The N1 domain of human lactoferrin is required for internalization by caco-2 cells and targeting to the nucleus.
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.
Suzuki
Yasushi A
YA
Department of Nutrition, University of California, Davis, California 95616, USA.
Wong
Henry
H
Ashida
Kin-Ya
KY
Schryvers
Anthony B
AB
Lönnerdal
Bo
B
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
D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D007781:Lactoferrin
FEBS J. 2007 Apr;274(8):2038-53
17371504
Blood. 1983 May;61(5):885-8
6831049
Microbiology (Reading). 2003 Jul;149(Pt 7):1729-1737
12855724
Biochim Biophys Acta. 1985 Dec 19;821(3):453-60
3000447
Adv Exp Med Biol. 1998;443:49-55
9781342
Mol Microbiol. 1999 Apr;32(1):111-21
10216864
Biochemistry. 2001 Dec 25;40(51):15771-9
11747454
J Pediatr Gastroenterol Nutr. 2003 Feb;36(2):190-9
12548053
Biochem J. 1988 Jan 15;249(2):435-41
2829858
FEBS Lett. 1989 Sep 11;255(1):201-4
2551729
Nature. 1995 Feb 23;373(6516):721-4
7854459
Eur J Biochem. 2004 Jan;271(2):303-17
14717698
J Biol Chem. 1996 Jan 12;271(2):1166-73
8557646
Biochem J. 1991 Jun 15;276 ( Pt 3):733-8
1648350
Cell Mol Life Sci. 2005 Nov;62(22):2560-75
16261254
J Cell Physiol. 1992 Dec;153(3):477-82
1447310
Protein Expr Purif. 1997 Mar;9(2):203-10
9056485
J Biol Chem. 2001 Nov 9;276(45):42575-9
11535608
Acta Paediatr Scand. 1987 Sep;76(5):733-40
3661174
J Biol Chem. 1993 Dec 25;268(36):27069-75
7903302
Clin Diagn Lab Immunol. 1995 Nov;2(6):763-5
8574844
Annu Rev Nutr. 1995;15:93-110
8527233
Biometals. 2004 Jun;17(3):325-9
15222485
Biochemistry. 1992 Sep 29;31(38):9243-51
1327110
J Pediatr Gastroenterol Nutr. 1995 Jul;21(1):37-43
8576812
Biochim Biophys Acta. 1983 Sep 13;759(3):229-35
6349699
Biometals. 2004 Jun;17(3):311-5
15222483
Am J Physiol. 1988 Apr;254(4 Pt 1):G580-5
2833117