The transcobalamin receptor, redux

In this issue of Blood, Quadros and colleagues have identified a novel high-affinity cellular receptor for transcobalamin, a serum B₁₂-binding protein that escorts the micronutrient to sites of cellular uptake. Importantly, they also identify the gene for the transcobalamin receptor.

Cobalamin (Cbl; B₁₂) is an essential micronutrient required by all cells in the body. For several decades, we have been aware that Cbl-binding proteins escort B₁₂ to sites of absorption and cellular uptake. Thus, intrinsic factor (IF), with its high-binding specificity for Cbl, mediates the absorption of B₁₂ in the gut. The distribution and delivery of Cbl to cellular uptake sites is mediated by transcobalamin (TC, transcobalamin II), a nonglycosylated serum Cbl-binding protein. In contrast, the mechanics of Cbl absorption and cellular uptake, both receptor driven, are only now being understood.

Dietary Cbl binds to IF in the duodenum and then escorts it to sites of uptake in the ileum. Recent work on the IF-Cbl receptor has identified cubilin as the protein that binds IF-Cbl and amnionless as the protein that tethers cubilin to the plasma membrane of the ileal enterocyte.¹  The IF-Cbl-cubilin-amnionless complex undergoes endocytosis followed by proteolytic degradation of IF in lysosomes and release of Cbl to the cytoplasm. Newly absorbed Cbl enters portal circulation as TC-Cbl and undergoes systemic distribution. Cellular requirements for B₁₂ are satisfied by expression of cell surface receptors for TC-Cbl.

Functional aspects of TC receptor dynamics and regulation of its expression were characterized in early studies using cultured mammalian cells.^2-4  High-affinity TC receptors capture TC-Cbl and internalize the complex by endocytosis involving clathrin-coated pits and vesicles, as shown in the figure. In acidic endosomes, TC-Cbl dissociates from the TC receptor, which then recycles back to the cell surface. The entire cycle takes approximately 20 minutes.⁴  Endosomes carrying TC-Cbl fuse to form lysosomes where TC is degraded by proteolysis, and Cbl is exported to the cytoplasm. Cobalamins are processed in the cytoplasm by mechanisms that remain largely unknown. However, intracellular escorts or chaperones are likely to be involved.⁵  In mitochondria, 5′-deoxyadenosyl-Cbl is synthesized from processed Cbl, and it serves as the coenzyme for the conversion of methylmalonyl-CoA to succinyl-CoA by methylmalonyl-CoA mutase. In the cytoplasm, methyl-Cbl is formed, probably on methionine synthase, and serves as a coenzyme for the remethylation of homocysteine to methionine using N⁵-methyltetrahydrofolate as the methyl group donor.

Although Quadros et al⁶  purified and characterized human TC in 1986 and later cloned its gene,⁷  complete characterization, including cloning of the TC receptor, has been more challenging and unfulfilled. However, in this issue of Blood, Quadros and colleagues appear to have met the challenge using an elaborate 3-tier affinity purification protocol. This technique has provided them with enough pure protein from human placenta to determine the amino acid sequences of TC receptor peptides and to identify a protein in the data bank. With this information in hand, it was a relatively simple matter to identify the TC receptor gene.

Quadros et al provide convincing evidence for a true high-affinity TC receptor. The protein contains 282 amino acids: 31 N-terminal residues form a signal peptide, 198 residues form an extracellular domain, 21 residues are in the transmembrane region, and 32 residues are cytoplasmic. The full-length receptor purified from human placenta has high-affinity binding for TC-Cbl (K_a = 2 × 10⁹ mol/L), as does the expressed extracellular domain (K_a = 3 × 10⁹ mol/L). A polyclonal antibody to the extracellular domain blocks the binding of TC-Cbl to the purified receptor and inhibits the cellular uptake of TC-Cbl. Heterozygous cells, in which 1 allele for the TC receptor gene has been knocked out, have greatly reduced uptake of TC-Cbl in comparison to wild-type cells. Finally, cells transfected with the TC receptor gene show a 3-fold increase in TC-Cbl uptake.

Quadros et al have finally identified an elusive high-affinity receptor for TC-Cbl and its gene. Although it remains to be seen if there is universal expression and tissue distribution of this receptor, it will now be possible to screen for mutations and polymorphisms that might result in diminished capacity to acquire this essential micronutrient.