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The N1 Domain of Human Lactoferrin Is Required for Internalization by Caco-2 Cells and Targeting to the Nucleus

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    The N1 Domain of Human Lactoferrin Is Required for Internalization by Caco-2 Cells and Targeting to the Nucleus†
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    Department of Nutrition, University of California, Davis, California 95616, and Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada
    †This work was partly supported by NIH Grant HD43240.
    * To whom correspondence should be addressed: Department of Nutrition, University of California, One Shields Ave., Davis, CA 95616. E-mail: [email protected]. Telephone: (530) 752-8347. Fax: (530) 752-3564.
    ‡University of California.
    §University of Calgary.
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    Biochemistry

    Cite this: Biochemistry 2008, 47, 41, 10915–10920
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    https://doi.org/10.1021/bi8012164
    Published September 12, 2008
    Copyright © 2008 American Chemical Society
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    Abstract

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    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.

    Copyright © 2008 American Chemical Society

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    Biochemistry

    Cite this: Biochemistry 2008, 47, 41, 10915–10920
    Click to copy citationCitation copied!
    https://doi.org/10.1021/bi8012164
    Published September 12, 2008
    Copyright © 2008 American Chemical Society
    Request reuse permissions

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