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 receptor's 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.
McAbee
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DD
Department of Chemistry and Biochemistry, California State University, Long Beach 90840-3903, USA. dmcabee@csulb.edu
Jiang
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Walsh
K B
KB
D000818:Animals; D037263:Asialoglycoprotein Receptor; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007781:Lactoferrin / Q000378:metabolism*; D037102:Lectins / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000378:metabolism*
D037263:Asialoglycoprotein Receptor; D037102:Lectins; D011956:Receptors, Cell Surface; D007781:Lactoferrin; D002118:Calcium