Iron uptake from transferrin by rat intestinal brush-border

and lactoferrin membrane vesicles

HIROSHI KAWAKAMI, SHUN’ICHI DOSAKO, AND BO LijNNERDAL Department of Nutrition, University of California, Davis, California 95616; and Technical Research Institute, Snow Brand Milk Products, 1-2, Minamidai, Kawagoe, Saitama 350, Japan

HIROSHI, SHUN’ICHI DOSAKO, AND Bo LONuptake from transferrin and lactoferrin by rat intestinal brush-border membrane vesicles. Am. J. Physiol. 258 (Gastrointest. Liver Physiol. 21): G535-G541, 1990.-Interaction of 5gFe-labeled rat transferrin, human lactoferrin, and bovine lactoferrin with rat small intestinal brush-border membrane vesicles was investigated with the use of a rapid filtration technique. Specific binding of 5gFe-labeled rat transferrin and bovine lactoferrin to brush-border membrane vesicles from suckling and adult rats was identified. In contrast, no binding of human lactoferrin occurred. The presence of transferrin receptors on the brush-border membrane of suckling rats was confirmed by immunoblotting, and the molecular mass of the receptor was 96 kDa under nonreducing conditions. Scatchard plot analysis indicated 2.4 x 1014 binding sites/mg of membrane protein with an affinity constant (Ka) of 4.9 X lo6 M-l for rat milk transferrin and 2.2 x 1014 binding sites/mg of membrane protein with a K, of 3.2 X lo6 M-l for bovine lactoferrin. Bovine lactoferrin competitively inhibited the binding of rat transferrin to the brush-border membrane vesicles. Deglycosylation of rat transferrin and bovine lactoferrin had no influence on the binding of these proteins. The results suggested that bovine lactoferrin bound to the receptor for rat transferrin on the brush-border membrane and that the polypeptide chain rather than the glycan moiety is responsible for the interaction of these proteins with the rat brush-border membrane.

Lf occurred. The binding kinetics strongly suggested the presence of a Lf receptor. Hu et al. (22) found that the mouse BBMVs have a specific receptor for human, bovine, and mouse Lf but not for mouse Tf. Huebers et al. (23) observed iron uptake from rat Tf by rat intestinal mucosa, but not from human Lf. This discrepancy is probably attributable to variations in experimental conditions such as species of the experimental animals and source of Tf or Lf. Our previous results in a rat model suggested that iron from iron-saturated bovine Lf is absorbed across the rat intestinal mucosa by an alternative mechanism from the transport of soluble iron salts (27). In this study, we showed the presence of Tf receptors on rat small intestinal BBMVs and investigated the interaction of ““Felabeled rat Tf, bovine Lf, and human Lf with the BBMVs from suckling and adult rats to better characterize the role of these proteins in iron uptake in this species. In addition, the role of the glycans of Tf and Lf in receptor binding was studied, since the carbohydrate chain of glycoproteins has been shown to have an effect on the ligand-receptor interaction (2, 38).

transferrin

Materials. Antibodies against rat serum Tf were purchased from Cappel Laboratories (West Chester, PA); monoclonal antibodies against rat Tf receptor were from Bioproducts for Science (Indianapolis, IN); alkaline phosphatase-conjugated antibodies against mouse immunoglobin (IgG), nitro blue tetrazolium (NBT), and 5bromo-4-chloro-3-indolyl-phosphate (BCIP) were from Promega (Madison, WI); rat serum Tf was from Rockland (Gilbertsville, PA); 5gFeC13(specific activity 2.4-2.9 mCi/mg) was from ICN Biochemicals (Irvine, CA); peptide N-glycosidase F was from Genzyme (Boston, MA); and periodic acid-Schiff (PAS) reagent was from Sigma Chemical (St. Louis, MO). Hydrophilic membrane filters (GVWP, 0.22 pm) were from Millipore (Bedford, CA); the affinity gel (Affi-Gel 10) was from BioRad Laboratories (Richmond, CA); and nitrocellulose membranes were from Schleicher & Schuell (Keene, NH). Preparation of lactoferrin and transferrin. Human Lf and bovine Lf were prepared through affinity chromatography from each skim milk by a one-step procedure with the use of immobilized monoclonal antibodies against human Lf or bovine Lf as described elsewhere (28) .

KAWAKAMI, NERDAL. Iron

receptor;

iron absorption

ON THE SPECIES, either transferrin (Tf) or lactoferrin (Lf) is the major iron-binding protein in milk. For example, rat and rabbit milk have a high Tf concentration (3,19), whereas human, monkey, and bovine milk are high in Lf (10, 20, 26). Although the iron content of milk is low, infants consuming maternal milk maintain adequate iron status (16). A high bioavailability of iron from human milk (16, 34, 39) suggests facilitated uptake of iron from these iron-binding proteins in the small intestine. This view is supported by the documented presence of Tf or Lf in duodenal fluids as well as at the surface of intestinal mucosa (21, 24, 32) and by the marked ability of Lf from human and bovine milk to resist proteolytic attack during transit through the digestive tract in infants (11, 41). However, the relative efficiency of these proteins to deliver iron in various species is still under debate. Davidson and Lonnerdal (12) demonstrated that human and monkey Lf bound to monkey intestinal brush-border membrane vesicles (BBMVs), but no binding of bovine

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Rat milk Tf was isolated from rat skim milk through Affi-Gel 10 immobilized with antibodies against rat serum Tf. Briefly, 2 ml of Affi-Gel 10 was mixed with an equal volume of antibody solution (10 mg/ml) and stirred gently at 4°C for 4 h. After excess antibodies were removed by centrifugation, unreacted sites of the gel were blocked with 0.1 M ethanolamine-HCl (pH 8.0). Approximately 9.4 mg of antibodies were incorporated into 1 ml of gel. The affinity gel with immobilized antibodies was packed into a column (IO x 60 mm). After passing 5 ml of rat skim milk through the column, components that did not interact with antibodies were washed out with 0.01 M sodium phosphate buffer containing 0.5 M sodium chloride (pH 7.2). Then Tf was eluted with 0.2 M acetate buffer containing 0.15 M sodium chloride at pH 3.7. The eluate was immediately adjusted to pH 7.0 with 1 M sodium hydroxide, dialyzed against deionized water, and concentrated with Ultrafree-PF filter units (Nihon Millipore, Tokyo, Japan). Iron-free Lf and Tf were prepared by subsequent dialysis against 0.1 M citrate (pH 2.2) containing 0.1% EDTA for 24 h and then against an excess of deionized water for 72 h. Labeling of Lf and Tf with 5gFe. 5gFe-citrate was prepared by adding a l,OOO-fold molar excess of citrate to “‘FeC& in 0.1 M hydrochloride. After 10 min, the pH was adjusted to 7.0 by addition of 0.1 M sodium hydroxide. Iron-free Lf and Tf were labeled by adding sufficient ““Fe-citrate to saturate the proteins, which were dissolved in 50 mM tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.5) containing 0.1% sodium bicarbonate, and were left overnight to ensure specific binding of iron. Unbound 5gFewas removed by passage through an Excellulose GF-5 desalting column (Pierce, Rockford, IL). Preparation of BBMVs from rat intestine. Small intestines were obtained from suckling rats (2 wk old) and the mother (14 wk old), respectively, perfused with icecold saline, and slit open. The mucosa was scraped with a glass slide and weighed. BBMVs were prepared by differential centrifugation techniques and magnesium precipitation technique as described by Davidson and Lonnerdal (12). The final vesicle suspension was used immediately or frozen and stored at -70°C until use. The purification of BBMVs was monitored by measuring protein concentration and sucrase activity. Protein concentrations of the initial mucosal homogenate and the purified BBMV preparation were determined by a modification of the Lowry assay (37) using bovine serum albumin as standard. Sucrase activity was assayed as the liberation of glucose on incubation with sucrose (9). Contamination of basolateral membrane in the BBMV preparations was assessed by the activity of Na+-K+ATPase (35). Isolation of Tf receptor. Tf receptor was isolated from pup BBMVs solubilized with Triton X-100 by affinity chromatography with the use of immobilized diferric Tf. Briefly, 5 mg of diferric Tf, which was prepared by the procedure described above for labeling of Tf with 5gFe, was coupled to 1 ml of Affi-Gel 10. The purified BBMVs, prepared as above, were suspended in 0.01 M potassium

AND

LACTOFERRIN

phosphate buffer (pH 7.5) containing 0.15 M sodium chloride, 0.5 mM phenylmethanesulfonyl fluoride, 0.02% sodium azide, and 2% Triton X-100 at a protein concentration of 10 mg/ml. The suspension was stirred gently for 3 h at 4°C and centrifuged at 17,000 g for 40 min. Five milliliters of the supernatant was incubated with 1 ml of Tf-immobilized gel for 2 h at 4OC. After the gel was packed into a column (10 x 60 mm) and washed thoroughly with 0.01 M potassium phosphate buffer (pH 7.5) containing 0.5 M sodium chloride and 0.01 M 3- [ (3Cholamidopropyl) -dimethylamino] -1 -propanesulfonate (CHAPS), Tf receptor was eluted with 0.1 M glycineHCI buffer (pH 2.8) containing 0.15 M sodium chloride and 0.01 M CHAPS. The eluate was immediately adjusted to pH 7.0 with 1 M sodium hydroxide and concentrated with the Ultrafree-PF filter unit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was performed in 4-20% polyacrylamide gradient gels containing 1% SDS (precasted gels; Integrated Separation Systems, Newton, MA) according to the method of Laemmli (30). Electrophoretic transfer of proteins to nitrocellulose membranes. Proteins were transferred to nitrocellulose membranes in 0.02 M Tris-0.15 M glycine buffer (pH 8.3) containing 20% methanol with transfer electrophoresis unit TE (Hoefer Scientific Instruments, San Francisco, CA) at 200 mA according to the method of Burnette (7) Visualization of proteins on nitrocellulose membranes. The lanes containing molecular weight standard proteins were cut off and stained with amido black. The lanes containing proteins bound to Tf-immobilized gel were visualized with mouse monoclonal antibodies against rat Tf receptor as primary antibody, alkaline phosphataseconjugated antibodies against mouse IgG as secondary antibody, and NBT and BCIP as substrate to identify the alkaline phosphatase reaction products. Briefly, the strips of nitrocellulose membrane were rinsed with 0.01 M Tris HCl buffer (pH 7.5) containing 0.15 M sodium chloride (TBS), and soaked in TBS containing 3% skim milk for 1 h with gentle shaking at room temperature to saturate additional protein binding sites. After the buffer was replaced by TBS containing 1% skim milk and monoclonal antibodies against rat Tf receptor at 1:3,000 dilution, the strips were incubated for 2 h at room temperature and rinsed six times for 5 min each in TBS containing 0.05% Tween 20. The strips were further incubated with TBS containing alkaline phosphataseconjugated antibodies against mouse IgG at l:5,OOO dilution for 1 h, and rinsed 6 times for 5 min each in TBS containing 0.05% Tween 20. Finally, the strips were incubated with 10 ml of Tris HCl buffer (pH 9.5) containing 66 ~1 of NBT substrate and 33 ~1 of BCIP substrate until color developed. Deglycosylation of Lf and Tf. One-half milligram of bovine Lf was dissolved in 1 ml of 0.25 M sodium phosphate buffer (pH 8.6) and incubated with 25 units of peptide N-glycosidase F at 37°C for 18 h according to the method of Tarentino et al. (43). After the incubation, deglycosylated bovine Lf was immediately separated from the enzyme through the affinity column with anti-

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bodies against bovine Lf and concentrated with Ultrafree-PF filter units. Deglycosylated rat Tf was prepared similarly. Removal of glycans from Tf and Lf was confirmed by an increase in migration of the deglycosylated proteins on SDS-PAGE and by the absence of staining with PAS reagent (14). Binding assays. Assays were performed in triplicate by incubation of the 5gFe-labeled proteins with 20 pg of BBMVs and 40 mM Tris-N-2-hydroxyethylpiperazineN’-2-ethanesulfonic acid (HEPES) buffer (pH 7.4) containing 0.1 M D-mannitol, 0.1 M sodium chloride, and 2 mM D-glucose to a final volume of 100 ~1. The concentration of 6gFe-labeled proteins was varied from 0.1 to 2 PM. The incubation was carried out in a 37°C water bath, and the reaction was terminated by the addition of 1 ml of ice-cold saline. This solution was immediately vacuum filtered through a prewetted 0.22-pm Millipore hydrophilic membrane filter and rinsed three times with 1 ml of ice-cold saline. The filters were counted in a gamma scintillation counter (Gamma 8500, Beckman Instruments, Fullerton, CA) to determine the amount of 5gFe associated with the BBMVs. Nonspecific binding of Lf or Tf to the BBMVs was determined by the addition of a lOO-fold excess of unlabeled Lf or Tf to the incubation mixture. Nonspecific binding to the filters was corrected for in all experiments by performing an incubation with 5gFe-labeled Lf or Tf in the absence of BBMVs. Competitive binding assay. The competitive binding assay between rat serum Tf, bovine Lf, human Lf, and 0.1 PM 5gFe-labeled rat milk Tf was performed by introducing to the incubation medium increasing concentrations of each of the proteins, ranging from 0.2 to 10 yM.

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Mr I x10m31 - 200 - 116 - 95

Tf receptor

-

55 43

-

36

FIG. 1. Identification of transferrin (Tf) receptor on pup brushborder membranes by immunoblotting technique with the use of monoclonal antibodies against rat Tf receptor, alkaline phosphatase conjugated antibodies against mouse immunoglobin, and color development substrates. A: transferrin receptor. B: molecular weight standard proteins.

0.3

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Characterization of rat intestinal BBMV. Enrichment of sucrase activity in the BBMVs was typically 20-fold over that of the original homogenate. Recoveries were generally 45%. No activity of the basolateral membrane marker Na+-K+-ATPase was detectable. Isolation and visualization of Tf receptor. Tf receptor was isolated from pup BBMVs solubilized with Triton X-100 by affinity chromatography with the use of immobilized Tf and was visualized on nitrocellulose membrane with mouse monoclonal antibodies against rat Tf receptor, alkaline phosphatase-conjugated antibodies against mouse IgG, and color development substrates. As shown in Fig. 1, Tf receptor appeared as a single major species with a molecular mass of 96 kDa under nonreducing conditions. Iron uptake from Tf and Lf. The time courses of iron binding from rat milk Tf, bovine Lf, and human Lf to the pup BBMVs are shown in Fig. 2. Each point represents the mean value of the specific binding, which was obtained by subtracting nonspecific binding from total binding to the BBMVs. Nonspecific binding to the BBMVs was

Iron uptake from transferrin and lactoferrin by rat intestinal brush-border membrane vesicles.

Interaction of 59Fe-labeled rat transferrin, human lactoferrin, and bovine lactoferrin with rat small intestinal brush-border membrane vesicles was in...
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