Isolation and partial characterization of a lactotransferrin receptor from mouse intestinal brush-border

Abstract

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)

Human Lactoferrin and Peptides Derived from a Surface-Exposed Helical Region Reduce ExperimentalEscherichia coliUrinary Tract Infection in Mice

“…Little is known about the metabolic pathway of ingested LF in mice. An intestinal LF receptor has been described and characterized for the mouse brush-border membrane (14,15). This receptor, a glycoprotein (130 kDa), binds both hLF and bLF (14,15).…”

“…An intestinal LF receptor has been described and characterized for the mouse brush-border membrane (14,15). This receptor, a glycoprotein (130 kDa), binds both hLF and bLF (14,15). LF receptors (110 kDa) have been reported to be present in the brush border membranes of infant rhesus monkey and infant human intestines (27).…”

Effect of lactoferrin on the production of tumor necrosis factor-? and nitric oxide

“…If lactoferrin itself acts as a transcriptional factor that binds to the promoter region of the specific gene, it must first be established that lactoferrin can be transported to the nucleus from the outside of the cell. Several researchers have reported on the lactoferrin-specific receptor to which lactoferrin binds, as well as the translocation of the intact lactoferrin into nucleus [Garre et al, 1992;Hu et al, 1990;Roiron et al, 1989;Mazurier et al, 1989]. Once it can be translocated into the nucleus, it binds easily to the DNA [Ravazzollo et al, 1988].…”

Undetectable apolipoproteinA-Igene expression suggests an unusual mechanism of dietary lipid mobilisation in the intestine ofCyprinus carpio

“…To obtain the peripheral membrane and integral membrane protein fractions, fresh BBMV or the pellet obtained after the binding assay were suspended in 50·µl of 50·mmol·l -1 Na 2 CO 3 , pH·11.5, 10·mmol·l -1 EDTA and incubated for 15·min on ice (Hu et al, 1997). The integral membrane protein fraction was recovered by centrifugation for 30·min at 14·000·g, where the supernatant corresponds to the peripheral membrane fraction.…”