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...

The screening of a bovine submaxillary gland cDNA library yielded 25 clones coding for bovine lactotransferrin. The nucleotide sequence of the longest insert contained a protein-coding region of 21 15 nucleotides and a 3' non-coding region of 194 nucleotides followed by a poly(A) tract of about 55 nucleotides. The predicted peptide sequence included a 16-amino-acid signal sequence upstream of the first amino acid of the native protein.The identity of the clone was confirmed by matching the amino acid sequence predicted from the cDNA with the N-terminal and tryptic peptide sequences derived from purified bovine milk lactotransferrin, and also by similarity with human and murine lactotransferrins. The cDNA described corresponds to a 705-amino-acid-long preprotein that lacks the start methionine. The sequence of the secreted protein is 689 amino acids long and contains five potential glycosylation sites. Bovine lactotransferrin is 69% and 64% identical to human and murine lactotransferrins, respectively.The transferrins are a family of non-haem, iron-binding proteins which includes serum transferrin, lactotransferrin and melanotransferrin. They are monomeric glycoproteins with a molecular mass of about 80 kDa constituted by two lobes, each possessing one iron-binding site with the capacity to bind reversibly one ferric ion (Fe3+) (reviewed in [l, 21). Lactotransferrins (also called lactoferrins) are present in various biological fluids such as human milk [3 -51, bovine milk [6], saliva [7, 81 and mucous secretions [8, 91. They are also present in leucocytes [S, 10, 111. Although lactotransferrins were first isolated in 1960 from both human and bovine milk, most of the studies concerning their structure and biological roles (reviewed in [l, 21 and [12 -141) were performed on the human protein. The sequence of human lactotransferrin was resolved using chemical methods [15] and cDNA analysis [16] (and cited by Anderson et al. in [17]). It consists of a 691-amino-acid polypeptide chain to which two biantennary glycans of the N-acetyllactosaminic type are linked [18]. This sequence is 70% identical to the sequence of murine lactotransferrin recently determined by cDNA sequencing [19] and resolved as a 688-amino-acid polypeptide chain to which a single glycan of the N-acetyllactosaminic type is conjugated 1201.The only information known about the primary structure of bovine lactotransferrin concerned its N-terminal amino acid sequence APRKNVRWXTISQPE [21] and its carboCorrespondence to A.

Cyclophilin B is a cyclosporin

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