Biological effects of Inactivating TCblR/CD320: The Gene Encoding the Receptor for Cellular Uptake of Transcobalamin-bound Cobalamin

Abstract

The clinical, haematological and neuropathological disorders that result from B12 deficiency have been well defined. The hematological presentations of B12 (Cbl) deficiency cannot be distinguished from that of folate deficiency. In addition, hyperhomocysteinemia resulting from B12 deficiency is considered as one of the risk factors for cardiovascular and neurodegenerative diseases. The recent identification of the gene (CD320) encoding the membrane receptor (TCblR) for cellular uptake of transcobalamin (TC) bound cobalamin (Cbl) has enabled us to study the cellular effects of TCblR gene knockdown in vitro and knockout in vivo and investigate potential TCblR gene mutations that could cause vitamin B12 deficiency. To accomplish the studies on TCblR, I proposed four aims: 1) to study TCblR expression in normal and cancer cell lines, 2) to study the effect of TCblR siRNA induced gene knockdown on cell proliferation, 3) to characterize a TCblR knockout mouse model for studying vitamin B12 deficiency related disorders, 4) to Identify of a mutation in the TCblR gene as the cause of neonatal methylmalonic aciduria. Our data showed that the mRNA level measured by quantitative real-time PCR (qRT-PCR) and receptor expression by TC-Cbl binding in HEK293 cells was similar with higher receptor expression during the log phase of cell growth and low expression in confluent cells. Also, the data from functional receptor assay and qRT-PCR showed higher TCblR expression in some cancer cell lines compared to two normal primary human fibroblast lines. These data suggest that TCblR could be a potential therapeutic target to treat cancer or deliver drug to tumors. To study the effect on cell proliferation by blocking vitamin B12 uptake, three siRNAs were tested and showed greater that 80% downregulation of TCblR. Our data showed that down-regulation of TCblR by gene-specific siRNAs caused HEK293 and SW48 cell lines to stop proliferating. However, HEK293 cells overexpressing TCblR showed no inhibition of cell proliferation, which indicated that the decrease of cell proliferation was specifically due to blocking vitamin B12 uptake by down regulating TCblR expression. To characterize TCblR/CD320 (TCblR-/-) knockout mice, we first demonstrated that the TCblR-/- mice lack TCblR expression from embryo to adult. Second, we determined levels of vitamin B12 in both WT and TCblR-/- mice. Surprisingly, we found that the B12 levels in the brain and spinal cord were most diminished in TCblR-/- mice. We confirmed that TCblR-/- mice develop B12 deficiency by showing elevated homocystyeine and MMA in TCblR-/- mouse serum. The diminished B12 levels in CNS resulted in decreased global DNA methylation and imbalanced homocysteine metabolism. These results led to studies that examined behavior and cognitive function in TCblR-/- mice utilizing novel object recognition and place avoidance tests. However, both tests showed that there is no difference on behavior and cognitive function between WT and TCblR-/- mouse groups, which indicated that the absence of vitamin B12 in brain does not affect memory and learning in the mouse. Furthermore, complete blood count (CBC) analysis showed no hematological deficits (anemia) in TCblR-/- mice. In a case of neonatal methylmalonic aciduria, to investigate a potential defect in the receptor as the likely cause of decreased Cbl uptake into cells, patient’s fibroblast were analyzed. The mRNA level of TCblR as well as the size of the transcript was normal. Sequence analysis of the cDNA identified a codon deletion at nt 262 – 264 (GAG). This resulted in a single glutamic acid deletion in the first LDLR type A domain, which is critical for TC-Cbl binding and appears to have contributed to a 50% decrease in affinity and uptake. Increasing B12 intake to maintain a higher cobalamin concentration may be advocated for patients with this gene defect. These studies provided critical information on TCblR and also demonstrated the important role of TCblR to B12 Homeostasis in CNS using TCblR-/- mouse model. However, TCblR-/- mouse model does not develop megaloblastic anemia, memory and learning deficits, further investigation will be needed to determine whether TCblR-/- mouse develops neuro-pathological changes seen in human B12 deficiency. On the other hand, in order to understand the role of TCblR in cancer, more in vivo studies are needed.