Wong S C, Zhang L, Proefke S A, Matherly L H
Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute and the Department of Pharmacology, School of Medicine, Wayne State University, 110 E. Warren Ave., Detroit, MI 48201, USA.
Biochim Biophys Acta. 1998 Oct 15;1375(1-2):6-12. doi: 10.1016/s0005-2736(98)00118-7.
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.
