 transfected cell plasma membrane was observed, which demonstrated that all the mutants had ability to target to cellular membrane and similar expression level compared with wt OATP-C -GFP fusion gene.3. 5 min [3H]ES uptake assay has shown that all the mutants import less radioactive substrate to cell plasma, which indicated an impaired uptake function of OATP-C mutants.4. Mutation site of Mut1 and Mut6 was predicted to localize on the outer domain of OATP-C molecule topological model, theoretically, which may be critical for OATP-C to transport solutes. To evaluate the function of Mut1 and Mut6, dynamic curve of Estrone Sulfate uptake was calculated when taking Wt as control: Wt: Km =0.26±0.03, Vmax =111.8±2.1; Mut1: Km =0.43±0.04, Vmax =50.5±2.6 ( Km P =0.005,Vmax P =0.000 ); Mut6: Km =1.20±0.06, Vmax =88.7±5.9 ( Km P =0.000,Vmax P =0.003 ) (Unit of Km isμM; Unit of Km is pmol.mg.protein-1.5min-1). Our data has shown that the ability of uptaking substrate ES by Mut1 and Mut6 were significantly reduced.Conclusion Our previous study has found that soatp gene of the ancient vertebrate skate has almost the same characteristics with human OATP-C. From the view of evolutionary comparative medicine, and based on our analysis of amino acid sequence of OATP-C with soatp and also the topological model of OATP-C molecule, it was postulated that the positive charge amino acid in the highly conserved homologous region may be critical to its function of uptake organic anion substrates. Our present data has confirmed our hypothesis. This study may provide us a new insight to understand the molecular mechanism transport the organic anion solutes by OATP family. It also might be important for design and screening the agonist and inhibitor of OATP-C transporter in clinical application. |
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