Yasumi Nakayama

The protein and the gene encoding the receptor for the cellular uptake of transcobalamin bound cobalamin

The transcobalamin (TC, TCII) receptor (TCblR) on the plasma membrane binds TC- cobalamin (Cbl) and internalizes the complex by endocytosis. This receptor was purified from human placental membranes by affinity chromatography. Tryptic digest of the protein extracted from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and subjected to liquid chromatography/mass spectrometry identified 4 peptides that matched with a membrane protein in the data bank. TCblR belongs to the low-density lipoprotein receptor family, with 2 low-density lipoprotein receptor type A domains separated by a complement-like cysteine-rich region. The 282-amino acid sequence includes a signal peptide of 31 residues, extracellular domain of 198 residues, a transmembrane region of 21 residues, and a cytoplasmic domain of 32 residues. The binding of TC-Cbl does not require the cytoplasmic domain or its orientation in the plasma membrane because the recombinant extracellular domain binds TC-Cbl with high affinity and specificity. The protein is heavily glycosylated and accounts for the 58-kDa size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The human gene first identified as 8D6A and more recently as CD 320 encoding TCblR is located at p13.2 on the short arm of chromosome 19, spans a length of 6.224 kb, and is composed of 5 exons and 4 introns.

Positive newborn screen for methylmalonic aciduria identifies the first mutation in TCblR/CD320, the gene for cellular uptake of transcobalamin‐bound vitamin B12

Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin‐bound cobalamin (holo‐TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo‐TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low‐density lipoprotein receptor type A‐like domain. Inserting the codon by site‐directed mutagenesis fully restored TCblR function. Hum Mutat 31:1–6, 2010.

The transcobalamin receptor knockout mouse: a model for vitamin B12 deficiency in the central nervous system

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.—Lai, S.‐C., Nakayama, Y., Sequeira, J. M., Wlodarczyk, B. J., Cabrera, R. M., Finnell, R. H., Bottiglieri, T., Quadros, E. V. The transcobalamin receptor knockout mouse: a model for vitamin B12 deficiency in the central nervous system. FASEB J. 27, 2468–2475 (2013). www.fasebj.org

Targeted Delivery of Saporin Toxin by Monoclonal Antibody to the Transcobalamin Receptor, TCblR/CD320

Cellular uptake of cobalamin (Cbl) occurs by endocytosis of transcobalamin saturated with Cbl by the transcobalamin receptor (TCblR/CD320). The cell cycle–associated overexpression of this receptor in many cancer cells provides a suitable target for delivering chemotherapeutic drugs and cytotoxic molecules to these cells while minimizing the effect on the normal cell population. We have used monoclonal antibodies to the extracellular domain of TCblR to deliver saporin-conjugated secondary antibody to various cell lines propagating in culture. A molar ratio of 2.5:10 nmol/L of primary:secondary antibody concentration was identified as the lowest concentration needed to produce the optimum cytotoxic effect. The effect was more pronounced when cells were seeded at lower density, suggesting lack of cell division in a fraction of the cells at higher density as the likely explanation. Cells in suspension culture, such as K562 and U266 cells, were more severely affected than adherent cultures, such as SW48 and KB cells. This differential effect of the anti–TCblR-saporin antibody conjugate and the ability of an anti-TCblR antibody to target proliferating cells were further evident by the virtual lack of any effect on primary skin fibroblasts and minimal effect on bone marrow cells. These results indicate that preferential targeting of some cancer cells could be accomplished through the TCblR. Mol Cancer Ther; 9(11); 3033–40. ©2010 AACR.

Down-regulation of transcobalamin receptor TCblR/CD320 by siRNA inhibits cobalamin uptake and proliferation of cells in culture

The clinical phenotype of cobalamin (Cbl) deficiency is dictated by the essential role of this vitamin in two key enzymatic reactions. Multiple proteins and receptors participate in the absorption, transport and delivery of this vitamin to tissue cells. Cellular uptake of Cbl is mediated by transcobalamin (TC), a plasma protein and a transmembrane receptor (TCblR) with high affinity for TC saturated with Cbl. Knockdown of TCblR with siRNA results in decreased TC-Cbl uptake. The ensuing Cbl deficiency leads to an increase in doubling time and decreased proliferation of these cells. The study confirms the seminal role of this receptor in the cellular uptake of Cbl and its down-regulation as a potential strategy to inhibit proliferation of cancer cells.

Characterizing monoclonal antibodies to antigenic domains of TCblR/CD320, the receptor for cellular uptake of transcobalamin-bound cobalamin.

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.

Mapping the functional domains of TCblR/CD320, the receptor for cellular uptake of transcobalamin-bound cobalamin

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 the receptor for TC‐Cbl ensures efficient uptake of Cbl into cells. The high‐affinity interaction of TCblR with TC‐Cbl (Ka=10 nM‐1) was investigated using deletions and mutations of amino acid sequences in TCblR. Only the extracellular 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 (Ka = 19–24 nM‐1). 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 the 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.—Jiang, W., Nakayama, Y., Sequeira, J. M.Quadros, E. V., Mapping the functional domains of TCblR/CD320, the receptor for cellular uptake of transcobalamin‐bound cobalamin. FASEB J. 27, 2988–2994 (2013). www.fasebj.org

Saporin Conjugated Monoclonal Antibody to the Transcobalamin Receptor TCblR/CD320 Is Effective in Targeting and Destroying Cancer Cells

Cobalamin uptake into cells is mediated by the CD320 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 CD320 were spared while tumor cell lines with higher CD320 expression were destroyed. Targeting the pathway for cellular uptake of vitamin B12 via the CD320 receptor with toxin-antibody conjugates appears to be a viable treatment strategy for certain cancers that over expresses this receptor.