Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting.
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.
Velho
Albertina M
AM
Department of Biosciences University of Kent, CT2 7NJ, UK.
Jarvis
Simon M
SM
D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D001205:Ascorbic Acid / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D006031:Glycosylation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D021381:Protein Transport; D060347:Sodium-Coupled Vitamin C Transporters; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000975:Antioxidants; D029363:Organic Anion Transporters, Sodium-Dependent; D010455:Peptides; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D001205:Ascorbic Acid