Receptor-mediated Transcytosis of Lactoferrin through the Blood-Brain Barrier

Abstract

Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation; it accumulates in the brain during neurodegenerative disorders. Before determining Lf function in brain tissue, we investigated its origin and demonstrate here that it crosses the blood-brain barrier. An in vitro model of the blood-brain barrier was used to examine the mechanism of Lf transport to the brain. We report that differentiated bovine brain capillary endothelial cells exhibited specific high (K d ‫؍‬ 37.5 nM; n ‫؍‬ 90,000/cell) and low (K d ‫؍‬ 2 M; n ‫؍‬ 900,000 sites/cell) affinity binding sites. Only the latter were present on nondifferentiated cells. The surface-bound Lf was internalized only by the differentiated cell population leading to the conclusion that Lf receptors were acquired during cell differentiation. A specific unidirectional transport then occurred via a receptor-mediated process with no apparent intraendothelial degradation. We further report that iron may cross the bovine brain capillary endothelial cells as a complex with Lf. Finally, we show that the low density lipoprotein receptor-related protein might be involved in this process because its specific antagonist, the receptor-associated protein, inhibits 70% of Lf transport. Lactoferrin (Lf)1 (1) is a mammalian cationic iron-binding glycoprotein belonging to the transferrin (Tf) family. Despite some striking differences, mainly in the glycan moiety, there are marked sequence and conformational homologies among Lfs from different species, as well as similar general functions (for review, see Ref.2). Many physiological roles have been ascribed to Lf, particularly in the host defense against infection and severe inflammation (for review, see Ref. 3). This broad spectrum of biological functions relies on the interaction of Lf with numerous cells. The binding of Lf to cells is independent of its degree of iron saturation and is mediated mainly via interaction of the cluster of basic amino acids at its NH 2 terminus with sulfated molecules (4, 5). However, Lf is also targeted to specific cell receptors, and only a few of these involved in its uptake have been clearly identified. The 105-kDa Lf receptor characterized on activated human T-cells (6) is expressed at the cell surface of platelets (7), megacaryocytes (8), dopaminergic neurons, and mesencephalon microvessels (9). Lf receptor internalizes Lf, which is subsequently degraded (30 -40%), whereas the remaining fraction is recycled (10). In addition, the low density lipoprotein receptor-related protein (LRP) displays a high affinity for Lf and is responsible for its clearance (11)(12)(13)(14). This is inhibited by RAP, the receptor-associated protein known to be an antagonist for LRP (15). Transcytosis of Lf was described for HT29 cells (16) and was a minor pathway, up-regulated during iron deprivation (17).Lf is produced by exocrine glands (1, 18) and is widely distributed in the body fluids. It is stored in specific granules of neutrophilic leukocytes (19) and is relea...

Multivalent Metal-Induced Iron Acquisition from Transferrin and Lactoferrin by Myeloid Cells

“…The transferrin receptor has been well described (9 -11); however, the identity of cellular lactoferrin receptor(s) remains poorly defined (28,30,(32)(33)(34)(35)(36)(37)(38)(39). Our data do not suggest that the mechanism responsible for the ability of the various metals to enhance surface binding of these proteins to the cell surface involves an increase in transferrin receptor expression.…”

“…Lactoferrin does not bind to the transferrin receptor (28 -31). It does bind to the surface of many cell types, but whether this occurs via a lactoferrin-specific receptor remains unclear (28,30,(32)(33)(34)(35)(36)(37)(38)(39)(40). Even when lactoferrin binding to cells occurs, resulting Fe acquisition has not been uniformly observed (37,38,41,42).…”

Enhanced gene expression in the brain following intravenous administration of lactoferrin-bearing polypropylenimine dendriplex

“…Firstly, it has been reported that the specific binding of Lf with Lf receptors on BBB was not affected by the low endogenous Lf under physiological conditions, which is not the case for Tf [28]. Furthermore, Lf-mediated penetration of BBB is unidirectional, which might increase accumulation of the dendriplexes in the brain [29] and thus makes Lf a highly promising targeting moiety for brain delivery.…”

Effect of lactoferrin- and transferrin-conjugated polymersomes in brain targeting: in vitro and in vivo evaluations

“…TfR and LfR have been demonstrated to exist on the BBB in different species and to be involved in Tf and Lf transport across the BBB in vitro and in vivo [11,12,14,15] . In the standard protocol, perfusion is performed before tissues are harvested, and in most of our past experiments, perfusion was performed.…”

“…Though Tf and Lf are quite similar overall in sequence and structure, and coordinate iron in the same manner, they differ in the structure of their inter-lobe linker, the salt bridge between the helical linker, their pattern of disulfide bonding, and their receptor binding properties [11] . Lf receptor (LfR) and Tf receptor (TfR) have been demonstrated to exist on the BBB in different species and to be involved in Lf and Tf transport across the BBB in vitro and in vivo [12][13][14][15] . There are interesting reports that the expression of LfR in the brain is increased under some disease conditions such as Parkinson's disease and Alzheimer's disease [16][17][18] and that TfR is more highly expressed on tumor cells than on ordinary cells [19] .…”