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The membrane receptor TCblR/CD320 binds transcobalamin (TC) saturated with vitamin B12 [cobalamin (Cbl)] and mediates cellular uptake of the vitamin. The specificity of TC for Cbl and of th receptor for TC-Cbl ensures efficient uptake of Cbl into cells. The high-affinity interaction of TCblR with TC-Cbl (K12= 10 nM-1 was investigated using deletions and mutations of amino acid sequences in TCblR. Only the extraceUular region (aa 32-229) is needed for TC-Cbl binding, but the N-glycosylation sites (N126, N195, and N213) are of no importance for this function. Deleting the cysteine-rich region (aa 95-141) that separates the two low-density lipoprotein receptor type A (LDLR-A) domains does not affect TC-Cbl binding (K12 = 19-24 nM-l). The two LDLR-A domains (aa 54-89 and 132-167) with the negatively charged acidic residues involved in Ca2+ binding are critical determinants of ligand binding. The cytoplasmic tail is apparently crucial for internalization of the ligand. Within this region, the RPL-GLL motif and the PDZ binding motifs (QERL/KESL appear to be involved in initiating and completing th process of ligand internalization. Mutations and deletions of these regions involved in binding and internalization of TC-Cbl are likely to produce the biochemical and clinical phenotype of Cbl deficiency. [ABSTRACT FROM AUTHOR]

The membrane receptor (TCbIR/CD320) for transcobalamin (TC)-bound cobalamin (Cbl) facilitates the cellular uptake of Cbl. A genetically modified mouse model involving ablation of the CD320 gene was generated to study the effects on cobalamin homeostasis. The nonlethal nature of this knockout and the lack of systemic cobalamin deficiency point to other mechanisms for cellular Cbl uptake in the mouse. However, severe cobalamin depletion in the central nervous system (CNS) after birth (P<0.01) indicates that TCbIR is the only receptor responsible for Cbl uptake in the CNS. Metabolic Cbl deficiency in the brain was evident from the increased methylmalonic acid (P< 0.01 - 0.04), homocysteine (P<0.01), cystathionine (P<0.01), and the decreased S-adenosylmethionine/S-adenosyl homocysteine ratio (P<0.01). The CNS pathology of Cbl deficiency seen in humans may not manifest in this mouse model; however, it does provide a model with which to evaluate metabolic pathways and genes affected. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) were generated to the extracellular domain of transcobalamin receptor (TCblR) and used to identify the regions of the receptor protein involved in antibody binding. Based on the effect of transcobalamin bound cobalamin (TC-Cbl) on antibody binding, this study identified both blocking and binding antibodies. Both types of antibodies bind apo as well as holo receptors, whereas the blocking antibody when bound to the apo receptor prevents the binding and cellular uptake of TC-Cbl. Binding of these antibodies to truncated receptor constructs has identified the peptide domains of the receptor involved in antibody binding. These antibodies have potential utility in blocking cellular uptake of Cbl and delivery of drugs via TCblR, which is over-expressed in many cancers. [ABSTRACT FROM AUTHOR]

Cellular uptake of vitamin B 12 (cobalamin, Cbl) is mediated by a cell surface receptor (TCblR/CD320) that binds transcobalamin (TC) saturated with Cbl. TC is secreted by the vascular endothelium, has a relatively short half-life, binds Cbl with high affinity and presents the vitamin to the receptor for cellular uptake. Here we show binding and internalization of the TC-Cbl complex along with its' receptor (TCblR) in several human cell lines. The expression of TCblR is linked to the cell cycle with highest expression in actively proliferating cells. Upon binding TC-Cbl, the receptors appear to segregate on the plasma membrane and are internalized over the course of 30-60 min. Subsequently, the receptors appear to be destroyed along with the TC, which results in the release of free Cbl in the lysosome. The appearance of TCblR on the cell surface is limited to newly synthesized protein without contribution from recycling of the receptor. Therefore, Cbl uptake into cells is fully dependent on the expression of newly synthesized TCblR that is up-regulated in actively proliferating cells. The cell cycle-associated up-regulation of TCblR in cancers provides a route for targeted drug delivery., (Published by Elsevier Inc.)

In humans, vitamin B 12 deficiency causes peripheral and CNS manifestations. Loss of myelin in the peripheral nerves and the spinal cord (SC) contributes to peripheral neuropathy and motor deficits. The metabolic basis for the demyelination and brain disorder is unknown. The transcobalamin receptor-knockout mouse ( Cd320 -/- ) develops cobalamin (Cbl) deficiency in the nervous system, with mild anemia. A decreased S-adenosylmethionine: S-adenosylhomocysteine ratio and increased methionine were seen in the brain with no significant changes in neurotransmitter metabolites. The structural pathology in the SC presented as loss of myelin in the axonal tracts with inflammation. The sciatic nerve (SN) showed increased nonuniform, internodal segments suggesting demyelination, and remyelination in progress. Consistent with these changes, the Cd320 -/- mouse showed an increased latency to thermal nociception. Further, lower amplitude of compound action potential in the SN suggested that the functional capacity of the heavily myelinated axons were preferentially compromised, leading to loss of peripheral sensation. Although the metabolic basis for the demyelination and the structural and functional alterations of the nervous system in Cbl deficiency remain unresolved, the Cd320 -/- mouse provides a unique model to investigate the pathologic consequences of vitamin B 12 deficiency. -Arora, K., Sequeira, J. M., Alarcon, J. M., Wasek, B., Arning, E., Bottiglieri, T., Quadros, E. V. Neuropathology of vitamin B 12 deficiency in the Cd320 -/- mouse.

Vitamin B 12 deficiency causes megaloblastic anemia and neurologic disorder in humans. Gene defects of transcobalamin (TC) and the transcobalamin receptor (TCblR), needed for cellular uptake of the TC-bound B 12 , do not confer embryonic lethality. TC deficiency can produce the hematologic and neurologic complications after birth, whereas TCblR/ CD320 gene defects appear to produce mild metabolic changes. Alternate maternofetal transport mechanisms appear to provide adequate B 12 to the fetus. To understand this mechanism, we evaluated the role of TC, TCblR/ CD320 , and megalin in maternofetal transport of B 12 in a TCblR/ CD320 -knockout (KO) mouse. Our results showed high expression of TCblR/ CD320 in the labyrinth of the placenta, embryonic brain, and spinal column in wild-type (WT) mice. Megalin expression was about the same in both WT and KO mouse visceral yolk sac, brain, and spinal column. Megalin mRNA was down-regulated in the KO embryonic spinal cord (SC) and kidneys. Megalin expression remained unaltered in adult WT and KO mouse brain, SC, and kidneys. Injected dsRed-TC-B 12 and TC- 57 CoB 12 accumulated in the visceral yolk sac of KO mice where megalin is expressed and provides an alternate mechanism for the maternofetal transport of Cbl during fetal development.-Arora, K., Sequeira, J. M., Quadros, E. V. Maternofetal transport of vitamin B 12 : role of TCblR/ CD320 and megalin., (© FASEB.)

Cobalamin uptake into cells is mediated by the CD 320 receptor for transcobalamin-bound cobalamin. Optimum receptor expression is associated with proliferating cells and therefore, in many cancers this receptor expression is up regulated. Delivering drugs or toxins via this receptor provides increased targeting to cancer cells while minimizing toxicity to the normal tissues. Saporin conjugated monoclonal antibodies to the extracellular domain of TCblR were effectively internalized to deliver a toxic dose of Saporin to some cancer cell lines propagating in culture. Antibody concentration of 2.5 nM was effective in producing optimum inhibition of cell proliferation. The cytotoxic effect of mAb-Saporin appears to be dictated primarily by the level of receptor expression and therefore normal primary cells expressing low levels of CD 320 were spared while tumor cell lines with higher CD 320 expression were destroyed. Targeting the pathway for cellular uptake of vitamin B 12 via the CD 320 receptor with toxin-antibody conjugates appears to be a viable treatment strategy for certain cancers that over expresses this receptor.

The membrane receptor (TCblR/CD320) for transcobalamin (TC)-bound cobalamin (Cbl) facilitates the cellular uptake of Cbl. A genetically modified mouse model involving ablation of the CD320 gene was generated to study the effects on cobalamin homeostasis. The nonlethal nature of this knockout and the lack of systemic cobalamin deficiency point to other mechanisms for cellular Cbl uptake in the mouse. However, severe cobalamin depletion in the central nervous system (CNS) after birth (P<0.01) indicates that TCblR is the only receptor responsible for Cbl uptake in the CNS. Metabolic Cbl deficiency in the brain was evident from the increased methylmalonic acid (P<0.01-0.04), homocysteine (P<0.01), cystathionine (P<0.01), and the decreased S-adenosylmethionine/S-adenosyl homocysteine ratio (P<0.01). The CNS pathology of Cbl deficiency seen in humans may not manifest in this mouse model; however, it does provide a model with which to evaluate metabolic pathways and genes affected.