Role of N-glycosylation in the structure and function of the methotrexate membrane transporter from CCRF-CEM human lymphoblastic leukemia cells.
The carrier protein for methotrexate and tetrahydrofolate cofactors (GP-MTX) in CCRF-CEM human lymphoblastic leukemia cells in a 117 kDa glycoprotein containing both N- and O-linked oligosaccharides (Matherly et al., J Biol Chem 267: 23253-23260, 1992). Tunicamycin, an inhibitor of N-glycosylation, was used to investigate the roles of asparagine-linked oligosaccharides in the structure, intracellular routing, and transport function of GP-MTX. Tunicamycin was growth inhibitory toward CCRF-CEM cells (IC50-0.80 micrograms/mL) and caused a potent suppression of [3H]mannose incorporation into nascent glycoproteins. From 1-3 micrograms/mL, inhibition of [3H]mannose incorporation was 66-87%, exceeding that for [35S]methionine incorporation by 2 to 4-fold. Tunicamycin (1 and 2 micrograms/mL) exposures decreased the median molecular masses of GP-MTX on immunoblots (to 82 and 67 kDa, respectively) and were accompanied by reduced maximal rates of methotrexate uptake (31 and 37%, respectively, of control levels). Conversely, the Ki values for methotrexate binding to the transporter were unaffected by tunicamycin treatments. The effects of tunicamycin on methotrexate influx closely correlated with lower levels of immunoreactive GP-MTX in plasma membranes and specific [3H]methotrexate binding to intact cells, suggesting that the transport effect was due to decreased numbers of carrier proteins at the membrane surface. The reduced molecular mass values for GP-MTX, which accompanied tunicamycin exposures, were further decreased (to 55 and 50 kDa at 1 and 2 micrograms/mL, respectively) by digestions with N-glycanase. Hence, despite the large loss of N-glycan from GP-MTX in tunicamycin-treated cells, residual core oligosaccharides remained. The sizes of hypoglycosylated GP-MTX following both treatments were similar to that of the functionally homologous methotrexate membrane carrier previously identified in L1210 murine leukemia cells.
Matherly
L H
LH
Developmental Therapeutics Program, Michigan Cancer Foundation, Detroit 48201.
Angeles
S M
SM
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007945:Leukemia, Lymphoid / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism*; D009363:Neoplasm Proteins; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D008727:Methotrexate