Isolation and partial characterization of a lactotransferrin receptor from mouse intestinal brush border.
Several lines of evidence have recently suggested the occurrence of a specific lactotransferrin receptor in the small intestinal brush-border membrane in several animal species, which is thought to be involved in lactotransferrin-mediated intestinal iron absorption. We report here for the first time the isolation and partial characterization of this receptor from mouse intestinal brush border. The receptor has been purified to homogeneity by affinity chromatography on an immobilized human lactotransferrin column. The purified receptor was found to be active in that it binds iron-free and iron-saturated lactotransferrin with a Kd of 0.1 microM. Anti-receptor antibodies were prepared, and the receptor was further isolated by immunoaffinity chromatography in higher yield but in a denatured form. The purified receptor was revealed by sodium dodecyl sulfate-polyacrylamide electrophoresis to be a protein of about Mr = 130,000, consisting of a single polypeptide chain. The isoelectric point was determined to be 5.8. The receptor was further shown to bear concanavalin A and phytohemagglutinin L binding glycans. Digestion by N-glycanase and endo-N-acetyl-beta-D-glucosaminidase B led to a decrease of Mr = 25,000, while the endo-N-acetyl-beta-D-glucosaminidase H was uneffective, suggesting that the lactotransferrin receptor is mainly glycosylated by bi- and triantennary glycans. To gain further insight into the interaction of the receptor with lactotransferrin, namely, the number of ligand molecules bound per molecule of receptor, mouse lactotransferrin was cross-linked to its membrane-bound enterocyte receptor by use of radiolabeled sulfosuccinimidyl 3-[[2-(p-azidosalicylamido)ethyl]dithio]propionate (SASD).(ABSTRACT TRUNCATED AT 250 WORDS)
Hu
W L
WL
Laboratoire de Chimie Biologique de l'Université des Sciences et Techniques de Lille Flandres-Artois, Villeneuve d'Ascq, France.
Mazurier
J
J
Montreuil
J
J
Spik
G
G
D000818:Animals; D001386:Azides; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003208:Concanavalin A / Q000378:metabolism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D007422:Intestines / Q000032:analysis* / Q000648:ultrastructure; D007526:Isoelectric Point; D007781:Lactoferrin / Q000378:metabolism; D051379:Mice; D008871:Microvilli / Q000032:analysis*; D008970:Molecular Weight; D010835:Phytohemagglutinins / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D013388:Succinimides
D001386:Azides; D003432:Cross-Linking Reagents; D010835:Phytohemagglutinins; D011956:Receptors, Cell Surface; D013388:Succinimides; C038340:lactoferrin receptors; C063086:sulfosuccinimidyl 3-((2-(4-azidosalicylamido)ethyl)dithio)propionate; D003208:Concanavalin A; D006026:Glycoside Hydrolases; D007781:Lactoferrin