Isolated Rat Hepatocytes Bind Lactoferrins by the RHL-1 Subunit of the Asialoglycoprotein Receptor in a Galactose-Independent Manner

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Abstract
Isolated rat hepatocytes bind and internalize the iron-binding protein lactoferrin (Lf) by a set of high-affinity, recycling, Ca2+-dependent binding sites. We have purified a 45-kDa membrane protein (p45) from rat hepatocytes that exhibits Ca2+-dependent receptor activity. In this study, we found p45 to be identical to the major subunit (RHL-1) of the rat asialoglycoprotein receptor. Two tryptic fragments of p45 showed 100% identity with RHL-1 internal sequences (Leu121 → Lys126 and Phe198 → Lys220), and monospecific antisera against p45 and RHL-1 cross-reacted equally well with each protein. Molar excesses of anti-p45 IgG, anti-RHL-1 IgG, asialoorosomucoid, and asialofetuin competitively blocked the binding of 125I-Lf to isolated rat hepatocytes at 4 °C. Similarly, either excess anti-p45 or Lf blocked the binding of 125I-asialoorosomucoid to cells at 4 °C. We did not detect the minor subunits of the rat asialoglycoprotein receptor (RHL-2/3) in p45 preparations from Triton X-100 extracts of hepatocytes and 125I-Lf bound to purified RHL-1 but not to RHL-2/3 immobilized on nitrocellulose. Nonetheless, anti-RHL-2/3 IgG reduced the binding of 125I-Lf to hepatocytes at 4 °C. Exoglycosidases were used to remove terminally-exposed N-acetylneuraminyl, α- and β-galactosyl, and N-acetylhexosaminyl sugars from human and bovine Lf glycans, and lectin blotting confirmed that glycosidase-treated Lfs lacked detectable terminal galactosyl sugars. Unexpectedly, these deglycosylated Lfs exhibited no loss in their ability to compete with unmodified Lfs for binding to isolated hepatocytes. In addition, molar excess of β-lactose but not sucrose competitively blocked the binding of 125I-Lf to cells, indicating that Lf bound at or very near the carbohydrate-recognition domain of RHL-1. We conclude that RHL-1 is the Ca2+-dependent Lf receptor on hepatocytes and that it binds Lf at its carbohydrate-recognition domain yet in a galactose-independent manner.