Title : Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane
protein with three N-linked carbohydrate chains
Abstract :
- Organic anion transport protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous transport proteins , is expressed on the basolateral (sinusoidal) surface of rat hepatocytes
- The organization of oatp1a1 within the plasma membrane has not been well defined, and computer-based models have predicted possible 12- as well as 10-transmembrane domain structures
- Which of oatp1a1's four potential N-linked glycosylation sites are actually glycosylated and their influence on transport function have not been investigated in a mammalian system
- In the present study, topology of oatp1a1 in the rat hepatocyte plasma membrane was examined by immunofluorescence analysis using an epitope-specific antibody designed to differentiate a 10- from a 12-transmembrane domain model
- To map glycosylation sites , the asparagines at the each of the four N-linked glycosylation consensus sites were mutagenized to glutamines
- Mutagenized oatp1a1 constructs were expressed in HeLa cells, and effects on protein expression and transport activity were assessed
- These studies revealed that oatp1a1 is a 12-transmembrane-domain protein in which the second and fifth extracellular loops are glycosylated at asparagines 124, 135, and 492 , whereas the potential glycosylation site at asparagine 62 is not utilized, consistent with its position in a transmembrane domain
- Constructs in which more than one glycosylation site were eliminated had reduced transport activity but not necessarily reduced transporter expression
- This was in accord with the finding that fully unglycosylated oatp1a1 was well expressed but located intracellularly with limited transport ability as a consequence of its reduced cell surface expression
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. protein, , protein, three N-linked carbohydrate chains, -
- 3. glycosylation, , -, -, sites
- 5. glycosylation, , -, -, sites
- 7. glycosylated, , protein, -, asparagines 124, 135, and 492
- 7. glycosylation, , -, -, asparagine 62
- 7. glycosylation, , -, -, site
- 7. utilized, , -, -, asparagine 62
- 7. utilized, , -, -, site
- 8. glycosylation, , -, -, site
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 7. oatp, -, asparagine 62
- 7. protein, -, asparagines 124, 135, and 492