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.
Chiao
J H
JH
Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Yang
C H
CH
Roy
K
K
Pain
J
J
Sirotnak
F M
FM
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