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.

Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells

“…19 Transport efficiency for both the wild-type and E45K hRFCs, therefore, clearly decreased at high levels of expression. Similar findings were previously reported in other human cells transfected with wild-type hRFC 33,34 and may reflect roles for additional regulatory elements in hRFC function. For the CEM R0/RFC transfectants, high levels of wild-type hRFC expression resulted in a near complete (within four-fold for MTX, 2.5-fold for raltitrexed) reversal of this highly resist- Leukemia ant phenotype.…”

“…33 For serial dilutions, protein was separated on 4-15% gradient gels (Bio-Rad) and probed with anti-RFC antibody (1:1000 dilution). hRFC protein was detected and imaged as previously described.…”

Topological and Functional Analysis of the Human Reduced Folate Carrier by Hemagglutinin Epitope Insertion

“…2). If the RFC is properly localized to the plasma membrane, one would expect to see green fluorescence at the periphery of the cells expressing each construct, similar to the localization reported recently for a carboxyl-terminal HA fusion of the human RFC (22). This plasma membrane localization was seen in most of the transfectants analyzed in this study.…”

“…The human RFC protein is 591 amino acids as deduced from its cDNA sequence, and the hydropathy profile predicts 12 transmembrane-spanning (TM) domains with the amino and carboxyl termini located in the cytoplasm. Recently, the cytoplasmic location of the carboxyl-terminal tail has been confirmed for the human RFC (22). The mouse and hamster homologues (518 amino acids each) have significantly shorter carboxyl-terminal tails than the human RFC (15)(16)(17)(18)(19)(20), although all three RFCs share about 68% identical or similar amino acid residues.…”

“…The mouse and hamster homologues (518 amino acids each) have significantly shorter carboxyl-terminal tails than the human RFC (15)(16)(17)(18)(19)(20), although all three RFCs share about 68% identical or similar amino acid residues. The human RFC is glycosylated at an asparagine residue in the first extracellular loop (9,22,23), and while the hamster RFC contains the conserved sequence in this loop (15), its glycosylation status has not been confirmed. The murine RFC lacks the conserved glycosylation site and is not glycosylated (20).…”