N-Glycosylation dictates proper processing of organic anion transporting polypeptide 1B1

PLoS One. 2012;7(12):e52563. doi: 10.1371/journal.pone.0052563. Epub 2012 Dec 20.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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
  • Organic Anion Transporters / metabolism*
  • Protein Processing, Post-Translational*
  • Protein Structure, Secondary

Substances

  • Liver-Specific Organic Anion Transporter 1
  • Mutant Proteins
  • Organic Anion Transporters
  • SLCO1B1 protein, human

Grant support

This work was supported by the Returned high-level talents from abroad grant of the Chinese Ministry of Human Resources and Social Security and National Natural Science Foundation of China grant (81141115) to MH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.