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Selected residues in a highly conserved 15-residue region, 174SVDTAAMAGLAFTC L188 of human transcobalamin (TC), a cobalamin (Cbl: vitamin B12) binding protein, were subjected to site-directed mutagenesis. The mutant constructs were expressed in TC-deficient fibroblasts or in vitro to assess the effect of these mutations on Cbl binding. Phylogenetic analyses and protein parsimony indicated that TC evolved earlier than other mammalian Cbl-binding proteins, intrinsic factor and haptocorrins, and divergence occurred between mouse/rat and human dispersing TC gene to different chromosomes. These studies show that (a) two of the three polar residues, S174, T177, or D176 and two of the three conserved alanine residues, A179 and A184 present in the 15-residue evolutionary conserved region are essential for Cbl-binding by human TC, and (b) TC gene is transferred in a syntenic manner to different chromosomes, at least before the divergence of mouse/rat and human. [Copyright &y& Elsevier]

Purified human placental transcobalamin II receptor (TC II-R) dimer of molecular mass 124 kDa bound to Sepharose-linked bacterial immunoglobulin (IgG) binding proteins protein A, protein G, and protein A/G. TC II-R dimer was detected directly, by blotting human placental and rabbit and rat kidney membrane proteins with 125I-protein A, or indirectly, using antiserum to TC II-R or IgG–Fc region and 125I-protein. TC II-R antiserum, but not protein A, protein G, protein A/G, or antiserum to the IgG–Fc region, when added to culture medium of human intestinal epithelial Caco-2 cells or umbilical vein endothelial cells, inhibited ligand binding. However, protein A, protein G, protein A/G, or antiserum to the Fc region inhibited the internalization of the ligand TC II-[57Co]cyanocobalamin. Taken together, these studies strongly suggest TC II-R is an IgG-like molecule that contains an Fc-like region which is important in ligand internalization but not binding. [Copyright &y& Elsevier]