Identification and Analysis of a Ca²⁺-Dependent Lactoferrin Receptor in Rat Liver

Summary

We identified a 45 kDa Ca²⁺-dependent Lf binding protein on rat hepatocytes. Dithiobis(sulfosuccimidylproprionate) (DTSSP)-crosslinked ¹²⁵I-Lf to a 45 kDa adduct in a Ca²⁺-dependent manner on intact cells. The ¹²⁵I-labeledcrosslinked complexes were absent when either surface-bound ¹²⁵I-Lf was stripped prior to crosslinking or an excess of unlabeled Lf was included in the DTSSP reaction. Triton X-100 extracts of hepatocyte membrane ghosts were chromatographed on Lf-agarose, and a 45 kDa polypeptide (p45) was eluted by EGTA. Anti-p45 sera blocked vigorously ¹²⁵I-Lf endocytosis to intact rat hepatocytes, confirming that p45 functions as the Ca²⁺-dependent Lf receptor on hepatocytes. Two tryptic fragments of p45 showed 100% identity with internal sequences (Leu¹²¹ →Lys ¹²⁶ and Phe¹⁹⁸-Lys²²⁰) of the major subunit (RHL- l) of the rat asialoglycoprotein receptor. Antisera against p45 and RHL-1 crossreacted equally well with each protein, and asialoorosomucoid blocked the binding of ¹²⁵I-Lf to hepatocytes. We did not detect the minor subunits (RHL2/3) of the rat asialoglycoprotein receptor in p45 preparations from Triton X-100-extracts of hepatocytes, and ¹²⁵I-Lf bound to immobilized RHL-1 but not to RHL-2/3. Exoglycosidases were used to remove terminally-exposed NeuNAc and α-and β-Gal from bovine Lf glycans, and lectin blotting confirmed that glycosidase-treated Lfs lacked detectable terminal Gal. Unexpectedly, deglycosylated Lf exhibited no loss in its ability to compete with unmodified Lf for binding to isolated hepatocytes. Moreover, β-lactose but not sucrose competed vigorously for ¹²⁵I-Lf endocytosis by hepatocytes, indicating that Lf binds at or near the carbohydrate-recognition domain of RHL-l. We conclude that RHL-1 is the Ca²⁺-dependent Lf receptor on hepatocytes and that it binds Lf in a Gal-independent manner.