title: The N1 domain of human lactoferrin is required for internalization by caco-2 cells and targeting to the nucleus.
abstract: Human lactoferrin (hLf) has been shown to interact with cells from the Caco-2 human small intestinal cell line. There currently is little information about the molecular details of its interaction. As a first step toward detailed characterization of this interaction, we used a series of Lf chimeras to analyze which part of Lf is responsible for the interaction with Caco-2 cells. Recombinant chimeric proteins consisting of segments of hLf and bovine transferrin (bTf) were produced in a baculovirus-insect cell system and purified by a combination of cation exchange chromatography and immobilized bTf antibody affinity chromatography. Each chimera was labeled with a green fluorescent dye to monitor its interaction with Caco-2 cells. Similarly, the intestinal Lf receptor (LfR), also known as intelectin, was probed with an anti-LfR antibody that was detected with a secondary antibody conjugated with a red-color fluorescent dye. The results demonstrated that chimeric proteins containing the N-lobe or the N1.1 subdomain of Lf bound as well as intact Lf to Caco-2 cells. Confocal microscopy analysis revealed that these proteins, along with the LfR, were internalized and targeted to the nucleus. These results indicate that the N1.1 subdomain of hLf is sufficient for binding, internalization, and targeting to the nucleus of Caco-2 cells.
authors.affiliation: Department of Nutrition, University of California, Davis, California 95616, USA.
authors.forename: Yasushi A
authors.initials: YA
authors.lastname: Suzuki
authors.forename: Henry
authors.initials: H
authors.lastname: Wong
authors.forename: Kin-Ya
authors.initials: KY
authors.lastname: Ashida
authors.forename: Anthony B
authors.initials: AB
authors.lastname: Schryvers
authors.forename: Bo
authors.initials: B
authors.lastname: Lönnerdal
mesh_terms: D000595:Amino Acid Sequence; D018938:Caco-2 Cells; D002467:Cell Nucleus / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007781:Lactoferrin / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D007781:Lactoferrin
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