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Intrinsic factor (IF(50)) is a cobalamin (Cbl)-transporting protein of 50 kDa, which can be cleaved into two fragments: the 30 kDa N-terminal peptide IF(30) and the 20 kDa C-terminal glycopeptide IF(20). Experiments on binding of Cbl to IF(30), IF(20), and IF(50) revealed comparable association rate constants (k(+)(Cbl) = 4 x 10(6), 14 x 10(6), and 26 x 10(6) M(-1) s(-1), respectively), but the equilibrium dissociation constants were essentially different (K(Cbl) = 200 microM, 0.2 microM, and <or=1 pM, respectively). The smaller fragment, IF(20), had unexpectedly high affinity for Cbl; however, efficient retention of the ligand required the presence of both fragments. Detailed schemes of the interaction of Cbl with IF(50) and with IF(30) and IF(20) are presented, where the sequential attachment of Cbl to the IF(20) and IF(30) domains plays the key role in recognition and retention of the ligand. Each isolated fragment of IF was tested for the binding to the specific receptor cubilin in the presence or absence of Cbl. Neither apo nor holo forms of IF(20) and IF(30) were recognized by the receptor. When two fragments were mixed and incubated with Cbl, they associated into a stable complex, IF(30+20).Cbl, which bound to cubilin as well as the noncleaved IF(50).Cbl complex. We suggest that formation of the cubilin recognition site on IF is caused by assembly of two distant domains, which allows the saturated protein to be recognized by the receptor. The obtained parameters for ligand and receptor binding indicate that both full-length IF(50) and the fragments may be involved in Cbl assimilation.
Intrinsic factor (IF) has been expressed previously in Baculovirus with a yield (0.1-1 mg/l) that was inadequate for structural and metabolic studies. IF cDNAs were cloned into the shuttle vector pPIC9 of P. pastoris, and the proteins were induced and purified by cobalamin (Cbl) affinity chromatography. Expression of recombinant proteins revealed a major band of 49 kDa for both human and rat IF. Expression of human IF was achieved at 1040 mg/l, but of rat IF at only 1-2 mg/l. Reaction of human IF with a photo-activatable derivative of Cbl was demonstrated by Western blotting, and detection of IF fragments by anti-Cbl monoclonal antibody and by amino-terminal sequencing revealed at least three regions (residues 129-151, 234-254, and +294) linked to Cbl. Both recombinant human and rat [125I]IF-Cbl bound to rat and guinea pig brush border membranes with similar affinity, but the binding capacity of human IF for the rat receptor was only 10% compared with rat IF. All six amino acids within the previously identified N-terminal binding region of human IF were mutated to be identical to rat IF, but the resulting chimeric IF still bound poorly to rat membranes. Mutations of residues 26/27 (Glu26 to Asp and Asn27 to Gln) and 32/34 (Ser32 to Thr and Tyr34 to Arg) showed changes in both Ka and Vmax, with great effects on Vmax. In conclusion, P. pastoris is an expression system that produces functional human IF at a higher yield than in the baculovirus system. Cbl binding was directly demonstrated at multiple sites along the linear sequence of human IF. The receptor binding function of the amino terminal sequence 25 62 has been confirmed, but it is insufficient to reproduce all the features of IF-Cbl binding.
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A 11 1/2-year-old girl of Turkish origin showed symptoms and signs of pernicious anaemia with low B12 level in blood but normal gastric acid secretion. There were no antibodies to mucosal cells or intrinsic factor. Quantitative determination of intrinsic factor in gastric juice, done by a RIA method, proved absence of the factor. Both parents had normal values for haemoglobin, normal B12-absorption tests and slightly decreased B12 blood levels. The gastric acid secretion was normal in both, but intrinsic factor in the gastric juice was decreased markedly. These findings support the hypothesis that congenital pernicious anaemia, e.g. congenital absence of intrinsic factor, is transmitted by an autosomal recessive trait.
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A method employing non-radioactive vitamin B(12) and microbiological assay is described for estimating intrinsic factor in gastric juice and for detecting antibody to intrinsic factor in serum. Satisfactory agreement was obtained between the results by this method and by a modification of the method of Ardeman and Chanarin (1963). During the first hour after gastric stimulation 11 patients with pernicious anaemia secreted between 0 and 240 units of intrinsic factor compared with between 1,600 and 39,000 units in 21 patients with other conditions. The results in three out of four patients with gastric atrophy were higher than those in pernicious anaemia but lower than in other conditions.
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A new radioisotope dilution assay for vitamin B12-intrinsic factor complex is described. The method is based on the use of the binding type intrinsic factor antibody (the binding reagent), which when combined with the intrinsic factor-vitamin B12 complex (labelled ligand), is quantitatively adsorbed onto zirconium phosphate gel at pH 6.25. The new assay has been shown to provide a measure of intrinsic factor comparable with other intrinsic factor assays, but it has the important advantage of being able to measure the unlabelled vitamin B12-intrinsic factor complex (unlabelled ligand), and will, therefore, be valuable in the study of physiological events in the gastrointestinal tract. During the study, it was found that there is some evidence for a least two types of binding intrinsic factor antibody: One which combines preferentially with the intrinsic factor-vitamin B12 complex and one which combines equally well with this complex or with free intrinsic factor.