Identification and analysis of a CA(2+)-dependent lactoferrin receptor in rat liver. Lactoferrin binds to the asialoglycoprotein receptor in a galactose-independent manner.
We identified a 45 kDa Ca(2+)-dependent Lf binding protein on rat hepatocytes. Dithiobis(sulfosuccimidylproprionate) (DTSSP)-crosslinked 125I-Lf to a 45 kDa adduct in a Ca(2+)-dependent manner on intact cells. The 125I-labeled crosslinked 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 Ca(2+)-dependent Lf receptor on hepatocytes. Two tryptic fragments of p45 showed 100% identity with internal sequences (Leu121-->Lys126 and Phe198-->Lys220) of the major subunit (RHL-1) 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 (RHL-2/3) 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 alpha- and beta-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, beta-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-1. We conclude that RHL-1 is the Ca(2+)-dependent Lf receptor on hepatocytes and that it binds Lf in a Gal-independent manner.
Department of Biological Sciences, University of Notre Dame, Indiana 46556, USA.
D D
DD
McAbee
D J
DJ
Bennatt
Y Y
YY
Ling
D000595:Amino Acid Sequence; D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D005690:Galactose / Q000378:metabolism; D007781:Lactoferrin / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000378:metabolism*
D001212:Asialoglycoproteins; D011956:Receptors, Cell Surface; C038340:lactoferrin receptors; D007781:Lactoferrin; D002118:Calcium; D005690:Galactose