Role of N-glycosylation in the structure and function of the methotrexate membrane transporter from CCRF-CEM human lymphoblastic leukemia cells

Abstract

The carrier protein for methotrexate and tetrahydrofolate cofactors (GP-MTX) in CCRF-CEM human lymphoblastic leukemia cells is a 117kDa 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 ( ic 50 = 0.80 μ g/mL ) and caused a potent suppression of [ 3H]mannose incorporation into nascent glycoproteins. From 1–3 μg/mL, inhibition of [ 3H]mannose incorporation was 66–87%, exceeding that for [ 35S]methionine incorporation by 2 to 4-fold. Tunicamycin (1 and 2 μg/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 K t 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 μg/mL, respectively) by digestions with N-glycanase. Hence, despite the large loss of N-glycan from GP-MTX in tunicamycin-treated cells, residual core oligosccharides 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.