Human intrinsic factor expressed in the plant <i>Arabidopsis thaliana</i>

Fedosov, Sergey N.; Laursen, Niels Bech; Nexø, Ebba; Moestrup, Søren K.; Petersen, Torben E.; Jensen, Erik Appel; Berglund, Lars

doi:10.1046/j.1432-1033.2003.03716.x

Cited by 46 publications

(49 citation statements)

References 26 publications

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“…The experiments were performed on the recombinant human proteins IF and TC purified from plants [17] and yeast [18], respectively. Both proteins were originally obtained as Cbl-saturated holo-forms, and preparation of the unsaturated apo-forms required their denaturing.…”

Section: Preparation Of the Proteinsmentioning

confidence: 99%

Application of a fluorescent cobalamin analogue for analysis of the binding kinetics

et al. 2006

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Fluorescent probe rhodamine was appended to 5′ OH‐ribose of cobalamin (Cbl). The prepared conjugate, CBC, bound to the transporting proteins, intrinsic factor (IF) and transcobalamin (TC), responsible for the uptake of Cbl in an organism. Pronounced increase in fluorescence upon CBC attachment facilitated detailed kinetic analysis of Cbl binding. We found that TC had the same affinity for CBC and Cbl (Kd = 5 × 10−15 m), whereas interaction of CBC with the highly specific protein IF was more complex. For instance, CBC behaved normally in the partial reactions CBC + IF30 and CBC + IF20 when binding to the isolated IF fragments (domains). The ligand could also assemble them into a stable complex IF30–CBC–IF20 with higher fluorescent signal. However, dissociation of IF30–CBC–IF20 and IF–CBC was accelerated by factors of 3 and 20, respectively, when compared to the corresponding Cbl complexes. We suggest that the correct domain–domain interactions are the most important factor during recognition and fixation of the ligands by IF. Dissociation of IF–CBC was biphasic, and existence of multiple protein–analogue complexes with normal and partially corrupted structure may explain this behaviour. The most stable component had Kd = 1.5 × 10−13 m, which guarantees the binding of CBC to IF under physiological conditions. The specific intestinal receptor cubilin bound both IF–CBC and IF–Cbl with equal affinity. In conclusion, the fluorescent analogue CBC can be used as a reporting agent in the kinetic studies, moreover, it seems to be applicable for imaging purposes in vivo.

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Section: Preparation Of the Proteinsmentioning

confidence: 99%

Application of a fluorescent cobalamin analogue for analysis of the binding kinetics

et al. 2006

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“…The recombinant IF was produced from plants as described in our previous publications (6,13). IF from gastric juice was purified according to the method of ref 14. Spectral Measurements and Molar Absorbance.…”

Section: Expression and Purification Of Human Ifmentioning

confidence: 99%

“…The optical response from H 2 OCbl was measured at the subsaturating concentrations of the ligand ( Figure 1A). This allowed us to establish the increments ∆ of the Cbl molar absorbance added to that of the apoprotein ( 0 , Figure 1A,B) and from that of the protein, which was not subjected to GdnHCl treatment (6).…”

Section: Spectral Characteristics Of the Unsaturated Ifmentioning

confidence: 99%

Composite Organization of the Cobalamin Binding and Cubilin Recognition Sites of Intrinsic Factor

et al. 2005

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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 × 10 6 , 14 × 10 6 , and 26 × 10 6 M -1 s -1 , respectively), but the equilibrium dissociation constants were essentially different (K Cbl ) 200 µM, 0.2 µM, and e1 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) 1 is one of three cobalamin (Cbl or vitamin B 12 )-transporting proteins present in a mammalian organism (1-3). Abundant secretion of IF into the intestinal tract is necessary for normal assimilation of the vitamin. IF is relatively resistant to proteolysis, and its binding activity exceeds by far the amount of Cbl liberated from food (1,3). High selectivity toward Cbl (4) and high affinity of the saturated protein for the specific receptor (5, 6) distinguish IF from other Cbl transporters. The above features guarantee (i) uptake of the physiologically active ligand and (ii) accessibility of the receptor, which is not blocked by an excess of unsaturated IF.IF is employed as an additive to many vitamin preparations as well as in the Schilling test. The binder is also used for measurement of Cbl levels in biological samples (7,8).Therefore, understanding the mechanisms behind IF action is important for proper application of this protein for both analytical and medical purposes. The continuing controversy over the affinity of specific binders for Cbl with exceptional dispersion of the results [K Cbl ) 10 -14 -10 -8 M (9-12)] emphasizes the significance and necessity of the thorough kinetic analysis.In our previous work (13), we investigated oligomerization of the full-length protein IF 50 and its two proteolytic fragments upon Cbl binding. The first fragment, IF 30 , represented the N-terminal peptide of 30 kDa with most ...

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“…In the present study, we tested only the absorption of free vitamin B 12 . Recombinant human IF is now available commercially (14,15 ), and as soon as it becomes available for human use, our design can be used to test whether IF can correct negative absorption of free vitamin B 12 . In our patient group, this would help distinguish those with vitamin B 12 malabsorption attributable to a defective receptor from those with inherited lack of IF.…”

Section: Discussionmentioning

confidence: 99%

Nonradioactive Vitamin B12 Absorption Test Evaluated in Controls and in Patients with Inherited Malabsorption of Vitamin B12

et al. 2005

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Background: Current tests for evaluation of vitamin B 12 absorption are problematic because they involve the use of radioactively labeled vitamin B 12 . We describe a vitamin B 12 absorption test that circumvents this problem. Methods: We measured cobalamin or transcobalamin saturated with cobalamin (holo-TC) 24 h after three 9-g doses of vitamin B 12 given orally at 6-h intervals. We studied 17 patients with inherited malabsorption of vitamin B 12 attributable to Imerslund-Grasbeck syndrome (n ‫؍‬ 13) or intrinsic factor deficiency (n ‫؍‬ 4), their obligate heterozygous biological parents (n ‫؍‬ 19), and healthy controls (n ‫؍‬ 44).

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Human intrinsic factor expressed in the plant Arabidopsis thaliana

Cited by 46 publications

References 26 publications

Application of a fluorescent cobalamin analogue for analysis of the binding kinetics

Application of a fluorescent cobalamin analogue for analysis of the binding kinetics

Composite Organization of the Cobalamin Binding and Cubilin Recognition Sites of Intrinsic Factor

Nonradioactive Vitamin B12 Absorption Test Evaluated in Controls and in Patients with Inherited Malabsorption of Vitamin B12

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