Title : Topological studies of
hSVCT1 , the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting
Abstract :
- The Na(+)-dependent transporters, hSVCT1 and hSVCT2 , were assessed in COS-1 cells for their membrane topology
- Antibodies to N- and C-termini of hSVCT1 and C-terminus of hSVCT2 identified positive immunofluorescence only after permeabilisation, suggesting these regions are intracellular
- PNGase F treatment confirmed that WT hSVCT1 (approximately 70-100 kDa) is glycosylated and site-directed mutagenesis of the three putative N-glycosylation sites, Asn138, Asn144, Asn230 , demonstrated that mutants N138Q and N144Q were glycosylated (approximately 68-90 kDa) with only 31-65% of WT l-ascorbic acid (AA) uptake while the glycosylation profile of N230Q remained unaltered (approximately 98% of WT activity)
- However, the N138Q/N144Q double mutant displayed barely detectable membrane expression at approximately 65 kDa, no apparent glycosylation and minimal AA uptake (<10%) with no discernible improvement in expression or activity when cultured at 28 degrees C or 37 degrees C. Marker protein immunocytochemistry with N138Q/N144Q identified intracellular aggregates with hSVCT1 localised at the nuclear membrane but absent at the plasma membrane thus implicating its role as a possible intracellular transporter and suggesting N-glycosylation is required for hSVCT1 membrane targeting
- Also, Lys242 on the same putative hydrophilic loop as Asn230 after biotinylation was inaccessible from the extracellular side when analysed by MALDI-TOF MS. A new hSVCT1 secondary structure model supporting these findings is proposed
Output (sent_index, trigger,
protein,
sugar,
site):
- 3. N-glycosylation, , -, -, sites, Asn138, Asn144, Asn230
- 3. and, , -, -, sites, Asn138, Asn144, Asn230
- 3. glycosylated, , WT hSVCT1, -, -
- 4. N-glycosylation, , hSVCT1, -, -
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 3. WT hSVCT1, -, sites, Asn138, Asn144, Asn230