Intrinsic Factor-cobalamin Complex Research Articles

Imerslund–Gräsbeck syndrome: New mutation in amnionless

Vitamin B12, cobalamin (Cbl), is one of the essential vitamins and its dietary sources are meat, poultry, liver, fish, shellfish, egg, and milk. Cbl deficiency is attributable to various causes including inadequate intake, defects in absorption and transport, and disorders of Cbl metabolism. Imerslund–Grasbeck syndrome (IGS), first reported in 1960, is characterized by Cbl deficiency and proteinuria. 1,2 Most of the initial cases were Finnish and Norwegian, 3 but this syndrome has since been reported throughout Europe, the mid-east and North America, and in patients of African ancestry. 4,5 Bi-allelic mutations in either the cubilin (CUBN )o ramnionless (AMN) gene are responsible for this syndrome. The encoded proteins cubilin and amnionless form the receptor to transport the intrinsic factor-cobalamin (IF-B12) complex into the enterocytes in the terminal ileum. Therefore, defects in either cubilin or amnionless result in Cbl malabsorption. We report a Thai boy with IGS in whom a deleterious mutation in AMN was confirmed. Case report

Luminal expression of cubilin is impaired in Imerslund-Grasbeck syndrome with compound AMN mutations in intron 3 and exon 7

Juvenile megaloblastic anaemia 1 (OMIM # 261100) is a rare autosomic disorder characterized by selective cobalamin mal-absorption and inconstant proteinuria produced by mutations in either CUBN or AMN genes. Amnionless, the gene product of AMN, is a transmembrane protein that binds tightly to the N-terminal end of cubilin, the gene product of CUBN. Cubilin binds to intrinsic factor-cobalamin complex and is expressed in the distal intestine and the proximal renal tubule. We report a compound AMN heterozygosity with c.742C>T, p.Gln248X and c.208-2A>G mutations in 2 siblings that led to premature termination codon in exon 7 and exon 6, respectively. It produced a dramatic decrease in receptor activity in urine, despite absence of CUBN mutation and normal affinity of the receptor for intrinsic factor binding. Heterozygous carriers for c.742T and c.208-2G had no pathological signs. These results indicate that amnionless is essential for the correct luminal expression of cubilin in humans.

A urinary radioisotope-binding assay to diagnose Gräsbeck-Imerslund disease.

Gräsbeck-Imerslund disease (congenital familial selective vitamin B12-malabsorption with proteinuria, MGA1, MIM No. 261100) is a rare disorder displaying autosomal recessive inheritance. This study was designed to investigate the usefulness of measuring the activity of the urinary receptor for the intrinsic factor-cobalamin complex as a tool to diagnose this disease. The receptor activity was measured by a radioisotope-binding assay, using phenyl-Sepharose gel as the adsorbant solid phase of the receptor. In 10 Finnish patients, urinary receptor activity was on the average 640 times (15-1400 times) lower than that in 13 healthy control subjects: mean values of 0.1 nmol/mol (range, 0.01-0.32 nmol/mol) and 6.4 nmol/mol (range, 3.8-12.4 nmol/mol) creatinine, respectively. The mean value of urinary receptor activity in 11 first-degree, healthy relatives of the patients was 4.6 nmol/mol (range, 1.1-10.4 nmol/mol) creatinine, a difference from levels in control subjects that is not statistically significant. When the first-degree relatives were divided into heterozygotes (parents and siblings heterozygous for the haplotype of genetic markers associated with the disease gene) and wild-type homozygotes (siblings not displaying the disease haplotype), no difference was seen. Determination of receptor activity in the urine is a highly accurate method for diagnosis of Gräsbeck-Imerslund disease at an early stage, but it does not detect carriers of the disorder.

Identification of rat yolk sac target protein of teratogenic antibodies, gp280, as intrinsic factor-cobalamin receptor.

Previous studies in the rat have shown that antibodies to gp280, a protein > 200 kD and closely associated with the early endocytic system can induce fetal malformations. Although gp280 is thought to act as a receptor, its ligand(s) is not known. In the current study, we report that purified gp280 from rat kidney, like the intrinsic factor-Cobalamin receptor (IFCR), binds to the intrinsic factor-cobalamin (IFCbl) complex with an association constant of 0.3 x 10(9) M-1 and mediates its internalization. Furthermore, antibodies raised to purified gp280 and IFCR inhibited the binding of IF-[57Co]Cbl complex to intestinal, renal, and yolk sac apical membranes and revealed a single identically sized protein on immunoblotting of the renal membranes. Both antibodies precipitated a single radiolabeled protein > 200 kD from cellular extract from [35S]methionine-labeled yolk sac epithelial cells, and antibody to gp280 inhibited the uptake and internalization of 125IF-Cbl. Immunoelectron microscopy using the two antibodies revealed that in the kidney, both proteins were colocalized. These observations suggest that IF-Cbl complex is a ligand for gp280 and that gp280 and IFCR are identical proteins.

Intrinsic factor-cobalamin receptor activity in a marsupial, the american opossum ( Didelphis virginiana)

1. 1. Significant and specific binding of intrinsic factor-cobalamin occurred in proximal but not in the distal half of the intestine in an adult marsupial, the American opossum. 2. 2. The purified opossum kidney receptor, like rat and canine kidney receptors, revealed a single band of M r≈ 230 on SDS-PAGE. However, unlike the rat and canine receptors, the opossum receptor was sensitive to both Endoglycosidase H and peptide- N-glycosidase F. 3. 3. The opossum intrinsic factor-cobalamin receptor demonstrated a ten-fold higher affinity for intrinsic factor-cobalamin complex when the source of IF was from the opossum pancreas, rather than rat stomach. 4. 4. The opossum kidney receptor had low immune-crossreactivity with anti-serum raised to rat and canine kidney receptor. 5. 5. These studies suggest that intrinsic factor-cobalamin receptor expressed in the American opossum, though conserved, appears to be structurally different from the rat and canine receptors.

The intrinsic factor (IF)-cobalamin receptor binding site is located in the amino-terminal portion of IF

Intrinsic factor has two binding sites, one each for cobalamin and for the ileal receptor recognizing the intrinsic factor-cobalamin complex. To obtain initial functional mapping of these domains, cDNAs encoding intact rat and human intrinsic factor or fragments thereof were expressed transiently in COS-1 cells or in an in vitro transcription/translation system. Deletion of as little as 12% of the amino acids from the carboxyl terminus resulted in loss of cobalamin binding activity. On the other hand, the receptor binding region of intrinsic factor appears localized to a restricted region in the amino-terminal portion of the protein. Only those transcription/translation fragments of rat or human intrinsic factor tested that contained amino acid residues 25 to 62 (out of 399) showed calcium-dependent binding to isolated kidney brush borders, the shortest sequence corresponding with 20 consecutive amino acids. In contrast, a 232-amino acid carboxyl-terminal fragment of rat intrinsic factor and 243- and 338-amino acid carboxyl-terminal fragments of human intrinsic factor showed no receptor binding activity.

Intrinsic factor receptor during fetal development of the human intestine.

Intrinsic factor receptor activity was observed in mucosal homogenates from whole small intestine and colon of 10-19-week fetuses, whereas it was only detected in the distal part of the small intestine of a 25-week fetus. The receptor-intrinsic factor-cobalamin complex was eluted into the void-volume position when ileum mucosal extract was assayed for receptor activity by gel filtration after incubation with either fetal gastric extract or human gastric juice. The intrinsic-factor-binding capacity of intestinal mucosal extracts ranged from 2.6 to 30.5 fmol/mg and was correlated with the gestational age of six fetuses. The dissociation constant of the receptor for the intrinsic factor-cobalamin complex was estimated at 0.24-0.36 nM at pH 7.4. In conclusion, intrinsic-factor-receptor activity was detected in the whole intestine in 10-19-week fetuses, whereas it was only present in the distal ileum at the end of fetal development.

Receptor-mediated endocytosis of the intrinsic factor—cobalamin complex in HT 29, a human colon carcinoma cell line

A HT 29 cell line derived from human colonic carcinoma was shown to express the intrinsic factor receptor, with about 5000 binding sites per cell and an association constant of 20 × 10 9 1/mol at pH 7.4 and 4°C. The number of binding sites increased dramatically between 7 and 10 days of culture time. Endocytosis of the intrinsic factor-cobalamin-receptor complex was inhibited by two ways: at 4°C and at 37°C by incubating the cells with vinblastine, monensin and chloroquine. The plasma membrane receptor was cross-linked to [ 57Co]cobalamin-intrinsic factor and solubilized with Triton X-100. The cross-linked complex had a relative molecular mass of 330 kDa in native PAGE.

Effect of lectins on the cobalamin-protein binding reactions: implications for the tissue uptake of cobalamin

Plant lectins have been thought to impair nutrient absorption, both by specific and nonspecific interference in the absorptive process. The main objective of this investigation was to study the effect of lectins on the various binding reactions involving cobalamin (cbl)-protein complexes and their receptors, and to identify the rate-limiting step important in maintaining tissue levels of cobalamin. Among the lectins tested in vivo, only concanavalin A (ConA) was able to inhibit the transport of cobalamin to the tissues and caused a 70% to 75% inhibition of [ 57Co] cobalamin transported to the liver and kidney. The inhibition of transport to the tissues was independent of route of administration of cobalamin, whether intragastric or systemic, and was not due to decreased gastrointestinal uptake. When tested in vitro, concanavalin A inhibited the binding of transcobalamin II-cbl to its receptor, but not the binding of cobalamin to intrinsic factor or intrinsic factor-cobalamin complex to the ileal receptor. These results suggest that late events during transcellular transport of cobalamin through the enterocytes is the rate-limiting step determining tissue levels of cobalamin and that ConA inhibits these latter events.

Monoclonal antibodies to human intrinsic factor

Mice were immunized with human intrinsic factor, and their lymph node cells were fused with a myeloma cell line by standard hybridoma techniques. Eleven of the resulting 227 hybridomas secreted immunoglobulin G capable of binding to intrinsic factor-cobalamin complex. Cloning by limiting dilution gave 6 clones secreting anti-intrinsic factor antibodies that bound human intrinsic factor-cobalamin complex with affinities of 13–116 nM; 3 antibodies also bound rabbit intrinsic factor-cobalamin complex. Five antibodies inhibited to some degree the binding of cobalamin by intrinsic factor, and 2 also prevented attachment of intrinsic factor-cobalamin complex to guinea pig ileal receptors. Anti-rabbit intrinsic factor antibodies specifically precipitated a peptide of molecular weight 53,000, corresponding to the molecular weight of rabbit intrinsic factor from homogenates of rabbit gastric mucosal explants biosynthetically labeled with [35S]methionine and from culture medium in which the expiants were incubated. Indirect fluorescence immunocytochemistry with the antibodies in human and rabbit gastric mucosal sections showed intense selective staining of parietal cells. These results (a) document species differences between human and rabbit intrinsic factors not previously demonstrable with polyclonal anti-intrinsic factor sera; (b) confirm earlier evidence that cobalamin binding and receptor functions occur at separate sites in intrinsic factor; and (c) provide a useful approach to studying structure-function relations of the intrinsic function molecule.

Lectin binding to the porcine and human ileal receptor of intrinsic factor-cobalamin

The purified porcine receptor for the intrinsic factor-cobalamin complex bound to concanavalin A, lentil lectin and wheat germ lectin covalently coupled to Sepharose and was eluted with the corresponding soluble sugars. In contrast, human intrinsic factor bound efficiently to concanavalin A, to some extent to lentil lectin, but only slightly to wheat germ agglutinin. The binding of IF-Cbl to the receptor was inhibited when the receptor was pre-incubated with soluble wheat germ agglutinin, with an inhibition constant estimated to be 1.9 mumol/l. After transfer of the purified receptor from SDS-PAGE to Immobilon, ligand blotting of the purified receptor with iodinated lectin showed that concanavalin A and lentil lectin bound to three (75, 56 and 43 kDa) components but that wheat germ agglutinin bound only to the 75 kDa component. These results showed that the alpha subunit of the receptor could bind to wheat germ agglutinin, resulting in an inhibition of its binding with intrinsic factor. Both binding sites of intrinsic factor and of wheat germ agglutinin could be located near to each other.

Radioautographic localisation of iodinated human intrinsic factor in the guinea pig ileum using electron microscopy.

The uptake of iodinated human intrinsic factor by guinea pig ileum was studied in vivo using electron microscopy radioautography. Iodination of intrinsic factor cobalamin complex with N-chlorobenzene-sulphonamide beads did not modify its physicochemical properties in gel filtration, nor in iso-electrofocusing. It didn't affect its biological activity either in vitro in presence of solubilised receptor, or in vivo in the Schilling test. The 125I-intrinsic factor cobalamin complex was instilled in vivo in ileal blind loops in presence of either CaCl2, EDTA, bile or intestinal juice. The labelled complex was predominantly located in the intermicrovillous pits of the apical membrane and in the apical cytoplasm. The uptake was about five-fold lower in presence of EDTA. On the apical membrane, the number of grains (per 80 micron length of epithelium) increased from 2.7 (0.2) up to 9.4 (1.7) respectively for 15 minutes and two hours of delay. This suggests a recycling of the intrinsic factor receptor complex. In the apical compartment, the silver grains were often detected over non-coated vesicles and over the infoldings of the lateral membrane. These results show that intrinsic factor is internalised into enterocytes during cobalamin absorption, and that a part of intrinsic factor enters the blood circulation through transcytosis.

Purification, properties, and immunochemical localization of a receptor for intrinsic factor-cobalamin complex in the rat kidney.

High levels of receptor for intrinsic factor-cobalamin (vitamin B12) were detected in human, canine, and rat kidneys. The ratio of specific activity (picomoles/mg of protein) for kidney relative to intestine was 116, 20, and 797, respectively, in these species. The receptor was purified about 3000-fold from 200 g of rat kidney with a recovery of 16% and exhibited a single band on nondenaturing gel electrophoresis. Quantitative amino acid analysis of the receptor gave a value of 457,310 g of amino acid/mol of intrinsic factor-cobalamin binding activity. The pure receptor revealed an Mr of 430,000, as assessed by filtration with Bio-Gel A-5m. Treatment with papain resulted in the production of active monomers of Mr to about 205,000-210,000. Electrophoresis in the presence of sodium dodecyl sulfate confirmed the monomer Mr to be 230,000. The monomer receptor did not reveal the presence of any further subunits upon reductive alkylation. Following cyanogen bromide cleavage the kidney receptor revealed three peptides of Mr 115,000, 60,000, and 54,000. The pI of these peptides was 5.17, 6.17, and 6.17, respectively. Western blot analysis using antiserum raised to the receptor demonstrated a protein with an Mr of 175,000 and 230,000 for intestinal and kidney membrane receptors, respectively. Immunologically, the rat kidney receptor was identical to the rat ileal receptor but was distinct from the canine ileal receptor. Ultrastructural localization revealed the presence of the receptor in the apical surface membrane of proximal tubular cells of the kidney and absorptive cells of the ileum. The kidney is the best source for obtaining this receptor in reasonable quantities.

Radioimmunoassay for assessing exocrine pancreatic insufficiency, based on the differential enzymatic degradation of cobalamin-binding proteins.

Pancreatic proteases degrade the protein moiety of the R protein-cobalamin complex but not the intrinsic factor-cobalamin complex. Accordingly, we used these two proteins as substrates in an in vitro enzymatic assay to assess pancreatic function by incubating basal jejunal fluids with a mixture of intrinsic factor and cyano[57Co]cobalamin coupled to R-type protein and then using immunoprecipitation to determine the distribution of isotopically labeled cobalamin bound to the two proteins. With normal jejunal fluids, 91.2 (SD 6.1)% and 4.5 (SD 5.5)% of cyano[57Co]cobalamin was precipitated with antisera to intrinsic factor and anti-R protein, respectively. In the patients' jejunal fluids, the cyano[57Co]cobalamin precipitated with the respective antisera was 5.3 (SD 10.0)% and 96 (SD 6.2)%. In patients with other gastrointestinal problems, the sequestration of cobalamin was indistinguishable from that observed with the normal fluids. The clearcut discrimination this radioimmunoassay provided between abnormal and normal samples was confirmed by parallel comparative chromatographic analysis.

Phase separation of rat intestinal brush border membrane proteins using Triton X-114

Rat intestinal microvillus membrane contains at least 24 polypeptides, of which 18 can be solubilized using Triton X-114 at 4°C. Upon phase separation at 32°C, 11 proteins separated nearly completely into the detergent-rich phase, while 9 proteins were found exclusively in the aqueous phase. Enzymes which were uniquely included in the detergent phase were alkaline phosphatase, leucine aminopeptidase, γ-glutamyl transpeptidase, and Ca 2+Mg 2+ ATPase. The proteins which were excluded from the detergent phase and found exclusively in the aqueous phase included the disaccharidases (glucoamylase, sucrase-isomaltase, trehalase, lactase) and the ileal receptor for the intrinsic factor-cobalamin complex. Integral membrane proteins can thus be separated during solubilization into two groups prior to further purification or characterization.

3] Isolation of native and proteolytically derived ileal receptor for intrinsic factor—cobalamin

This chapter describes the assay method, purification, and isolation of native and proteolytically derived ileal receptor for intrinsic factor–cobalamin. Receptor activity during the purification was assessed by measuring the binding of the hog IF-[ 57 Co]Cbl complex made with purified hog intrinsic factor (IF). Hog IF can be replaced by human IF. However the important consideration is that IF preparations should be free of any possible contamination of other Cbl-binding proteins (nonIF). The supernatant fraction containing unbound IF-[ 57 Co]Cbl and the pellet containing receptor bound IF-[ 57 Co]Cbl are assayed for radioactivity. Receptor specific binding is calculated as the difference between the binding observed in the presence of CaCl 2 and in the absence of calcium with EDTA. The most important step in the purification is the immunoaffinity chromatography using rabbit anti-hog intrinsic factor bound to Sepharose 4B. For the purification of the dog receptor attempts to use either intrinsic factor alone or the intrinsic factor-cobalamin complex as an affinity absorbent resulted in very poor binding of the receptor compared to rabbit anti-hog intrinsic factor antiserum. The advantage of this method is the recycling of both the intrinsic factor-cobalamin complex and the affinity ligand. After elution of the receptor the column can be washed with 0.2 M glycine-HCl buffer, pH 3.0, to remove the intrinsic factor-cobalamin complex which was bound to the antibody. The eluate is neutralized to pH 7 and can be reused after assessing the amount of complex present.

Isolation and characterization of two species of receptor which bind intrinsic factor

The intrinsic factor receptor from guinea pig ilea has been characterized following purification by affinity chromatography. The purified receptor complexed to intrinsic factor-cobalamin (holo-receptor) had an anhydrous molecular weight of 680,000, a Stokes radius of 10.9 nm, and a sedimentation coefficient of 15.1 S. In contrast, unsaturated receptor (apo-receptor) resolved into distinct “large” and “small” molecular species having, respectively, (i) molecular weights of 700,000 and 350,000; (ii) Stokes radii of 11.1 and 7.06 nm; (iii) sedimentation coefficients of 15.5 S and 11.9 S; (iv) association constants for binding the intrinsic factor-cobalamin complex of 7.3 and 2.5 × 10 10 liters mol −1; and (v) two isoproteins for the larger species (p I's 4.05 and 4.80), and one isoprotein for the smaller species (p I 4.90). A rabbit anti-receptor serum gave only one precipitation line in immunodiffusion against either the large or the small receptor, and each one of these two lines fused completely with the one of two lines which formed with the purified preparation containing both receptor species. Autoradiography of the precipitin lines obtained when the receptor was coupled to intrinsic factor-cyano[ 57Co]cobalamin demonstrated that both species of receptor were functional. The reaction of complete immunologie identity, the similar electrical properties and similar kinetics for binding to intrinsic factor, and the observed molecular weight differences indicate that the small and large apo-receptors are chemically interrelated, and suggest that the large receptor may consist of two small functional proteins.

The preparation of human intrinsic factor-cobalamin complex from human gastric juice by immunoadsorption

1. (1) Immune complexes of human intrinsic factor were prepared by mixing gastric juice saturated with vitamin B 12, and sera from patients with pernicious anaemia that had a high proportion of binding (Type II) antibody. 2. (2) The complexes were isolated by sodium sulphate precipitation followed by Sephadex G-150 gel filtration. 3. (3) Acid conditions dissociated the immune complexes and allowed separation of specific antibody and purified antigen bound to vitamin B 12 by Sephadex G-200 gel filtration. 4. (4) Specific antibody was covalently attached to Protein A Sepharose CL-4B by coupling with water soluble carbodiimide which allowed intrinsic factor-B 12 complex to be purified directly from gastric juice. 5. (5) The intrinsic factor obtained after iodination, ran as a single band on SDS-polyacrylamide gel electrophoresis and was biologically active.

Immunocytochemical localization of the intrinsic factor-cobalamin receptor in dog-ileum: distribution of intracellular receptor during cell maturation.

Absorption of cobalamin is facilitated by the binding of the intrinsic factor-cobalamin complex (IF-cbl) to specific receptors in the ileum. The physical and biochemical characteristics of this ligand-receptor binding reaction have been extensively studied, but little is known about the cellular mechanisms or receptor synthesis, intracellular transport, and expression on the microvillus surface membrane. We attempted to delineate these mechanisms by using ultrastructural immunocytochemistry to localize the IF-cbl receptor in the crypt, mid-villus, and villus tip regions of mucosal biopsies obtained from the ileum of anesthetized dogs. Prior to initiating the ileal localization studies, the antisera to purified canine IF-cbl receptor that was employed in our studies was shown to have specificity for site (e.g., ileal enterocytes vs. other cells within the gastrointestinal tract) and immunohistochemical specificity. Receptor synthesis in endoplasmic reticulum begins in crypt enterocytes, but continues in cells throughout the villus. In the mid-villus region synthesized receptor translocates vectorially to the microvillus surface associated with membranous vesicles and then inserts into the microvillus pit. Receptor remains fixed to the microvillus pit and does not distribute uniformly over the brush border membrane. All villus tip enterocytes contained IF-cbl receptor in microvillus pits, vesicles, and endoplasmic reticulum, but in addition extensive perinuclear membrane staining was evident as well as re-internalized receptor associated with multivesicular bodies. Basolateral membranes contained no receptor at any level of the villus. These observations suggest that the IF-cbl receptor (a) translocates to the apical cell surface at the mid-villus region by transport in vesicles, (b) directly inserts into and then remains fixed in microvillus pits, (c) is elaborated on the luminal surface most extensively in villus tip cells, and (d) although reinternalized, does not move IF and/or cbl to the basolateral cell surface.

Effect of bile and bile acids on binding of intrinsic factor to cobalamin and intrinsic factor-cobalamin complex to ileal receptor.

Human bile or commercially available bile acids bind [57Co]cobalamin (Cbl) due to the presence of "R"-type binders, which are normally present in the former and present as a contaminant in the latter. The competition of these R proteins for the binding of Cbl by intrinsic factor (IF) could explain the in vivo inhibition attributed previously to the bile acids themselves. R protein seems to be involved in the inhibition of Cbl binding because protease digestion of either bile or bile acid abolishes the Cbl binding ability. Moreover, antibody to R protein abolishes the inhibition. Bile or bile acids do not have a direct effect on either purified IF or the IF-Cbl receptor molecule, even though bile acids increase the attachment of IF-[57Co]Cbl to ileal brush-border membranes. These data demonstrate two steps where components of bile could affect Cbl absorption: the binding of Cbl to IF and of IF-Cbl to its ileal receptor. It is not clear whether these in vitro phenomena are important for the normal absorption of Cbl in vivo.