Weifang Hong

Yunnan Agricultural University, Panlong, Shaanxi, China

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Publications (3)11.19 Total impact

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    ABSTRACT: 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±2.8μM) compared to the high affinity component of wild-type OATP1B1 (Km=0.38±0.06μM). 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±0.76μM and 30.8±4.4μM, 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 eight-fold increase in the Km value compared with that of the wild-type and W258A. Our results suggested that Trp258 and Trp259 may play different roles for the uptake of different substrates by OATP1B1.
    Molecular pharmacology 07/2013; · 4.12 Impact Factor
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    ABSTRACT: 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.
    PLoS ONE 12/2012; 7(12):e52563. · 3.53 Impact Factor
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    ABSTRACT: As an important structure in membrane proteins, transmembrane domains have been found to be crucial for properly targeting the protein to cell membrane as well as carrying out transport functions in transporters. Computer analysis of OATP sequences revealed transmembrane domain 2 (TM2) is among those transmembrane domains that have high amino acid identities within different family members. In the present study, we identify four amino acids (Asp70, Phe73, Glu74, and Gly76) that are essential for the transport function of OATP1B1, an OATP member that is specifically expressed in the human liver. A substitution of these four amino acids with alanine resulted in significantly reduced transport activity. Further mutagenesis showed the charged property of Asp70 and Glu74 is critical for proper function of the transporter protein. Comparison of the kinetic parameters indicated that Asp70 is likely to interact with the substrate while Glu74 may be involved in stabilizing the binding site through formation of a salt-bridge. The aromatic ring structure of Phe73 seems to play an important role because substitution of Phe73 with tyrosine, another amino acid with a similar structure, led to partially restored transport function. On the other hand, replacement of Gly76 with either alanine or valine could not recover the function of the transporter. Considering the nature of a transmembrane helix, we proposed that Gly76 may be important for maintaining the proper structure of the protein. Interestingly, when subjected to transport function analysis of higher concentration of esteone-3-sulfate (50 µM) that corresponds to the low affinity binding site of OATP1B1, mutants of Phe73, Glu74, and Gly76 all showed a transport function that is comparable to that of the wild-type, suggesting these amino acids may have less impact on the low affinity component of esteone-3-sulfate within OATP1B1, while Asp 70 seems to be involved in the interaction of both sites.
    PLoS ONE 05/2012; 7(5):e36647. · 3.53 Impact Factor

Publication Stats

17 Citations
11.19 Total Impact Points

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  • 2013
    • Yunnan Agricultural University
      Panlong, Shaanxi, China
  • 2012
    • South China Agricultural University
      • College of Life Sciences
      Guangzhou, Guangdong Sheng, China