Importance of extracellular disulfide bonds for the surface expression and function of human organic anion transporting polypeptide 1B1

Organic anion transporting polypeptide 1B1 (OATP1B1) is an important member of the solute carrier organic anion transporter family and is exclusively expressed on the basolateral membrane of human hepatocytes. As a membrane protein, its extracellular cysteine residues are very likely forming disulfide bonds. The cryo-electron microscopy structure of OATP1B1 reveals that 16 of its 20 cysteine residues are located on the extracellular side. Our present study showed that OATP1B1 has no free cysteines to react with the sulfhydryl-reactive biotinylation reagent maleimide-PEG₂-biotin. However, mutation of 1 of the 2 cysteines in each disulfide bond pair would release a free cysteine that can be labeled by maleimide-PEG₂-biotin. These results indicate that all 16 extracellular cysteine residues in OATP1B1 are involved in the formation of disulfide bonds. Among the 8 extracellular disulfide bonds, C430-C530 and C599-C613 are essential for the surface expression of OATP1B1, because their breakage makes OATP1B1 almost entirely retained intracellularly; disulfide bonds C142-C463, C465-C485, C474-C524, C489-C504, and C506-C459 have a significant effect on both surface expression and transport activity of OATP1B1 per se, whereas C162-C607 is dispensable for both surface expression and function of OATP1B1. In addition, the N-glycosylation status of OATP1B1 would be changed on disulfide bond breakage, which may play a role in rescuing the surface expression of disulfide bond-disrupted OATP1B1. Taken together, most extracellular disulfide bonds are crucial for normal surface expression and function of OATP1B1. SIGNIFICANCE STATEMENT: This study revealed that all extracellular cysteine residues in organic anion transporting polypeptide 1B1 (OATP1B1) form disulfide bonds and play important roles in the expression and function of OATP1B1. Nonsynonymous single nucleotide variations for some key cysteine residues of OATP1B1 such as C430, C530, C599, and C613 have been observed. Our current findings may provide a good basis to predict the in vivo function of OATP1B1 cysteine variants and potential OATP1B1-mediated adverse drug reactions in those individuals who carry these genetic variants.

Disulfide bond; Extracellular cysteine; Maleimide biotinylation; Organic anion transporting polypeptide; Post-translational modification; Surface biotinylation.