1 Department of Cancer and Inflammation Research, Department of Molecular Medicine, Det Sundhedsvidenskabelige Fakultet, SDU2 unknown3 Department of Cancer and Inflammation Research, Department of Molecular Medicine, Det Sundhedsvidenskabelige Fakultet, SDU
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.
Biochemistry, 2005, Vol 44, Issue 9, p. 3604-14
Binding Sites; Cobalt Radioisotopes; Humans; Intrinsic Factor; Kinetics; Peptide Fragments; Protein Binding; Protein Processing, Post-Translational; Receptors, Cell Surface; Recombinant Proteins; Spectrometry, Fluorescence; Surface Plasmon Resonance; Vitamin B 12