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

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.

Figure 1. OATP1B1 is glycosylated in HEK293 cells.

A. The effect of tunicamycin on protein size of OATP1B1. B. Transport of E-3-S (0.1 μM) in OATP1B1-expressed HEK293 cells with or without tunicamycin treatment. C. Plasma membrane proteins from cells expressing OATP1B1 treated with N-glycosidase F. Cells expressing OATP1B1 were treated with 0.5 μg/ml of tunicamycin for 42 h before analysis. For protein expression, cells were lysed with RIPA buffer, separated by SDS-PAGE, followed by Western blotting with anti-HA antibody. Fifty micrograms of protein was loaded for each lane. Transport function of tunicamycin treated cells was expressed as a percentage of the uptake measured in the untreated control. The results represent data from three experiments, with triplicate measurements for each sample. The results shown are means ± S.E. (n  = 3). For glycosidase treatment, cell surface proteins were biotinylated and precipitated with streptavidin beads. Proteins were then denatured with 0.5%SDS and 1% β-mercaptoethanol at 75°C for 15 min. The denatured proteins were incubated with or without N-glycosidase F overnight at 37°C before subjected to SDS-PAGE.

Figure 2. Secondary structure model of OATP1B1.

Putative glycosylation sites were identified with NetNGlyc 1.0 Server and compared with membrane protein topology prediction analysis TopPred (Kyte-Doolittle hydrophobicity scale), only asparagines located extracellularly were considered as candidates for glycosylation sites. Putative sites were marked as black diamonds and each position was indicated with arrows.

Figure 3. Effect of single disruption of putative glycosylation sites.

A. Western blot analysis of single mutants. Plasma membrane proteins were isolated from cells expressing wild-type OATP1B1 and mutants through biotinylation. Same blot was probed with integrin antibody as surface protein loading control. B. Transport of E-3-S (0.1 μM) in OATP1B1 and five single mutants. Transport function of mutants was expressed as a percentage of the uptake measured in wild-type. The results represent data from three experiments, with triplicate measurements for each mutant. The results shown are means ± S.E. (n  = 3). C. Plasma membrane proteins of wild-type OATP1B1 and N134Q treated with N-glycosidase F. Glycosidase treatment was performed as described above.

Figure 4. Effect of multiple disruption of OATP1B1 glycosylation sites.

A. Western blot analysis for plasma membrane protein expression of double mutants. B. Transport of E-3-S (0.1 μM) in OATP1B1 and double mutants. C. Western blot analysis for plasma membrane protein expression of triple mutants. D. Plasma membrane proteins of wild-type OATP1B1 and N134/503/516Q treated with N-glycosidase F. E. Total protein expression of OATP1B1 and triple mutants. Same blot was probed with actin antibody as loading control. F. Western blot analysis of wild-type OATP1B1 and triple mutant N134/503/516Q treated with proteasome inhibitor MG132. Cells were treated with 10 μM MG132 for 6 h before being lysed with RIPA buffer and subjected to Western blotting. Fifty micrograms of protein was loaded for each lane.

Figure 5. Functional analysis of triple glycosylation site mutants.

Cells were incubated with 0.1 μM E-3-S for 2 min at 37°C. Transport function of mutants was expressed as a percentage of the uptake measured in wild-type OATP1B1. The results represent data from three experiments, with triplicate measurements for each mutant. The results shown are means ± S.E. (n  = 3). Asterisks indicate values significantly different (p<0.05) from that of wild-type OATP1B1.

Figure 6. Immunofluorescence study of triple mutant N134/503/516Q.

Cells expressing OATP1B1 wild-type and N134/503/516Q were stained with anti-HA antibody (1∶100) and anti-calnexin antibody (1∶100) and reacted with Alexa Fluor 488 goat anti-mouse IgG or Alexa Fluor 555 goat anti-rabbit IgG antibody. Specific immunostaining shown as green (OATP1B1) or red (calnexin) fluorescence.