Title : Effects of the loss of capacity for N-glycosylation on the transport activity and cellular localization of the human
reduced folate carrier
Abstract :
- The role of N-glycosylation in reduced folate carrier ( RFC ) transport and membrane targeting was examined in transport-deficient K562 (K500E) cells transfected with human RFC cDNAs
- Treatment of cells expressing wild-type RFC with tunicamycin (0-3 microg) resulted in a progressive shift of the approximately 85 kDa RFC on western blots to 65 kDa
- At 3 microg/ml tunicamycin , the nearly complete loss of glycosylated RFC was accompanied by a approximately 25% decreased rate of methotrexate uptake
- A deglycosylated RFC cDNA construct in which asparagine-58 was replaced by glutamine ( Gln58- RFC ) was expressed in K500E cells as a 65 kDa protein and restored transport capacity for methotrexate and (6S)5-formyl tetrahydrofolate
- With both wild-type and Gln58- RFC constructs, expression of cDNA-encoded RFC protein far exceeded relative levels of RFC uptake
- Wild-type and Gln58-RFCs containing a hemagglutinin (HA) epitope at the carboxyl terminus were similarly functional and, by immunofluorescence staining with rhodamine-conjugated anti-HA antibody, were localized to plasma membranes
- Collectively, our results demonstrate that N-glycosylation of human RFC plays no significant role in either transport function or membrane targeting
- The discrepancy between the stoichiometries of RFC expression and transport activity for both wild-type RFC and Gln58- RFC implies that identical regulatory controls and/or non- RFC transport components are necessary to completely restore transport function in the transfected cells