Multiple folate transport systems in L1210 cells

Adv Enzyme Regul. 1992:32:3-15. doi: 10.1016/0065-2571(92)90005-k.

Abstract

Biotin derivatives of methotrexate (biotin-SS-MTX) and folate (biotin-SS-folate), in which the functional components are joined by a dissociable disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by HPLC, elemental analysis and mass spectrometry. These compounds provide a convenient means for the single-step purification of the folate transporters from L1210 cells. Parental L1210 murine leukemia cells, which contain only the microM transporter (the reduced folate/MTX transport protein) were treated with the N-hydroxysulfosuccinimide ester of biotin-SS-MTX, and a detergent extract of the plasma membranes was exposed to streptavidin-agarose beads to adsorb the labeled protein. Dithiothreitol cleavage of the disulfide linkage released the transporter, which migrated as a well-defined component (43 kDa) on SDS-PAGE gels; no other proteins were present. An L1210 subline (JF), obtained by adapting cells to grow on nanomolar concentrations of folate, contains both the microM transporter and the nM transporter (high-affinity folate binding protein). When these cells were treated with the N-hydroxysulfosuccimide ester of biotin-SS-folate and processed as described above, analysis on SDS-PAGE gels revealed the presence of two proteins, the microM transporter (43 kDa) and the nM transporter (39 kDa). Both transporters were characterized with respect to amino acid content; blocked N-termini precluded Edman sequencing. Treatment of the nM transporter with peptide:N-glycosidase F produced a smaller component (32 kDa); the microM transporter, conversely, was unchanged by this procedure. When the microM transporter in parental L1210 cells was labeled with fluorescein-MTX and then treated with phosphoinositol-specific phospholipase C (PI-PLC), no change in fluorescence was detected. Alternatively, when the nM transporter in the JF subline was labeled with fluorescein-folate and then treated with PI-PLC, complete loss of fluorescence was observed. These results indicate that the L1210 microM transporter is a non-glycosylated, integral membrane protein, while its nM counterpart is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Biotin / analogs & derivatives*
  • Biotin / chemical synthesis
  • Biotin / metabolism
  • Carrier Proteins / chemistry
  • Carrier Proteins / isolation & purification
  • Carrier Proteins / metabolism
  • Chromatography, Affinity / methods
  • Folate Receptors, GPI-Anchored
  • Folic Acid / analogs & derivatives
  • Folic Acid / chemical synthesis
  • Folic Acid / metabolism*
  • Leukemia L1210 / metabolism*
  • Methotrexate / analogs & derivatives*
  • Methotrexate / chemical synthesis
  • Methotrexate / metabolism
  • Mice
  • Receptors, Cell Surface / isolation & purification
  • Receptors, Cell Surface / metabolism
  • Tumor Cells, Cultured

Substances

  • Carrier Proteins
  • Folate Receptors, GPI-Anchored
  • Receptors, Cell Surface
  • biotin-SS-folate
  • biotin-SS-methotrexate
  • Biotin
  • Folic Acid
  • Methotrexate