title: Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor and transport to endosomes.
abstract: LDL receptor related protein (LRP) is a ubiquitously expressed cell surface receptor that binds, at least in vitro, a plethora of ligands among them alpha 2-macroglobulin and lactoferrin (Lf). The function of LRP in internalisation and distribution of ligands within cellular metabolism is still unclear. We here investigated by combined ligand- and immunoblotting the participation of LRP/alpha 2MR and its associated protein (RAP) in receptor mediated endocytosis of Lf into rat liver. We found LRP highly enriched in sucrose density gradient fractions around density 1.10 g/ml, previously characterised as endosomal fractions. RAP was concentrated in distinct fractions around density 1.14 g/ml. This separation of RAP from LRP/alpha 2MR is physiologically meaningful as RAP avidly binds to LRP/alpha 2MR and by that shuts off all ligand binding function. In endosomal fractions we found one single binding protein for 125I-labelled Lf. With a specific anti LRP/alpha 2MR antibody and ligand blotting with 125I-labelled RAP this endosomal Lf binding site was verified to be LRP/alpha 2MR. Endosomes did not bind labelled Lf when prepared from rats that received an intravenous injection of Lf (20 mg per animal) 20 min prior to preparation. Surprisingly we immunodetected Lf in these endosomes at a position around 600 kDa, comigrating with LRP/alpha 2MR. We determined Lf binding to be optimal at pH 5.8, what led us to suggest the existence of a very stable LF-LRP/alpha 2MR complex in endosomes. These data support the idea of effective binding of Lf at pH as found in inflamed tissue environment where Lf is reported to be involved in leukocyte mediated inflammation regulation.
authors.affiliation: Department of Medical Chemistry, University of Vienna, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Meilinger
authors.forename: M
authors.initials: M
authors.lastname: Haumer
authors.forename: K A
authors.initials: KA
authors.lastname: Szakmary
authors.forename: F
authors.initials: F
authors.lastname: Steinböck
authors.forename: B
authors.initials: B
authors.lastname: Scheiber
authors.forename: H
authors.initials: H
authors.lastname: Goldenberg
authors.forename: M
authors.initials: M
authors.lastname: Huettinger
mesh_terms: D000818:Animals; D001692:Biological Transport; D002451:Cell Compartmentation; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007781:Lactoferrin / Q000097:blood*; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008297:Male; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D011971:Receptors, Immunologic / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism*
chemical_list: D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011971:Receptors, Immunologic; D000511:alpha-Macroglobulins; D007781:Lactoferrin