ABSTRACT
Organic anion transporting polypeptide 1B1 (OATP1B1) is specifically expressed at the basolateral membrane of human hepatocytes and plays important roles in the uptake of various endogenous and exogenous compounds including many drugs. The proper functioning of OATP1B1, hence, is essential for the bioavailability of various therapeutic agents and needs to be tightly regulated. Dileucine-based signals are involved in lysosomal targeting, internalization, and trans-Golgi network to endosome transporting of membrane proteins. In the current study, we analyzed the 3 intracellular and 13 transmembrane dileucine motifs (DLMs) within the sequence of OATP1B1. It was found that the simultaneous replacement of I332 and L333 with alanine resulted in a significantly reduced level of the mature form of OATP1B1. The cell surface expression of I332A/L333A could be partially rescued by MG132, as well as agents that prevent clathrin-dependent protein internalization, suggesting that this dileucine motif may be involved in the endocytosis of OATP1B1. On the other hand, I376/L377 and I642/L643, which are localized at transmembrane helices (TM) 8 and 12, respectively, are involved in the interaction of the transporter with its substrates. I642A/L643A exhibited a significantly decreased protein level compared to that of the wild-type, implying that the motif is important for maintaining the stability of OATP1B1 as well.
ABSTRACT
N-glycosylation plays critical roles in protein secretion, sorting, stability, activity modulation, and interactions to other molecules in the eukaryotic organisms. Fungal ß-1,4-mannanases have been widely used in the agri-food industry and contribute to the pathogenesis on plants. However, the information on N-glycosylation of a specific fungal carbohydrate-active enzyme (CAZyme) is currently limited. Herein, a cDNA was cloned from Aspergillus aculeatus QH1, displaying a full length of 1302 bp with an open reading frame of 1134 bp encoding for a GH5 subfamily 7 ß-1, 4-mannanase, namely AacMan5_7A. The enzyme was purified and exhibited an optimal activity at pH 4.6 and 60 °C, hydrolyzing glucomannan and galactomannan, but not yeast mannan. AacMan5_7A is an N-glycosylated protein decorated with a high-mannose type glycan. Further through UPLC-ESI-MS/MS analysis, one of the four predicted N-glycosylation sites at N255 position was experimentally verified. The present study expands the information of N-glycosylation in fungal CAZymes, providing scientific bases for enhancing the production of fungal enzymes and their applications in food, feed, and plant biomass conversions.
ABSTRACT
Organic anion transporting polypeptides (OATPs, gene symbol SLCO) mediate sodium-independent transport of endogenous compounds such as bile salts, hormones and their conjugates as well as toxins and drugs. OATP1B1 is the major OATP specifically expressed at the basolateral membrane of human hepatocytes and many clinically important drugs have been shown to be substrates of the transporter. According to the computer-based hydropathy analysis, a large intracellular loop 3 (IL3) is situated between transmembrane domain 6 and 7 of OATPs, in which a conserved NPxY motif is found. In the current study, HEK293 cells expressing the HA-tagged OATP1B1 was utilized to investigate the role of the NPxY motif for the function and expression of the transporter. Alanine replacement of N335 or P336 retained substantial uptake function; while simultaneous mutation of these residues resulted in a double mutant that lost almost all the transport activity. On the other hand, Y338A showed >80% reduction for estrone-3-sulfate uptake. Plasma membrane protein analysis revealed that N335/P336A completely lost its cell surface protein expression; while that of Y338A is dramatically reduced. Further investigation with pharmacological inhibitors and immunocytochemistry demonstrated that N335/336A is detained in the Golgi apparatus and Y338A exhibited accelerated protein degradation rate compared to that of the wild-type. Conservative replacement of Y338 with phenylalanine fully recovered uptake and expression of the transporter. In summary, a new role was observed for the NPxY motif located in the IL3 of OATP1B1, which may affect processing and stability of the transporter.
Subject(s)
Amino Acid Motifs , Liver-Specific Organic Anion Transporter 1/metabolism , Amino Acid Sequence , Golgi Apparatus/metabolism , HEK293 Cells , Humans , Liver-Specific Organic Anion Transporter 1/chemistry , Proteolysis , Sequence Homology, Amino AcidABSTRACT
Organic anion transporting polypeptides (OATPs, gene symbol SLCO) are important membrane transporter proteins that mediate the uptake of wide ranges of endogenous and exogenous compounds. OATP2B1 has been found in multiple organs and tissues, including the liver, small intestine, kidney, brain, placenta, heart, skin, as well as skeletal muscle, and is proposed to be involved in the uptake of orally administered drugs. Quite a few reports have demonstrated that transmembrane domains (TMs) are crucial for proper functions of OATP family members. Comparative modeling proposed that TM1, along with TM2, 4, and 5 of the N-terminal half of OATP2B1, may be localized within the substrate interaction pocket and are important for uptake function of the transporter. Alanine scanning of the putative transmembrane domain 1 of OATP2B1 revealed that substitution of L58 with alanine dramatically altered the Km value, and mutation of V52, H55, Q59, and L69 resulted in significantly reduced substrate turnover number, whereas A61V, Q62A, and S66A exhibited significant change in both Km and Vmax values. In addition, phenylalanine at position 51 seems to play an important role in maintaining proper folding of OATP2B1 because alanine replacement of F51 caused accelerated degradation of the transporter protein. Although proteasome and lysosome inhibitors could partially recover protein level, the mutant transporter remained nonfunctional. Taken together, the identification of nine essential amino acid residues within TM1 of OATP2B1 suggested that the transmembrane domain is important for maintaining proper function of the transporter.
Subject(s)
Amino Acid Substitution , Organic Anion Transporters/chemistry , Organic Anion Transporters/metabolism , Amino Acid Motifs , Binding Sites , Biological Transport , HEK293 Cells , Humans , Models, Molecular , Organic Anion Transporters/genetics , Protein Domains , Protein Folding , Protein Structure, TertiaryABSTRACT
Organic anion-transporting polypeptides play important roles in the uptake of various endogenous and exogenous compounds. It has been proposed that OATP family members, as membrane proteins, may form oligomers. However, oligomerization status of OATPs is still largely unclear. In the present study, HEK293 cells stably expressing OATP1B1 were generated to investigate the oligomerization status of the transporter. Chemical cross-linking and coimmunoprecipitation experiments revealed that OATP1B1 may form homo-oligomers, possibly through disulfide bonds. When wild-type OATP1B1 was coexpressed with a loss-of-function mutant W258A, cells showed reduced uptake of prototypic substrate estrone-3-sulfate (ES). Interestingly, such a coexpression did not affect OATP1B1 transport activity of high concentrations ES, implicating that oligomerization status may affect only the high affinity component of ES. OATP1B1 possesses three GXXXG motifs that have been associated with protein dimerization in other membrane proteins. When glycine residues were replaced with alanine, G219A and G393A showed drastically reduced uptake function. Further studies revealed that G219A has a similar association capability to that of the wild-type, while mutation at Gly393 may affect oligomerization status of the transporter. Kinetic analysis showed that both G219A and G393A have a dramatically reduced Vmax for ES uptake. Km of G219A was increased while that of G393A exhibited a decreased value for high affinity component of ES binding. Our studies demonstrated that OATP1B1 may function as oligomers in the high affinity site of ES while acting as monomers for the low affinity binding component of the substrate.
Subject(s)
Liver-Specific Organic Anion Transporter 1/metabolism , Organic Anion Transporters/metabolism , Biological Transport/physiology , Cell Line , Cell Membrane/metabolism , Estrone/analogs & derivatives , Estrone/metabolism , HEK293 Cells , Humans , Kinetics , Peptides/metabolism , PolymerizationABSTRACT
Organic anion transporting polypeptides (OATPs, gene symbol SLCO) are membrane proteins that mediate the sodium-independent transport of a wide range of endogenous and exogenous compounds. Due to their broad substrate specificity, wide tissue distribution, and involvement in drug-drug interactions, OATPs have been considered as key players in drug absorption, distribution, and excretion. Transmembrane domains (TMs) are crucial structural features involved in proper functions of many transporters. According to computer-based modeling and previous studies of our laboratory and others, TM11 of OATP1B1 may face the substrate interaction pocket and thus play an important role in the transport function of the protein. Alanine-scanning of the transmembrane domain identified seven critical amino acid residues within the region. Further analysis revealed that alanine substitution of these residues resulted in reduced protein stability, which led to significantly decreased protein expression on the plasma membrane. In addition, all mutants exhibited an altered Km for ES uptake (either high affinity or low affinity component, or both), though Km for taurocholate transport only changed in R580A, G584A, and F591A. These results suggested that critical residues in TM11 not only affect protein stability of the transporter, but its interaction with substrates as well. The identification of seven essential residues out of 21 TM amino acids highlighted the importance of this transmembrane domain in the proper function of OATP1B1.
Subject(s)
Amino Acids/metabolism , Organic Anion Transporters/metabolism , Protein Binding/physiology , Protein Transport/physiology , Amino Acid Sequence , Cell Line , Cell Membrane/metabolism , HEK293 Cells , Humans , Kinetics , Liver-Specific Organic Anion Transporter 1 , Molecular Sequence Data , Peptides/metabolism , Protein Structure, Tertiary , Substrate SpecificityABSTRACT
Organic anion-transporting polypeptides are members of the solute carrier (SLC) family and key determinants for the transmembrane transport of a wide variety of compounds. OATP1B1 is predominantly expressed at the basolateral membrane of human hepatocytes and play an important role in drug clearance from the body. It has been demonstrated to be responsible for the hepatic uptake of various drugs. Computer-based hydropathy analysis predicted several putative phosphorylation sites at the amino and carboxyl termini and at intracellular loop 3 of OATP family members. Therefore, their transport functions may be regulated by phosphorylation. Previous studies have demonstrated that uptake function of OATP2B1 and OATP1A2 is regulated by protein kinase C (PKC). In the present study, we treated HEK293 cells stably expressing OATP1B1 with different PKC modulators and measured their transport activity for prototypic substrate estrone-3-sulfate. It was found that OATP1B1 uptake function was reduced upon PKC activation. Further studies indicated that PKC may affect OATP1B1 activity through regulation of the cell surface protein level. Moreover, we found out that PKC activator phorbol 12-myristate 13-acetate (PMA) not only affects the internalization of OATP1B1 but its recycling as well. Immunocytochemistry analysis revealed that internalized OATP1B1 co-localized with early and recycling endosomal markers and the co-localization of OATP1B1 with recycling endosome is dependent on PKC activation. Taken together, our present study demonstrated that PKC regulates the function of OATP1B1 by affecting internalization and recycling of the transporter protein.
Subject(s)
Cell Membrane/metabolism , Models, Biological , Organic Anion Transporters/metabolism , Protein Kinase C/metabolism , Computer Simulation , HEK293 Cells , Humans , Liver-Specific Organic Anion Transporter 1 , Protein Transport/physiologyABSTRACT
Because of their wide distribution and capability of transporting a large variety of compounds, organic anion-transporting polypeptides (OATPs) have been extensively recognized as crucial players in absorption, distribution, and excretion of various drugs. OATP1A2 was the first cloned human OATP and has been found to transport wide range of endogenous and exogenous compounds. Bovine Oatp1a2 (bOatp1a2) shares high homology with human OATP1A2 and is considered the functional ortholog of the latter. Previous study in our laboratory demonstrated that bOatp1a2 transport of estrone-3-sulfate (ES) exhibited biphasic saturation kinetics. In the present study, we investigated the transport function of bOatp1a2 for four different quinolone antibacterial agents (enrofloxacin, levofloxacin, norfloxacin, and ciprofloxacin) and found that all the tested fluoroquinolones can be transported by bOatp1a2. Further studies showed that different binding sites are responsible for the transport of different fluoroquinolones. Both ciprofloxacin and norfloxacin exhibited biphasic saturation kinetics. The Kms of the high- and low-affinity components for ciprofloxacin were 3.80 ± 0.85 µM and 182 ± 31 µM, respectively, while those for norfloxacin were 24.7 ± 0.1 µM and 393 ± 79 µM, respectively. Enrofloxacin and levofloxacin showed an inhibitory effect on the uptake of only the high concentration of ES and thus may be transported by the low-affinity site for ES. Interestingly, enrofloxacin and levofloxacin demonstrated an activation effect on ES uptake at the high-affinity binding site. These results suggested that multiple binding sites within the structure of bOatp1a2 may be responsible for the uptake of different quinolone antimicrobial agents.
Subject(s)
Binding Sites/physiology , Biological Transport/physiology , Fluoroquinolones/metabolism , Organic Anion Transporters/metabolism , Animals , Anti-Bacterial Agents/metabolism , Cattle , Cell Line , HEK293 Cells , Humans , KineticsABSTRACT
The organic anion-transporting polypeptides (OATPs, gene symbol SLCO) are a family of transporters that play important roles in the absorption, distribution, metabolism, and excretion of various drugs. Although substrate specificity of transporter proteins is under extensive study, the underlying mechanisms for substrate binding and/or recognition remain largely unknown. Transmembrane domain 6 (TM6) is a relatively conserved region within OATP family members, and several amino acid residues on its extracellular half are part of the OATP family signature sequence D-X-RW-(I,V)-GAWWX-G-(F,L)-L. In the present study, two adjacent tryptophan residues (Trp258 and Trp259) within TM6 were identified as critical amino acids for the transport function of OATP1B1. Kinetic studies showed that substitution of Trp258 with alanine resulted in monophasic kinetics for estrone-3-sulfate uptake, with a significantly higher Km value (Km = 12.0 ± 2.8 µM) than the high-affinity component of wild-type OATP1B1 (Km = 0.38 ± 0.06 µM). On the other hand, W259A retained the biphasic characteristic of the transporter. Km values of the high- and low-affinity components for estrone-3-sulfate of W259A are 1.93 ± 0.76 µM and 30.8 ± 4.4 µM, respectively. Further studies revealed that W258A retained transport function of another prototypic substrate, taurocholate, while W259A displayed a dramatically reduced uptake of the substrate and exhibited an 8-fold increase in the Km value compared with that of the wild-type and W258A. Our results suggest that Trp258 and Trp259 may play different roles in the uptake of different substrates by OATP1B1.
Subject(s)
Cell Membrane/genetics , Cell Membrane/metabolism , Organic Anion Transporters/genetics , Organic Anion Transporters/metabolism , Tryptophan/genetics , Tryptophan/metabolism , Amino Acid Sequence , Conserved Sequence , HEK293 Cells , Humans , Liver-Specific Organic Anion Transporter 1 , Molecular Sequence Data , Organic Anion Transporters/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary/physiology , Protein Transport/physiology , Tryptophan/chemistryABSTRACT
PURPOSE: Some members of the organic anion transporting polypeptide (OATP) family may play critical roles in determining the concentration of anticancer drugs in targeted organs/cells. The purpose of the present study was to investigate whether radiation can alter the expression of OATP in cancer cells so that OATP can be used as a specific drug target for anti-tumor treatment in combination with radiotherapy. MATERIALS AND METHODS: Two cancer cell lines (HepG2 and MCF7) were irradiated with three doses of X-ray and the expression levels of six OATP were assessed. RESULTS: Expressions of several OATP were altered after irradiation. OATP1A2, an OATP that was found to transport anti-tumor drugs and was undetectable in untreated cells, showed dramatically elevated level of expression after irradiation in MCF7 cells. The combined treatment of X-rays and the OATP1A2 substrate methotrexate exerted a more significant effect on growth rate and cell death compared with radiation or methotrexate treatment alone. At certain time points, X-rays increased OATP1A2 level while suppressing the expression of efflux transporters. CONCLUSIONS: Radiation may simultaneously increase the expression of uptake transporters and decrease the level of efflux transporters, suggesting OATP may be considered a novel target for delivery of anti-neoplastic agents in combination with radiotherapy.
Subject(s)
Organic Anion Transporters/genetics , Transcriptome/radiation effects , Hep G2 Cells , Humans , MCF-7 Cells , X-RaysABSTRACT
Organic anion transporting polypeptides (OATP) have been extensively recognized as key determinants of absorption, distribution, metabolism, and excretion of various drugs because of their broad substrate specificity, wide tissue distribution, and the involvement of drug-drug interaction. As the first cloned human OATP, OATP1A2 has been found to transport a wide spectrum of endogenous and exogenous compounds. Bovine Oapt1a2 shared high homology with the human transporter and is considered as its functional ortholog. In the present study, we expressed bovine Oatp1a2 in human embryonic kidney 293 cells and found that, unlike human OATP1A2, the transport of estrone-3-sulfate (E-3-S) exhibited biphasic saturation kinetics. The K(m) values are 0.25 ± 0.08 and 46.6 ± 18.5 µM, and V(max) values were 24.5 ±4.4 and 375 ± 142 pmol/mg protein/min for high- and low-affinity sites, respectively, suggesting the presence of multiple binding sites. Further study on other Oatp1a2 substrates showed that the high affinity component for E-3-S is responsible for the interaction with taurocholate, bromsulphthalein, and rifampicin and is sensitive to proton concentration change, whereas the low affinity binding site is only involved in the binding of the antitumor drug methotrexate and had no response to change of pH.
Subject(s)
Estrone/analogs & derivatives , Organic Anion Transporters/metabolism , Amino Acid Sequence , Animals , Binding Sites , Binding, Competitive , Cattle , Estrone/metabolism , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Kinetics , Methotrexate/metabolism , Organic Anion Transporters/chemistry , Organic Anion Transporters/genetics , Protein Structure, Secondary , Recombinant Proteins/metabolism , Rifampin/metabolism , Sulfobromophthalein/metabolism , Taurocholic Acid/metabolism , TransfectionABSTRACT
Organic anion transporting polypeptides (OATPs) have been extensively recognized as key determinants of absorption, distribution, metabolism and excretion (ADME) of various drugs, xenobiotics and toxins. Putative N-glycosylation sites located in the extracellular loops 2 and 5 is considered a common feature of all OATPs and some members have been demonstrated to be glycosylated proteins. However, experimental evidence is still lacking on how such a post-translational modification affect the transport activity of OATPs and which of the putative glycosylation sites are utilized in these transporter proteins. In the present study, we substituted asparagine residues that are possibly involved in N-glycosylation with glutamine residues and identified three glycosylation sites (Asn134, Asn503 and Asn516) within the structure of OATP1B1, an OATP member that is mainly expressed in the human liver. Our results showed that Asn134 and Asn516 are used for glycosylation under normal conditions; however, when Asn134 was mutagenized, an additional asparagine at position 503 is involved in the glycosylation process. Simultaneously replacement of all three asparagines with glutamines led to significantly reduced protein level as well as loss of transport activity. Further studies revealed that glycosylation affected stability of the transporter protein and the unglycosylated mutant was retained within endoplasmic reticulum.
Subject(s)
Organic Anion Transporters/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , Endoplasmic Reticulum/metabolism , Fluorescent Antibody Technique , Glycosylation , HEK293 Cells , Humans , Liver-Specific Organic Anion Transporter 1 , Molecular Sequence Data , Mutant Proteins/metabolism , Mutation/genetics , Organic Anion Transporters/chemistry , Protein Structure, SecondaryABSTRACT
Glycolate oxidase (GO) was purified to homogeneity from leaves of spinach (Spinacia oleracea). Through detecting the consumption of oxygen and the formation of hydrogen peroxide in the assay solution, it was found that GO could also oxidize glycerate, another metabolite in the photorespiratory pathway, and use FMN and FAD, but not riboflavin and lumiflavin, as its cofactors. The optimum reaction pH, Km for glycerate, k(cat) and activation energy of this oxidizing reaction were determined to be 8.0, 7.14 mmol/L, 1.04 s(-1) and 17.29 kJ/mol, respectively. Oxalate and pyruvate at 5.0 mmol/L could inhibit the glycerate-oxidizing activity by 34% and 26%, and oxalate acted as a competitive inhibitor of the glycerate oxidation reaction with a K(i) of 0.75 mmol/L. By the competition plotting with mixed-substrates, it was indicated that glycolate-oxidizing activity and glycerate-oxidizing activity of GO shared the same active site.
Subject(s)
Alcohol Oxidoreductases/metabolism , Glyceric Acids/metabolism , Plant Leaves/enzymology , Plant Proteins/metabolism , Spinacia oleracea/enzymology , Hydrogen-Ion Concentration , Kinetics , Oxidation-Reduction , Plant Leaves/metabolism , Spinacia oleracea/metabolismABSTRACT
The dependence of glycolate oxidase apoprotein (apoGO) activity on flavin analogs was surveyed in 9 higher plants from 7 families. Activities of all apoGOs depended not only on flavin mononucleotide (FMN) but also on flavin adenine dinucleotide (FAD), but not on riboflavin. The kinetic analysis showed that FMN was the optimum cofactor for apoGO from leaves of Brassica campestris. In plant kingdom, FMN, FAD and riboflavin are three flavin analogs with very similar structure, and they could coexist and be inter-converted from each other, so the question is how the apoprotein of glycolate oxidase (GO) recognized these flavin analogs. No inhibition effect of riboflavin on the activity of apoGO with FMN or FAD was found and no obvious quenching of riboflavin or apoGO protein fluorescence was detected with the addition of apoGO or riboflavin, respectively. These results indicated that riboflavin did not bind to apoGO tightly like FMN and FAD. Inorganic phosphate (Pi) did inhibit the activity of GO, and kinetic analysis revealed that this inhibition was caused by the competitive binding to apoGO between Pi and FMN. This competitive binding was further confirmed by the inhibition of Pi to the quenching of FMN and apoGO protein fluorescence with apoGO and FMN, respectively. It was suggested that the 5'-phosphate group of FMN or FAD may play a key role in the recognition and binding of riboflavin analog cofactors with apoGO.