Cellular internalization of lactoferrin in intestinal epithelial cells.
We studied the cellular internalization of lactoferrin (Lf) in an intestinal epithelial cell line, Caco-2, to investigate the mechanism of biological actions of ingested Lf. RT-PCR and Western blotting analyses revealed that differentiated Caco-2 cells express LfR mRNA and its protein with a 34 kD molecular weight under reducing conditions. Biotin-labeled Lf showed specific binding to the cellular membrane of differentiated Caco-2 cells with a dissociation constant (Kd) of 0.16 microM. The cellular internalization of Lf was studied in differentiated Caco-2 cells grown as monolayers on Transwell inserts, and compared to that of human transferrin (Tf). After labeling with fluorescent dye, either Lf or Tf was added to Caco-2 cells from the apical side or the basolateral one. Laser scanning confocal microscopy showed that labeled Lf was internalized only from the apical side and localized to the nuclei. On the other hand, labeled Tf was internalized from the basolateral side, not from the apical side, and localized in the cytoplasm. The internalization of labeled Lf was inhibited by excess of unlabeled Lf, but not of Tf. The internalization of labeled Lf, but not of labeled Tf, was also suppressed by heparin. This indicates that a heparin-binding site in the N-terminal region of Lf could be important for the internalization of Lf. These findings suggest that ingested Lf might be internalized by the intestinal epithelium in a manner different from Tf and might function in the nucleus.
Ashida
Kinya
K
Department of Nutrition, University of California, Davis, CA 95616, USA.
Sasaki
Hajime
H
Suzuki
Yasushi A
YA
Lönnerdal
Bo
B
D000818:Animals; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology*; D007781:Lactoferrin / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism
D011956:Receptors, Cell Surface; D014168:Transferrin; C038340:lactoferrin receptors; D006493:Heparin; D007781:Lactoferrin