Cubilin P1297L mutation associated with hereditary megaloblastic anemia 1 causes impaired recognition of intrinsic factor-vitamin B-12 by cubilin

Megaloblastic anemia 1 (MGA1) is an autosomal recessive disorder caused by the selective intestinal malabsorption of intrinsic factor (IF) and vitamin B-12/cobalamin (Cbl) in complex. Most Finnish patients with MGA1 carry the disease-specific P1297L mutation (FM1) in the IF-B-12 receptor, cubilin, B y site-directed mutagenesis, mammalian expression, and functional compariso n of the purified wild-type and FM1 mutant forms of the If-Cbl-binding cubi lin region (CUB domains 5-8, amino acid 928-1386), we have investigated the functional implications of the P1297L mutation. Surface plasmon resonance analysis revealed that the P1297L substitution specifically increases the K -d for IF-Cbl binding several-fold, largely by decreasing the association r ate constant. In agreement with the binding data, the wild-type protein, bu t not the FM1 mutant protein, potently inhibits 37 degrees C uptake of iodi ne 125-IF-Cbl in cubilin-expressing epithelial cells. In conclusion, the da ta presented show a substantial loss in affinity of the FM1 mutant form of the IF-Cbl binding region of cubilin, This now explains the malabsorption o f Cbl and Cbl-dependent anemia in MGA1 patients with the FM1 mutation. (C) 2000 by The American Society of Hematology.