Ligand-directed immunoaffinity purification and properties of the one-carbon, reduced folate transporter. Interspecies immuno-cross-reactivity and expression of the native transporter in murine and human tumor cells and their transport-altered variants.
Almost complete purification (> 95%) of the 46-kDa murine, one-carbon, reduced folate transporter (RFT) at a recovery of 20% was obtained by ligand-directed immunoaffinity fractionation from transporter overproducing L1210/R83 cells. These cells were labeled with the N-hydroxysuccinimide ester of [3H]aminopterin (AMT), the isolated plasma membrane alkaline washed to remove nonintegral membrane proteins, detergent-solubilized, and RFT-separated on an anti-AMT antibody-protein G-Sepharose column followed by preparative SDS-polyacrylamide gel electrophoresis. Anti-RFT antibody, subsequently derived, differentially blotted (L1210/R83 >> L1210/0) a 46-kDa protein during SDS-polyacrylamide gel electrophoresis of plasma membrane from L1210/R83 and L1210 cells and in L1210/R83 cells after trichloroacetic acid precipitation. In contrast to that reported for human tumor cells, glycosidase treatment of RFT revealed no common N- or O-linked core oligosaccharides associated with this protein. The same 46-kDa protein at different relative levels was revealed in a Western blot of plasma membrane from other murine tumors. Blotting of plasma membrane from methotrexate resistant, transport defective L1210 cell variants exhibited wild-type levels of a less electrophoretically mobile RFT or greater levels of the same 46-kDa RFT which could not be affinity labeled with N-hydroxysuccinimide-[3H]AMT. The same antibody differentially blotted a 83-kDa plasma membrane protein from human HL-60 and CCRF-CEM cells with different levels of reduced folate transport and affinity labeling of RFT, verifying the conserved nature of this protein consistent with earlier functional studies.
Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
J H
JH
Chiao
C H
CH
Yang
K
K
Roy
J
J
Pain
F M
FM
Sirotnak
D000630:Aminopterin / Q000378:metabolism*; D000818:Animals; D000906:Antibodies; D000937:Antigen-Antibody Reactions; D015153:Blotting, Western / Q000379:methods; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D058973:Folate Receptors, GPI-Anchored; D006026:Glycoside Hydrolases; D006801:Humans; D007938:Leukemia; D007939:Leukemia L1210 / Q000378:metabolism; D008024:Ligands; D051379:Mice; D011817:Rabbits / Q000276:immunology; D011956:Receptors, Cell Surface; D013045:Species Specificity; D014316:Tritium; D014407:Tumor Cells, Cultured
D000906:Antibodies; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D008024:Ligands; D011956:Receptors, Cell Surface; D014316:Tritium; D006026:Glycoside Hydrolases; D000630:Aminopterin