title: 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 Vmaxvalues 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; Kmvalues 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.
authors.affiliation: Faculty of Pharmacy, The University of Sydney , Sydney, NSW 2006, Australia.
authors.forename: Ting
authors.initials: T
authors.lastname: Chan
authors.affiliation: Faculty of Pharmacy, The University of Sydney , Sydney, NSW 2006, Australia.
authors.forename: Xiaoxi
authors.initials: X
authors.lastname: Lu
authors.affiliation: Faculty of Pharmacy, The University of Sydney , Sydney, NSW 2006, Australia.
authors.forename: Tahiatul
authors.initials: T
authors.lastname: Shams
authors.affiliation: Retinal Therapeutics Research Group, Save Sight Institute, The University of Sydney , Sydney, NSW 2000, Australia.
authors.forename: Ling
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Pharmacogenomics and Drug Development Group, Discipline of Pharmacology, School of Medical Sciences, The University of Sydney , Sydney, NSW 2006, Australia.
authors.forename: Michael
authors.initials: M
authors.lastname: Murray
authors.affiliation: Faculty of Pharmacy, The University of Sydney , Sydney, NSW 2006, Australia.
authors.forename: Fanfan
authors.initials: F
authors.lastname: Zhou
mesh_terms: D001692:Biological Transport / Q000235:genetics / Q000502:physiology; D019921:Biotinylation; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D000074011:Peptide Transporter 1; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D027981:Symporters / Q000235:genetics / Q000378:metabolism*
chemical_list: D000074011:Peptide Transporter 1; D020033:Protein Isoforms; C082186:SLC15A1 protein, human; D027981:Symporters
keywords: N-glycosylation; human oligopeptide transporter 1; post-translational modification; transport activity; transporter expression