Identification of high affinity folate binding proteins in human erythrocyte membranes.

Abstract

Mature human erythrocyte membranes contained specific, high affinity (Kd 33 X 10-11 M) folate binding moieties. Folate binding was pH, time-and temperature-dependent, saturable, and was much greater for pteroylmonoglutamate and 5-methyltetrahydrofolate than 5-formyltetrahydrofolate and amethopterin. On detergent solubilization of membranes, two peaks of specific folate binding with M, 2 200,000 and 160,000 were identified on Sephacryl S-200 gel filtration chromatography in Triton X-100, and this corresponded to two similar peaks of immunoprecipitated material when solubilized iodinated membranes were probed with anti-human placental folate receptor antiserum. Age-dependent separation of erythrocytes by Stractan density gradients revealed a sevenfold greater folate binding capacity in membranes purified from younger compared with aged erythrocytes. Since this difference was not reflected in proportionately higher immunoreactive folate binding protein, (as determined by a specific radioimmunoassay for these proteins) or differences in affinity in younger than aged cells, these findings indicate that erythrocyte folate binding proteins become progressively nonfunctional at the onset of red cell aging.

Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition

“…1; Fig. S1) (25,26,54). Two human folate receptors, hFRα and hFRβ, have been examined extensively as potential vectors for the delivery of folate-based drugs (6,55).…”

Megaloblastic hematopoiesis in vitro. Interaction of anti-folate receptor antibodies with hematopoietic progenitor cells leads to a proliferative response independent of megaloblastic changes.

“…Externally-oriented proteins on 9 x I07 intact LDMNC were iodinated with 1 mCi ['25I]NaI and 20 Mg iodogen, as previously described (21). After five centrifuge-wash cycles (1,000 g for 10 min) using 50 ml PBS per wash, cells were assessed for viability, after which membrane proteins were solubilized with 5 ml of 10 mM KPO4, pH 7.5, containing 1.5% Triton X-100, 0.1% sodium azide, and 10 mM EDTA for 24 h at 4C.…”

“…After washing the IgGsorb pellets four cycles with 30 vols of PBS, they were boiled in 5% SDS for 5 min. After centrifugation in a microfuge, the boiled supernatants were subjected to 15% SDS-PAGE and autoradiography, as described (21). A similar experiment where GammaBind G-agarose-purified IgG (120 Mg) from nonimmune serum and anti-PFR antiserum were substituted for crude sera was also carried out.…”

“…The efficiency of detritonization was > 95% after eight cycles based on the residual amount of radioactivity in the sample. After drying, the sample was resuspended in I ml of H20 and iodinated (21) with 100 Mg iodogen and I mCi ['25I]NaI for 20 min at 22°C. After quenching the reaction mixture with 0.1 M unlabeled Nal, the sample was resuspended in 1% Triton X-100 and applied to a calibrated I X 30 cm Sephadex G-25 column which was equilibrated and eluted with 10 mM KPO4, pH 7.5, containing 1% Triton X-100.…”

“…Earlier, we determined that the majority of FBPs on intact erythrocytes were nonfunctional (20,21); however, when rabbit anti-PFR antiserum (but not nonimmune or PFR-neutralized anti-PFR antiserum) was incubated with developing erythroid progenitor cells in vitro, we observed that burst forming unit (BFU)-erythroid (E)-derived normoblasts exhibited increased cloning efficiency and had biochemical evidence of intracellular folate deficiency as well as megaloblastic morphology (22). Thus, we concluded that normal human cells also relied on folate receptors for cellular folate uptake.…”