The Role of N-Glycosylation in Maintaining the Transporter Activity and Expression of Human Oligopeptide Transporter 1.

ABSTRACT

Human oligopeptide transporter 1 (hPepT1) mediates the absorption of dietary peptides and a range of clinically relevant drugs. According to the predicted topological structure, hPepT1 contains multiple asparagine residues in putative N-glycosylation sites. This study investigated the influence of the six putative N-glycosylation sites within the extracellular region between transmembrane domains 9 and 10 on hPepT1 transporter function and expression in HEK-293T cells. Our study confirmed that hPepT1 is N-glycosylated in HEK-293T cells with the glycosylated and fully deglycosylated isoforms exhibiting apparent molecular masses of ∼78 and ∼55 kDa, respectively. Transport uptake of Glycylsarcosine (Gly-sar) by the hPepT1-N562Q variant, but not by other single mutants, was moderately impaired. We also constructed multiple N-glycosylation mutants based on the hPepT1-N562Q mutant by mutagenizing the additional asparagine residues N404Q, N408Q, N439Q, N509Q, and N514Q. Transport function showed a graded decrease as the number of mutagenized residues increased and simultaneous removal of all six asparagine residues essentially abolished transport activity. Kinetic studies indicated that the Vmax values for Gly-sar transport by low activity mutants were decreased compared to those of wild-type, which suggested that the cell surface expression and/or turnover rate of hPepT1 mutants was impaired; Km values were unchanged in most cases. Using immunoblotting and immunofluorescence, the plasma membrane and total cellular expression of the mutant transporters were decreased in accordance with functional impairments. In summary, we provide the first molecular evidence that hPepT1 is modified by N-glycosylation and that all six asparagine residues in the large extracellular loop between transmembrane domains 9 and 10 are subject to N-glycosylation. This information enhances our understanding of the role of the large extracellular loop in hPepT1 regulation and could facilitate the development of new hPepT1 substrate drugs with improved bioavailability.