Transcobalamin Research Articles

The structure of the rat vitamin B12 transporter TC and its complex with glutathionylcobalamin

Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor (IF) and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxo-Cbl (HOCbl) form can coordinate various nucleophiles, e.g., glutathione (GSH), giving rise to glutathionylcobalamin (GSCbl), a naturally-occurring form of vitamin B12. Currently there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B12-transporter and GSCbl, which compared to HOCbl, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, HC and IF are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl.

Determination of plasma transcobalamin-II and zinc levels in newly-diagnosed and long-standing grand mal epileptic patients.

The changes of plasma transcobalamin-II (TCII) and Zinc (Zn) Levels in epileptic patients are not clearly understood. The aim of the current study was to evaluate the plasma contents of TCII and Zn levels in newly-diagnosed epileptic seizure patients, long-standing grand mal epileptic patients following treatment with sodium valproate and healthy control group. Thirty patients aged 36.76±12.91 years with newly-diagnosed and thirty long-standing grand mal epileptic patients aged 35.56 ±12.77 years were diagnosed based on the clinical symptoms. The control subjects were picked out from healthy individuals and matched to the patients, aged 36.30 ±12.80 years. Plasma Zn and TCN-2 was evaluated via spectrophotometry at 546 nm and 450 nm, respectively, using chimerical kits. Plasmalevel of TCII in the newly-diagnosed epileptic seizures patients and long-standing grand mal epileptic patients were significantly increased, compared to the healthy controls [14.89 ±3.24 and 21.84± 2.73 vs. 9.55±1.24, (n=30)], respectively.Plasma level of Zn was decreased in the newly-diagnosed epileptic seizure patients, while it was increased in long-standing grand mal epileptic patients compared to the control group [69.28± 6.41and 80.56 ±6.12 and vs.75.80±1.59, (n=30)], respectively. This study suggests that sodium valproate may disrupt the homeostatic balance of TCII and Zn, and cause abnormality of their serum level in newly-diagnosed epileptic seizure patients and long-standing grand mal epileptic patients. Further research is recommended to identify the underpinning for these changes.

Early recognition and treatment of TC II deficiency

Background: Transcobalamin (TC) is a carrier protein and it delivers vitamin B12 to the cellular TC receptor. TC II deficiency is a very rare disease and is life-threatening if left untreated. It is an Autosomal recessive disease and needs lifelong treatment. The clinical presentations are variable, started at early infancy, and sometimes mimic severe combined immunodeficiency or acute leukemia. It includes failure to thrive, diarrhea, anemia and or pancytopenia, hypotonia, developmental delay, and recurrent infection. Diagnosis of TC II deficiency is suspected based on clinical presentations with megaloblastic anemia, the elevation of plasma homocysteine, and urine methylmalonic acid level with a normal vitamin B12 and folate level. Molecular analysis of the TCN 2 gene is needed for confirmation of the diagnosis. Case Presentation: We present a case of 2 years old Saudi boy who was admitted to the hospital with a history of fever, recurrent chest infection, failure to thrive with diarrhea, and hypotonia, and his complete blood counts showed Pancytopenia. Though, normal vitamin B12 level and folate level, homocysteine, and urine methylmalonic acid lever were elevated. Peripheral smear and bone marrow aspiration and biopsy revealed Hypersegmented neutrophils and megaloblastic change.

Early recognition and treatment of TC II deficiency

Transcobalamin (TC) is a carrier protein and it delivers vitamin B12 to the cellular TC receptor. TC II deficiency is a very rare disease and is life-threatening if left untreated. It is an Autosomal recessive disease and needs lifelong treatment. The clinical presentations are variable, started at early infancy, and sometimes mimic severe combined immunodeficiency or acute leukemia. It includes failure to thrive, diarrhea, anemia and or pancytopenia, hypotonia, developmental delay, and recurrent infection. Diagnosis of TC II deficiency is suspected based on clinical presentations with megaloblastic anemia, the elevation of plasma homocysteine, and urine methylmalonic acid level with a normal level of vitamin B12 and folate. Molecular analysis of the TCN 2 gene is needed for confirmation of the diagnosis. We present a case of 2 years old Saudi boy who was admitted to the hospital with a history of fever, recurrent chest infection, failure to thrive with diarrhea, and hypotonia, and his complete blood counts showed Pancytopenia. Though, normal vitamin B12 level and folate level, homocysteine, and urine methylmalonic acid lever were elevated. Peripheral smear and bone marrow aspiration and biopsy revealed Hypersegmented neutrophils and megaloblastic change.

Vitamin B12 binding to mutated human transcobalamin, in-silico study of TCN2 alanine scanning and ClinVar missense mutations/SNPs

Many missense mutations/SNPs of the TCN2 gene (which yield Transcobalamin (TC)) were reported in the literature but no study is available about their effect on binding to vitamin B12(B12) at the structural level experimentally nor computationally. Predict the effect of TC missense mutations/SNPs on binding affinity to B12 and characterize their contacts to B12 at the structural level. TC-B12 binding energy difference from the wildtype (ΔΔGmut) was calculated for 378 alanine scanning mutations and 76 ClinVar missense mutations, repeated on two distinct X-ray structures of holoTC namely 2BB5 and 4ZRP. Destabilizing mutations then went through 100 ns molecular dynamics simulation to study their effect on TC-B12 binding at the structural level employing 2BB5 structure. Out of the studied 454 mutations (378 alanine mutations + 76 ClinVar mutations), 19 were destabilizing representing 17 amino acid locations. Mutation energy results show a neutral effect on B12 binding of several missense SNPs reported in the literature including I23V, G94S, R215W, P259R, S348F, L376S, and R399Q. Compared to the wildtype, all the destabilizing mutations have higher average RMSD-Ligand in the last 25% of the MD simulation trajectories and lower average hydrogen bond count while the other parameters vary. Previously reported TCN2 SNPs with an unknown effect on TC-B12 binding were found to have a neutral effect in the current study based on mutation energy calculations. Also, we reported 17 possible amino acids that destabilize TC-B12 binding upon mutation (four listed in ClinVar) and studied their structural effect computationally. Communicated by Ramaswamy H. Sarma

Generation of nanobodies targeting the human, transcobalamin-mediated vitamin B12 uptake route.

Cellular uptake of vitamin B12 in humans is mediated by the endocytosis of the B12 carrier protein transcobalamin (TC) via its cognate cell surface receptor TCblR, encoded by the CD320gene. Because CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells including cancer cells, this uptake route is a potential target for cancer therapy. We developed and characterized four camelid nanobodies that bind holo-TC (TC in complex with B12 ) or the interface of the human holo-TC:TCblR complex with nanomolar affinities. We determined X-ray crystal structures of these nanobodies bound to holo-TC:TCblR, which enabled us to map their binding epitopes. When conjugated to the model toxin saporin, three of our nanobodies caused growth inhibition of HEK293T cells and therefore have the potential to inhibit the growth of human cancer cells. We visualized the cellular binding and endocytic uptake of the most potent nanobody (TC-Nb4) using fluorescent light microscopy. The co-crystal structure of holo-TC:TCblR with another nanobody (TC-Nb34) revealed novel features of the interface of TC and the LDLR-A1 domain of TCblR, rationalizing the decrease in the affinity of TC-B12 binding caused by the Δ88mutation in CD320.

Response of Human Glioblastoma Cells to Vitamin B12 Deficiency: A Study Using the Non-Toxic Cobalamin Antagonist.

Simple SummaryThe most important biological function of vitamin B12 (cobalamin) is to accomplish DNA synthesis, which is necessary for cell division. Cobalamin deficiency may be especially acute for rapidly dividing cells, such as glioblastoma cells. Therefore, cobalamin antagonists offer a medicinal potential for developing anti-glioma agents. In the present study, we revealed, for the first time, that the induction of cobalamin deficiency by vitamin B12 antagonist with affinity to key transporter of cobalamins, inhibited glioblastoma cells growth and promoted cell cycle arrest. The effect was observed for non-toxic concentration of the agent, as demonstrated on zebrafish. Moreover, as compared to our previous study, the cytostatic effect of the agent was more pronounced in glioblastoma cells than in normal astrocytes. We believe that the study may become the basis for further in vitro and in vivo experiments concerning cobalamin deprivation as a potential therapeutic strategy for glioblastoma.The most important biological function of vitamin B12 is to accomplish DNA synthesis, which is necessary for cell division. Cobalamin deficiency may be especially acute for rapidly dividing cells, such as glioblastoma cells. Therefore, cobalamin antagonists offer a medicinal potential for developing anti-glioma agents. In the present study, we developed an in vitro model of cobalamin deficiency in glioblastoma cells. Long-term treatment of cells with the cobalamin analogue, hydroxycobalamin [c-lactam] (HCCL) was applied to induce an increase of hypocobalaminemia biomarker. Cytometric assays demonstrated that vitamin B12 promoted glioblastoma cells proliferation, whereas the treatment of cells with HCCL caused a dramatic inhibition of cell proliferation and an induction of cell cycle arrest at the G2/M phase. Vitamin B12 counteracted all the observed effects of HCCL. In the in silico study, we characterized the molecular interactions between HCCL and transcobalamin II (TCII). We have demonstrated that HCCL shares similar interactions with TCII as naturally occurring cobalamins and therefore may act as a competitive inhibitor of this key transporter protein. We assessed the impact of HCCL on the mortality or developmental malformations of zebrafish embryos. Collectively, our findings suggest that the use of cobalamin transport antagonists as potential anti-glioma agents would be worth exploring further.

Cellular uptake of vitamin B12: Role and fate of TCblR/CD320, the transcobalamin receptor

Cellular uptake of vitamin B12 (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.

Assessment of cellular cobalamin metabolism in Gaucher disease

BackgroundGaucher disease (GD) is a lysosomal disorder caused by biallelic pathogenic mutations in the GBA1 gene that encodes beta-glucosidase (GCase), and more rarely, by a deficiency in the GCase activator, saposin C. Clinically, GD manifests with heterogeneous multiorgan involvement mainly affecting hematological, hepatic and neurological axes. This disorder is divided into three types, based on the absence (type I) or presence and severity (types II and III) of involvement of the central nervous system. At the cellular level, deficiency of GBA1 disturbs lysosomal storage with buildup of glucocerebroside. The consequences of disturbed lysosomal metabolism on biochemical pathways that require lysosomal processing are unknown. Abnormal systemic markers of cobalamin (Cbl, B12) metabolism have been reported in patients with GD, suggesting impairments in lysosomal handling of Cbl or in its downstream utilization events.MethodsCultured skin fibroblasts from control humans (n = 3), from patients with GD types I (n = 1), II (n = 1) and III (n = 1) and an asymptomatic carrier of GD were examined for their GCase enzymatic activity and lysosomal compartment intactness. Control human and GD fibroblasts were cultured in growth medium with and without 500 nM hydroxocobalamin supplementation. Cellular cobalamin status was examined via determination of metabolomic markers in cell lysate (intracellular) and conditioned culture medium (extracellular). The presence of transcobalamin (TC) in whole cell lysates was examined by Western blot.ResultsCultured skin fibroblasts from GD patients exhibited reduced GCase activity compared to healthy individuals and an asymptomatic carrier of GD, demonstrating a preserved disease phenotype in this cell type. The concentrations of total homocysteine (tHcy), methylmalonic acid (MMA), cysteine (Cys) and methionine (Met) in GD cells were comparable to control levels, except in one patient with GD III. The response of these metabolomic markers to supplementation with hydroxocobalamin (HOCbl) yielded variable results. The content of transcobalamin in whole cell lysates was comparable in control human and GD patients.ConclusionsOur results indicate that cobalamin transport and cellular processing pathways are overall protected from lysosomal storage damage in GD fibroblasts. Extending these studies to hepatocytes, macrophages and plasma will shed light on cell- and compartment-specific vitamin B12 metabolism in Gaucher disease.

Vitamin B12 and its binding proteins in milk from cow and buffalo in relation to bioavailability of B12

Milk is an important source of highly bioavailable vitamin B12 (cobalamin) in human nutrition. In most animal products, vitamin B12 is strongly bound to various specific protein carriers. The 2 vitamin B12-specific proteins, predominantly transcobalamin (TC) and haptocorrin (HC), were earlier found in milk from Holstein Friesian cows and in human or sow milk, respectively. As the type of vitamin B12 binders may influence bioavailability of the vitamin, we examined vitamin B12 carriers in pooled milk specimens derived from European and Indian cow and buffalo herds. The total endogenous vitamin B12 concentration was comparable in all milk pools (≈3 nM), but the vitamin carriers varied considerably: TC + caseins in Danish cows, TC + HC in Indian cows and buffaloes, and mainly HC in Italian buffaloes. Danish cow milk contained half as much TC as vitamin B12, and the surplus vitamin was all attached via a single coordination bond to abundantly available histidine residues of casein. The specific binding proteins in Indian cow milk (TC + HC) approximately matched the molar content of vitamin B12. Milk from the 2 buffalo breeds contained more specific binders than vitamin B12, and the surplus proteins included the unsaturated TC ≈ 3 nM (Indian stock), or both TC ≈ 4 nM and HC ≈ 23 nM (Italian stock). The abundant HC of the latter sample bound nearly all endogenous vitamin B12. We tested (in vitro) the transfer of vitamin B12 from milk proteins to human carriers, involved in the intestinal uptake. The bovine TC-vitamin B12 complex rapidly dissociated at pH 2 (time of half reaction, τ1/2 < 1 min, 37°C) and was susceptible to digestion with trypsin + chymotrypsin (pH 7.5). Transfer of vitamin B12 from the precipitated bovine casein (pH 2) to human carriers proceeded with τ1/2 ≈ 7 min (37°C) and τ1/2 ≈ 35 min (20°C). Liberation of vitamin B12 from buffalo HC was hampered because of its pH stability and slow proteolysis. Nutritional availability of vitamin B12 is expected to be high in cow milk (with TC-vitamin B12 and casein-vitamin B12 complexes) but potentially constrained in buffalo milk (with HC-vitamin B12). This especially concerns the Italian buffalo milk, where a high excess of HC was found. We speculate whether the isolated stock of Italian buffalo maintained the ancestral secretion of carriers (HC ≫ vitamin B12, TC ≈ 0), whereas intensive crossbreeding of cows and buffaloes from other regions caused a change to TC ≤ vitamin B12, with low or absent HC. The substitution of HC by less sturdy carriers is apparently more beneficial to human consumers as far as vitamin B12 bioavailability is concerned.

Functional and phylogenetic characterization of noncanonical vitamin B12–binding proteins in zebrafish suggests involvement in cobalamin transport

In humans, transport of food-derived cobalamin (vitamin B12) from the digestive system into the bloodstream involves three paralogous proteins: transcobalamin (TC), haptocorrin (HC), and intrinsic factor (IF). Each of these proteins contains two domains, an α-domain and a β-domain, which together form a cleft in which cobalamin binds. Zebrafish (Danio rerio) are thought to possess only a single cobalamin transport protein, referred to as Tcn2, which is a transcobalamin homolog. Here, we used CRISPR/Cas9 mutagenesis to create null alleles of tcn2 in zebrafish. Fish homozygous for tcn2-null alleles were viable and exhibited no obvious developmentally or behaviorally abnormal phenotypes. For this reason, we hypothesized that previously unidentified cobalamin-carrier proteins encoded in the zebrafish genome may provide an additional pathway for cobalamin transport. We identified genes predicted to code for two such proteins, Tcn-beta-a (Tcnba) and Tcn-beta-b (Tcnbb), which differ from all previously characterized cobalamin transport proteins as they lack the α-domain. These β-domain-only proteins are representative of an undescribed class of cobalamin-carrier proteins that are highly conserved throughout the ray-finned fishes. We observed that the genes encoding the three cobalamin transport homologs, tcn2, tcnba, and tcnbb, are expressed in unique spatial and temporal patterns in the developing zebrafish. Moreover, exogenously expressed recombinant Tcnba and Tcnbb bound cobalamin with high affinity, comparable with binding by full-length Tcn2. Taken together, our results suggest that this noncanonical protein structure has evolved to fully function as a cobalamin-carrier protein, thereby allowing for a compensatory cobalamin transport mechanism in the tcn2-/- zebrafish.

Mice lacking the transcobalamin-vitamin B12 receptor, CD320, suffer from anemia and reproductive deficits when fed vitamin B12-deficient diet.

In humans, poor nutrition, malabsorption and variation in cobalamin (vitamin B12) metabolic genes are associated with hematological, neurological and developmental pathologies. Cobalamin is transported from blood into tissues via the transcobalamin (TC) receptor encoded by the CD320 gene. We created mice carrying a targeted deletion of the mouse ortholog, Cd320. Knockout (KO) mice lacking this TC receptor have elevated levels of plasma methylmalonic acid and homocysteine but are otherwise healthy, viable, fertile and not anemic. To challenge the Cd320 KO mice we maintained them on a vitamin B12-deficient diet. After 5 weeks on this diet, reproductive failure develops in Cd320 KO females but not males. In vitro, homozygous Cd320 KO embryos from cobalamin-deficient Cd320 KO dams develop normally to embryonic day (E) 3.5, while in vivo, few uterine decidual implantation sites are observed at E7.5, suggesting that embryos perish around the time of implantation. Dietary restriction of vitamin B12 induces a severe macrocytic anemia in Cd320 KO mice after 10-12 months while control mice on this diet are anemia-free up to 2 years. Despite the severe anemia, cobalamin-deficient KO mice do not exhibit obvious neurological symptoms. Our results with Cd320 KO mice suggest that an alternative mechanism exists for mice to transport cobalamin independent of the Cd320 encoded receptor. Our findings with deficient diet are consistent with historical and epidemiological data suggesting that low vitamin B12 levels in humans are associated with infertility and developmental abnormalities. Our Cd320 KO mouse model is an ideal model system for studying vitamin B12 deficiency.

Investigation of Receptor-Mediated Cyanocobalamin (Vitamin B12) Transport across the Inner Blood-Retinal Barrier Using Fluorescence-Labeled Cyanocobalamin.

The blood-to-retina supply of cyanocobalamin (vitamin B12) across the blood-retinal barrier (BRB) was investigated by synthesizing a fluorescence-labeled cyanocobalamin (Cy5-cyanocobalamin). In the in vivo analysis following internal jugular injection of Cy5-cyanocobalamin, confocal microscopy showed the distribution of Cy5-cyanocobalamin in the inner plexiform layer (IPL), the outer plexiform layer (OPL), and the retinal pigment epithelium (RPE). In the in vitro analysis with TR-iBRB2 cells, an in vitro model cell line of the inner BRB, Cy5-cyanocobalamin uptake by TR-iBRB2 cells exhibited a time-dependent increase after preincubation with transcobalamin II (TCII) protein, during its residual uptake without preincubation with TCII protein. The Cy5-cyanocobalamin uptake by TR-iBRB2 cells was significantly reduced in the presence of unlabeled cyanocobalamin, chlorpromazine, and chloroquine and was also significantly reduced under Ca2+-free conditions. Confocal microscopy of the TR-iBRB2 cells showed fluorescence signals of Cy5-cyanocobalamin and GFP-TCII protein, and these signals merged with each other. The RT-PCR, Western blot, and immunohistochemistry clearly suggested the expression of TCII receptor (TCII-R) in the inner and outer BRB. These results suggested the involvement of receptor-mediated endocytosis in the blood-to-retina transport of cyanocobalamin at the inner BRB with implying its possible involvement at the outer BRB.

Organometallic DNA-B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins.

The synthesis and structural characterization of Co-(dN)25 -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN)39 -Cbl, which are organometallic DNA-B12 conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN)25 -Cbl, or radiolabeled vitamin B12 (57 Co-CNCbl) and Co-(dN)25 -Cbl or Co-(dN)39 -Cbl. Binding of the new DNA-Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA-Cbl, Co-(dN)18 -Cbl. The contrasting affinities of TC versus IF and HC for the DNA-Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA-Cbl, which is first bound to the respective β domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA-Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells.

89Zr-Cobalamin PET Tracer: Synthesis, Cellular Uptake, and Use for Tumor Imaging.

Vitamin B12, or cobalamin (Cbl), is an essential nutrient. Acquisition, transport, and cellular internalization of Cbl are dependent on specific binding proteins and associated receptors. The circulating transport protein transcobalamin (TC) promotes cellular uptake via binding to specific receptors such as CD320, a receptor upregulated in several cancer cell lines. In this study, we report the successful synthesis of 89Zirconium-labeled Cbl that was derivatized with desferrioxamine (89Zr-Cbl). We document the purity of the tracer and its binding to TC compared with that of unmodified cyano-Cbl (CN-Cbl). In vitro studies employing the CD320 receptor-positive breast cancer cell line MDA-MB-453 showed a 6- to 10-fold greater uptake of 89Zr-Cbl when compared with the uptake in the presence of 200-fold excess of CN-Cbl at 37 °C. We used nude mice with MDA-MB-453 tumors to study the feasibility of employing the tracer to visualize CD320 positive tumors. In vivo positron emission tomography images displayed a clear visualization of the tumor with 1.42 ± 0.48 %ID/g uptake (n = 3) at 4 h after injection (p.i.) with the tracer retained at 48 h p.i. Ex vivo biodistribution studies using 89Zr-Cbl exhibited the highest uptake in kidney and liver at 48 h p.i. Results document the feasibility of synthesizing a Cbl-based tracer suitable for both in vivo and ex vivo studies of Cbl trafficking and with the potential to visualize tumors expressing TC receptors, such as CD320.

Structure of the human transcobalamin beta domain in four distinct states.

Vitamin B12 (cyanocobalamin, CNCbl) is an essential cofactor-precursor for two biochemical reactions in humans. When ingested, cobalamins (Cbl) are transported via a multistep transport system into the bloodstream, where the soluble protein transcobalamin (TC) binds Cbl and the complex is taken up into the cells via receptor mediated endocytosis. Crystal structures of TC in complex with CNCbl have been solved previously. However, the initial steps of holo-TC assembly have remained elusive. Here, we present four crystal structures of the beta domain of human TC (TC-beta) in different substrate-bound states. These include the apo and CNCbl-bound states, providing insight into the early steps of holo-TC assembly. We found that in vitro assembly of TC-alpha and TC-beta to a complex was Cbl-dependent. We also determined the structure of TC-beta in complex with cobinamide (Cbi), an alternative substrate, shedding light on the specificity of TC. We finally determined the structure of TC-beta in complex with an inhibitory antivitamin B12 (anti-B12). We used this structure to model the binding of anti-B12 into full-length holo-TC and could rule out that the inhibitory function of anti-B12 was based on an inability to form a functional complex with TC.

Measurement of total Transcobalamin employing a commercially available assay for Active B12

IntroductionVitamin B12 deficiency is mostly caused by insufficient gastro-intestinal absorption and in rare conditions by Transcobalamin (TC) deficiency. Unsaturated Transcobalamin (apoTC) can be measured by a binding assay using radiolabeled cobalamin. The Active B12 test analyzes saturated Transcobalamin (holoTC) and we hypothesize that this test can be used to measure total TC by additional in vitro saturation with cobalamin. MethodsSerum was saturated in vitro (16 times dilution) with a cyanocobalamin solution and total TC was selectively measured with the Abbott Active B12 test. ApoTC was calculated by subtracting endogenous holoTC from total TC after correction for dilution. Linearity was determined with a pool serum dilution series. Precision was investigated according to the CLSI EP15 protocol. Method comparison was performed against a binding assay using radiolabeled cobalamin. Reference values were determined in 100 healthy controls. ResultsThe method was linear in the range of 240 to 1933pmol/L (R2=0.997, lack of fit F=1.61). Precision of low- and high-pool total TC in serum were; 5.2% and 4.3% respectively. Method comparison against a radiolabeled cobalamin binding assay showed a proportional bias of 30% (y=0.70x+126). Total TC reference values were determined at 500–1276pmol/L. ConclusionWe describe a rapid method to quantify total TC, which can be implemented on routine platforms using commercial Active B12 tests. In addition, apoTC can be assessed by subtracting endogenous holoTC concentration which can be measured in the same run, securing the same calibration level for all three parameters (holoTC, apoTC and total TC). This method is applicable in clinical diagnostics and in larger epidemiological studies.

Maternofetal transport of vitamin B12: role of TCblR/CD320 and megalin.

Vitamin B12 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 B12, 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 B12 to the fetus. To understand this mechanism, we evaluated the role of TC, TCblR/CD320, and megalin in maternofetal transport of B12 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-B12 and TC-57CoB12 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 B12: role of TCblR/CD320 and megalin.

Structural basis of transcobalamin recognition by human CD320 receptor.

Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway.

Vitamin B12 and placental expression of transcobalamin in pregnant adolescents

BackgroundTranscobalamin is a key placental protein involved in transport of vitamin B12 to the fetus. However, few data currently exist on the ability of the placenta to modify vitamin B12 transporter expression, particularly in high-risk populations such as pregnant adolescents. ObjectiveThis study was conducted to determine the impact of maternal and neonatal serum vitamin B12 concentrations on placental transcobalamin (TC) expression in a cohort of healthy pregnant adolescents in the United States. DesignSerum vitamin B12 concentrations were measured in maternal blood samples at mid-gestation (26.4 ± 2.8 weeks) and delivery (39.8 ± 1.4 weeks) and infant cord blood samples at birth. Placentas were collected at delivery and TC mRNA expression (ΔΔCt) and TC protein abundance (TC:α-actin) were evaluated. Linear and binomial regression models were used to examine the associations of maternal serum (mid-gestation, delivery) and cord blood vitamin B12 concentrations with placental TC mRNA expression and protein abundance (n = 63). ResultsMaternal serum vitamin B12 concentrations at mid-gestation or delivery were not significantly associated with placental TC mRNA expression or TC protein abundance (p > 0.05). Higher placental TC protein abundance was associated with increased cord blood vitamin B12 concentrations (p = 0.003). ConclusionsHigher placental TC protein abundance was associated with higher cord blood vitamin B12 concentrations, suggesting a potential role in vitamin B12 transport to the fetus.