Douglas D. McAbee

Lactoferrin binding to the rat asialoglycoprotein receptor requires the receptor's lectin properties.

Lactoferrin binds to rat hepatic lectin 1 (RHL1), the major subunit of the asialoglycoprotein (ASGP) receptor, with high affinity, by a galactose-independent mechanism. To better understand the molecular basis of this novel interaction, we compared the binding of lactoferrin and asialo-orosomucoid (ASOR) to isolated rat hepatocytes and to purified ASGP receptors as a function of pH, Ca(2+) and receptor acylation. Binding of (125)I-lactoferrin and (125)I-ASOR to isolated rat hepatocytes at 4 degrees C decreased sharply at pH<6, following similar titration curves. Binding of (125)I-lactoferrin and (125)I-ASOR to hepatocytes was Ca(2+)-dependent. Binding increased progressively at > or =300 microM CaCl(2), in the presence of 1 mM EDTA. Monensin treatment of hepatocytes, which causes hepatocytes to accumulate inactive ASGP receptors, reduced surface binding of (125)I-lactoferrin and (125)I-ASOR by 46 and 49%, respectively, with only a 16% loss of immunodetectable receptor protein from the cell surface. Finally, deacylation of purified ASGP receptors in vitro with 1 M hydroxylamine abolished receptor lectin activity as reflected by the loss of (125)I-ASOR binding as well as the complete loss of specific (125)I-lactoferrin binding. Treatment with 1 M Tris had no effect on binding of either ligand. We conclude from these data that galactose-independent lactoferrin binding to the ASGP receptor requires the receptors carbohydrate-recognition domain to be in an active configuration. An active configuration is promoted by neutral pH and Ca(2+), and also requires the receptor subunits to be acylated.

Iron-Loading of Cultured Adult Rat Hepatocytes Reversibly Enhances Lactoferrin Binding and Endocytosis

Isolated rat hepatocytes bind and internalize bovine lactoferrin (Lf) protein and Lf‐bound Fe3+ via Ca2+‐dependent recycling Lf binding sites (McAbee, 1995, Biochem. J., 311:603–609). In this study, we determined if iron loading of primary cultures of adult rat hepatocytes altered their ability to bind and internalize Lf. Rat hepatocytes were cultured 16–24 h with or without ferric ammonium citrate (FAC) and then assayed for Ca2+‐dependent 125I‐Lf binding at 4°C or 125I‐Lf endocytosis at 37°C. Cells pretreated with FAC (5 μg/mL) internalized two‐ to sixfold more 125I‐Lf than did control cells. The FAC‐induced increase in 125I‐Lf endocytosis required 4–8 h of culture at 37°C and was fully reversible if cells were incubated an additional 24 h without FAC either in the presence or absence of the Fe3+ chelator desferrioxamine. Maximal endocytic rates for untreated and FAC‐treated cells were 370 and 2,300 molecules 125I‐Lf cell‐1 sec‐1, respectively. Both 125I‐Lf binding at 4°C and endocytosis at 37°C increased up to sixfold between 0.3–10 μg/mL FAC, indicating that iron‐induced enhancement of 125I‐Lf uptake was due to an increase in the number of Lf receptors present on the cells. 125I‐Lf bound to untreated and FAC‐treated cells at 4°C with similar affinities (Kd ∼ 1.5 μM). Cycloheximide but not actinomycin D blocked the FAC‐induced increase in 125I‐Lf binding, indicating that the increase in the number of Lf binding sites required translation but not transcription. Notably, iron loading blocked endocytosis of asialoorosomucoid by hepatocytes by up to 80%, reducing the number of active intracellular asialoglycoprotein receptors >65% without altering the number of active cell surface receptors. We conclude from these studies that Lf receptor activity on hepatocytes is regulated posttranscriptionally by the iron status of the cells. J. Cell. Physiol. 171:75–86, 1997.

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

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.

Copper and Zinc Ions Differentially Block Asialoglycoprotein Receptor-mediated Endocytosis in Isolated Rat Hepatocytes

Asialoglycoprotein receptors on hepatocytes lose endocytic and ligand binding activity when hepatocytes are exposed to iron ions. Here, we report the effects of zinc and copper ions on the endocytic and ligand binding activity of asialoglycoprotein receptors on isolated rat hepatocytes. Treatment of cells at 37 °C for 2 h with ZnCl2 (0–220 μm) or CuCl2 (0–225 μm) reversibly blocked sustained endocytosis of 125I-asialoorosomucoid by up to 93% (t 1/2 = 62 min) and 99% (t 1/2 = 54 min), respectively. Cells remained viable during such treatments. Zinc- and copper-treated cells lost ∼50% of their surface asialoglycoprotein receptor ligand binding activity; zinc-treated cells accumulated inactive asialoglycoprotein receptors intracellularly, whereas copper-treated cells accumulated inactive receptors on their surfaces. Cells treated at 4 °C with metal did not lose surface asialoglycoprotein receptor activity. Exposure of cells to copper ions, but not to zinc ions, blocked internalization of prebound 125I-asialoorosomucoid, but degradation of internalized ligand and pinocytosis of the fluid-phase marker Lucifer Yellow were not blocked by metal treatment. Zinc ions reduced diferric transferrin binding and endocytosis on hepatocytes by ∼33%; copper ions had no inhibitory effects. These findings are the first demonstration of a specific inhibition of receptor-mediated endocytosis by non-iron transition metals.